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Induction of Exocytosis from Permeabilized Mast Cells by the Guanosine Triphosphatases Rac and Cdc42
Abstract
We applied recombinant forms of the Rho-related small guanosine triphosphatases (GTPases) Rac2 and Cdc42/G25K to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells permeabilized with streptolysin-O leak soluble (cytosol) proteins over a period of 5 min and become refractory to stimulation by Ca2+ and guanosine triphosphate (GTP)gammaS over about 20-30 min. This loss of sensitivity is likely to be due to loss of key regulatory proteins that are normally tethered at intracellular locations. Exogenous proteins that retard this loss of sensitivity to stimulation may be similar, if not identical, to those secretory regulators that are lost. Recombinant Rac and Cdc42/G25K, preactivated by binding GTPgammaS, retard the loss of sensitivity (run-down) and, more importantly, enable secretion to be stimulated by Ca2+ alone. Investigation of the concentration dependence of each of these two GTPases applied individually to the permeabilized cells, and of Cdc42/G25K applied in the presence of an optimal concentration of Rac2, has provided evidence for a shared effector pathway and also a second effector pathway activated by Cdc42/G25K alone. Dominant negative mutant (N17) forms of Rac2 and Cdc42/G25K inhibit secretion induced by Ca2+ and GTPgammaS. Our data suggest that Rac2 and Cdc42 should be considered as candidates for GE, GTPases that mediate exocytosis in cells of hematopoeitic origin.
... The effect of the βγsubunits upon cell responsiveness always took a few minutes to become apparent (see Figure 3). This appears to be a general characteristic of all proteins modulating the run-down process, both negatively and positively Brown et al., 1998), and most probably registers the time taken for diffusion into the cells through the permeability lesions and then for interaction with intracellular targets. ...
... We recently have reported that the Rho-related GTPases Rac and Cdc42 can retard the rate of run-down of permeabilized mast cells (Brown et al., 1998). When preactivated by binding GTPγS, they are both capable of inducing substantial secretion in the absence of free guanine nucleotide, and this indicates that they can assume the role of the endogenous GTPases which normally mediate the activating signals for exocytosis. ...
... Confidence in this conclusion is found in the inhibition of exocytosis by RhoGDI (Mariot et al., 1996;O'Sullivan et al., 1996) which binds to all of the Rho-related GTPases including Rac and Cdc42 (Tanaka et al., 1995). When permeabilized mast cells are treated with pre-activated Rac or Cdc42 together with free GTPγS, a further increment in secretion can be induced (Brown et al., 1998). Depending on which Rho-related GTPase is applied as the stimulus, the effect of free GTPγS either amplifies the response, as with Rac, or is interactive, sensitizing the system so that secretion occurs at a reduced concentration of the applied GTPase, as with Cdc42. ...
We applied G protein-derived beta gamma-subunits to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells permeabilized with streptolysin-O leak soluble (cytosol) proteins over a period of 5 min and become refractory to stimulation by Ca2+ and GTPgammaS over approximately 20-30 min. beta gamma-Subunits applied to the permeabilized cells retard this loss of sensitivity to stimulation (run-down) and it can be inferred that they interact with the regulatory mechanism for secretion. While alpha-subunits are without effect, beta gamma-subunits at concentrations >10(-8 )M enhance the secretion due to Ca2+ and GTPgammaS. Unlike the small GTPases Rac and Cdc42, beta gamma-subunits cannot induce secretion in the absence of an activating guanine nucleotide, and thus further GTP-binding proteins (likely to be Rho-related GTPases) must be involved. The enhancement due to beta gamma-subunits is mediated largely through interaction with pleckstrin homology (PH) domains. It remains manifest in the face of maximum activation by PMA and inhibition of PKC with the pseudosubstrate inhibitory peptide. Soluble peptides mimicking PH domains inhibit the secretion due to GTPgammaS and block the enhancement due to beta gamma-subunits. Our data suggest that beta gamma-subunits are components of the pathway of activation of secretion due to receptor-mimetic ligands such as mastoparan and compound 48/80.
... In secretory cells, cytoskeletal rearrangements are a prerequisite for exocytosis, enabling docking and fusion of secretory granules with the plasma membrane (Linstedt and Kelly, 1987;Sontag et al., 1988;Norman et al., 1994;Vitale et al., 1995). Indeed, it has been recently reported that GTPactivated Rac and Cdc42 can induce exocytosis in permeabilized mast cells (Brown et al., 1998). A role for Rac and Cdc42 has also been described in the late steps of calciuminduced insulin secretion from pancreatic β cells (Kowluru et al., 1997). ...
... It is tempting to speculate that active Cdc42 supports exocytosis by inducing specific actin-based structures. Accordingly, the active participation of Cdc42 in the exocytotic reaction in pancreatic β cells (Kowluru et al., 1997) and mast cells (Brown et al., 1998) has been recently described. ...
... Indeed, in agreement with our previous observations (Gasman et al., 1997), we found here that inactivation of RhoA by C. botulinum does not modify secretagogue-evoked secretion in chromaffin cells even under conditions that caused large ADPribosylation of Rho. Furthermore, despite the evidence pointing to a key regulatory function for Rho-related GTPases in secretion in mast cells (Brown et al., 1998), basophilic leukemia cells (Prepens et al., 1996) and pancreatic β cells (Kowluru et al., 1997), the active role of Rho itself in the exocytotic machinery has been excluded in these secretory cell models. Rho may be needed in resting cells to connect secretory granules with the actin cytoskeleton and restrict granule movement, thus preventing any uncontrolled secretory responses. ...
The Rho GTPase family, including Rho, Rac and Cdc42 proteins, is implicated in various cell functions requiring the reorganization of actin-based structures. In secretory cells, cytoskeletal rearrangements are a prerequisite for exocytosis. We previously described that, in chromaffin cells, the trimeric granule-bound Go protein controls peripheral actin and prevents exocytosis in resting cells through the regulation of RhoA. To provide further insight into the function of Rho proteins in exocytosis, we focus here on their intracellular distribution in chromaffin cells. By confocal immunofluorescence analysis, we found that Rac1 and Cdc42 are exclusively localized in the subplasmalemmal region in both resting and nicotine-stimulated cells. In contrast, RhoA is associated with the membrane of secretory granules. We then investigated the effects of clostridial toxins, which differentially impair the function of Rho GTPases, on the subplasmalemmal actin network and catecholamine secretion. Clostridium difficile toxin B, which inactivates Rho, Rac and Cdc42, markedly altered the distribution of peripheral actin filaments. Neither Clostridium botulinum C3 toxin, which selectively ADP-ribosylates Rho, nor Clostridium sordellii lethal toxin, which inactivates Rac, affected cortical actin, suggesting that Cdc42 plays a specific role in the organization of subplasmalemmal actin. Indeed, toxin B strongly reduced secretagogue-evoked catecholamine release. This effect on secretion was not observed in cells having their actin cytoskeleton depolymerized by cytochalasin E or Clostridium botulinum C2 toxin, suggesting that the inhibition of secretion by toxin B is entirely linked to the disorganization of actin. C. sordellii lethal toxin also inhibited catecholamine secretion, but this effect was not related to the actin cytoskeleton as seen in cells pretreated with cytochalasin E or C2 toxin. In contrast, C3 exoenzyme did not affect secretion. We propose that Cdc42 plays an active role in exocytosis by coupling the actin cytoskeleton to the sequential steps underlying membrane trafficking at the site of exocytosis.
... In endocrine cells, members of the Rho family have been proposed to regulate exocytosis. Indeed, Cdc42 and Rac control regulated secretion in pancreatic beta cells (12), basophilic leukemia cells (13), and mast cells (14). In chromaffin cells, RhoA localized on secretory granules controls subplasmalemmal actin and exocytosis by regulating a granule-associated phosphatidylinositol 4-kinase (15,16). ...
... Interestingly, functional Rac is needed for regulated exocytosis in rat basophilic leukemia cells (13), pancreatic beta cells (12), and mast cells (14). On the other hand, Rho is not considered to be an active regulator of secretion (12)(13)(14)(15), despite the finding that activated RhoA stabilizes actin filaments around the secretory granules (15,16). ...
... Interestingly, functional Rac is needed for regulated exocytosis in rat basophilic leukemia cells (13), pancreatic beta cells (12), and mast cells (14). On the other hand, Rho is not considered to be an active regulator of secretion (12)(13)(14)(15), despite the finding that activated RhoA stabilizes actin filaments around the secretory granules (15,16). Thus, our hypothesis that Rac1 is involved in evoked neurotransmitter release correlates well with these data obtained in endocrine cells. ...
Rho, Rac, and Cdc42 monomeric GTPases are well known regulators of the actin cytoskeleton and phosphoinositide metabolism
and have been implicated in hormone secretion in endocrine cells. Here, we examine their possible implication in Ca2+-dependent exocytosis of neurotransmitters. Using subcellular fractionation procedures, we found that RhoA, RhoB, Rac1, and
Cdc42 are present in rat brain synaptosomes; however, only Rac1 was associated with highly purified synaptic vesicles. To
determine the synaptic function of these GTPases, toxins that impair Rho-related proteins were microinjected into Aplysia neurons. We used lethal toxin from Clostridium sordellii, which inactivates Rac; toxin B from Clostridium difficile, which inactivates Rho, Rac, and Cdc42; and C3 exoenzyme from Clostridium botulinum and cytotoxic necrotizing factor 1 from Escherichia coli, which mainly affect Rho. Analysis of the toxin effects on evoked acetylcholine release revealed that a member of the Rho
family, most likely Rac1, was implicated in the control of neurotransmitter release. Strikingly, blockage of acetylcholine
release by lethal toxin and toxin B could be completely removed in <1 s by high frequency stimulation of nerve terminals.
Further characterization of the inhibitory action produced by lethal toxin suggests that Rac1 protein regulates a late step
in Ca2+-dependent neuroexocytosis.
... Rho family members also regulate secretion. In mast cells RhoA, Rac1, and Cdc42 stimulate exocytosis of secretory granules, while C-3 transferase and dominant inactive RhoA and Rac1 inhibit GTPγS-stimulated secretion (Brown et al., 1998;Mariot et al., 1996;Norman et al., 1996;O'Sullivan et al., 1996;Price et al., 1995). New evidence indicates that Cdc42 may be important in regulating delivery of newly synthesized proteins to the basolateral domain of polarized Madin-Darby canine kidney (MDCK) epithelial cells (Kroschewski et al., 1999). ...
... While there have been several reports of Rho family members regulating endocytosis and exocytosis (Brown et al., 1998;Kroschewski et al., 1999;Lamaze et al., 1996;Mariot et al., 1996;Norman et al., 1994;Norman et al., 1996;O'Sullivan et al., 1996;Price et al., 1995;Schmalzing et al., 1995), little is known about the roles of these proteins in post-endocytic or biosynthetic traffic. ...
The endocytic and postendocytic trafficking pathways in polarized epithelial cells was examined. First, sorting of fluid and membrane internalized from the apical plasma membrane was analayzed. Internalized fluid and membrane were intially delivered to apical early endosomes (AEE). Fluid remained in the AEE while membrane was rapidly sorted and delivered to the Rab11+ apical recycling endosome (ARE). The delivery of membrane markers to the ARE was microtubule-dependent. Transferrin, a marker of basolateral recycling pathway, had access to the AEE but not the ARE. Next, the role of Rac1 and RhoA in endocytosis and postendocytic was determined. Both Rac1 and RhoA were involved in regulation of endocytosis from both plasma membrane domains. Furthermore, Rac1 was implicated in regulation of apically targeted traffic from both the endocytic and secretory pathways. Expression of dominant active Rac1 (Rac1V12) caused formation of a central aggregate of membranes composed in part of the ARE. Markers targeted for the apical plasma membrane were trapped within this aggregate. RhoA was involved in the regulation of traffic from basolateral early endosomes (BEE). Expression of dominant active RhoA (RhoAV14) trapped ligands internalized from the basolateral plasma membrane in BEE that were also associated with filamentous actin (F-actin). A subset of BEE from control cells were also f-actin positive. Colocalization of BEE with proteins involved in actin polymerization based propulsion (APBP) and myosin motor-based propulsion was determined. Proteins involved in APBP were not associated with BEE bu MIc, a type I myosin, did colocalize with a subset of BEE. This suggests that BEE are transported along associated f-actin to the level of the stress fibers carried by MIc. Finally, the role of SNAP-23 in basolateral recycling of transferrin was determined. SNAP-23 was localized to the endosomes throughout the cell and to the basolateral plasma membrane. Treatment of permeabilized MDCK cells with botulinum neurotoxin E or addition of exogenous SNAP-23 or anti-SNAP-23 antibodies all inhibited transferrin recycling. This suggests that SNAP-23 is important for basolateral recycling in polarized epithelial cells.
... Indeed, we found that inactivation of RhoA by C. botulinum does not modify secretagogueevoked secretion in chromaffin cells [46]. Furthermore, despite the evidence pointing to a key regulatory function for Rho-related GTPases in secretion, the active role of Rho itself in the exocytotic machinery has been excluded in various secretory cell models [53][54][55]. To probe the actual role of other members of the Rho family in the exocytotic pathway in chromaffin cells, we recently used various clostridial toxins that selectively modify specific GTP-binding proteins [56]. ...
... Our results suggest that Cdc42 plays an active role in chromaffin cell secretion, most likely by mediating specific actin rearrangements in the subplasmalemmal cytoskeleton [56]. Accordingly, the active participation of Cdc42 in the exocytotic reaction in pancreatic cells [54] and mast cells [55] has been recently described. These findings suggest a dual role for actin filaments: one inhibiting secretory granule movement by a trapping mechanism and a second which positively stimulates exocytosis. ...
Catecholamine secretion from chromaffin cells has been used for a long time as a general model to study exocytosis of large dense core secretory granules. Permeabilization and microinjection techniques have brought the possibility to dissect at the molecular level the multi-protein machinery involved in this complex physiological process. Regulated exocytosis comprises distinct and sequential steps including the priming of secretory granules, the formation of a docking complex between granules and the plasma membrane and the subsequent fusion of the granule with the plasma membrane. Key proteins involved in the exocytotic machinery have been identified. For instance, SNAREs which participate in the docking events in most intracellular transport steps along the secretory pathway, play a role in exocytosis in both neuronal and endocrine cells. However, in contrast to intracellular transport processes for which the highest fusion efficiency is required after correct targeting of the vesicles, the number of exocytotic events in activated secretory cells needs to be tightly controlled. We describe here the multistep control exerted by heterotrimeric and monomeric G proteins on the progression of secretory granules from docking to fusion and the molecular nature of some of their downstream effectors in neuroendocrine chromaffin cells.
... It has no nuclear localization domain but regulates gene expression (Gu et al. 2002). Both, Rac1 and Rac2, induce secretion in mast cells (Brown et al. 1998). If Rac2 is knocked out, defects in mast cell proliferation, survival and function occur (Schrader et al. 2002). ...
... Moreover, it was published that Rho GTPases, mainly Rac and Cdc42, are involved in mast cell secretion independent from PLCγ (Brown et al. 1998;Djouder et al. 2000;Hong-Geller et al. 2001). Here it is shown that IP 3 -induced calcium mobilization and translocation of Stim1 depends on Rac activity. ...
Mast cells are an important part of the immune system and play a pivotal role in allergy and anaphylaxis. These hematopoetic cells occur in connective tissue, in mucosa of the gastrointestinal tract and the respiratory tract, heart, nerve cells and uterus. Crosslinking of high affine IgE-receptors on cell surface results in release of mediators like histamine, tryptase and several cytokines which causes symptoms of an allergic reaction.
This study comprises two parts to determine the role of Rac in FceRI signaling pathways in mast cells. Modification and activation of Rho GTPases by the TAT-fusion construct DNT-TAT and the resulting effects on different signaling proteins of the FceRI pathway were analyzed in the first part of the thesis. The TAT-protein of HIV1 penetrates cellular membranes and is used for transport of proteins or protein-domains into cells. Initial studies showed, that Rho GTPase activating toxins like CNF1 and DNT are not taken up by mast cells. Therefore, the catalytic domain of Bordetella dermonecrotic toxin (DNT) was fused with TAT to deliver the toxin into mast cells and to activate Rho GTPases.
Characterization of DNT-TAT revealed that the toxin activates recombinant Rac and Rho by covalent modification and selectively activates Rac in RBL cells and BMMC. DNT-TAT was taken up into cytoplasm and transported into the nucleus of mast cells within 15 min. Activation of Rac by DNT-TAT resulted in activation of phospholipase Cg and subsequently in accumulation of inositol phosphates. DNT-TAT induced calcium mobilization and degranulation of mast cells. Moreover, activation of Rac by the toxin revealed a role of Rac in regulation of ERK-activation, which is believed to be essential in cytokine production.
The second part focused on the role of Rac in downstream FceRI pathways, especially on calcium mobilization and degranulation. Therefore, A23187, a calcium ionophore, and membrane permeable InsP3 were used to induce calcium depletion of the ER independently of the IgE-receptor and PLCgamma.
The study showed that Rac was activated by antigen and calcium ionophore in a concentration dependent manner. Inhibition of PLCgamma-independent calcium mobilization and degranulation by Rho GTPase-inactivating toxins indicate a role of Rac in downstream pathways. Rac-induced translocation of Stim1, which is involved in regulation of membrane calcium channels, to the plasma membrane underlines this hypothesis and might be a hint for Rac-mediated transport of Stim1. At last, it was demonstrated that Rac is probably involved in phosphorylation of ERK and AKT via Ras-Rac-crosstalk.
... Recent findings have indicated that two isoforms of Rac, Rac1 and Rac2, are functionally interchangeable in inducing secretion from mast cells, and that an additional Rhorelated protein, Cdc42, is equipotent to Rac [44]. These findings have yet to be confirmed in eosinophils, although preliminary experiments using bovine brain RhoGDI have shown an inhibitory effect by this protein on eosinophil degranulation induced by GTPgS and Ca 2þ (unpublished observations). ...
... These findings have yet to be confirmed in eosinophils, although preliminary experiments using bovine brain RhoGDI have shown an inhibitory effect by this protein on eosinophil degranulation induced by GTPgS and Ca 2þ (unpublished observations). Mast cells express Cdc42 and both isoforms of Rac [44,45], while eosinophils have so far been found to express Rac2 [46]. Taken together, these results suggest that it is likely that mast cells and eosinophils employ more than one GTP-binding protein to trigger degranulation. ...
... Rho family members also regulate secretion. In mast cells, RhoA, Rac1, and Cdc42 stimulate exocytosis of secretory granules, whereas C-3 transferase and dominant inactive RhoA and Rac1 inhibit guanosine 5-O-(3-thiotriphosphate)-stimulated secretion (Norman et al., 1994Norman et al., , 1996 Price et al., 1995; Mariot et al., 1996; O'Sullivan et al., 1996; Brown et al., 1998 ). New evidence indicates that Cdc42 may be important in regulating the delivery of newly synthesized proteins to the basolateral domain of polarized MDCK epithelial cells (Kroschewski et al., 1999). ...
... Although there have been several reports of Rho family members regulating endocytosis and exocytosis (Norman et al., 1994Norman et al., , 1996 Price et al., 1995; Schmalzing et al., 1995; Lamaze et al., 1996; Mariot et al., 1996; O'Sullivan et al., 1996; Brown et al., 1998; Kroschewski et al., 1999), little is known about the roles of these proteins in postendocytic or biosynthetic traffic. In cells expressing Rac1V12, we observed that apically directed membrane protein traffic was selectively impaired compared with traffic directed toward the basolateral membrane. ...
Madin-Darby canine kidney (MDCK) cells expressing constitutively active Rac1 (Rac1V12) accumulate a large central aggregate of membranes beneath the apical membrane that contains filamentous actin, Rac1V12, rab11, and the resident apical membrane protein GP-135. To examine the roles of Rac1 in membrane traffic and the formation of this aggregate, we analyzed endocytic and biosynthetic trafficking pathways in MDCK cells expressing Rac1V12 and dominant inactive Rac1 (Rac1N17). Rac1V12 expression decreased the rates of apical and basolateral endocytosis, whereas Rac1N17 expression increased those rates from both membrane domains. Basolateral-to-apical transcytosis of immunoglobulin A (IgA) (a ligand for the polymeric immunoglobulin receptor [pIgR]), apical recycling of pIgR-IgA, and accumulation of newly synthesized GP-135 at the apical plasma membrane were all decreased in cells expressing Rac1V12. These effects of Rac1V12 on trafficking pathways to the apical membrane were the result of the delivery and trapping of these proteins in the central aggregate. In contrast to abnormalities in apical trafficking events, basolateral recycling of transferrin, degradation of EGF internalized from the basolateral membrane, and delivery of newly synthesized pIgR from the Golgi to the basolateral membrane were all relatively unaffected by Rac1V12 expression. Rac1N17 expression had little or no effect on these postendocytic or biosynthetic trafficking pathways. These results show that in polarized MDCK cells activated Rac1 may regulate the rate of endocytosis from both membrane domains and that expression of dominant active Rac1V12 specifically alters postendocytic and biosynthetic membrane traffic directed to the apical, but not the basolateral, membrane.
... First, secretion efficiency in permeabilized mast cells was enhanced upon incubation with recombinant activated forms of Rho and Rac and reduced under conditions that inhibit Rho and Rac activity (Price et al., 1995; Norman et al., 1996 ). In a similar system, recombinant Rac and Cdc42 were shown to delay the onset of the progressive decrease in secretion efficiency normally observed after streptolysin-O treatment (Brown et al., 1998 ). The ability to retard the gradual loss of responsiveness to stimulation indicated that Rac and Cdc42 were able to functionally replace proteins that had diffused out of the permeabilized cell, either as regulators or direct components of degranulation. ...
... Since these phenotypes were indicative of aberrant cellular behavior and physiology, we discontinued further study of RhoA in the context of degranulation in RBL- 2H3 cells. A role for the small GTPases, Cdc42, Rac, and Rho, in exocytosis had been proposed based on earlier work in which the addition of activated, recombinant GTPases enhanced degranulation in permeabilized cells (Norman et al., 1994; Price et al., 1995; Brown et al., 1998 ). The majority of these studies had made the presumption that the GTPases were stimulating degranulation by primarily inducing the actin cytoskeletal rearrangements that normally accompany secretion. ...
We have expressed dominant-active and dominant-negative forms of the Rho GTPases, Cdc42 and Rac, using vaccinia virus to evaluate the effects of these mutants on the signaling pathway leading to the degranulation of secretory granules in RBL-2H3 cells. Dominant-active Cdc42 and Rac enhance antigen-stimulated secretion by about twofold, whereas the dominant-negative mutants significantly inhibit secretion. Interestingly, treatment with the calcium ionophore, A23187, and the PKC activator, PMA, rescues the inhibited levels of secretion in cells expressing the dominant-negative mutants, implying that Cdc42 and Rac act upstream of the calcium influx pathway. Furthermore, cells expressing the dominant-active mutants exhibit elevated levels of antigen-stimulated IP(3) production, an amplified antigen-stimulated calcium response consisting of both calcium release from internal stores and influx from the extracellular medium, and an increase in aggregate formation of the IP(3) receptor. In contrast, cells expressing the dominant-negative mutants display the opposite phenotypes. Finally, we are able to detect an in vitro interaction between Cdc42 and PLCgamma1, the enzyme immediately upstream of IP(3) formation. Taken together, these findings implicate Cdc42 and Rac in regulating the exocytosis of secretory granules by stimulation of IP(3) formation and calcium mobilization upon antigen stimulation.
... The low molecular mass GTP-binding proteins of the Rho family (Rho, Rac, Cdc42) appear to be involved in activation of mast cells and RBL cells (20). Introduction of Rac into permeabilized mast cells cause secretion (21), and expression of dominant inhibitory forms of Cdc42 and Rac1 inhibits antigeninduced degranulation (22). ...
... Secretion from permeabilized mast cells were increased by dominant active Rac and Rho proteins (20). Gomperts and co-workers suggested Rac and Cdc42 as candidates for "G E ," a GTP-binding protein, mediating exocytosis in cells of hematopoietic origin (21). A role of small GTPases of the Rho family in RBL and mast cell activation was supported by recent findings that toxin B, which glucosylates and inactivates Rho GTPases, completely blocks secretion in these cells (36). ...
Using large clostridial cytotoxins as tools, the role of Rho GTPases in activation of RBL 2H3 hm1 cells was studied. Clostridium difficile toxin B, which glucosylates Rho, Rac, and Cdc42 and Clostridium sordellii lethal toxin, which glucosylates Rac and Cdc42 but not Rho, inhibited the release of hexosaminidase from RBL cells mediated by the high affinity antigen receptor (FcepsilonRI). Additionally, toxin B and lethal toxin inhibited the intracellular Ca(2+) mobilization induced by FcepsilonRI-stimulation and thapsigargin, mainly by reducing the influx of extracellular Ca(2+). In patch clamp recordings, toxin B and lethal toxin inhibited the calcium release-activated calcium current by about 45%. Calcium release-activated calcium current, the receptor-stimulated Ca(2+) influx, and secretion were inhibited neither by the Rho-ADP-ribosylating C3-fusion toxin C2IN-C3 nor by the actin-ADP-ribosylating Clostridium botulinum C2 toxin. The data indicate that Rac and Cdc42 but not Rho are not only involved in late exocytosis events but are also involved in Ca(2+) mobilization most likely by regulating the Ca(2+) influx through calcium release-activated calcium channels activated via FcepsilonRI receptor in RBL cells.
... Small Rho-related GTPases are well established as transducers of signals from the receptor to the actin cytoskeleton [Hall, 1998]. They were also found to be essential to secretion of inflammatory mediators from mast cells Prepens et al., 1996;Brown et al., 1998;Hong-Geller and Cerione, 2000]. So far, however, experimental evidence suggests that the pathways leading from Rho GTPases to secretion are distinct and independent from those leading to the cytoskeleton [Norman et al., 1996;Guillemot et al., 1997;Sullivan et al., 1999]. ...
... In mast cells, small Rho-related GTPases are essential to the exocytotic function Prepens et al., 1996;Brown et al., 1998;Hong-Geller and Cerione 2000]. These GTPases are well established as transducers of signals from the receptor to the actin cytoskeleton [Hall, 1998]. ...
To investigate the role of the actin cytoskeleton in exocytosis, we have tested the effects of latrunculin B, a microfilament-disrupting drug, on secretion from intact and permeabilised rat peritoneal mast cells. The toxin strongly inhibited secretion from intact cells (attached or in suspension) responding to a polybasic agonist, compound 48/80. However, this effect was revealed only after a profound depletion of actin filaments. This was achieved by a long (1 h) exposure of cells to the drug before activation, together with its presence during activation. Maximal inhibition of secretion by such treatment was 85% at 40 microgram/ml latrunculin B. These results indicate that minimal actin structures are essential for the exocytotic response. In contrast, stimulus-induced cell spreading was prevented by latrunculin (5 microgram/ml) applied either before or after activation. The effects of the toxin on intact cells were fully reversible. The responses of permeabilised cells were affected differentially: secretion induced by calcium was more sensitive to latrunculin than that induced by GTP-gamma-S. The calcium response, therefore, is more dependent upon the integrity of the actin cytoskeleton than the response induced by GTP-gamma-S. Again, maximal inhibitory effects (approximately 65 and 25% at 40 microgram/ml) were observed only when cells were exposed to the toxin both before and after permeabilisation. Since the permeabilised cells system focuses on the final steps of exocytosis, the incomplete inhibition suggests that actin plays a modulatory rather than a central role at this stage.
... This triggers a downstream signaling cascade via the Lyn-Syk-LAT-PLCγ and the Fyn-Gab2-PI3K signaling pathways [4][5][6][7]. Studies from our lab and others have revealed that Rho GTPases are downstream targets of FcεRI signaling and part of the regulatory mechanism of mast cell degranulation [8][9][10][11]. Rho proteins are monomeric G proteins belonging to the Ras superfamily of GTPases that play diverse roles in many cellular processes, particularly those involved in cytoskeletal dy-namics [12]. We have shown that antigen activation of mast cells triggers profound morphological transitions that generate cell protrusions which require Rho GTPase function [10,11]. ...
When antigen-stimulated, mast cells release preformed inflammatory mediators stored in cytoplasmic granules. This occurs via a robust exocytosis mechanism termed degranulation. Our previous studies revealed that RhoA and Rac1 are activated during mast cell antigen stimulation and are required for mediator release. Here, we show that the RhoGEF, GEF-H1, acts as a signal transducers of antigen stimulation to activate RhoA and promote mast cell spreading via focal adhesion (FA) formation. Cell spreading, granule movement, and exocytosis were all reduced in antigen-stimulated mast cells when GEF-H1 was depleted by RNA interference. GEF-H1-depleted cells also showed a significant reduction in RhoA activation, resulting in reduced stress fiber formation without altering lamellipodia formation. Ectopic expression of a constitutively active RhoA mutant restored normal morphology in GEF-H1-depleted cells. FA formation during antigen stimulation required GEF-H1, suggesting it is a downstream target of the GEF-H1-RhoA signaling axis. GEF-H1 was activated by phosphorylation in conjunction with antigen stimulation. Syk kinase is linked to the FcεRI signaling pathway and the Syk inhibitor, GS-9973, blocked GEF-H1 activation and also suppressed cell spreading, granule movement, and exocytosis. We concluded that during FcεRI receptor stimulation, GEF-H1 transmits signals to RhoA activation and FA formation to facilitate the exocytosis mechanism.
... The permeabilized mast cells released histamine and b-N-acetylglucosaminidase, dependent on the presence of nucleoside triphosphate and micromolar concentrations of Ca 2+ . SLO-permeabilized mast cells have been used as a simplified system for studies to understand the molecular mechanisms of exocytosis (114)(115)(116)(117)(118) and the role of plasma membrane repair mechanisms (65,76,119). In these experiments, relatively high SLO doses were used, allowing formation of numerous pores in the plasma membrane of target cells. ...
Mast cells are potent immune sensors of the tissue microenvironment. Within seconds of activation, they release various preformed biologically active products and initiate the process of de novo synthesis of cytokines, chemokines, and other inflammatory mediators. This process is regulated at multiple levels. Besides the extensively studied IgE and IgG receptors, toll-like receptors, MRGPR, and other protein receptor signaling pathways, there is a critical activation pathway based on cholesterol-dependent, pore-forming cytolytic exotoxins produced by Gram-positive bacterial pathogens. This pathway is initiated by binding the exotoxins to the cholesterol-rich membrane, followed by their dimerization, multimerization, pre-pore formation, and pore formation. At low sublytic concentrations, the exotoxins induce mast cell activation, including degranulation, intracellular calcium concentration changes, and transcriptional activation, resulting in production of cytokines and other inflammatory mediators. Higher toxin concentrations lead to cell death. Similar activation events are observed when mast cells are exposed to sublytic concentrations of saponins or some other compounds interfering with the membrane integrity. We review the molecular mechanisms of mast cell activation by pore-forming bacterial exotoxins, and other compounds inducing cholesterol-dependent plasma membrane perturbations. We discuss the importance of these signaling pathways in innate and acquired immunity.
... Filopodia are actinbased structures that arise from neuronal growth cones and function in neuronal pathfinding (Davenport et al., 1993;Rosentreter et al., 1998). The recent demonstration that Cdc42 is associated with coatamer proteins in the Golgi apparatus, that it regulates exit of apical and basolateral proteins from the Golgi network and is involved in exocytosis of secretory granules in mast cells is indicative of the diverse roles that Cdc42 plays in cells (Brown et al., 1998;Hong-Geller and Cerione, 2000;Wu et al., 2000;Müsch et al., 2001). The downstream effectors of Cdc42 fall into six families most of which contain a CRIB-binding domain and include Cdc42binding kinase, myotonic dystrophy kinase-related Cdc42binding kinase, mixed lineage kinase, p21-activated kinase (PAK), WASP (Wiscot-Aldrich Syndrome Protein), IQGAP and MSE55/BORG/CEP (Burbelo et al., 1995). ...
Melanosomes are specialized melanin-synthesizing organelles critical for photoprotection in the skin. Melanosome transfer to keratinocytes, which involves whole organelle donation to another cell, is a unique biological process and is poorly understood. Time-lapse digital movies and electron microscopy show that filopodia from melanocyte dendrites serve as conduits for melanosome transfer to keratinocytes. Cdc42, a small GTP-binding protein, is known to mediate filopodia formation. Melanosome-enriched fractions isolated from human melanocytes expressed the Cdc42 effector proteins PAK1 and N-WASP by western blotting. Expression of constitutively active Cdc42(Cdc42V12) in melanocytes co-cultured with keratinocytes induced a highly dendritic phenotype with extensive contacts between melanocytes and keratinocytes through filopodia, many of which contained melanosomes. These results suggest a unique role for filopodia in organelle transport and, in combination with our previous work showing the presence of SNARE proteins and rab3a on melanosomes, suggest a novel model system for melanosome transfer to keratinocytes.
... Other candidates for Gg include Rac and Cdc42. RacI, complexed to RhoGDI is able to support secretion in permeabilised mast cells (O 'Sullivan et al., 1996), and activated Rac or Cdc42 induce exocytosis in the presence o f calcium (Brown et al., 1998a). It is possible that such a Gg protein would be localised on the granule membrane. ...
In recent years great strides have been made in our understanding of the secretory pathway. Much of the new information regarding regulated exocytosis has come from biochemical studies of neuronal cells and tissues. The aim of the work described in this thesis was to determine if cells of the haematopoietic lineage use the same granule docking and fusion machinery as that used for neurotransmitter release and to investigate the function of candidate genes involved in granulocyte degranulation. Western blotting across eosinophil subfractions failed to identify known components of the synaptic vesicle fusion machinery. cDNA library screening resulted in the isolation of VAMP2 and cellubrevin cDNA clones from eosinophil and RBL-2H3 cDNA libraries. Tagged VAMP2 or cellubrevin cDNA constructs transiently expressed in RBL-2H3 cells localised to the endocytic pathway. To determine if key gene products thought to play a role in regulated exocytosis affected RBL-2H3 cell degranulation, a functional assay utilising flow cytometry was developed. This enabled the effect of overexpression of GFP-tagged ARF and PLD 1b on RBL-2H3 degranulation to be determined. Using this approach the GDP-bound mutant of ARF6 inhibited ionomycin stimulated degranulation while overexpression of wild-type PLD 1b increased granule fusion and localised to RBL-2H3 granules. Therefore PLD1b and ARF6 are implicated in granulocyte degranulation.
... Cdc42 was first identified by its role in the polarization of yeast cells during bud formation (Adams et al., 1990). Over time, Cdc42 was found to stimulate actin polarization and reorganization (Li et al., 1995), vesicular Bereitgestellt von | Slovak Academy of Science Angemeldet Heruntergeladen am | 13.02.20 18:06 transport (Brown et al., 1998), and to have an effect on transcription regulation (Coso et al., 1995). ...
The septins constitute a conserved family of guanosine phosphate-binding and filamentforming proteins widespread across eukaryotic species. Septins appear to have two principal functions. One is to form a cortical diffusion barrier, like the septin collar at the bud neck of Saccharomyces cerevisiae, which prevents movement of membrane-associated proteins between the mother and daughter cells. The second is to serve as a polymeric scaffold for recruiting the proteins required for critical cellular processes to particular subcellular areas. In the last decade, structural information about the different levels of septin organization has appeared, but crucial structural determinants and factors responsible for septin assembly remain largely unknown. This review highlights recent findings on the architecture and function of septins and their remodelling with an emphasis on mitotically-dividing budding yeasts.
... Activation of GPCRs on MCs can additionally induce or inhibit the release of diverse inflammatory mediators (Pundir & Kulka, 2010). It is well documented that reorganization of actin cytoskeleton by activation of small GTPases such as Rac, Rho, and Cdc42, is necessary for exocytosis in MCs with both positive and negative roles (Brown, O'Sullivan, & Gomperts, 1998;Pendleton & Koffer, 2001;Price, Norman, Ridley, & Koffer, 1995). As described before, stimulation of different GPCRs leads to the activation of the PI3K/Akt pathway, which in turn can activate different small GTPases (Hayre, Degese, Gutkind, & O'Hayre, 2014;Papakonstanti & Stournaras, 2008). ...
A multitude of physiological processes regulated by G protein-coupled receptors (GPCRs) signaling are accomplished by the participation of active rearrangements of the cytoskeleton. In general, it is common that a cross talk occurs among networks of microfilaments, microtubules, and intermediate filaments in order to reach specific cell responses. In particular, actin-cytoskeleton dynamics regulate processes such as cell shape, cell division, cell motility, and cell polarization, among others.This chapter describes the current knowledge about the regulation of actin-cytoskeleton dynamic by diverse GPCR signaling pathways, and also includes some protocols combining immunofluorescence and confocal microscopy for the visualization of the different rearrangements of the actin-cytoskeleton. We report how both the S1P-GPCR/G12/13/Rho/ROCK and glucagon-GPCR/Gs/cAMP axes induce differential actin-cytoskeleton rearrangements in epithelial cells. We also show that specific actin-binding molecules, like phalloidin and LifeAct, are very useful to analyze F-actin reorganization by confocal microscopy, and also that both molecules show similar results in fixed cells, whereas the anti-actin antibody is useful to detect both the G- and F-actin, as well as their compartmentalization. Thus, it is highly recommended to utilize different approaches to investigate the regulation of actin dynamics by GPCR signaling, with the aim to get a better picture of the phenomenon under study.
... Several pieces of experimental evidence also argue in favor of this model, including the enhanced neutrophil secretion in the presence of cytochalasin B (32), and a direct effect of gelsolin on Ca 2ϩ -dependent secretion in permeabilized mast cells (15). As gelsolin is regulated by the GTP-binding protein rac (5), and rac activates secretion in mast cells (33), the activation of secretion by GTP␥S (23) might also involve a gelsolin-dependent pathway. ...
Article:Gelsolin is involved in IgG-mediated phagocytosis
... After this, the cells are able to take a repeat stimulus and to release MVs once more. Refractory periods where cells lose their sensitivity to calcium stimulation are common, as with mast cells permeabilized with streptolysin-O that lose their capacity to be stimulated over 20e30 min [20]; Listeriolysin O, the toxin from Listeria monocytogenes has the same effect on mast cells [21]. The latter work further showed the refractory stimulation by calcium to be due to reduced intracellular release of calcium rather than to be influenced by extracellular calcium influx. ...
... 121 In endocrine cells, Cdc42 and Rac have been proposed to regulate exocytosis in pancreatic beta cells, basophilic leukaemia cells, and mast cells. [122][123][124] Rho, together with Rac, has also been shown to inhibit non-receptor-mediated endocytosis in Xenopus oocytes and receptor-mediated endocytosis in mammalian cells, as reviewed by Ridley and Symons. 125,126 Kroschewski et al. showed that Cdc42 controls transport to the basolateral membrane in MDCK cells, as deletion of this protein results in an impaired transport to this compartment. ...
Despite the importance of bacterial lipopolysaccharide (LPS) in infection and inflammation, many aspects of LPS action remain poorly understood. Especially, the mechanisms by which cells recognise and react to endotoxins or endotoxin-containing particles and how cellular responses are translated into systemic effects have long remained obscure. However, the recent identification of Toll-like receptors as essential participants in endotoxin signal transduction has provided the first answers in clarifying cellular LPS responses. In this review, we discuss the consequences of the clarification of the cellular effects of LPS. Furthermore, for LPS to exert its effects, it has to be transported to its target cells and be recognised before signalling may be induced, and we shall review the current state of affairs with regard to these recognition processes. Finally, we shall investigate how current knowledge may explain endotoxin neutralisation and subsequent detoxification, either through LPS internalisation or via LPS immobilisation, or through the actions of LPS-binding molecules.
... 5,6 Moreover, Cdc42 that localizes to the golgi apparatus promotes the targeting of secretory vesicles to the cell surface. [7][8][9] Studies have also shown that Rho and Rac are critical in the release of secretory granules by adrenal chromaffin, mast and neuronal cells. [10][11][12] Though Rho GTPases are the master players of actin cytoskeleton and membrane trafficking, their role in controlling vesicle fusion has remained largely mysterious. ...
Vesicle trafficking is crucial for delivery of membrane compartments as well as signaling molecules to specific sites on the plasma membrane for regulation of diverse processes such as cell division, migration, polarity establishment and secretion. Rho GTPases are well-studied signaling molecules that regulate actin cytoskeleton in response to variety of extracellular stimuli. Increasing amounts of evidence suggest that Rho proteins play a critical role in vesicle trafficking in both the exocytic and endocytic pathways; however, the molecular mechanism underlying the process remains largely unclear. We recently defined a mechanism of action for RhoA in membrane trafficking pathways through regulation of the octameric complex exocyst in a manuscript published in Developmental Cell. We have shown that microtubule-associated RhoA-activating factor GEF-H1 is involved in endocytic and excocytic vesicle trafficking. GEF-H1 activates RhoA in response to RalA GTPase, which in turn regulates the localization and the assembly of exocyst components and exocytosis. Our work defines a mechanism for RhoA activation in response to RalA signaling and during vesicle trafficking. These results provide a framework for understanding how RhoA/GEF-H1 regulates the coordination of actin and microtubule cytoskeleton modulation and vesicle trafficking during migration and cell division.
... Rho-type small GTPases are signal transduction proteins associated with the regulation of the actin network in many cellular processes. Recent findings also suggest connections between Rho family GTPases and vesicle trafficking, e.g. in cells of haematopoietic origin (Price et al., 1995;Yonei et al., 1995;Prepens et al., 1996;Hackam et al., 1997;Brown et al., 1998;Hong-Geller and Cerione, 2000), in adrenal chromaffin cells (Komuro et al., 1996;Gasman et al., 1999), in synaptosomes (Doussau et al., 2000), in pancreatic acinar cells (Rosado et al., 1998), in epithelial cells (Kroschewski et al., 1999) in Xenopus laevis oocytes (Schmalzing et al., 1995;Lamaze et al., 1996), in Dictyostelium discoideum (Seastone et al., 1998) and in yeast (Adamo et al., 1999;Robinson et al., 1999). The effects of Rho proteins on vesicle trafficking may be connected to their effects on the actin cytoskeleton, the reorganization of which has been considered to be essential for exocytosis. ...
We recently isolated from the filamentous fungus Trichoderma reesei (Hypocrea jecorina) a gene encoding RHOIII as a multicopy suppressor of the yeast temperature-sensitive secretory mutation, sec15-1. To characterize this gene further, we tested its ability to suppress other late-acting secretory mutations. The growth defect of yeast strains with sec1-1, sec1-11, sec3-2, sec6-4 and sec8-9 mutations was suppressed. Expression of rho3 also improved the impaired actin organization of sec15-1 cells at +38°C. Overproduction of yeast Rho3p using the same expression vector as T. reesei RHOIII appeared to be toxic in sec3-101, sec5-24, sec8-9, sec10-2 and sec15-1 cells. When expressed from the GAL1 promoter, RHO3 suppressed the growth defect of sec1 at the restrictive temperature and inhibited the growth of sec3-101 at the permissive temperature. Disruption of the rho3 gene in the T. reesei genome did not affect the hyphal or colony morphology nor the cellular cytoskeleton organization. Furthermore, the growth of T. reesei was not affected on glucose by the rho3 disruption. Instead, both growth and protein secretion of T. reesei in cellulose cultures was remarkably decreased in rho3 disruptant strains when compared with the parental strain. These results suggest that rho3 is involved in secretion processes in T. reesei.
... In mast cells, small Rho-related GTPases are essential to the exocytotic function Brown et al., 1998). Expression of dominant negative mutant forms of the Cdc42 and Rac1 in RBL-2H3 cells substantially affected the microfilament reorganization. ...
Mast cell activation mediated by the high affinity receptor for IgE (FcεRI) is a key event in allergic response and inflammation. Other receptors on mast cells, as c-Kit for stem cell factor and G protein-coupled receptors (GPCRs) synergistically enhance the FcεRI-mediated release of inflammatory mediators. Activation of various signaling pathways in mast cells results in changes in cell morphology, adhesion to substrate, exocytosis, and migration. Reorganization of cytoskeleton is pivotal in all these processes. Cytoskeletal proteins also play an important role in initial stages of FcεRI and other surface receptors induced triggering. Highly dynamic microtubules formed by αβ-tubulin dimers as well as microfilaments build up from polymerized actin are affected in activated cells by kinases/phosphatases, Rho GTPases and changes in concentration of cytosolic Ca²⁺. Also important are nucleation proteins; the γ-tubulin complexes in case of microtubules or Arp 2/3 complex with its nucleation promoting factors and formins in case of microfilaments. The dynamic nature of microtubules and microfilaments in activated cells depends on many associated/regulatory proteins. Changes in rigidity of activated mast cells reflect changes in intermediate filaments build up from vimentin. This review offers a critical appraisal of current knowledge on the role of cytoskeleton in mast cells signaling.
... Thus, Cdc42 may function either upstream of KCl in the classical stimulus-secretion pathway or may function in a parallel pathway exclusive to glucose activation that regulates vesicle/granule mobilization and/or targeting to the plasma membrane. This proposed role for Cdc42 in insulin secretion would be consistent with the reports of others showing that Cdc42 regulates targeting of secretory vesicles (46,60) and enhances exocytosis by mast cells (61,62). Future investigations are aimed at better defining the pathway in which Cdc42 participates in vesicle targeting in glucose-stimulated insulin secretion. ...
In pancreatic beta cells, insulin granule exocytosis is regulated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein (SNAP) receptor) proteins, and this is coupled to cortical F-actin reorganization via the Rho family GTPase Cdc42 by an unknown mechanism. We investigated interactions among the target SNARE protein Syntaxin 1A and the vesicle-associated membrane SNARE protein (VAMP2) with Cdc42 and compared these structural interactions with their functional importance to glucose-stimulated insulin secretion in MIN6 beta cells. Subcellular fractionation analyses revealed a parallel redistribution of Cdc42 and VAMP2 from the granule fraction to the plasma membrane in response to glucose that temporally corresponded with the glucose-induced activation of Cdc42. Moreover, within these fractions Cdc42 and VAMP2 were found to co-immunoprecipitate under basal and glucose-stimulated conditions, suggesting that they moved as a complex. Furthermore, VAMP2 bound both GST-Cdc42-GTPgammaS and GST-Cdc42-GDP, indicating that the Cdc42-VAMP2 complex could form under both cytosolic GDP-bound Cdc42 and plasma membrane GTP-bound Cdc42 conformational conditions. In vitro binding analyses showed that VAMP2 bound directly to Cdc42 and that a heterotrimeric complex with Syntaxin 1A could also be formed. Deletion analyses of VAMP2 revealed that only the N-terminal 28 residues were required for Cdc42 binding. Expression of this 28-residue VAMP2 peptide in MIN6 beta cells resulted in the specific impairment of glucose-stimulated insulin secretion, indicating a functional importance for the Cdc42-VAMP2 interaction. Taken together, these data suggest a mechanism whereby glucose activates Cdc42 to induce the targeting of intracellular Cdc42-VAMP2-insulin granule complexes to Syntaxin 1A at the plasma membrane.
... There are believed to be 20 members in mammals, 18 so far described (Schultz et al. 1998, Govek et al. 2005). Rho proteins are key regulators of the actin cytoskeleton in all eukaryotic cells and have additional roles in microtubule cytoskeleton reorganization (Wittmann and WatermanStorer 2001), gene expression (Coso et al. 1995, Hill et al. 1995, Perona et al. 1997) and membrane transport processes (Brown et al. 1998, Komuro et al. 1996, Lamaze et al. 1996). The three most intensively studied Rho proteins; RhoA, Rac1 and Cdc42, have their own niches in the regulation of the actin cytoskeleton. ...
Rab8 and its interacting proteins as regulators of cell polarization During the development of a multi-cellular organism, progenitor cells have to divide and migrate appropriately as well as organize their differentiation with one another, in order to produce a viable embryo. To divide, differentiate and migrate cells have to undergo polarization, a process where internal and external components such as actin, microtubules and adhesion receptors are reorganized to produce a cell that is asymmetric, with functionally different surfaces. Also in the adult organism there is a continuous need for these processes, as cells need to migrate in response to tissue damage and to fight infection. Improper regulation of cell proliferation and migration can conversely lead to disease such as cancer. GTP-binding proteins function as molecular switches by cycling between a GTP-bound (active) conformation and a GDP-bound (inactive) conformation. The Ras super-family of small GTPases are found in all eukaryotic cells. They can be functionally divided into five subfamilies. The Ras family members mainly regulate gene expression, controlling cell proliferation and differentiation. Ras was in fact the first human oncogene to be characterized, and as much as 30% of all human tumors may be directly or indirectly caused by mutations of Ras molecules The Rho family members mainly regulate cytoskeletal reorganization. Arf proteins are known to regulate vesicle budding and Rab proteins regulate vesicular transport. Ran regulates nuclear transport as well as microtubule organization during mitosis. The focus of the thesis of Katarina Hattula, is on Rab8, a small GTPase of the Rab family. Activated Rab8 has previously been shown to induce the formation of new surface extensions, reorganizing both actin and microtubules, and to have a role in directed membrane transport to cell surfaces. However, the exact membrane route it regulates has remained elusive. In the thesis three novel interactors of Rab8 are presented. Rabin8 is a Rab8-specific GEF that localizes to vesicles where it presumably recruits and activates its target Rab8. Its expression in cells leads to remodelling of actin and the formation of polarized cell surface domains. Optineurin, known to be associated with a leading cause of blindness in humans (open-angle glaucoma), is shown to interact specifically with GTP-bound Rab8. Rab8 binds to an amino-terminal region and interestingly, the Huntingtin protein binds a carboxy-terminal region of optineurin. (Aberrant Huntingtin protein is known to be the cause Huntington s disease in humans.) Co-expression of Huntingtin and optineurin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures. Furthermore, optineurin promoted cell polarization in a similar way to Rab8. A third novel interactor of Rab8 presented in this thesis is JFC1, a member of the synaptogamin-like protein (Slp) family. JFC1 interacts with Rab8 specifically in its GTP-bound form, co-localizes with endogenous Rab8 on tubular and vesicular structures, and is probably involved in controlling Rab8 membrane dynamics. Rab8 is in this thesis work clearly shown to have a strong effect on cell shape. Blocking Rab8 activity by expression of Rab8 RNAi, or by expressing the dominant negative Rab8 (T22N) mutant leads to loss of cell polarity. Conversely, cells expressing the constitutively active Rab8 (Q67L) mutant exhibit a strongly polarized phenotype. Experiments in live cells show that Rab8 is associated with macropinosomes generated at ruffling areas of the membrane. These macropinosomes fuse with or transform into tubules that move toward the cell centre, from where they are recycled back to the leading edge to participate in protrusion formation. The biogenesis of these tubules is shown to be dependent on both actin and microtubule dynamics. The Rab8-specific membrane route studied contained several markers known to be internalized and recycled (1 integrin, transferrin, transferrin receptor, cholera toxin B subunit (CTxB), and major histocompatibility complex class I protein (MHCI)). Co-expression studies revealed that Rab8 localization overlaps with that of Rab11 and Arf6. Rab8 is furthermore clearly functionally linked to Arf6. The data presented in this thesis strongly suggests a role for Rab8 as a regulator for a recycling compartment, which is involved in providing structural and regulatory components to the leading edge to participate in protrusion formation. Rab8 reglerar cellens form och utseende. En avhandling vid medicinska fakulteten, Helsingfors universitet beskriver hur det GTP bindande proteinet Rab8 reglerar cellens form och utseende (cellpolarisering). I en flercellig organism måste celler specialicera sig och röra på sig för att organismen skall utvecklas korrekt. För att detta skall kunna ske måste de först ändra form i en process kallad polarisering. Cellens komponenter organiseras då assymetriskt för att bilda funktionellt skilda delar. Att en cell kan röra på sig har inte bara en essentiell funktion under utvecklingsskedet, det är också en process som behövs under hela organismens livstid. Det behövs t.ex. rörliga celler för att bekämpa infektioner och för att sår skall kunna läkas. Det är också viktigt att celler som inte skall röra på sig förblir stilla. Strikt kontroll av vilka celler som skall och inte skall röra på sig behövs kontinuerligt. Ett exempel på vad som kan hända om denna kontroll inte fungerar är cancer. Små GTPaser är en stor grupp proteiner som fungerar som omkopplare eller strömbrytare i vidareföring av signaler inuti cellen. Deras funktioner i cellen är diversa och inkluderar bland annat kontroll av celltillväxt, celldelning, och membrantransport. Denna stora grupp (fler än 100 proteiner) kan delas upp i fem familjer: Ras, Rho, Rab, Arf och Ran. Ras var den första humana onkogenen att beskrivas 1981, och i uppskattningsvis en tredjedel av all cancer finns mutationer som leder till ett permanent aktiverat Ras-protein. Rab-familjen av små GTPaser kontrollerar membrantransport i cellen och det är en medlem av denna familj, Rab8, som är fokuset av Katarina Hattulas avhandling. Tidigare forskning har inte klart kunnat visa vilken roll Rab8 har i cellen. I avhandlingen ges bevis på att Rab8 kraftigt påverkar cellens form och utseende. Resultatet av den forskning som presenteras visar en roll för Rab8 i återvinning av material som behövs för att en cell skall kunna bilda och upprätthålla en polariserad fenotyp. Något som bland annat behövs för att cellen skall kunna röra på sig. Avhandlingen presenterar också i detalj hur man sökt efter proteiner som specifikt kan binda till och påverka Rab8s funktion. Tre nya Rab8 bindande protein beskrivs. Ett av dessa är optineurin, som på annat håll har visats vara knutet till glaukom, (en av de främsta anledningarna till blindhet i världen med 33 miljoner drabbade). En koppling mellan optineurin och Huntingtin, det protein vars gen är muterad i personer med Huntingtons sjukdom visas också. Ett annat Rab8 bindande protein som beskrivs är Rabin8. Det aktiverar Rab8 och behövs för att Rab8 ska kunna utföra sin uppgift vid cellpolartisering. Sammantaget ger den forskning som presenteras en ny insikt i hur membrantransport reglerad av Rab8 aktivt bidrar till att ändra cellens form och utseende. Detta kan ge en ny insikt i cellulära processer som cellmigration såväl som en rad sjukdomsförlopp till exempel glaukom, Huntingtons sjukdom och cancer.
... transcriptional regulation (Huang et al., 1993; Laudanna et al., 1996; Erickson et al., 1997; Brown et al., 1998). Rac1, Rac2, and Rac3 proteins share greater than 90% amino acid identity but differ in their carboxyl-terminal sequences, which may confer distinct sub-cellular localizations and regulatory molecule interactions (Tao et al., 2002). ...
Rac2 is a Rho family GTPase that is widely expressed in hematopoietic cells and plays a critical role in host defense. This study investigates the mechanisms responsible for increased Rac2 gene expression during myeloid cell differentiation. Treatment of K562 chronic myelogenous leukemia cells with phorbol-12-myristate-13-acetate (PMA) induces megakaryocytic differentiation and Rac2 gene transcription following a lag of 6-12 h. Promoter/luciferase reporter gene assays reveal that a 135 bp cis-element located between -4223 and -4008 bp upstream of the Rac2 transcription start site is necessary and sufficient for PMA-induced gene expression. The AP1 transcription factor binds to three cis-elements within the 135 bp Rac2 gene regulatory region both in vitro and in vivo following PMA treatment, and mutagenesis of the AP1 binding sites ablates the PMA responsiveness of the 135 bp Rac2 gene regulatory region. Over-expression of AP1 is sufficient to induce expression of a transiently transfected Rac2 promoter/luciferase plasmid, but not the endogenous Rac2 gene. Induction of AP1 in vitro DNA-binding activity is apparent within 1 h of PMA stimulation. However, AP1 binding to the endogenous Rac2 promoter exhibits a lag of 5-9 h, which correlates with reduced histone H3-Lys9 methylation, increased histone H3 acetylation, and increased nuclease accessibility within the 135 bp Rac2 gene regulatory region. These results demonstrate that PMA induction of Rac2 expression during terminal myeloid differentiation requires the coordinate induction of transcription factors and remodeling of Rac2 gene chromatin structure.
... The rho family member RhoD induces process formation, the disappearance of stress fibers and focal contacts and is localized to the plasma membrane and early endosomes where it regulates endosome morphology, fusion and movement (Murphy et al., 1996). Rho proteins have also been shown to regulate exocytosis from mast cells (Norman et al., 1996; Brown et al., 1998). Rho family proteins are therefore positioned to regulate not only the actin polymerization events involved in pseudopodial protrusion but also these related to the role of the microtubule cytoskeleton and membrane traffic in cell motility. ...
Polarization of the motile cell is associated with the formation of a distinct plasma membrane domain, the pseudopod, whose stabilization determines the directionality of cell movement. The rapid movement of cells over a substrate requires that an essential aspect of cell motility must be the supply of the necessary molecular machinery to the site of pseudopodial extension. Renewal of this pseudopodial domain requires the directed delivery to the site of pseudopodial protrusion of proteins which regulate actin cytoskeleton dynamics, cell-substrate adhesion, and localized degradation of the extracellular matrix. Polarized targeting mechanisms include the targeted delivery of beta-actin mRNA to the leading edge and microtubule-based vesicular traffic. The latter may include Golgi-derived vesicles of the biosynthetic pathway as well as clathrin-dependent and clathrin-independent endocytosis and recycling. Coordination of protrusive activities and supply mechanisms is critical for efficient cellular displacement and may implicate small GTPases of the Rho family. While the specific molecular mechanisms underlying pseudopodial protrusion of the motile cell are well-characterized, discussion of these diverse mechanisms in the context of cellular polarization has been limited.
... Rac regulates the secretion of RBL cells (22,48). Recently, it has been shown that the glucosylating clostridial cytotoxins (e.g., toxin B) completely inhibit the degranulation of RBL cells after stimulation of the Fc⑀RI receptor (32). ...
FcepsilonRI signaling in rat basophilic leukemia cells depends on phosphatidylinositol 3-kinase (PI3-kinase) and the small GTPase Rac. Here, we studied the functional relationship among PI3-kinase, its effector protein kinase B (PKB), and Rac using inhibitors of PI3-kinase and toxins inhibiting Rac. Wortmannin, an inhibitor of PI3-kinase, blocked FcepsilonRI-mediated tyrosine phosphorylation of phospholipase Cgamma, inositol phosphate formation, calcium mobilization, and secretion of hexosaminidase. Similarly, Clostridium difficile toxin B, which inactivates all Rho GTPases including Rho, Rac and Cdc42, and Clostridium sordellii lethal toxin, which inhibits Rac (possibly Cdc42) but not Rho, blocked these responses. Stimulation of the FcepsilonRI receptor induced a rapid increase in the GTP-bound form of Rac. Whereas toxin B inhibited the Rac activation, PI3-kinase inhibitors (wortmannin and LY294002) had no effect on activation of Rac. In line with this, wortmannin had no effect on tyrosine phosphorylation of the guanine nucleotide exchange factor Vav. Wortmannin, toxin B, and lethal toxin inhibited phosphorylation of PKB on Ser(473). Similarly, translocation of the pleckstrin homology domain of PKB tagged with the green fluorescent protein to the membrane, which was induced by activation of the FcepsilonRI receptor, was blocked by inhibitors of PI3-kinase and Rac inactivation. Our results indicate that in rat basophilic leukemia cells Rac and PI3-kinase regulate PKB and suggest that Rac is functionally located upstream and/or parallel of PI3-kinase/PKB in FcepsilonRI signaling.
Transformation by Ras and other oncogenes is generally accompanied by dramatic alterations in cell morphology and re-organisation of the actin cytoskeleton, suggesting a role for Rho GTPases. There is now accumulating evidence for the requirement of Rho GTPases in Ras-induced transformation of rodent fibroblasts, but at present little is known about their involvement in human cancers. Therefore, the role played by Rho GTPases in the transformation of human breast and colon epithelial cells was investigated. Mutant active and dominant negative forms of Rho proteins were injected into cells to study the short-term effects of each protein on cell behaviour. Each protein induced morphological changes as predicted from previous research, although the extent of the response was dependent on the initial morphology of individual cell lines. In summary, RhoA induced stress fibre formation, Racl injection resulted in membrane ruffling and Cdc42 gave rise to filopodia. The effects of Rho proteins on cell motility were studied in a motile Ras-transformed breast epithelial cell line. Active forms of RhoA, Racl and Cdc42 each reduced cell motility, while inhibition of either RhoA or Racl had no effect. However, injection of dominant negative Cdc42 significantly reduced the migration speed of the Ras-transformed cells. A tetracycline-regulated expression system was used to study the long-term effects of mutant active or dominant negative Rho proteins in DLD-1 cells derived from a human colon carcinoma. Expression of active forms of RhoA, Racl and Cdc42 inhibited colony formation in soft agar and disrupted growth on Matrigel. Their expression also altered the cellular organisation of DLD-1 cells. RhoA disrupted nuclear division through deregulated centrosome duplication, resulting in cells that exhibited multi-septated nuclei and continued to synthesise DNA without undergoing division. Racl altered membrane trafficking as illustrated by cells showing surface blebs and increased levels of pinocytosis. Finally, Cdc42 altered cell morphology, although in a manner more associated with a Racl phenotype, as cells spread and showed large lamellipodia and membrane ruffles. These observations differ from those obtained previously in other cell types and indicate that the effects of Rho family proteins are cell-type dependent. This work emphasises the importance of analysing protein function in a range of cell types as well as the effects of both short- and long-term protein expression, in order to understand the precise roles of proteins in specific situations.
Purpose: The aim of this study was investigated whether low-dose ionizing radiation attenuates mast cell migration by modulating migration-associated signaling pathways and the expression of chemotactic cytokines.
Materials and Methods: IgE-sensitized RBL-2H3 mast cells were exposed with ionizing radiation at 0.01, 0.05, 0.1, or 0.5 Gy using a ¹³⁷Cs γ-irradiator and stimulated with 2,4-dinitrophenol (DNP)-human serum albumin (HSA). Cell migration was determined using a transwell assay system, F-actin distribution using Alex Fluor 488-conjugated phalloidin, expression of various signaling proteins by Western blotting, mRNA expression by RT-PCR.
Results: Low-dose ionizing radiation significantly suppressed mast cell migration induced by IgE-mediated mast cell activation. Furthermore, low-dose ionizing radiation altered cell morphology, as reflected by changes in F-actin distribution, and inhibited the activation of PI3K, Btk, Rac1, Cdc42. These effects were mediated by Nr4a2, an immune-modulating factor. Knockdown of Nr4a2 reduced mast cell migration, inhibited the PI3K and Btk signaling pathways, and reduced expression of the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1). We further demonstrated that direct blockade of MCP-1 using neutralizing antibodies inhibits mast cell migration.
Conclusion: Low-dose ionizing radiation inhibits mast cell migration through the regulation production of MCP-1 by Nr4a2 in the activated mast cell system.
Purpose of review: To provide an overview on the present understanding of roles of oxidative DNA damage repair in cell signaling underlying bronchoconstriction common to, but not restricted to various forms of asthma and chronic obstructive pulmonary disease. Recent findings: Bronchoconstriction is a tightening of smooth muscle surrounding the bronchi and bronchioles with consequent wheezing and shortness of breath. Key stimuli include air pollutants, viral infections, allergens, thermal and osmotic changes, and shear stress of mucosal epithelium, triggering a wide range of cellular, vascular, and neural events. Although activation of nerve fibers, the role of G-proteins, protein kinases and Ca++, and molecular interaction within contracting filaments of muscle are well defined, the overarching mechanisms by which a wide range of stimuli initiate these events are not fully understood. Many, if not all, stimuli increase levels of reactive oxygen species, which are signaling and oxidatively modifying macromolecules, including DNA. The primary reactive oxygen species target in DNA is guanine, and 8-oxoguanine is one of the most abundant base lesions. It is repaired by 8-oxoguanine DNA glycosylase1 during base excision repair processes. The product, free 8-oxo-7,8-dihydro-2′-deoxyguanosine base, is bound by 8-oxoguanine DNA glycosylase1 with high affinity, and the complex then functions as an activator of small guanosine triphosphatases, triggering pathways for inducing gene expression and contraction of intracellular filaments in mast and smooth muscle cells. Summary: Oxidative DNA damage repair-mediated cell activation signaling result in gene expression that ‘primes’ the mucosal epithelium and submucosal tissues to generate mediators of airway smooth muscle contractions.
Clostridium difficile toxins A and B (TcdA and TcdB) belong to the class of large clostridial cytotoxins and inactivate by glucosylation some low molecular mass GTPases of the Rho-family (predominantly Rho-Rac and Cdc42), known as regulators of the actin cytoskeleton. TcdA and B also represent the main virulence factors of the anaerobic gram-positive bacterium that is the causal agent of pseudomembranous colitis. In our study, TcdB was chosen instead of TcdA for the well-known higher cytotoxic potency. Inactivation of Rho-family GTPases by this toxin in our experimental conditions induced morphological changes and reduction of electron-dense mast cell-specific granules in Human Mast Cell line-1 (HMC-1) cells, but not cell death or permeabilization of plasma-membranes. Previously reported patch-clamp dialysis experiments revealed that high intracellular free-Ca(2+) and GTPγS concentrations are capable of inducing exocytosis as indicated by significant membrane capacitance (Cm) increases in HMC-1 cells. In this study, we investigated the direct effects of TcdB upon HMC-1 cell "stimulated" Cm increase, as well as on mediated "constitutive" secretion of hexosaminidase and interleukin-16 (IL-16). Compared to untreated control cells, HMC-1 cells incubated with TcdB for 3-24hours exhibited a significant reduction of the mean absolute and relative Cm increase in response to free-Ca(2+) and GTPγS suggesting an inhibition of secretory processes by TcdB. In conclusion, the HMC-1 cell line represents a suitable model for the study of direct effects of C. difficile toxins on human mast cell secretory activity.
Copyright © 2014. Published by Elsevier Ireland Ltd.
ADP-ribosylation factors (ARFs) constitute a family of structurally related proteins that forms a subset of the Ras superfamily of regulatory GTP-binding proteins. Like other GTPases, activation of ARFs is facilitated by specific guanine nucleotide exchange factors (GEFs). In chromaffin cells, ARF6 is associated with the membrane of secretory granules. Stimulation of intact cells or direct elevation of cytosolic calcium in permeabilized cells triggers the rapid translocation of ARF6 to the plasma membrane and the concomitant activation of phospholipase D (PLD) in the plasma membrane. Both calcium-evoked PLD activation and catecholamine secretion in permeabilized cells are strongly inhibited by a synthetic peptide corresponding to the N-terminal domain of ARF6, suggesting that the ARF6-dependent PLD activation near the exocytotic sites represents a key event in the exocytotic reaction in chromaffin cells. In the present study, we demonstrate the occurrence of a brefeldin A-insensitive ARF6-GEF activity in the plasma membrane and in the cytosol of chromaffin cells. Furthermore, reverse transcriptase-polymerase chain reaction and immunoreplica analysis indicate that ARNO, a member of the brefeldin A-insensitive ARF-GEF family, is expressed and predominantly localized in the cytosol and in the plasma membrane of chromaffin cells. Using permeabilized chromaffin cells, we found that the introduction of anti-ARNO antibodies into the cytosol inhibits, in a dose-dependent manner, both PLD activation and catecholamine secretion in calcium-stimulated cells. Furthermore, co-expression in PC12 cells of a catalytically inactive ARNO mutant with human growth hormone as a marker of secretory granules in transfected cells resulted in a 50% inhibition of growth hormone secretion evoked by depolarization with high K(+). The possibility that the plasma membrane-associated ARNO participates in the exocytotic pathway by activating ARF6 and downstream PLD is discussed.
Mast cells are activated by Ag-induced clustering of IgE bound to FcεRI receptors or by basic secretagogues that stimulate pertussis toxin-sensitive heterotrimeric G proteins. The cell response includes the secretion of stored molecules, such as histamine, through exocytosis and of de novo synthesized mediators, such as arachidonate metabolites. The respective roles of G proteins α and βγ subunits as well as various types of phospholipase C (PLC) in the signaling pathways elicited by basic secretagogues remain unknown. We show that a specific Ab produced against the C-terminus of Gαi3 and an anti-recombinant Gαi2 Ab inhibited, with additive effects, both exocytosis and arachidonate release from permeabilized rat peritoneal mast cells elicited by the basic secretagogues mastoparan and spermine. A specific Ab directed against Gβγ dimers prevented both secretions. Anti-PLCβ Abs selectively prevented exocytosis. The selective phosphatidylinositol 3-kinase inhibitor LY 294002 prevented arachidonate release without modifying exocytosis. Gβγ coimmunoprecipitated with PLCβ and phosphatidylinositol 3-kinase. The anti-PLCγ1 and anti-phospholipase A2 Abs selectively blocked arachidonate release. Protein tyrosine phosphorylation was inhibited by anti-Gβγ Abs, LY294002, and anti PLCγ1 Abs. These data show that the early step of basic secretagogue transduction is common to both signaling pathways, involving βγ subunits of Gi2 and Gi3 proteins. Activated Gβγ interacts, on one hand, with PLCβ to elicit exocytosis and, on the other hand, with phosphatidylinositol 3-kinase to initiate the sequential activation of PLCγ1, tyrosine kinases, and phospholipase A2, leading to arachidonate release.
Gi3, a member of the Gi family of heterotrimeric GTP-binding proteins, regulates vesicle trafficking along both the constitutive and regulated pathways. In mast cells, specialized secretory cells which secrete a variety of inflammatory mediators by regulated exocytosis, activation of Gi3 provides a sufficient signal for exocytosis [Aridor, M., Rajmilevich, G., Beaven, M. A. & Sagi-Eisenberg, R. (1993) Science 262, 1569−1572]. Such activation can be achieved in patch-clamped or streptolysin-O (SLO)-permeabilized mast cells by a combination of Ca2+ and nonhydrolyzable analogs of GTP. In contrast, Ca2+-activated exocytosis in intact cells is Gi3 independent. We show here that overexpression of a GTPase-deficient mutant (Gαi3Q204L), but not of the wild-type form of Gαi3, in rat basophilic leukemia cells (RBL-2H3), a tumor analog of mucosal mast cells, resulted in marked potentiation of exocytosis and release of arachidonic acid in intact cells activated by a Ca2+ ionophore alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. In contrast, exocytosis and arachidonic acid release stimulated by aggregation of the cell surface receptors for immunoglobulin E (IgE) were unaffected. These results strongly suggest that the intracellular receptor, responsible for the activation of Gi3, is a low-affinity Ca2+-binding protein that can only be activated during Ca2+ ionophore stimulation. Moreover, these results also suggest that the propagation of the Ca2+-activated and Gi3-mediated signaling pathway requires the blocking of Gi3 GTPase activity. Finally, our results indicate that release of arachidonic acid is at least one of the downstream effectors of Gi3.
Membrane trafficking includes a highly dynamic and intricate set of intracellular pathways responsible for the transport of
molecules in and out of the cell, and between the different intracellular compartments. A lot of attention has been paid in
the past decades to the role played by distinct classes of small GTPases on the regulation of membrane trafficking, with special
emphasis on the Rab and Arf families. More recently, Rho GTPases have been implicated in several important aspects of membrane
trafficking. The initial indications that Rho proteins might be involved in membrane trafficking came from the observation
of the localization of some of these proteins at specific intracellular compartments. These observations are corroborated
by the findings of specific effects of these proteins on different membrane transport pathways. The role played by Rho family
members in different aspects of membrane trafficking will be considered in this chapter.
Localized disassembly of cortical F-actin has long been considered necessary for facilitation of exocytosis. Exposure of permeabilized
mast cells to calcium/ATP induces cortical F-actin disassembly (calmodulin-dependent) and secretion (calmodulin-independent).
The delay in the onset of secretion is characteristic for the calcium/ATP response and is abolished by GTP. Here we report
that a constitutively active mutant of Rho (V14RhoA) enhanced both secretion and cortical F-actin disassembly. In addition,
V14RhoA mimicked GTP by abolishing the delay in secretion. Inhibition of Rho by C3 transferase prevented both secretion (∼80%)
and F-actin disassembly (∼20%). Thus, both Rho GTPase and calcium/calmodulin contribute to the control of cortical F-actin
disassembly. Stabilization of actin filaments by high concentrations of phalloidin or by a calmodulin-inhibitory peptide (based
on the calmodulin-binding domain of myosin light chain kinase) did not affect the extent of secretion or the secretion-enhancing
effects of V14RhoA. These results further support the existence of divergent, Rho-dependent, pathways regulating actin and
exocytosis. Furthermore, compound Y-27632, a specific inhibitor of Rho-associated protein kinase (p160ROCK), attenuated the Rho-induced loss of cortical F-actin without affecting secretion. A model is presented in which Rho regulates
secretion and cortical F-actin in a manner dependent on and/or synergistic with calcium.
The transmembrane adaptor protein NTAL (non-T-cell activation linker) participates in signalosome assembly in hematopoietic cells, but its exact role in cell physiology remains enigmatic. We report here that BM-derived mast cells from NTAL-deficient mice, responding to Ag alone or in combination with SCF, exhibit reduced spreading on fibronectin, enhanced filamentous actin depolymerization and enhanced migration towards Ag relative to WT cells. No such differences between WT and NTAL(-/-) BM-derived mast cells were observed when SCF alone was used as activator. We have examined the activities of two small GTPases, Rac and Rho, which are important regulators of actin polymerization. Stimulation with Ag and/or SCF enhanced activity of Rac(1,2,3) in both NTAL(-/-) and WT cells. In contrast, RhoA activity decreased and this trend was much faster and more extensive in NTAL(-/-) cells, indicating a positive regulatory role of NTAL in the recovery of RhoA activity. After restoring NTAL into NTAL(-/-) cells, both spreading and actin responses were rescued. This is the first report of a crucial role of NTAL in signaling, via RhoA, to mast cell cytoskeleton.
Indiana University-Purdue University, Indianapolis This dissertation examines the molecular mechanisms regulating Rac2 gene expression during cell differentiation and identification of a minimal cis-element required for the induction of Rac2 gene expression during K562 cell differentiation. The Rho family GTPase Rac2 is expressed in hematopoietic cell lineages and is further up-regulated upon terminal myeloid cell differentiation. Rac2 plays an important role in many hematopoietic cellular functions, such as neutrophil chemotaxis, superoxide production, cytoskeletal reorganization, and stem cell adhesion. Despite the crucial role of Rac2 in blood cell function, little is known about the mechanisms of Rac2 gene regulation during blood cell differentiation. Previous studies from the Skalnik lab determined that a human Rac2 gene fragment containing the 1.6 kb upstream and 8 kb downstream sequence directs lineage-specific expression of Rac2 in transgenic mice. In addition, epigenetic modifications such as DNA methylation also play important roles in the lineage-specific expression of Rac2. The current study investigated the molecular mechanisms regulating human Rac2 gene expression during cell differentiation using chemically induced megakaryocytic differentiation of the human chronic myelogenous leukemia cell line K562 as the model system. Phorbol 12-myristate 13-acetate (PMA) stimulation of K562 cells resulted in increased Rac2 mRNA expression as analyzed by real time-polymerase chain reaction (RT-PCR). Luciferase reporter gene assays revealed that increased transcriptional activity of the Rac2 gene is mediated by the Rac2 promoter region. Nested 5’- deletions of the promoter region identified a critical regulatory region between -4223 bp and -4008 bp upstream of the transcription start site. Super shift and chromatin immunoprecipitation assays indicated binding by the transcription factor AP1 to three distinct binding sites within the 135 bp minimal regulatory region. PMA stimulation of K562 cells led to extensive changes in chromatin structure, including increased histone H3 acetylation, within the 135 bp Rac2 cis-element. These findings provide evidence for the interplay between epigenetic modifications, transcription factors and cis-acting regulatory elements within the Rac2 gene promoter region to regulate Rac2 expression during K562 cell differentiation.
The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. In this report, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses (< or = 10 microM) of the actin-depolymerizing drugs latrunculin B (Lat B) or cytochalasin B (CB) enhanced both formyl peptide receptor- and Ca(2+) ionophore-stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP), inhibited primary granule exocytosis measured as myeloperoxidase release but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly before primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin, which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodeling was highly polarized when cells were primed with CB; however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis were blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor NSC23766 also inhibited actin remodeling and primary granule exocytosis induced by Lat B/fMLF or CB/fMLF, but not by Ca(2+) ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis.
For regulated secretion, mast cells and several other cell types utilize compound exocytosis, a combination of granule-plasma membrane and granule-granule fusions. The molecular machinery that controls this massive export process has not been identified. We report that SNAP-23, a t-SNARE related to SNAP-25, relocates in response to stimulation from plasma membrane lamellipodia-like projections to granule membranes in permeabilized mast cells. While relocation is a prerequisite for secretion, it can occur without membrane fusion and will expedite a subsequent secretory response. After relocation, SNAP-23 is required for exocytosis, implying a crucial role in promoting membrane fusion. Thus, relocation of this SNARE regulates compound exocytosis and links granule-plasma membrane and granule-granule fusions.
Studies of pancreatic islet function in the pathogenesis of type 2 diabetes mellitus have tended to focus on the short-term control of insulin secretion. However, the long-term control of beta-cell mass is also relevant to diabetes, since this parameter is reduced substantially even in non-insulin-dependent diabetes in humans. In animal models of type 2 diabetes, the normal balance between beta-cell proliferation and programmed cell death is perturbed. We take the perspective in this overview that inosine monophosphate dehydrogenase (IMPDH; EC 1.1.1. 205) may represent a previously neglected molecular integrator or sensor that exerts both functional (secretory) and anatomical (proliferative) effects within beta-cells. These properties reflect the fact that IMPDH is a rate-limiting enzyme in the new synthesis of the purine guanosine triphosphate (GTP), which modulates both exocytotic insulin secretion and DNA synthesis, as well as a number of other critical cellular functions within the beta-cell. Alterations in the expression or activity of IMPDH may be central to beta-cell replication, cell cycle progression, differentiation, and maintenance of adequate islet mass, effects that are probably mediated both by GTP directly, and indirectly via low molecular mass GTPases. If GTP becomes depleted, a hierarchy of beta-cell functions becomes progressively paralyzed, until eventually the effete cell is removed via apoptosis.
ADP-ribosylation factor 6 (ARF6) appears to play an essential role in the endocytic/recycling pathway in several cell types. To determine whether ARF6 is involved in insulin-regulated exocytosis, 3T3-L1 adipocytes were infected with recombinant adenovirus expressing wild-type ARF6 or an ARF6 dominant negative mutant (D125N) that encodes a protein with nucleotide specificity modified from guanine to xanthine. Overexpression of these ARF6 proteins affected neither basal nor insulin-regulated glucose uptake in 3T3-L1 adipocytes, nor did it affect the subcellular distribution of Glut1 or Glut4. In contrast, the secretion of adipsin, a serine protease specifically expressed in adipocytes, was increased by the expression of wild-type ARF6 and was inhibited by the expression of D125N. These results indicate a requirement for ARF6 in basal and insulin-regulated adipsin secretion but not in glucose transport. Our results suggest the existence of at least two distinct pathways that undergo insulin-stimulated exocytosis in 3T3-L1 adipocytes, one for adipsin release and one for glucose transporter translocation.
Calcium, initially considered as the universal link between receptor stimulation and the onset of exocytosis in secretory cells, is now recognised as only one of a number of intracellular activators. In cells of haematopoietic origin (including mast cells), the key activator is one or more GTPases. Cells of this class, stimulated with GTPgammaS can undergo exocytosis in the effective absence of Ca(2+). A number of GTP-binding proteins that mediate exocytosis (G(E)) have been proposed but the best evidence supports roles for members of the Rho family of monomeric GTPases and for betagamma-subunits derived from G(i3). While preactivated Rac and Cdc42 can induce secretion from permeabilised mast cells in the absence of a guanine nucleotide betagamma-subunits only act to enhance the secretion induced by other GTP-binding proteins (likely to be members of the Rho family of monomeric GTPases). Further work is required to identify downstream effectors activated by these GTP-binding proteins and to show how they interact with the SNAP and SNARE isoforms known to be present in these cells.
Here we review evidence that actin and its binding partners are involved in the release of neurotransmitters at synapses. The spatial and temporal characteristics of neurotransmitter release are determined by the distribution of synaptic vesicles at the active zones, presynaptic sites of secretion. Synaptic vesicles accumulate near active zones in a readily releasable pool that is docked at the plasma membrane and ready to fuse in response to calcium entry and a secondary, reserve pool that is in the interior of the presynaptic terminal. A network of actin filaments associated with synaptic vesicles might play an important role in maintaining synaptic vesicles within the reserve pool. Actin and myosin also have been implicated in the translocation of vesicles from the reserve pool to the presynaptic plasma membrane. Refilling of the readily releasable vesicle pool during intense stimulation of neurotransmitter release also implicates synapsins as reversible links between synaptic vesicles and actin filaments. The diversity of actin binding partners in nerve terminals suggests that actin might have presynaptic functions beyond synaptic vesicle tethering or movement. Because most of these actin-binding proteins are regulated by calcium, actin might be a pivotal participant in calcium signaling inside presynaptic nerve terminals. However, there is no evidence that actin participates in fusion of synaptic vesicles.
Phagocytosis and the microbicidal functions of neutrophils require dynamic changes of the actin cytoskeleton. We have investigated the role of gelsolin, a calcium-dependent actin severing and capping protein, in peripheral blood neutrophils from gelsolin-null (Gsn-) mice. The phagocytosis of complement opsonized yeast was only minimally affected. In contrast, phagocytosis of IgG-opsonized yeast was reduced close to background level in Gsn- neutrophils. Thus, gelsolin is essential for efficient IgG- but not complement-mediated phagocytosis. Furthermore, attachment of IgG-opsonized yeast to Gsn- neutrophils was reduced ( approximately 50%) but not to the same extent as ingestion ( approximately 73%). This was not due to reduced surface expression of the Fcgamma-receptor or its lateral mobility. This suggests that attachment and ingestion of IgG-opsonized yeast by murine neutrophils are actin-dependent and gelsolin is important for both steps in phagocytosis. We also investigated granule exocytosis and several steps in phagosome processing, namely the formation of actin around the phagosome, translocation of granules, and activation of the NADPH-oxidase. All these functions were normal in Gsn- neutrophils. Thus, the role of gelsolin is specific for IgG-mediated phagocytosis. Our data suggest that gelsolin is part of the molecular machinery that distinguishes complement and IgG-mediated phagocytosis. The latter requires a more dynamic reorganization of the cytoskeleton.
It has been proposed that the cortical actin filament networks act as a cortical barrier that must be reorganized to enable docking and fusion of the synaptic vesicles with the plasma membranes. We identified a novel neuron-associated developmentally regulated protein, designated as Nadrin. Expression of Nadrin is restricted to neurons and correlates well with the differentiation of neurons. Nadrin has a unique structure; it contains a GTPase-activating protein (GAP) domain for Rho family GTPases, a potential coiled-coil domain, and a succession of 29 glutamines. In vitro the GAP domain activates RhoA, Rac1, and Cdc42 GTPases. Expression of Nadrin in NIH3T3 cells markedly reduced the number of the actin stress fibers and the formation of the ruffled membranes, suggesting that Nadrin regulates actin filament reorganization. In PC12 cells, Nadrin colocalized with synaptotagmin in the neurite termini and also with cortical actin filaments in the subplasmalemmal regions. Expression of Nadrin or its mutant composed of the coiled-coil and GAP domain enhanced Ca(2+)-dependent exocytosis of PC12 cells, but a mutant lacking the GAP domain inhibited exocytosis. These results suggest that Nadrin plays a role in regulating Ca(2+)-dependent exocytosis, most likely by catalyzing GTPase activity of Rho family proteins and by inducing the reorganization of the cortical actin filaments.
This chapter describes the Rho guanosine 5'-triphosphates (GTPases) secretion and actin dynamics in permeabilized mast cells. Permeabilized mast cells proved to be a valuable tool for studying the function of Rho-related GTPases. Rho, Rac, and Cdc42 were shown to be crucial for regulation of the late steps of exocytosis. The chapter discusses the use of primary mast cells—permeabilized by streptolysin O (SLO)—for studying Rho family GTPase. The chapter discusses the preparation of recombinant GTPases, an assay for GTP-binding activity, and subsequent loading of recombinant proteins with GTP. The chapter describes various procedures for introducing GTPase into mast cells and evaluation of their effects on the cytoskeleton and exocytosis. The chapter also discusses the isolation, permeabilization, and stimulation of rat peritoneal mast cells. The chapter concludes with a discussion of the analysis of F-actin content and morphology.
Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.
The rapid, ultrasensitive silver stains that have been developed recently for detecting proteins in polyacrylamide gels show variation in staining from gel to gel and do not stain certain proteins at all. It was found that treatment of gels with dithiothreitol prior to impregnation with silver nitrate results in more reproducible staining patterns that are also qualitatively similar to those obtained with Coomassie blue. In addition, it obviates the need for treatment with intense light, and results in sensitivities at least as high as those obtained with previously published methods.
Cytoplasmic Ca2+ is a major regulator of exocytosis in secretory cells; however, the Ca(2+)-dependent mechanisms that trigger secretion have not been elucidated. Protein kinase C (PKC) has been proposed to be an important Ca(2+)-dependent component of this regulation; however, the effects of this enzyme on the exocytotic apparatus have not been identified. We developed a PKC-deficient, semi-intact PC12 cell system in which direct stimulatory effects of purified PKC on Ca(2+)-dependent norepinephrine secretion were studied. The reconstitution of optimal Ca(2+)-activated norepinephrine secretion by semi-intact PC12 cells required the addition of MgATP and cytosolic proteins. PKC-deficient cytosol exhibited reduced reconstituting activity that was fully restored by the addition of purified PKC. The restoration of Ca(2+)-dependent norepinephrine secretion by PKC required the presence of other proteins in the cytosol, in particular, a high molecular weight protein. The high molecular weight protein was identified as p145, a recently characterized 145-kDa brain protein. The addition of PKC enhanced phosphorylation of p145 under conditions of fully reconstituted Ca(2+)-activated norepinephrine secretion. The results indicate that 1) PKC is neither necessary nor sufficient for Ca(2+)-activated secretion, whereas other cytosolic proteins are required; and 2) the stimulation of Ca(2+)-activated secretion by PKC is dependent upon cytosolic proteins such as p145 and may be largely mediated through the phosphorylation of p145.
rac1 and rac2 p21s are ras p21-like small GTP-binding proteins which are implicated in the NADPH oxidase-catalyzed superoxide generation in phagocytes. rac1 and rac2 p21s have a Cys-A-A-Leu (A = aliphatic amino acid) structure in their C-terminal region which may undergo post-translational processing including prenylation, proteolysis, and carboxyl methylation. We studied the function of this post-translational processing of rac p21s in their interaction with the stimulatory and inhibitory GDP/GTP exchange proteins for rac p21s, named smg GDS and rho GDI, and in their NADPH oxidase activation. We produced human recombinant rac1 and rac2 p21s in insect cells and purified them from the membrane and soluble fractions as the post-translationally processed and unprocessed forms, respectively. Post-translationally processed rac1 and rac2 p21s were sensitive to both smg GDS and rho GDI, but post-translationally unprocessed rac1 and rac2 p21s were insensitive to them. The GTP gamma S (guanosine 5'-(3-O-thio)triphosphate)-bound form of post-translationally processed rac1 and rac2 p21s stimulated the NADPH oxidase activity, but post-translationally unprocessed rac1 and rac2 p21s were far less effective. These results indicate that both rac1 and rac2 p21s stimulate the NADPH oxidase activity and that their post-translational processing is important not only for their interaction with smg GDS and rho GDI but also for their NADPH oxidase activation.
The mechanism of eosinophil secretion was studied in guinea pig eosinophils by measuring release of hexosaminidase from cell suspensions (greater than 98% pure) permeabilized with streptolysin-O and by whole-cell patch-clamp capacitance measurements. It is shown that release of eosinophil granule components occurs by an exocytotic mechanism in which individual granules fuse with the plasma membrane. Exocytosis can be induced by intracellular application of the nonhydrolyzable GTP analog guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), suggesting the involvement of a GTP-binding protein. The activation is modulated by the intracellular calcium concentration, with activation by GTP-gamma-S inducing transient elevations in the concentration of Ca2+. Thus, the nature and regulation of the release mechanism appear to be very similar to that of the mast cell and neutrophil.
A new family of ras-related proteins, designated rac (ras-related C3 botulinum toxin substrate) has been identified. rac1 and rac2 cDNA clones were isolated from a differentiated HL-60 library and encode proteins that are 92% homologous and share 58% and 26-30% amino acid homology with human rhos and ras, respectively. Nucleotide sequence analysis predicts both rac1 and rac2 proteins to contain 192 amino acids with molecular masses of 21,450 and 21,429 daltons, respectively. rac1 and rac2 possess four of the five conserved functional domains in ras associated with binding and hydrolysis of guanine nucleotides. They also contain the COOH-terminal consensus sequence Cys-X-X-X-COOH which localizes ras to the inner plasma membrane and the residues Gly12 and Ala59, at which sites mutations elicit transforming potential to ras. The rac transcripts, particularly rac2, display relative myeloid tissue selectivity. Both rac1 transcripts (2.4 and 1.1 kilobases (kb] increase when HL-60 cells differentiate to neutrophil-like morphology. In contrast, differentiation of U937 cells to monocyte-like morphology causes no change in the 2.4-kb mRNA and a decrease in the 1.1-kb mRNA species. rac2 mRNA (1.45 kb) increases 7-9-fold and 3-fold upon differentiation of HL-60 and U937 cells, respectively. Neither rac mRNAs are present in a Jurkat T cell line, and unlike rac1, rac2 mRNA is absent in human brain and liver tissue. Transfection experiments permitted the demonstration that rac1 and rac2 are substrates for ADP-ribosylation by the C3 component of botulinum toxin. The data suggest that racs are plasma membrane-associated GTP-binding proteins which could regulate secretory processes, particularly in myeloid cells.
This study addresses the question of the role of cytoplasmic proteins in exocytosis from permeabilised rat mast cells. We have used two different methods of cell permeabilisation (ATP4- and streptolysin O) to regulate the size of the plasma membrane lesions, and thus to dictate the rate and extent of efflux of the cytosolic proteins, and compared the secretory response of the two preparations. We report evidence for the existence of two factors present in the cytosol, which affect the exocytotic mechanism in opposing manners. One of these is required for the maintenance of cell responsiveness; it is retained for more than 120 min by ATP4- -permeabilised cells but lost within 60 min from cells permeabilised by streptolysin O. The other factor, which leaks immediately from cells treated from streptolysin O, but only gradually from cells treated with ATP4-, has the effect of suppressing the affinity for both Ca2+ and guanine nucleotide in the exocytotic reaction.
The secretory process is a coordinated cellular response, initiated by occupation of surface receptors and comprising an ordered sequence of biochemical steps subject to multiple controls. Conceptually we can divide the sequence into two main sections comprising early, receptor-mediated events leading to generation of intracellular second messengers, and later events leading to membrane fusion and exocytosis. With the discovery that occupation of Ca2+ mobilising receptors leads to activation of polyphosphoinositide phosphodiesterase (PPI-pde) through the mediation of a G-protein (Gp), all the early events can be ascribed to the plasma membrane. Investigation of the exocytotic stage of secretion has been simplified by the use of permeabilised cells in which the composition of the cytosol can be precisely controlled. We have used streptolysin-O, a bacterial cytolysin which generates protein-sized pores in the plasma membrane, to investigate the exocytotic mechanism of rat mast cells. We find that in addition to the activation of PPI-dpe, GTP also acts in concert with Ca2+ at, or close to, the exocytotic site. Exocytosis can occur after substantial depletion of cytosol lactate dehydrogenase and 3-phosphoglycerate kinase indicating that soluble cytosol proteins are unlikely to play any role. There is no absolute requirement for ATP or phosphorylating nucleotide in exocytosis though when present the effective affinities of the two obligatory effectors (i.e. Ca2+ and GTP) are substantially enhanced.
1. Mitochondrial and microsomal fractions were prepared from rat parotid glands. Both fractions were able to take up (45)Ca. The mitochondrial (45)Ca-uptake system could be driven by ATP (energy-coupled Ca(2+) uptake) or by ADP+succinate (respiration-coupled Ca(2+) uptake). Energy-coupled Ca(2+) uptake was blocked by oligomycin but not by carbonyl cyanide m-chlorophenylhydrazone; respiration-coupled Ca(2+) uptake was blocked by carbonyl cyanide m-chlorophenylhydrazone but not by oligomycin. Microsomal Ca(2+) uptake was dependent on the presence of ATP; the ATP-dependent Ca(2+) uptake was not affected by oligomycin or carbonyl cyanide m-chlorophenylhydrazone. Ca(2+) uptake by both fractions was inhibited by Ni(2+). 2. Incubation of parotid pieces with adrenaline increased the rate of release of amylase and the uptake of (45)Ca. The adrenaline-stimulated release of amylase was not dependent on the presence of extracellular Ca(2+). 3. The effect of adrenaline on the subcellular distribution of (45)Ca in parotid pieces incubated with (45)Ca was studied. In parotid tissue incubated with (45)Ca, both mitochondrial and microsomal fractions contained (45)Ca. Incubation with adrenaline increased the amount of (45)Ca incorporated into the mitochondrial fraction but not the microsomal fraction. In parotid tissue preloaded with (45)Ca subsequent incubation with adrenaline caused a decrease in the amount of (45)Ca found in both the mitochondrial and microsomal fractions. 4. From these data we conclude that the regulation of the cytosolic Ca(2+) concentration in the parotid may involve both mitochondrial and microsomal Ca(2+)-uptake systems. We suggest that the action of adrenaline on the parotid may be to increase the movement of Ca(2+) to the cytosol by increasing the flux of Ca(2+) across mitochondrial, microsomal and plasma membranes.
Rho, Rac, and Cdc42 are small GTPases that regulate the formation of a variety of actin structures and the assembly of associated
integrin complexes, but little is known about the target proteins that mediate their effects. Here we have used a motif-based
search method to identify putative effector proteins for Rac and Cdc42. A search of the GenBank data base for similarity with the minimum Cdc42/Rac interactive binding (CRIB) region of a potential effector protein p65 has identified over 25 proteins containing a similar motif from a range of different species. These candidate Cdc42/Rac-binding
proteins include family members of the mixed lineage kinases (MLK), a novel tyrosine kinase from Drosophila melanogaster (DPR2), a human protein MSE55, and several novel yeast and Caenorhabditis elegans proteins. Two murine p65 isoforms and a candidate protein from C. elegans, F09F7.5, interact strongly with the GTP form of both Cdc42 and Rac, but not Rho in a filter binding assay. Three additional
candidate proteins, DPR2, MSE55, and MLK3 showed binding to the GTP form of Cdc42 and weaker binding with Rac, and again no
interaction with Rho. These results indicate that proteins containing the CRIB motif bind to Cdc42 and/or Rac in a GTP-dependent
manner, and they may, therefore, participate in downstream signaling.
The brain-enriched p21cdc42/rac1-activated serine/threonine kinase, p65PAK, was identified and purified on the basis of overlays with [gamma-32P]GTP-Cdc42 onto SDS-fractionated proteins (Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-S., and Lim, L. (1994) Nature 367, 40-46). In this study, the ubiquitously expressed p21cdc42/rac1 binding protein with relative molecular weight of 62,000 was purified from rat testes and shown to contain peptides related to PAK. It has thus been designated as the gamma-PAK isoform (alpha- and beta-isoforms being brain enriched). Isolation of gamma-PAK cDNAs show that the kinase is highly conserved with alpha-PAK in both the p2 binding and kinase domains. The purified protein exhibited kinase activity that was activated by GTP-Cdc42 or GTP-Rac1 in vitro. In platelets, a p62 in situ renaturable kinase was recognized by antibodies raised against gamma-PAK. This thrombin-activated protein kinase appears to coprecipitate with another kinase of M(r) 86,000, suggesting that PAK may be part of a thrombin-responsive signaling complex.
The Ras-related protein Cdc42 plays a role in yeast cell budding and polarity. Two related proteins, Rac1 and RhoA, promote
formation in mammalian cells of membrane ruffles and stress fibers, respectively, which contain actin microfilaments. We now
show that microinjection of the related human Cdc42Hs into Swiss 3T3 fibroblasts induced the formation of peripheral actin
microspikes, determined by staining with phalloidin. A proportion of these microspikes was found to be components of filopodia,
as analyzed by time-lapse phase-contrast microscopy. The formation of filopodia was also found to be promoted by Cdc42Hs microinjection.
This was followed by activation of Rac1-mediated membrane ruffling. Treatment with bradykinin also promoted formation of microspikes
and filopodia as well as subsequent effects similar to that seen upon Cdc42Hs microinjection. These effects of bradykinin
were specifically inhibited by prior microinjection of dominant negative Cdc42HsT17N, suggesting that bradykinin acts by activating
cellular Cdc42Hs. Since filopodia have been ascribed an important sensory function in fibroblasts and are required for guidance
of neuronal growth cones, these results indicate that Cdc42Hs plays an important role in determining mammalian cell morphology.
Activation of the NADPH oxidase of phagocytes involves the small GTP-binding protein p21rac. In this paper we report that neutrophil cytosol contains predominantly p21rac2 rather than p21rac1, and that the P21rac2 is almost entirely complexed with rhoGDI (GDP dissociation inhibitor) to form a heterodimer with a molecular mass of 45-50 kDa. Activation of superoxide production by phorbol 12-myristate 13-acetate or formylmethionyl-leucyl-phenylalanine in whole cells, and by SDS in the cell-free assay, led to the dissociation of some of the p21rac2 from rhoGDI and its movement to the plasma membrane together with p47phox and p67phox. The appearance of these proteins at the plasma membrane was related to the dose of the agonist and to the rate of superoxide generation. The nucleotide bound to p21rac2 in this complex following isolation was almost exclusively GDP, with less than 2% GTP, and the complex was active in the cell-free assay. Although the rac/GDI complex could activate the NADPH oxidase in the absence of exogenous GTP, the rate of superoxide production was increased 3-fold by the addition of GTP and was almost completely inhibited by GDP. Our findings confirm that rhoGDI serves as GDP dissociation inhibitor and that the release of p21rac2 from this inhibitor is an important step in activation of the NADPH oxidase.
The Ras(17N) dominant negative antagonizes endogenous Ras function by forming stable, inactive complexes with Ras guanine nucleotide exchange factors (GEFs; e.g., SOS1). We have used the growth-inhibitory phenotype of Ras(17N) to characterize two aspects of Ras interaction with GEFs. First, we used a nonprenylated version of Ras(17N), designated Ras(17N/186S), which no longer associates with the plasma membrane and lacks the growth-inhibitory phenotype, to address the importance of Ras subcellular location and posttranslational modification for its interaction with GEFs. We observed that addition of an N-terminal myristylation signal to Ras(17N/186S) restored the growth-inhibitory activity of nonprenylated Ras(17N). Thus, membrane association, rather than prenylation, is critical for Ras interaction with Ras GEFs. Second, we used a biological selection approach to identify Ras residues which are critical for Ras(17N) growth inhibition and hence for interaction with Ras GEFs. We identified mutations at residues 75, 76, and 78 that abolished the growth-inhibitory activity of Ras(17N). Since GEF interaction is dispensable for oncogenic but not normal Ras function, our demonstration that single-amino-acid substitutions at these three positions impaired the transforming activity of normal but not oncogenic Ras provides further support for the role of these residues in Ras-GEF interactions. Finally, Ras(WT) proteins with mutations at these residues were no longer activated by mammalian SOS1. Altogether, these results suggest that the Ras intracellular location and Ras residues 75 to 78 are critical for Ras-GEF interaction.
Rac1 and Rac2 are closely related, low molecular weight GTP-binding proteins that have both been implicated in regulation of phagocyte NADPH oxidase. This enzyme system is composed of multiple membrane-bound and cytosolic subunits and when activated catalyzes the one-electron reduction of oxygen to superoxide. Superoxide and its highly reactive derivatives are essential for killing microorganisms. Rac proteins undergo posttranslational processing, primarily the addition of an isoprenyl group to a carboxyl-terminal cysteine residue. We directly compared recombinant Rac1 and Rac2 in a human neutrophil cell-free NADPH oxidase system in which cytosol was replaced by purified recombinant cytosolic components (p47-phox and p67-phox). Processed Rac1 and Rac2 were both highly active in this system and supported comparable rates of superoxide production. Under different cell-free conditions, however, in which suboptimal amounts of cytosol were present in the assay mixture, processed Rac2 worked much better than Rac1 at all but the lowest concentrations. This suggests that a factor in the cytosol may suppress the activity of Rac1 but not of Rac2. Unprocessed Rac proteins were only weakly able to support superoxide generation in either system, but preloading of Rac1 or Rac2 with guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S) restored activity. These results indicate that processing is required for nucleotide exchange but not for interaction with oxidase components.
n-Chimaerin is a GTPase-activating protein (GAP) mainly for Rac1 and less so for Cdc42Hs in vitro. The GAP activity of n-chimaerin is regulated by phospholipids and phorbol esters. Microinjection of Rac1 and Cdc42Hs into mammalian cells induces formation of the actin-based structures lamellipodia and filopodia, respectively, with the former being prevented by coinjection of the chimaerin GAP domain. Strikingly, microinjection of the full-length n-chimaerin into fibroblasts and neuroblastoma cells induces the simultaneous formation of lamellipodia and filopodia. These structures undergo cycles of dissolution and formation, resembling natural morphological events occurring at the leading edge of fibroblasts and neuronal growth cones. The effects of n-chimaerin on formation of lamellipodia and filopodia were inhibited by dominant negative Rac1(T17N) and Cdc42Hs(T17N), respectively. n-Chimaerin's effects were also inhibited by coinjection with Rho GDP dissociation inhibitor or by treatment with phorbol ester. A mutant n-chimaerin with no GAP activity and impaired p21 binding was ineffective in inducing morphological changes, while a mutant lacking GAP activity alone was effective. Microinjected n-chimaerin colocalized in situ with F-actin. Taken together, these results suggest that n-chimaerin acts synergistically with Rac1 and Cdc42Hs to induce actin-based morphological changes and that this action involves Rac1 and Cdc42Hs binding but not GAP activity. Thus, GAPs may have morphological functions in addition to downregulation of GTPases.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The mechanism of eosinophil secretion was studied in guinea pig eosinophils by measuring release of hexosaminidase from cell suspensions (greater than 98% pure) permeabilized with streptolysin-O and by whole-cell patch-clamp capacitance measurements. It is shown that release of eosinophil granule components occurs by an exocytotic mechanism in which individual granules fuse with the plasma membrane. Exocytosis can be induced by intracellular application of the nonhydrolyzable GTP analog guanosine-5'-O-(3-thiotriphosphate) (GTP- gamma-S), suggesting the involvement of a GTP-binding protein. The activation is modulated by the intracellular calcium concentration, with activation by GTP-gamma-S inducing transient elevations in the concentration of Ca2+. Thus, the nature and regulation of the release mechanism appear to be very similar to that of the mast cell and neutrophil.
This chapter discusses the purification method of recombinant Rho/Rac/G25K from Escherichia coli. The purification of Ras-related GTP-binding proteins from recombinant sources has proved to be invaluable for studying their biochemical properties and biological effects. The simplest expression systems have made use of Escherichia coli, although Ras-like GTPases produced in this way are not posttranslationally modified. Yeast and baculovirus-Sf9 (Spodoptera frugiperda, fall armyworm ovary) insect cells have also been used, and because they are eukaryotic hosts, the GTPases expressed are at least partially modified. The mammalian Rho subfamily consists of RhoA, B, and C; Rac1l and 2; G25K/CDC42; RhoG; and TC10. These proteins are 30% identical to Ras in amino acid sequence and 55% identical to each other, and their overall three-dimensional structure is similar to that of Ras. Like all small GTPases, the Rho-related proteins are regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), and the characterization of these regulatory proteins relies on a source of a recombinant protein.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Studies of intracellular traffic in yeast and mammalian systems have implicated members of the Rab family of small GTP-binding proteins as regulators of membrane fusion. We have used the patch clamp technique to measure exocytotic fusion events directly and investigate the role of GTP-binding proteins in regulating exocytosis in mast cells. Intracellular perfusion of mast cells with GTP-gamma S is sufficient to trigger complete exocytotic degranulation in the absence of other intracellular messengers. Here we show that GTP is a potent inhibitor of GTP-gamma S-induced degranulation, indicating that sustained activation of a GTP-binding protein is sufficient for membrane fusion. We have found that synthetic oligopeptides, corresponding to part of the effector domain of Rab3a, stimulate complete exocytotic degranulation, similar to that induced by GTP-gamma S. The response is selective for Rab3a sequence and is strictly dependent on Mg2+ and ATP. This suggests that sustained activation of a Rab3 protein causes exocytotic fusion. The peptide response can be accelerated by GDP-beta S, suggesting that Rab3a peptides compete with endogenous Rab3 proteins for a binding site on a target effector protein, which causes fusion on activation.
Human cloned CD8+ cytotoxic T lymphocytes permeabilized with alpha-toxin of Staphylococcus aureus can be triggered by the guanosine triphosphate (GTP) analogue GTP gamma S to release the contents of their granula by exocytosis. To localize the guanosine nucleotide-binding protein (G-protein) activated by GTP gamma S in the sequence of events after T-lymphocyte triggering we have used several inhibitors of T-cell activation that inhibit early stages in T-cell triggering. The protein kinase C-inhibitor staurosporine, the immunosuppressants cyclosporin A and FK-506 and genistein, an inhibitor of tyrosine kinases, all inhibited esterase release triggered in intact cells by anti-T-cell receptor antibodies but not GTP gamma S-induced release from permeabilized cells. Cyclosporin A, FK-506 and genistein also blocked exocytosis triggered in intact cells by a combination of phorbolester and the calcium ionophore A23187. In addition, cytochalasin B, an inhibitor of actin polymerization, inhibited exocytosis in intact cells but enhanced exocytosis from permeabilized cells. These data show that the G-protein effecting exocytosis is localized distally in the cascade of events after T-cell activation.
Exocytosis from metabolically depleted permeabilized rat mast cells was measured in response to provision of Ca2+ and guanine nucleotide [GTP or guanosine 5'-[gamma-thio]triphosphate (GTP[S])]. For cells permeabilized in simple salt solutions (NaCl), both of these effectors were required to induce secretion. Exclusion of Mg2+ caused an increase in both the sensitivity of the system to GTP and the extent of secretion elicited, while having no such effects on secretion induced by GTP[S]. The effect of Mg2+ depletion on the ability of GTP to stimulate secretion is probably due to the dependence on Mg2+ of the GTPase activity of GE (a postulated GTP-binding protein which mediates exocytosis). This argues that a persistent stimulus to the G-protein is required to support secretion. Affinity for both GTP[S] and GTP is enhanced when the cells are permeabilized in zwitterionic electrolytes (glutamate, gamma-aminobutyric acid, glycine) instead of NaCl. Under these conditions, secretion occurs in response to provision of either GTP[S] [in the effective absence of Ca2+ (pCa 9)] or Ca2+ (in the absence of guanine nucleotide). Secretion induced by GTP[S] is strongly promoted by the presence of Mg2+ at concentrations in the millimolar range; this promotion by Mg2+ declines as the concentration of Ca2+ is elevated towards pCa 7. At pCa 6, Mg2+ is without effect. Ca(2+)-induced secretion requires the provision of MgATP. Since this is further enhanced by low concentrations (< 100 microM) and then inhibited by high concentrations of GDP, the essential role of ATP is likely to be in the maintenance of GTP via transphosphorylation by a nucleoside diphosphate kinase reaction. Thus, under conditions of high affinity (glutamate environment), GTP[S] alone is capable of inducing exocytosis. Ca2+ acts in concert with guanine nucleotides: it enhances the rate and extent of secretion and increases the affinity for Mg2+ and guanine nucleotides in the activation of the GTP-binding protein (GE) which regulates exocytosis.
This chapter report techniques for studying the exocytotic process in rat peritoneal mast cells. It also investigated other myeloid cells and these methods, and more importantly the basic considerations, are widely applicable. Regulated exocytosis is controlled by a sequence of tightly coupled, biochemical steps and membrane-membrane interactions that occur within the cell interior. Investigations using whole cells or tissues, and using agonists, ionophores, and various pharmacological agents, have in many cases revealed a requirement for Ca²⁺ and an intact cellular metabolism. However, as the sites at which the membrane fusions occur are intracellular, it is necessary to gain experimental access to the cytosol to investigate the processes that lead up to the exocytotic event. This has been achieved by various methods of cell permeabilization.
The mechanisms of granule protein secretion have been studied in streptolysin-O-permeabilized guinea pig eosinophils. Secretion of the granule-associated enzyme N-acetyl-beta-D-glucosaminidase was dependent on both Ca2+ and a nonhydrolyzable GTP analogue, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), suggesting roles for both calcium and GTP binding proteins. Secretion was maximal by 7 min, and varied between 35 and 60% of the total enzyme activity. Other GTP analogues also elicited secretion, with rank order GTP-gamma-S greater than guanylyl-imidophosphate greater than guanylyl (beta-gamma-methylene-diphosphate). Unrelated nucleotide triphosphates showed little or no effect confirming the specificity of the G protein. Transmission electronmicroscopy confirmed that permeabilization alone did not result in loss of granules and that exocytosis was dependent on the addition of the effectors, Ca2+ and GTP-gamma-S. ATP enhanced the magnitude of the secretory response and also enhanced the effective affinities for both Ca2+ and GTP-gamma-S. In the presence of 10(-5) M GTP-gamma-S the ED50 (Ca2+) was pCa 5.57 +/- 0.04 (2.69 microM) in the absence of ATP and declined to pCa 6.16 +/- 0.03 (0.69 microM) in the presence of ATP (p less than 0.0001). Furthermore, ATP served to restore responsiveness in cells that had been rendered refractory by delaying stimulation after permeabilization. Pretreatment with PMA (an activator of PKC) inhibited the induction of a refractory state, whereas inhibition of PKC partially countered the ability of ATP to restore responsiveness, both observations pointing to a requirement for a specific component of the secretory mechanism to be in a phosphorylated state in order to condone the secretion process. These observations show that secretory mechanisms in eosinophils are similar to those in other myeloid cells, in particular neutrophils and mast cells, although the time course of secretion is more protracted.
Metabolically stable analogues of GTP, e.g. guanosine 5′‐[γ‐thio]trio]triphosphate (GTP[S]) and guanosine 5′‐[β‐γ‐imido]triphosphate (pp[NH]pG), enhance the extent of Ca ²⁺ ‐dependent secretion of β‐ N ‐acetylgluco‐saminidase and β‐galactosidase from electropermeabilised human platelets in the presence of less than 5 μM Ca ²⁺ .A similar effect is observed on addition either of 1,2‐dioctanoin or of GTP in the presence or absence of thrombin.
In the presence of higher Ca ²⁺ concentrations the extent of enhancement of lysosomal secretion declines and little, or no, enhancement is observed at a [Ca ²⁺ ] of 30–40 μM. Addition of leupeptin or antipain prevents this decrease in lysosomal secretion and enhances the extent of Ca ²⁺ ‐dependent lysosomal secretion obtained in the presence or absence of guanine nucleotides, thrombin or 1,2‐dioctanoin.
The concentration of GTP[S] or pp[NH]pG required to obtain half‐maximal enhancement of lysosomal secretion is dependent on [Ca ²⁺ ] for secretion of 5‐hydroxytryptamine, β‐ N ‐acetylglucosaminidase and β‐galactosidase. At two fixed [Ca ²⁺ ] the median effective concentration (EC 50 ) values for GTP[S] and pp[NH]pG which characterise enhancement of 5‐hydroxytryptamine secretion are significantly different from those characterising enhancement of the secretion of β‐ N ‐acetylglucosaminidase and β‐galactosidase.
In the presence of a saturating concentration of GTP[S] marked 5‐hydroxytryptamine and β‐ N ‐acetylglucosaminidase secretion is observed at nanomolar [Ca ²⁺ ] and these responses show little dependence on [Ca ²⁺ ] over the attainable range. Secretion of β‐ N ‐acetylglucosaminidase is also induced at nanomolar Ca ²⁺ concentrations by addition of activators of protein kinase C.
Guanosine 5′‐[β‐thio]diphosphate inhibits enhancement of β‐ N ‐acetylglucosaminidase secretion induced by GTP[S] but has no effect on secretion of this enzyme induced by Ca ²⁺ when added alone.
Our data provide some support for a model in which addition of metabolically stable guanine nucleotides enhances Ca ²⁺ ‐dependent platelet lysosomal secretion by activating a guanine‐nucleotide‐binding protein (G E ) located close to the exocytotic site. However, not all the data are consistent with this postulate.
Ras proteins can be modified at their COOH-terminal cysteine in the motif Cys-Ali-Ali-Xaa by a farnesyl isoprenoid. This modification is essential for membrane association and biological activity of ras proteins. A similar COOH-terminal amino acid sequence, Cys-Xaa-Ali-Xaa, exists in the ras-related GTP-binding proteins rac 1 and rac 2. To determine whether these proteins were similarly modified, COS cells were transfected with rac 1 and rac 2 cDNA and expressed proteins were labeled with [3H]mevalonic acid. We report here that both rac 1 and rac 2 are post-translationally modified by addition of an isoprenoid group, the likely site of which is the COOH-terminal cysteine. Isoprenylation was found only in racs associated with particulate cell fractions, suggesting that this modification may be associated with membrane localization of the proteins. These data specifically identify mammalian low molecular mass GTP-binding proteins other than ras that undergo post-translational modification and further define the COOH-terminal consensus sequence, Cys-Ali-Ali-Xaa, as an isoprenylation signal. This sequence may identify a larger family of low molecular mass GTP-binding proteins which are isoprenylated.
We have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated Gp (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human Gp protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins (approximately 50% identical), and the rac proteins (approximately 70% identical). The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell-division-cycle protein CDC42. The human placental gene, which we designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein.
Calpactin, or calpactin heavy chain (p36), reconstitute secretion in digitonin-permeabilized adrenal chromaffin cells after a reduction in their secretory potential resulting from the loss of cytosolic components. We have characterized the stimulatory effect of p36, which resulted in an increase in both the extent and the rate of exocytosis. A mixture of other annexins (p70 and p32) did not have any effect on secretion at similar or greater concentrations than p36. Controlled proteolysis of p36 using chymotrypsin was carried out, and the 33,000 molecular weight core and 3000 molecular weight tail peptide isolated. In contrast to p36, p33 had no effect on exocytosis, even at high calcium concentrations. The N-terminal tail peptide and a synthetic peptide based on the tail of p36 [Ac-calpactin-(1-15)-NH2] had no effect on endogenous secretion, or secretion stimulated by exogenous p36. The results show that both the tail and core domains are required for p36 to stimulate exocytosis. The tail domain is unlikely to be required for interaction with cellular components but probably has a regulatory effect on the core domain. Endogenous secretion and the stimulatory effect of p36 were markedly inhibited by depletion of ATP. The ATP requirement for p36 action was not due to a requirement for phosphorylation by protein kinase C (PKC), since the PKC inhibitor staurosporine partially inhibited endogenous secretion but did not affect the stimulation of exocytosis due to exogenous p36.
Rat peritoneal mast cells have been permeabilised by treatment with streptolysin O which generates membrane lesions of macromolecular dimensions. In the presence of Ca2+ buffered at concentrations in the micromolar range, the permeabilised mast cells release histamine, beta-N-acetylglucosaminidase and lactate dehydrogenase. Release of the two secretory components (but not lactate dehydrogenase) has an obligatory requirement for a nucleoside triphosphate and micromolar concentrations of Ca2+. Inosine triphosphate (ITP) supports the release reaction better than ATP does. It is concluded that the secretory materials are released from the cells by an exocytotic mechanism, while lactate dehydrogenase leaks from the cells through the toxin-generated lesions. By initially withholding and then supplying Ca2+ to the permeabilised cells, it is shown that the exocytotic secretory reaction can persist even when the cytosol is depleted of the bulk of soluble proteins. The streptolysin O treated mast cell preparation represents a simplified system with which to study the mechanism of exocytosis.
The term 'stimulus-secretion coupling' has, since first enunciated, been held to involve the mobilization of cytosol Ca2+, which in turn is sufficient to trigger exocytotic secretory processes in metabolically competent cells. However, recent studies on a wide range of secretory cell types indicate that a role for Ca2+ can be obviated: examples are stimulation with phorbol ester (phorbol myristate acetate, PMA) which causes the activation of protein kinase C or the stimulation of platelets with collagen. Ca2+-independent exocytosis also occurs when analogues of GTP are injected through the lumen of patch pipettes directly into the cytosol of mass cells. The results presented here suggest that GTP analogues can activate secretory processes by actions at two distinct locations: one may be at the level of the receptor involving the activation of polyphosphoinositide (PPI) phosphodiesterase with consequent liberation of diacylglycerol (DG); the other involves direct activation of the exocytotic mechanism. These conclusions are based on measurements of exocytotic secretion from permeabilized neutrophils into which we have been able to introduce, individually and in combination, Ca2+ chelators (EGTA and BAPTA), Ca2+ (buffered at micromolar concentrations with EGTA), analogues of GTP and GDP and the direct activator of protein kinase C, PMA.
Exocytosis is the release of intracellular vesicular contents directly to the cell exterior after fusion of the vesicular and plasma membranes. It is generally accepted as the process by which transmitters and hormones are released from neurons and neurosecretory cells. There is overwhelming biochemical evidence that exocytosis is the mechanism by which catecholamines are released from adrenal chromaffin cells. With the exception of the hamster, however, there is little ultrastructural evidence to support such a mechanism. We have used a modified in vitro tannic-acid method to visualize exocytosis by transmission electron microscopy in intact and saponin-permeabilized bovine chromaffin cells. When cells are exposed to tannic-acid-containing medium, the content of vesicles involved in exocytosis is coagulated in situ as the vesicle opens to the exterior. Numerous exocytotic profiles were observed. The exposed vesicle contents appeared more granular than those of vesicles in the cell interior. Tannic acid also made the plasma membrane more distinct. Small holes were apparent in the plasma membrane of saponin-treated cells, with little disruption of underlying cytoplasmic structure. Furthermore, when these cells were stimulated with calcium, exocytosis was evident only at regions of intact plasma membrane, not at the holes. Parallel measurements of secretion showed no secretion in the presence of tannic acid. Pretreatment with tannic acid prevented subsequent secretion by intact cells and markedly reduced that of permeabilized cells, indicating a probable change in the nature of the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Mast cells undergo an extensive and violent morphological transformation on stimulation. Here we describe the dynamics of fusion of the secretory granules in individual mast cells during exocytosis. The cell membrane capacitance (proportional to the cell surface area) was measured using the whole-cell patch-pipette technique, in which the intracellular space is dialysed with the solutions used to fill the patch pipette. Our results show that degranulation occurs spontaneously and reproducibly if the GTP analogue, GTP-gamma-S, and Mg-ATP are present in the pipette filling solution. Contrary to previous reports, in these conditions Ca2+ (and/or Ca2+ buffers) is not required for degranulation. Although electrogenic Ca2+ entry was not detected before or during degranulation and membrane conductance remained low, the capacitance, and by implication the area of the membrane of degranulating cells, increased sigmoidally and stepwise. We conclude that stepwise increases of capacitance are due to the fusion of individual secretory granules with the plasma membrane, and that guanine nucleotide regulatory proteins are involved in the control of this process.
By subjecting isolated adrenal medullary cells to intense electric fields of brief duration it is possible to gain access to the cell interior without impairing the ability of the cell to undergo exocytosis. After a single exposure to a field of 2 kV/cm, τ=200 μsec, adrenal medullary cells behave as if their plasma membrane contains two pores of effective radius 2 nm. At 37°C these ‘equivalent pores’ remain patent for up to 1 hr. The formation and stability of these ‘pores’ is not affected by the Ca content of the bathing solution. The ‘pores’ permit externally applied catecholamine and Ca-EGTA to equilibrate rapidly with the cell water.
Cells rendered ‘leaky’ in K glutamate medium containing 5mm Mg-ATP and EGTA to give an ionized Ca close to 10−8m release less than 1% of their total catecholamine. These same cells can release up to 30% of their catecholamine when exposed to 10−5m Ca. This Ca-dependent release is unaffected by Ca-channel blockers such as D600. Catecholamine release in response to a calcium challenge only seems to occur during the first few minutes whilst the Ca concentration is changing, and the extent of release depends on the final Ca concentration achieved. Half-maximal release occurs at about 1 μm Ca, and this value is independent of the EGTA concentration used to buffer the ionized Ca. The relation between ionized Ca and catecholamine release is best fitted by a requirement for 2 Ca ions.
Calcium-evoked release of catecholamine is associated with the release of dopamine-β-hydroxylase (DβH) but not lactate dehydrogenase. The ratio DβH/catecholamine released is the same as that in stimulated intact cells and perfused glands. The time course of appearance in the external medium of DβH and catecholamine is identical. Transmission electron microscopy of ‘leaky’ cells exposed to 10−8m Ca reveals no marked differences from unstimulated intact cells. The cytoplasm of ‘leaky’ cells exposed to 10−5m Ca contains large membrane-bounded vacuoles. When secretion is caused to take place in the presence of horseradish peroxidase, this marker is found within the vacuoles.
Ca-dependent release of both catecholamine and DβH requires Mg-ATP. Cells equilibrated with Ca in the absence of Mg-ATP can be triggered to undergo exocytosis by the addition of Mg-ATP. In the absence of Mg, ATP alone is ineffective. Of a variety of other nucleotides tested, none is as effective as ATP. Mg-ATP affects the extent of exocytosis and not its apparent affinity for Ca.
Replacement of glutamate as the major anion by chloride results in a marked reduction in Ca-dependent release of both catecholamine and DβH. Chloride causes a small increase in Ca-independent release of catecholamine, a large reduction in the extent of exocytosis, and a decrease in the apparent affinity of exocytosis for Ca. Of a variety of anions examined, their order of effectiveness at supporting Ca-dependent exocytosis is glutamate−>acetate−>Cl−>Br−>SCN−.
Exocytosis is not obviously affected by replacing K by Na or sucrose or by altering the pH over the range pH 6.6 to 7.8. Raising the free Mg concentration reduces the extent of Ca-dependent exocytosis and also its apparent affinity for calcium. Calcium-dependent exocytosis in ‘leaky’ cells is largely unaffected by (i) a variety of agonists and antagonists of the nicotinic receptor; (ii) agents that disrupt microtubules and microfilaments; (iii) phalloidin; (iv) vanadate; (v) inhibitors of anion permeability; (vi) protease inhibitors; and (vii) agents that dissipate the vesicle pH gradient and potential. It is partially inhibited by (i) certain antipsychotic drugs; (ii) a rise in osmotic pressure, (iii) lowering the temperature below 20°C, and (iv) N-ethyl maleimide.
This chapter describes methods to measure nucleotide exchange rates and GTP hydrolysis rates of Rho, Rac, and G25K. Like all GTP-binding proteins, Rho-related GTPases exist in two conformational states: an inactive GDP-bound form and an active GTP-bound form; their interconversion occurs through a cycle of guanine nucleotide exchange and GTP hydrolysis. The intrinsic nucleotide exchange and GTPase activities of all members of the Ras superfamily are relatively slow, and in vivo, they are stimulated by guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs and GAPs for the members of the Rho subfamily have been identified, although their cellular roles and exact specificities are still unclear. The first GAP described for this family (Rho–GAP) was purified biochemically from human spleen and was shown to be active on Rho, but it is also active on Rac and CDC42/G25K. Another important regulator of the Rho family of proteins is the Rho–GDP-dissociation inhibitor (GDI), Rho–GDI. Guanine nucleotide off rates are discussed in the chapter.
Secretagogues of rat peritoneal mast cells, such as mastoparan and compound 48/80, induce mast cell exocytosis by activating directly the guanosine triphosphate-binding proteins that are required for exocytosis. The introduction of a synthetic peptide that corresponds to the carboxyl-terminal end sequence of G alpha i3 into the cells specifically blocked this secretion. Similar results were obtained when antibodies to this peptide were introduced. The G alpha i3 was located in both the Golgi and the plasma membrane, but only the latter source of G alpha i3 appeared to be essential for secretion. These results indicate that G alpha i3 functions to control regulated exocytosis in mast cells.
Members of the Rho family of small guanosine triphosphatases (GTPases) regulate the organization of the actin cytoskeleton;
Rho controls the assembly of actin stress fibers and focal adhesion complexes, Rac regulates actin filament accumulation at
the plasma membrane to produce lamellipodia and membrane ruffles, and Cdc42 stimulates the formation of filopodia. When microinjected
into quiescent fibroblasts, Rho, Rac, and Cdc42 stimulated cell cycle progression through G1 and subsequent DNA synthesis.
Furthermore, microinjection of dominant negative forms of Rac and Cdc42 or of the Rho inhibitor C3 transferase blocked serum-induced
DNA synthesis. Unlike Ras, none of the Rho GTPases activated the mitogen-activated protein kinase (MAPK) cascade that contains
the protein kinases c-Raf1, MEK (MAPK or ERK kinase), and ERK (extracellular signal-regulated kinase). Instead, Rac and Cdc42,
but not Rho, stimulated a distinct MAP kinase, the c-Jun kinase JNK/SAPK (Jun NH2-terminal kinase or stress-activated protein
kinase). Rho, Rac, and Cdc42 control signal transduction pathways that are essential for cell growth.
A new brain serine/threonine protein kinase may be a target for the p21ras-related proteins Cdc42 and Rac1. The kinase sequence is related to that of the yeast protein STE20, implicated in pheromone-response pathways. The kinase complexes specifically with activated (GTP-bound) p21, inhibiting p21 GTPase activity and leading to kinase autophosphorylation and activation. Autophosphorylated kinase has a decreased affinity for Cdc42/Rac, freeing the p21 for further stimulatory activities or downregulation by GTPase-activating proteins. This bimolecular interaction provides a model for studying p21 regulation of mammalian phosphorylation signalling pathways.
The role of calmodulin in exocytotic secretion was studied using digitonin-permeabilized bovine adrenal chromaffin cells to examine the effect of calmodulin directly introduced into the cells and using tetanus toxin as a specific inhibitor of exocytotic secretion. Addition of calmodulin to the permeabilized cells increased Ca(2+)-dependent norepinephrine release in a dose-dependent manner. The enhancement of release by calmodulin was specific to calmodulin: bovine serum albumin, actin, and caldesmon had no such effect. Enhancement of release by calmodulin occurred at Ca2+ concentrations of more than 10(-6) M and increased with an increase of Mg2+ concentration. The release of norepinephrine enhanced by calmodulin was inhibited by tetanus toxin. These results indicate directly that calmodulin plays an important role in exocytotic secretion from chromaffin cells. Exocytosis is known to occur by fusion of plasma membrane with limiting membranes of secretory vesicles following an increase in intracellular Ca2+. We used the enterotoxin of Clostridium perfringens type A as a specific tool to modify plasma membrane permeability to induce calcium influx. Multigranular exocytosis was recognized electron-microscopically in addition to the single-granular exocytosis in rat anterior pituitary cells and pancreatic acinar cells treated with the enterotoxin in the presence of extracellular Ca2+. The treatment with the enterotoxin did not induce any drastic change in the fine membrane structures of both types of cells. The enterotoxin-treated anterior pituitary cells and pancreatic acinar cells should provide a useful system for studying the molecular mechanism of fusion of membranes in exocytosis.
Depending on the size of the pores one wishes to produce in plasma membranes, the choice will probably fall on one of the three toxins discussed above. S. aureus alpha-toxin should be tried first when pores of 1-1.5 nm diameter are required. This is generally the case when Ca2+ and nucleotide dependence of a given process is being studied. If alpha-toxin does not work, this is probably due to the fact that the toxin either does not produce pores, or that the pores are too small. In this case, high concentrations of alpha-toxin should be tried. If this still does not work, we recommend the use of HlyA. When very large pores are to be created, e.g. for introduction of antibodies into the cells, SLO or another member of this toxin family are the agents of choice. SLO preparations need to be checked for presence of protease contaminants. Tetanolysin currently offers advantages since it is protease-free, and the size of the pores can probably be controlled by varying the toxin dose. Methods for assessing the size of pores created by such agents have been published in the recent literature, and the appropriate papers can be consulted whenever the need arises.
Hepatocyte growth factor (HGF) induced motility of cultured mouse keratinocytes (308R cells). This HGF-induced cell motility was inhibited by microinjection of either rho GDI, an inhibitory GDP/GTP exchange protein for rho p21 small GTP-binding protein, or a botulinum exoenzyme C3 which is known to selectively impair the function of rho p21 by ADP-ribosylating its effector domain. The rho GDI action was prevented by comicroinjection with the guanosine 5'-(3-0-thio)triphosphate (GTP gamma S)-bound active form of rhoA p21, and the C3 action was prevented by comicroinjection with a rhoA p21 mutant (rhoAIle41 p21) which is resistant to the C3 action. The HGF-induced cell motility was not inhibited by microinjection of a dominant negative rac1 p21 mutant (rac1Asn17 p21) or a dominant negative Ki-ras p21 mutant (Ki-rasAsn17 p21). Microinjection of the GTP gamma S-bound form of rac1 p21 or a dominant active Ki-ras p21 mutant (Ki-rasVal12 p21) did not induce cell motility. These results indicate that both rho p21 and rho GDI, but neither rac p21 nor ras p21, are involved in the HGF-induced cell motility. However, microinjection of the GTP gamma S-bound form of rhoA p21 alone did not induce cell motility in the absence of HGF, suggesting that activation of rho p21 is necessary but not sufficient for the HGF-induced cell motility. The HGF-induced cell motility was mimicked by 12-0-tetradecanoyl-phorbol-13-acetate, a protein kinase C-activating phorbol ester, but not by Ca2+ ionophore. The phorbol ester-induced cell motility was also inhibited by microinjection of rho GDI or C3. These results indicate that both rho p21 and rho GDI are also involved in the phorbol ester-induced cell motility.
Superoxide production by phagocytic white blood cells requires the assembly of an NADPH oxidase from membrane and cytosolic proteins. Recombinant cytosolic proteins p47phox and p67phox and neutrophil membranes were used to purify a third cytosolic component that is necessary and sufficient for cell-free reconstitution of NADPH oxidase. The component was isolated as a complex of rho-GDP dissociation inhibitor (rho-GDI) and two members of the rho subfamily of ras-related guanine nucleotide binding proteins, rac2 and CDC42Hs. Oxidase reconstitution with these pure cytosolic proteins was unaffected by GTP gamma S but was inhibited by GDP beta S, suggesting that the active complex contained endogenous bound GTP. Direct binding of rho-GDI to the GTP gamma S-bound forms of these G-proteins was demonstrated by gel filtration following exchange with radiolabeled guanine nucleotide. rho-GDI was shown to be nonessential for cell-free oxidase reconstitution in experiments that compared the activities of pure recombinant forms of these G-proteins. Recombinant rac augmented superoxide production, while recombinant CDC42Hs, which shares 70% amino acid sequence identity with rac, did not. Three highly conserved regions of rac1 and rac2 were noted as markedly divergent in CDC42Hs. It is proposed that one or more of these regions of rac may be involved in the specific interaction of rac with the other NADPH oxidase protein(s).
Treatment of rat basophilic leukemia (RBL) 2H3-hm1 cells with Clostridium difficile toxin B (2 ng/ml), which reportedly depolymerizes the actin cytoskeleton, blocked [3H]serotonin release induced by 2,4-dinitrophenyl-bovine serum albumin, carbachol, mastoparan, and reduced ionophore A23187-stimulated degranulation by about 55-60%. In lysates of RBL cells, toxin B 14C-glucosylated two major and one minor protein. By using two-dimensional gel electrophoresis and immunoblotting, RhoA and Cdc42 were identified as protein substrates of toxin B. In contrast to toxin B, Clostridium botulinum transferase C3 that selectively inactivates RhoA by ADP-ribosylation did not inhibit degranulation up to a concentration of 150 microg/ml. Antigen-stimulated tyrosine phosphorylation of a 110-kDa protein was inhibited by toxin B as well as by the phosphatidylinositol 3-kinase inhibitor wortmannin. Depolymerization of the microfilament cytoskeleton of RBL cells by C. botulinum C2 toxin or cytochalasin D resulted in an increased [3H]serotonin release induced by antigen, carbachol, mastoparan, or by calcium ionophore A23187, but without affecting toxin B-induced inhibition of degranulation. The data indicate that toxin B inhibits activation of RBL cells by glucosylation of low molecular mass GTP-binding proteins of the Rho subfamily (most likely Cdc42) by a mechanism not involving the actin cytoskeleton.
We have used ultrastructural techniques to investigate secretion in permeabilized eosinophils. As each exocytotic event is rapid we have used tannic acid incubation to trap the maximum number of fusion figures; tannic acid has been used previously in other secretory systems to arrest exocytosis at the cell surface whilst still allowing the preceding events to occur. Using this approach, in conjunction with ultrathin sectioning and cryoreplication, it is possible to demonstrate clear evidence of exocytosis in permeabilized eosinophils after stimulation by GTP-gamma-S. Large numbers of arrested fusion sites are found, including early fusion pedestals, visible in freeze-fracture replicas, having single narrow necked pores as small as 12 x 43 nm. Both individual and compound exocytoses are found, with retention of the secretory product, in particular the crystalline granule core, occurring at many sites. Large numbers of coated pits are also found in cells following extended tannic acid incubation, membrane coats even occurring on arrested granule membranes, suggesting a role in post-fusion membrane recovery. The accessibility of the cell interior and the large number of arrested fusion sites, particularly the presence of very early stages of exocytosis (evident as pedestals in freeze-fracture replicas), makes this a suitable preparation for the localization of key regulators of exocytosis at their sites of action. Although this approach, utilizing permeabilization coupled with tannic acid incubation is not without inherent problems-as with any electron microscopic technique care must be taken to understand the potential for artefacts-there are a number of advantages, particularly with regard to labeling studies, over techniques utilizing ultra rapid freezing.
Streptolysin-O is widely used in cell biological investigations in order to make large (>12 nm) pores in the plasma membrane and so to render the cytosol directly accessible to experimental manipulation. We have compared the effect of streptolysin-O commercially formulated (Murex Diagnostics) as a diagnostic reagent in pathology with two pure reagents (a conventional purified protein, and a recombinant protein generated in E.coli) on exocytotic secretion from mast cells. For mast cells permeabilised by streptolysin obtained from the commercial source, exocytosis (of beta-D-N-acetylglucosaminidase) is dependent on provision of both Ca(2)+ and a guanine nucleotide. In contrast, for cells permeabilised by either of the two pure proteins, a substantial extent of Ca(2)+-independent exocytosis can be elicited. When the Murex material is subject to dialysis or ultrafiltration, some secretion can be induced in the absence of Ca(2)+, indicating a modulatory function of the low mol wt additives of formulation, mainly phosphate and cysteine. However, Ca(2)+-independent exocytosis is still manifest when the pure proteins are reconstituted with ultrafiltrates from the Murex material. These observations indicate that reagents used to permeabilise cells should be characterised thoroughly and used with great care. Confirmation that the cytolytic activity of the Murex material derives from a cholesterol directed factor was demonstrated by inhibition of exocytosis when red blood cell derived (and hence cholesterol containing) sonicated liposomes were provided.
Mast cells permeabilized by treatment with streptolysin-O in the presence of Ca2+ and GTP-gamma-S can secrete almost 100% of their contained N-acetyl-beta-D-glucosaminidase. If these stimuli are provided to the permeabilized cells after a delay, the response is diminished and the ability of the cells to undergo secretion runs down progressively over a period of about 30 min. This is thought to be due to the loss of key proteins involved in the exocytotic mechanism. Using this effect as the basis of a biological assay, we have isolated a protein from bovine brain cytosol that retards the loss of responsiveness to stimulation by Ca2+ and GTP-gamma-S. Purification of this protein and peptide sequencing have enabled us to identify it as the small GTP-binding protein rac complexed to the guanine nucleotide exchange inhibitor rhoGDI. Both proteins are required to retard the loss of the secretory response, while purified rhoGDI applied alone accelerates the rundown.