Journal of Cell Science

Published by Company of Biologists
Online ISSN: 1477-9137
Print ISSN: 0021-9533
Publications
Nuclear envelopes of somatic cells have at least two different major proteins in the 60-70(X 10(3] molecular weight range (lamins A(C) and B) that seem to be involved in chromatin attachment. In contrast, nuclear envelopes from clam germinal vesicles have only a single major protein of the same size class (approximately 67 X 10(3) Mr) and have no chromatin attached to them. The data presented in this report show that this 67 X 10(3) Mr clam protein shares a variety of physical properties with lamins A(C) and B, derived from rat liver nuclei. These properties include similar size, although different isoelectric points; phosphorylated forms; strong tendencies to cross-link by disulphide bonds; presence of carbohydrates, demonstrated by direct incorporation of mannose and labelling with borohydride; and shared epitopes, demonstrated using both monoclonal and polyclonal antibodies. Taken together, these observations identify the clam 67 X 10(3) Mr protein, the major structural protein of a nuclear envelope that lacks attached chromatin, as being lamin-like and demonstrate that it is more closely related to lamin A(C) than to lamin B.
 
We have examined the relationship of externally accessible proteins associated with the internal cytoskeleton of procyclic Trypanosoma brucei. Two approaches were taken. First, externally disposed glycoproteins were identified with lectins and examined for their persistence and location in isolated cytoskeletons. Second, proteins containing tyrosine residues available for chemical modification on the outer surface were identified in isolated cytoskeletons and probed for glycosylation. The procyclic form of T. brucei that was employed does not express the variable surface glycoprotein. The lectin concanavalin A (ConA) bound to the outer surface of T. brucei in two discrete locations; one a narrow line close to the flagellum attachment zone on the cell body, the other at the distal tip of the flagellum itself. Of these, only the cell body labelling was detected when isolated cytoskeletons were probed with fluorescein isothiocyanate-labelled ConA. When cytoskeletons were prepared from cells labelled with gold-conjugated ConA, a narrow line of label was detected parallel to the flagellum attachment zone but was distinct from it. Only one cytoskeletal protein, of Mr 88,000, could be labelled at the cell surface by the 125I/iodogen procedure. This protein could be precipitated from SDS-solubilized cytoskeletons with ConA-agarose. These data indicate the existence of a previously undetected cytoskeletal structure, situated in the cell body, close to the point of flagellum attachment, which has a transmembrane association with an external Mr 88,000 glycoprotein.
 
The epidermal blistering disease, pemphigus vulgaris (PV), is caused by circulating autoantibodies that react with a desmosomal glycoprotein desmoglein (Dsg3). This antigen is expressed only in stratified epithelial tissues. Here we show that the simple epithelial canine kidney cell line, MDCK, expresses at least two desmoglein isoforms recognised by different monoclonal antibodies. One of these isoforms is a 130 x 10(3) M(r) polypeptide that is recognised by both PV autoantisera and a monoclonal antibody reactive with a cytoplasmic domain of human Dsg3. Antibodies in PV sera bind to the surface of MDCK cells but not cause loss of intercellular adhesion. This is the first demonstration of the expression of a polypeptide related to human PV antigen by a simple epithelial cell type.
 
When the intermediate filament proteins vimentin and desmin were reacted for a short period of time with the arginine-specific reagent 1,2-cyclohexanedione, the modification had a severe, inhibitory effect on the assembly of intermediate filaments and on the susceptibility of the basic, amino-terminal polypeptide of both proteins to degradation by the intermediate filament-specific, Ca2+-activated proteinase. However, it had only a slightly inhibitory effect on the binding of vimentin and desmin to ribosomal RNA from Ehrlich ascites tumour cells. Since the Ca2+-activated proteinase is very likely to be a trypsin-like enzyme, with a preference for arginyl and lysyl peptide bonds, the results indicate that the arginine residues of the amino-terminal polypeptide of vimentin and desmin are highly essential for filament assembly but largely dispensable for the binding of both proteins to nucleic acids. This was supported by the observation that two breakdown products of vimentin lacking a 5 X 10(3) Mr and an 8 X 10(3) Mr polypeptide from the amino terminus, respectively, did not assemble into intermediate filaments but were still capable of binding to rRNA. Both polypeptides also bound to single-stranded DNA-cellulose under non-denaturing conditions, but passed the affinity column in the presence of 6 M-urea. Thus, the binding of vimentin to nucleic acids appears to be based on two components: a non-specific electrostatic interaction mediated by the positively charged arginine residues of the amino-terminal polypeptide that is insensitive to denaturation by urea, and a specific interaction that is sensitive to denaturation by urea.
 
Schizosaccharomyces pombe contains four putative (1,3)beta-D-glucan synthase (GS) catalytic subunits, Bgs1p-4p. In this work, we cloned bgs4+ and show that Bgs4p is the only subunit found to be a part of the GS enzyme and essential for maintaining cell integrity during cytokinesis and polarized growth. Here we show that bgs4+, cwg1+ (cwg1-1 shows reduced cell-wall beta-glucan and GS catalytic activity) and orb11+ (orb11-59 is defective in cell morphogenesis) are the same gene. bgs4+ is essential for spore germination and bgs4+ shut-off produces cell lysis at growing poles and mainly at the septum prior to cytokinesis, suggesting that Bgs4p is essential for cell wall growth and to compensate for an excess of cell wall degradation during cytokinesis. Shut-off and overexpression analysis suggest that Bgs4p forms part of a GS catalytic multiprotein complex and that Bgs4p-promoted cell-wall beta-glucan alterations induce compensatory mechanisms from other Bgs subunits and (1,3)alpha-D-glucan synthase. Physiological localization studies showed that Bgs4p localizes to the growing ends, the medial ring and septum, and at each stage of wall synthesis or remodeling that occurs during sexual differentiation: mating, zygote and spore formation, and spore germination. Bgs4p timing and requirements for proper positioning during cytokinesis and its localization pattern during spore maturation differ from those of Bgs1p. Bgs4p localizes overlapping the contractile ring once Bgs1p is present and a Calcofluor white-stained septum material is detected, suggesting that Bgs4p is involved in a late process of secondary or general septum synthesis. Unlike Bgs1p, Bgs4p needs the medial ring but not the septation initiation network proteins to localize with the other septation components. Furthermore, Bgs4p localization depends on the polarity establishment proteins. Finally, F-actin is necessary for Bgs4p delocalization from and relocalization to the growing regions, but it is not needed for the stable maintenance of Bgs4p at the growing sites, poles and septum. All these data show for the first time an essential role for a Bgs subunit in the synthesis of a (1,3)beta-D-glucan necessary to preserve cell integrity when cell wall synthesis or repair are needed.
 
Schizosaccharomyces pombe cells divide by medial fission throughout contraction of an actomyosin ring and deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Although many studies have focused on the actomoysin ring and septum assembly, little information is available concerning the mechanism of cell separation. Here we describe the characterization of eng1+, a new gene that encodes a protein with detectable endo-beta-1,3-glucanase activity and whose deletion is not lethal to the cells but does interfere in their separation. Electron microscopic observation of mutant cells indicated that this defect is mainly due to the failure of the cells to degrade the primary septum, a structure rich in beta-1,3-glucans, that separates the two sisters cells. Expression of eng1+ varies during the cell cycle, maximum expression being observed before septation, and the protein localizes to a ring-like structure that surrounds the septum region during cell separation. This suggests that it could also be involved in the cleavage of the cylinder of the cell wall that covers the division septum. The expression of eng1+ during vegetative growth is regulated by a C2H2 zinc-finger protein (encoded by the SPAC6G10.12c ORF), which shows significant sequence similarity to the Saccharomyces cerevisiae ScAce2p, especially in the zinc-finger region. Mutants lacking this transcriptional regulator (which we have named ace2+) show a severe cell separation defect, hyphal growth being observed. Thus, ace2p may regulate the expression of the eng1+ gene together with that of other genes whose products are also involved in cell separation.
 
Genetic interactions of S. pombe cps1-12 mutant with the actin mutant cps8-188 and with cdc septation mutants. Phasecontrast and Calcofluor white UV staining micrographs of log-phase cells grown on YES liquid medium at 25°C and shifted to different temperatures. Cells are shown grown at the temperature that reveals in each case the most apparent difference between single and double mutant phenotypes (8 hours at 30°C for cps8-188, 8 hours at 31°C for cdc16-116 and 6 hours at 34°C for cdc14-118 single and double cps1-12 mutants). The phenotype of the cps1-12 cps8-188 double mutant is more aggravated than that of the corresponding single mutants, while the phenotype of the cps1-12 cdc14-118 double mutant resembles that of the cdc14-118 single mutant, and the phenotype of the cps1-12 cdc16116 double mutant is similar to that of the cps1-12 single mutant. The cps1-12 single mutant cells were grown at 34°C, but a weaker phenotype was obtained at 30 or 31°C. Cells were centrifuged, suspended in 50 µg/ml Calcofluor white and directly observed for phase-contrast and Calcofluor white staining. 
Bgs1p localizes to all around the cell and septum and to regions of altered cell wall deposition in the actin mutant cps8-188. GFP-bgs1 + bgs1∆ mutant cells were grown as in Fig. 2, shifted to 32°C for 5 hours or to 37°C for 3 or 6 hours, and examined for GFP and Calcofluor white staining. Cells representative of the different mutant phenotypes at each temperature and incubation time are shown. 
Schizosaccharomyces pombe Bgs1p/Cps1p has been identified as a putative (1,3)beta-D-glucan synthase (GS) catalytic subunit with a possible function during cytokinesis and polarized growth. To study this possibility, double mutants of cps1-12 and cdc septation mutants were made. The double mutants displayed several hypersensitive phenotypes and altered actin distribution. Epistasis analysis showed mutations prior to septum synthesis were dominant over cps1-12, while cps1-12 was dominant over the end of septation mutant cdc16-116, suggesting Bgs1p is involved in septum cell-wall (1,3)beta-D-glucan synthesis at cytokinesis. We have studied the in vivo physiological localization of Bgs1p in a bgs1delta strain containing a functional GFP-bgs1(+) gene (integrated single copy and expressed under its own promoter). During vegetative growth, Bgs1p always localizes to the growing zones: one or both ends during cell growth and contractile ring and septum during cytokinesis. Bgs1p localization in cdc septation mutants indicates that Bgs1p needs the medial ring and septation initiation network (SIN) proteins to localize properly with the rest of septation components. Bgs1p localization in the actin mutant cps8-188 shows it depends on actin localization. In addition, Bgs1p remains polarized in the mislocalized growing poles and septa of tea1-1 and tea2-1 mutants. During the meiotic process of the life cycle, Bgs1p localizes to the mating projection, to the cell-to-cell contact zone during cell fusion and to the neck area during zygote formation. Also, Bgs1p localization suggests that it collaborates in forespore and spore wall synthesis. During spore germination, Bgs1p localizes first around the spore during isotropic growth, then to the zone of polarized growth and finally, to the medial ring and septum. At the end of spore-cell division, the Bgs1p displacement to the old end occurs only in the new cell. All these data show that Bgs1p is localized to the areas of polarized cell wall growth and so we propose that it might be involved in synthesizing the lineal (1,3)beta-D-glucan of the primary septum, as well as a similar lineal (1,3)beta-D-glucan when other processes of cell wall growth or repair are needed.
 
Characterization of a nucleolar pool of IP5K. (A-C) Confocal immunofluorescence was used to detect endogenous IP5K (green) and fibrillarin (red) in H1299 cells (A), primary human skin fibroblasts (B) and MCF-7 cells (C). In addition to using fibrillarin as a nucleolar marker, differential interference contrast (DIC) imaging distinguished nucleoli by their distinct morphology. (D) Output after IP5K was threaded through a nucleolar localization sequence (NoLS) detector (Scott et al., 2011); the threshold for a candidate nucleolar localization sequence is 0.8 (marked in red). (E) Confocal immunofluorescence images of endogenous IP5K and TCOF1 in H1299 cells. (F) H1299 cell lysate protein ('input', approx. 30 mg per lane) was incubated with immobilized GST or GST-IP5K, and bound proteins were identified as described in the Materials and Methods. The anti-TCOF1 antibody was not sufficiently sensitive to detect endogenous TCOF1, so in these experiments (the top western blot) a TCOF1-GFP fusion protein was expressed in H1299 cells 24 hours prior to lysis. (G,H) Confocal immunofluorescence images of endogenous IP5K and fibrillarin in H1299 cells that were serum-starved for 48 hours in HAM F-10 media (G) and then re-fed with 10% fetal calf serum for 8 hours (H). (I) Confocal immunofluorescence images of endogenous IP5K (green), BrU incorporation into rRNA in the presence of 1 mg/ml a-amanitin (red) and Pol I (purple) in MCF-7 cells. (J) Magnified images of the nucleolar region shown in I, which also includes an IP5K/BrU merge. Scale bars: 10 mm (A-I); 2 mm (J).
A non-catalytic role for IP5K in regulating the spatial distributions of UBF, TCOF1 and Pol I. (A,B) Confocal immunofluorescence was used to determine the localization of endogenous UBF (A) or TCOF1 (B), in either non-transfected H1299 cells (left) or in H1299 cells that were transfected for 24 hours with either IP5K-GFP (middle) or catalytically dead IP5K C162Y-GFP (right). (C-F) In further experiments, either control cells (C,E) or cells in which IP5K-GFP was expressed for 24 hours (D,F) were analyzed using multichannel immunofluorescence to identify the location of either IP5K-GFP, TCOF1, UBF or Pol I, as indicated. Scale bars: 10 mm.
rRNA synthesis assayed in situ. The synthesis of rRNA was quantified using ImageJ to determine the degree of BrU incorporation in permeabilized H1299 cells in the presence of 1 mg/ml a-amanitin (see supplementary material Fig. S4; and Materials and Methods section). Where indicated, cells were transfected with cDNAs encoding either DsRedNuc, TCOF1, IP5K or kinase-dead IP5K C162Y . The data (means and s.e.m.) were obtained from the nuclei of 70 transfected and 100 non-transfected cells, obtained from three independent experiments. **P,0.001 versus the DsRedNuc control.  
An RKK tripeptide in IP5K mediates binding of the kinase to UBF. (A) Sequence alignment of hIP5K and AtIP5K. The predicted secondary structure of hIP5K is shown in magenta shapes (tube, alpha helix; arrow, beta sheet; broken line, loop). The RKK tripeptide (highlighted in red) is aligned in a variant loop of AtIP5K (green), which is disordered in the crystal structures. (B) The structure of AtIP5K (PDB accession code: 2XAM) is shown in yellow and green. The structure of residues 41 RKK 43 (ball-and-stick model; carbon, magenta; oxygen, red; nitrogen, blue) is modeled on the basis of primary sequence alignment and secondary structure fold recognition. (C) Time-course of InsP 6 production by 0.3 mg of either wild-type GST-IP5K, or the GST-IP5K QQQ mutant. Data are means 6 s.e.m. from 3–5 experiments. (D) H1299 cell lysate protein ('input', approx. 30 mg per lane) was incubated with immobilized GST-IP5K QQQ , and bound proteins were identified as described in the Materials and Methods. The anti-TCOF1 antibodies were not sufficiently sensitive to detect endogenous TCOF1, so in these experiments (the top western blot) a TCOF1-GFP fusion protein was expressed in H1299 cells 24 hours prior to lysis. (E,F) Confocal immunofluorescence images of H1299 cells in which IP5K QQQ -GFP was expressed for 24 hours; the distribution of endogenous UBF (E) or endogenous TCOF1 (F) are also shown.  
Fundamental to the life and destiny of every cell is the regulation of protein synthesis through ribosome biogenesis, which begins in the nucleolus with the production of ribosomal RNA (rRNA). Nucleolar organization is a highly dynamic and tightly-regulated process; the structural factors that direct nucleolar assembly and disassembly are just as important in controlling rRNA synthesis as the catalytic activities that synthesize rRNA. Here, we report that a signalling enzyme, inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5K) is also a structural component in the nucleolus. We demonstrate that IP5K has functionally-significant interactions with three proteins that regulate rRNA synthesis: protein kinase CK2 (CK2), TCOF1 and upstream-binding-factor (UBF). Through molecular modelling and mutagenic studies, we identified an Arg-Lys-Lys tripeptide located on the surface of IP5K that mediates its association with UBF. Nucleolar IP5K spatial dynamics were sensitive to experimental procedures (serum starvation, or addition of actinomycin D) that inhibited rRNA production. We show that IP5K makes stoichiometrically-sensitive contributions to the architecture of the nucleoli in intact cells, thereby influencing the degree of rRNA synthesis. Our study adds significantly to the biological significance of IP5K; previously, it was this protein's kinase activity that had attracted attention. Our demonstration that IP5K "moonlights" as a molecular scaffold offers an unexpected new example of how the biological sophistication of higher organisms can arise from gene products acquiring multiple functions, rather than by an increase in gene number.
 
Cell growth and differentiation are influenced by intercellular contact, suggesting that cell adhesion molecules may be instrumental in triggering these events. F9 embryonal carcinoma cells are an ideal system in which to examine the function of cell adhesion molecules in growth and differentiation, since the relevant cell adhesion molecules and differentiation markers are well defined. Intercellular adhesion in F9 cells is mediated by uvomorulin, or E-cadherin, and cell surface beta-(1,4)-galactosyltransferase. Since previous studies suggested that neither F9 cell growth nor differentiation is directly dependent on uvomorulin function, in this study we examined whether cell surface galactosyltransferase plays any role in F9 cell growth or differentiation. A variety of galactosyltransferase perturbants, including anti-galactosyltransferase antibodies, UDPgalactose, and the substrate modifier protein alpha-lactalbumin, inhibited the growth of F9 cells, whereas control reagents did not. To examine this in more detail, we analyzed the effects of perturbing surface galactosyltransferase on progression through the F9 cell cycle. Anti-galactosyltransferase IgG treatment inhibited ornithine decarboxylase activity and lengthened the F9 cell cycle during G1 and G2, the latter mimicking the effects of retinoic acid, a reagent known to prolong the F9 cell cycle and induce differentiation. In contrast, anti-uvomorulin antibodies had no effect on F9 cell growth, ornithine decarboxylase activity, or progression through the cell cycle. Furthermore, perturbation of surface galactosyltransferase adhesions in F9 cell aggregates induced precocious F9 cell differentiation, as assayed by increased laminin synthesis, whereas control reagents had no effect. Thus, perturbing surface galactosyltransferase adhesions in F9 cells both decreases growth and stimulates synthesis of laminin. These results imply that interactions between surface galactosyltransferase and its oligosaccharide ligand during cell adhesion may affect the normal growth-regulatory and differentiation-inducing signals, as is seen, in part, during treatment with retinoic acid.
 
The surface distribution of the alpha 2/delta subunit of the 1,4-dihydropyridine receptor and its topographical relationship with the neural cell adhesion molecule (N-CAM) were investigated during early myogenesis in vitro, by double immunocytochemical labeling with the monoclonal antibody 3007 and an anti-N-CAM polyclonal antiserum. The monoclonal antibody 3007 has been previously shown to immunoprecipitate dihydropyridine receptor from skeletal muscle T-tubules. In further immunoprecipitation experiments on such preparations and muscle cell cultures, it was demonstrated here that the monoclonal antibody 3007 exclusively recognizes the alpha 2/delta subunit of the 1,4-dihydropyridine receptor. In rabbit muscle cell cultures, the labeling for both alpha 2/delta and N-CAM was first detected on myoblasts, in the form of spots on the membrane and perinuclear patches. Spots of various sizes organized in aggregates were then found on the membrane of myotubes. At fusion (T0), aggregates of N-CAM spots alone were found at the junction between fusing cells. At T6 and later stages, all alpha 2/delta aggregates present on myotubes co-localized with N-CAM, while less than 3% of N-CAM aggregates did not co-localize with alpha 2/delta. A uniform N-CAM staining also made its appearance. At T12, when myotubes showed prominent contractility, alpha 2/delta-N-CAM aggregates diminished in size. Dispersed alpha 2/delta spots of a small regular size spread over the whole surface of the myotubes and alignments of these spots became visible. Corresponding N-CAM spots were now occasionally seen, and uniform N-CAM staining was prominent. These results show that alpha 2/delta and N-CAM are co-localized and that their distributions undergo concomitant changes during early myogenesis until the T-tubule network starts to be organized. This suggest that these two proteins might jointly participate in morphogenetic events preceding the formation of T-tubules.
 
Neurite outgrowth on cellular and extracellular matrices is mediated by a variety of cell surface receptors. Some of these receptors recognize peptide determinants, whereas others bind oligosaccharide ligands. Previous studies have suggested that cell surface beta 1.4-galactosyltransferase functions as one of these receptors during neurite outgrowth on basal lamina by binding to N-linked oligosaccharides in the E8 domain of laminin. However, these previous investigations have been limited to the use of galactosyltransferase inhibitory reagents to block neurite formation. Therefore, in this study, we investigated whether the level of surface galactosyltransferase directly affects the efficiency of neurite outgrowth, or rather, is incidental to neurite formation. Northern blot analysis and cell surface galactosyltransferase assays were used to select two stable PC12 transfectants that overexpress surface galactosyltransferase by approximately four-fold. Radiolabeled antibody binding to intact cells and indirect immunofluorescence confirmed the higher expression of surface galactosyltransferase on transfected cells, compared to controls. Both galactosyltransferase transfected cell lines exhibited markedly enhanced neurite initiation, neurite formation, and rates of neurite elongation by two- to three-fold. These studies demonstrate that the expression of laminin receptors can be rate-limiting during neurite outgrowth, and that the level of surface galactosyltransferase can modulate the frequency and rate of neurite formation from PC12 cells on laminin.
 
The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the signalling molecules inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, 5)P(4)) in a signal-terminating reaction. We have utilised cell lines that stably underexpress the 43 kDa 5-phosphatase, as a model system to investigate whether Ins(1,4,5)P(3) can control the rate of its own formation by regulating the resupply of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). A sustained 2.6-fold elevation in the basal concentration of Ins(1,4,5)P(3), in cell lines underexpressing the 43 kDa 5-phosphatase, correlated with a 32% reduction in the total cellular mass of PtdIns(4,5)P(2). The depletion in cellular PtdIns(4,5)P(2) was confined to a Triton-insoluble cell compartment, enriched in caveolin. In resting cells with elevated Ins(1,4,5)P(3) concentrations resulting from underexpression of the 43 kDa 5-phosphatase, phosphatidylinositol (PtdIns) and phosphatidylinositol 4-phosphate (PtdIns(4)P) were depleted by 50% and PtdIns(4,5)P(2) by 61% in the caveolin-enriched Triton-insoluble compartment. Agonist stimulation resulted in the rapid turnover of phosphoinositides in the caveolin-enriched Triton-insoluble fraction of vector-transfected cells, but not in cells with high basal Ins(1,4,5)P(3) concentrations. Depletion of phosphoinositides from the caveolin-enriched Triton-insoluble pool in cells underexpressing the 43 kDa 5-phosphatase did not result from activation of phospholipase C isoenzymes, or inhibition of PtdIns 4-kinase or PtdIns(4)P 5-kinase activities. Significant inhibition of phosphatidylinositol transfer protein (PITP) activity (up to 70%) was observed in cells with elevated basal Ins(1,4,5)P(3) concentrations; however, no reduction in PITP(&agr;) protein expression was detected. These studies indicate that chronic elevation in cellular Ins(1,4,5)P(3) concentrations decreases the PITP-mediated resupply of phosphoinositides in the caveolin-enriched agonist-sensitive pool.
 
Several lines of evidence indicate that increases in nuclear Ca(2+) have specific biological effects that differ from those of cytosolic Ca(2+), suggesting that they occur independently. The mechanisms involved in controlling nuclear Ca(2+) signaling are both controversial and still poorly understood. Using hypotonic shock combined with mechanical disruption, we obtained and characterized a fraction of purified nuclei from cultured rat skeletal myotubes. Both immunoblot studies and radiolabeled inositol 1,4,5-trisphosphate [IP(3)] binding revealed an important concentration of IP(3) receptors in the nuclear fraction. Immunofluorescence and immunoelectron microscopy studies localized type-1 and type-3 IP(3) receptors in the nucleus with type-1 receptors preferentially localized in the inner nuclear membrane. Type-2 IP(3) receptor was confined to the sarcoplasmic reticulum. Isolated nuclei responded to IP(3) with rapid and transient Ca(2+) concentration elevations, which were inhibited by known blockers of IP(3) signals. Similar results were obtained with isolated nuclei from the 1B5 cell line, which does not express ryanodine receptors but releases nuclear Ca(2+) in an IP(3)-dependent manner. Nuclear Ca(2+) increases triggered by IP(3) evoked phosphorylation of cAMP response element binding protein with kinetics compatible with sequential activation. These results support the idea that Ca(2+) signals, mediated by nuclear IP(3) receptors in muscle cells, are part of a distinct Ca(2+) release component that originates in the nucleus and probably participates in gene regulation mediated by cAMP response element binding protein.
 
Subcellular localization of IP3R1 and IP3R3 in A7r5 cells. Cells were fixed, permeabilized and incubated with (A,B,D,E) Rbt03 polyclonal antibody (1/750) or (C,F) MMAtype 3 monoclonal antibody (1/100), and subsequently treated with anti-rabbit FITC (1/750) or anti-mouse FITC (1/500) secondary antibodies, respectively. IP3R localization was investigated in (A-C) resting cells or (D-F) after stimulation with AVP (3 µM, 5 hours). A flattened Zstack image of the confocal images of IP3R1 was generated (A) before and (D) after stimulation with AVP (3 µM). As a control, cells were incubated either (G) with the pre-immune serum of Rbt03 (1/750) or (H) with PBS before treatment with secondary antibodies. Bar, 10 µm. 
Time course of IP3R1 redistribution. The percentage of cells with IP3R1 located in the perinuclear region (squares) and in the cytoplasm (circles) after addition of 3 µM AVP is depicted for different time periods. The dashed line represents the removal of AVP (3 µM) after 2 hours, after which the cells where incubated in PBS without Ca 2+ for the indicated time periods. Each result is the mean±s.e.m. from three independent experiments. The s.e.m. is not indicated when smaller than the symbol. 
Effect of Ca 2+-mobilizing agents on IP3R1 redistribution. The percentage of cells with a perinuclear IP3R1 is depicted before and after incubation with AVP (3 µM), thapsigargin (1 µM) and CPA (50 µM) for 5 hours. Each result is the mean±s.e.m. from three independent experiments. *Significantly different from the control. 
Visualization of the microtubular and actin cytoskeleton. Cells were fixed, permeabilized and incubated (A,C,E) with anti-α tubulin (1/2000) or (B,D,F) with rhodamine-phalloidin (1 unit/ml). (A,C,E) Anti-mouse FITC (1/400) was used as secondary antibody. The microtubular and actin cytoskeleton was visualized (A,B) before and after (C,D) AVP (3 µM) or (E,F) OAG (50 µM) treatment for 5 hours. Bar, 10 µm. 
Visualization of SERCA pumps in A7r5. Cells were fixed, permeabilized and incubated with AS809-27 polyclonal antibody (1/300). Anti-rabbit FITC (1/750) was used as secondary antibody. SERCA was visualized in (A) resting cells and (B) after stimulation with AVP (3 µM, 2 hours). Bar, 10 µm. 
In A7r5 vascular smooth muscle cells, the two expressed inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms were differentially localized. IP(3)R1 was predominantly localized in the perinuclear region, whereas IP(3)R3 was homogeneously distributed over the cytoplasm. Prolonged stimulation (1-5 hours) of cells with 3 microM arginine-vasopressin induced a redistribution of IP(3)R1 from the perinuclear region to the entire cytoplasm, whereas the localization of IP(3)R3 appeared to be unaffected. The redistribution process occurred independently of IP(3)R downregulation. No structural changes of the endoplasmic reticulum were observed, but SERCA-type Ca(2+) pumps redistributed similarly to IP(3)R1. The change in IP(3)R1 localization induced by arginine-vasopressin could be blocked by the simultaneous addition of nocodazole or taxol and depended on Ca(2+) release from intracellular stores since Ca(2+)-mobilizing agents such as thapsigargin and cyclopiazonic acid could induce the redistribution. Furthermore, various protein kinase C inhibitors could inhibit the redistribution of IP(3)R1, whereas the protein kinase C activator 1-oleoyl-2-acetyl-sn-glycerol induced the redistribution. Activation of protein kinase C also induced an outgrowth of the microtubules from the perinuclear region into the cytoplasm, similar to what was seen for the redistribution of IP(3)R1. Finally, blocking vesicular transport at the level of the intermediate compartment inhibited the redistribution. Taken together, these findings suggest a role for protein kinase C and microtubuli in the redistribution of IP(3)R1, which probably occurs via a mechanism of vesicular trafficking.
 
In the present study, the contribution of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] generation on the mechanical-stimulation-induced Ca(2+) response was investigated in HSY-EA1 cells. Mechanical stimulation induced a local increase in the cytosolic concentration of Ins(1,4,5)P(3) ([IP(3)](i)), as indicated by the Ins(1,4,5)P(3) biosensor LIBRAvIII. The area of this increase expanded like an intracellular Ins(1,4,5)P(3) wave as [IP(3)](i) increased in the stimulated region. A small transient [IP(3)](i) increase was subsequently seen in neighboring cells. The phospholipase C inhibitor U-73122 abolished these Ins(1,4,5)P(3) responses and resultant Ca(2+) releases. The purinergic receptor blocker suramin completely blocked increases in [IP(3)](1) and the Ca(2+) release in neighboring cells, but failed to attenuate the responses in mechanically stimulated cells. These results indicate that generation of Ins(1,4,5)P(3) in response to mechanical stimulation is primarily independent of extracellular ATP. The speed of the mechanical-stimulation-induced [IP(3)](i) increase was much more rapid than that induced by a supramaximal concentration of ATP (1 mM). The contribution of the Ins(1,4,5)P(3)-induced Ca(2+) release was larger than that of Ca(2+) entry in the Ca(2+) response to mechanical stimulation in HSY-EA1 cells.
 
The subcellular localization of inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ signals is important for the activation of many physiological functions. In epithelial cells the spatial distribution of InsP3 receptor is restricted to specific areas, but little is known about the relationship between the receptor's distribution and cell polarity. To investigate this relationship, the best known polarized cell model, MDCK, was examined. This cell line is characterized by a strong expression of the type 3 InsP3 receptor and the subcellular localization of this receptor was followed during cell polarization using immunofluorescence and confocal analysis. In non-polarized cells, including ras transformed f3 MDCK cells, the type 3 InsP3 receptor was found to co-localize with markers of the endoplasmic reticulum in the cytoplasm. In contrast, in polarized cells, this receptor was mostly distributed at the apex of the lateral plasma membrane with the markers of tight junctions, ZO-1 and occludin. The localization of the type 3 InsP3 receptor in the vicinity of tight junctions was confirmed by immunogold electron microscopy. The culture of MDCK cells in calcium-deprived medium, led to disruption of cell polarity and receptor redistribution in the cytoplasm. Addition of calcium to these deprived cells induced the restoration of polarity and the relocalization of the receptor to the plasma membrane. MDCK cells were stably transfected with a plasmid coding the full-length mouse type 1 InsP3 receptor tagged with EGFP at the C-terminus. The EGFP-tagged type 1 receptor and the endogenous type 3 co-localized in the cytoplasm of non-polarized cells and at the tight junction level of polarized cells. Thus, the localization of InsP3 receptor in MDCK depends on polarity.
 
We reported that a plasmalemmal inositol 1,4,5-trisphosphate receptor-like protein (PM InsP3R-L) is localized in caveolae of various non-neuronal cells in vivo (Fujimoto et al. (1992) J. Cell Biol. 119, 1507-1513). In the present study, we investigated the distribution of PM InsP3R-L in cultured cells. In mouse epidermal keratinocytes (Pam 212) cultured in standard Ca2+ (1.8 mM), PM InsP3R-L was distributed densely in the vicinity of cell-to-cell contacts. In contrast, when Pam cells were cultured in low Ca2+ (0.06 mM) without making cell-to-cell contacts, PM InsP3R-L was observed randomly; by restoring the Ca2+ concentration, the circumferential actin filaments became obvious and the density of PM InsP3R-L increased in the contact region. Treatment of Pam cells with cytochalasin D caused aggregation of caveolae where PM InsP3R-L as well as F-actin and fodrin were localized. In bovine aortic endothelial cells, PM InsP3R-L was aligned along actin filaments crossing the cytoplasm in various directions. PM InsP3R-L of Pam cells was hardly extracted by treatment with 0.5% Triton X-100 or 60 mM octyl-glucoside in a cytoskeleton-stabilizing buffer for 15 minutes at 4 degrees C. The results show that the distribution of caveolae bearing PM InsP3R-L changes when the actin cytoskeleton is modified. They also indicate that the association of PM InsP3R-L with actin filaments may mediate the redistribution of caveolae. Since caveolae are thought to be related to signal transduction, their location defined by the actin cytoskeleton may affect the site where cellular reaction is to occur in response to various stimuli.
 
Intercellular propagation of signals through connexin32-containing gap junctions is of major importance in physiological processes like nerve activity-dependent glucose mobilization in liver parenchymal cells and enzyme secretion from pancreatic acinar cells. In these cells, as in other organs, more than one type of connexin is expressed. We hypothesized that different permeabilities towards second messenger molecules could be one of the reasons for connexin diversity. In order to investigate this, we analyzed transmission of inositol 1,4,5-trisphosphate-mediated calcium waves in FURA-2-loaded monolayers of human HeLa cells expressing murine connexin26, -32 or -43. Gap junction-mediated cell coupling in different connexin-transfected HeLa cells was standardized by measuring the spreading of microinjected Mn(2+) that led to local quenching of FURA-2 fluorescence. Microinjection of inositol 1,4,5-trisphosphate into confluently growing HeLa connexin32 transfectants induced propagation of a Ca(2+) wave from the injected cell to neighboring cells that was at least three- to fourfold more efficient than in HeLa Cx26 cells and about 2.5-fold more efficient than in HeLa Cx43 transfectants. Our results support the notion that diffusion of inositol 1,4,5-trisphosphate through connexin32-containing gap junctions is essential for the optimal physiological response, for example by recruiting liver parenchymal cells that contain subthreshold levels of this short lived second messenger.
 
Localized depletion of the Ins(1,4,5)P 3-sensitive Ca 2+ store depletes the entire store of Ca 2+. At-70 mV, locally photolysed Ins(1,4,5)P 3 (F, B) at a 10-mdiameter region (photolysis site 1; bright spot in A, left-hand panel; see also patch electrode, left side) evoked Ca 2+ transients (B). Results from photolysis site 1 are indicated by the red bars below the [Ca 2+ ] c traces in B. When repositioned to site 2 (A, right-hand panel), subsequent photolysis ~90 seconds later produced reproducible [Ca 2+ ] c increases (B). Results from photolysis site 2 are indicated by the blue line below the [Ca 2+ ] c trace (B). In a Ca 2+-free solution [containing EGTA (1 mM) and MgCl 2 (3 mM); unfilled line above the [Ca 2+ ] c trace], the [Ca 2+ ] c increase evoked by Ins(1,4,5)P 3 at photolysis site 2 (A) declined in amplitude as the store was depleted of Ca 2+ (B). When the store content had been substantially reduced at photolysis site 2 (A) (as revealed by the smaller Ca 2+ transients, B), Ins(1,4,5)P 3 was liberated by photolysis at site 1 (A). Again, as at photolysis site 2, the response was now almost abolished compared with that of the control. On restoring external Ca 2+ (B, right-hand side), the Ca 2+ increase evoked by Ins(1,4,5)P 3 at photolysis site 1 was restored towards control values. These results suggest that the SR is lumenally continuous and within it Ca 2+ can diffuse freely throughout. [Ca 2+ ] c measurements (B) have been derived from fluorescence intensity changes occurring in a circle of diameter 5 m in the center of the photolysis region. Thus, those results from photolysis site 1 are from a circle of diameter 5 m positioned at site 1; results from photolysis site 2 are from a circle of diameter 5 m at site 2. (C) Local photolysed Ins(1,4,5)P 3 (F) at photolysis site 1 (A, lefthand panel) increased [Ca 2+ ] c (C, right-hand panel), which was maximal at, and decreased with each 10 m increment away from, the release site (C, right panel); region 1 is the photolysis site. [Ca 2+ ] c measurements were made at lines of width 1 pixel. The position of each measurement (regions 1-9) line is shown (C, left panel) at a width of two pixels to facilitate visualization. The second photolysis site lies between regions 8-9-that is, ~75 m away from photolysis site 1. 
Ca 2+ can move through the SR to replenish a site previously depleted of the ion. (A-C) At-70 mV, locally photolysed Ins(1,4,5)P 3 (F, C) at a 10-mdiameter region [bright spot in A, left-hand panel; see also whole-cell electrode (left side) and the Ca 2+-containing shadow of the electrode (right side, 'Ca 2+ electrode')] increased [Ca 2+ ] c (B,C). The [Ca 2+ ] c images (B) are derived from the time-points indicated by the corresponding roman numerals in C. [Ca 2+ ] c changes in B are represented by colour; blue low and red high [Ca 2+ ] c (i-xii). A second photolysis of Ins(1,4,5)P 3 ~90 seconds later at the same site (C) generated an approximately comparable [Ca 2+ ] c increase. In a Ca 2+-free solution (containing 1 mM EGTA and 3 mM MgCl 2 ), the [Ca 2+ ] c increase evoked by Ins(1,4,5)P 3 declined and was abolished as the store became depleted of Ca 2+. [Ca 2+ ] c changes as before in B are represented by colour: (v-vii; note the cell position was moved by the solution exchange, A middle panel). When the Ca 2+-containing electrode ('Ca 2+ electrode') was subsequently sealed onto the cell (A, right-hand panel; C, blue bar) the local [Ca 2+ ] c , at the site of the Ca 2+ electrode attachment, increased, as indicated by the colour changes as before (B, right-hand panels, ix-xii), presumably as a consequence of store-operated Ca 2+ entry. [Ca 2+ ] c was at basal levels by 30 m from the patch pipette, the photolysis site was 77 m from the pipette. [Ca 2+ ] c at the photolysis site remained low (B,C). The Ca 2+ increase to Ins(1,4,5)P 3 at the photolysis region (A) was subsequently increased towards that of the control (C). The position of the region of measurement is shown as a white line in B (i,v,ix), left-hand corner. Measurements were made from a 1-pixel line; the line is drawn at a 2-pixel width to facilitate its visualization. 
Ins(1,4,5)P 3-sensitive Ca 2+ release at different SR Ca 2+ contents. At-70 mV, photolysed Ins(1,4,5)P 3 increased [Ca 2+ ] c (F, A). A second photolysis of Ins(1,4,5)P 3 ~90 seconds later at the same site generated an approximately comparable [Ca 2+ ] c increase (A). In a Ca 2+-free bath solution (containing 1 mM EGTA and 3 mM MgCl 2 ), this [Ca 2+ ] c increase declined in amplitude and rate of rise as the store was depleted of Ca 2+ (A). The velocity of release increased during the release process, as revealed by the increasing steepness of the slope during release (B,C), and acceleration increased (D). C and D are the first and second derivatives, respectively, of the upstroke of the transients numbered 1-4 in A. As the increase in velocity is also evident when the first derivative of the upstroke is plotted against [Ca 2+ ] c (E) rather than time (C), nonlinear Ca 2+ buffering does not provide an explanation for these results. Had it done so, the velocities derived from each transient would have been similar when examined as a function of [Ca 2+ ] c. The numbered traces in B-E correspond to the Ca 2+ transients numbered in A. The amplitudes of the transient (B) have been scaled and normalized to facilitate comparison. One explanation for these results is that Ins(1,4,5)P 3-mediated Ca 2+ release is itself facilitated by Ca 2+ released via the channel in a positive-feedback process. As lumenal [Ca 2+ ] declines (in the Ca 2+-free solution) and with it Ca 2+ release, so does the extent of the Ca 2+-dependent positive feedback. (F) The peak velocity of release (a measure of the extent of positive feedback) (C) determines the peak [Ca 2+ ] c achieved after Ins(1,4,5)P 3-mediated Ca 2+ release. Here, the peak velocity is plotted against the peak [Ca 2+ ] c obtained from the same cell. In this figure, the results from three separate cells, each indicated by the different-coloured symbols, are shown and the [Ca 2+ ] c was calibrated as described in Materials and Methods. (G) Ins(1,4,5)P 3-evoked Ca 2+ release from the transients numbered 1-4 in A that have been scaled to facilitate comparison of their time-course. As the peak [Ca 2+ ] c achieved decreases (see A), the time required to reach their peak increased (G). This result would suggest that Ins(1,4,5)P 3-mediated inactivation of Ins(1,4,5)P 3 R is unlikely to explain the termination of release as the [Ins(1,4,5)P 3 ] is similar in each case. 
BAPTA (AM form) prevents the acceleration of Ins(1,4,5)P 3-mediated Ca 2+ release and limits the rise in [Ca 2+ ] c. Depolarization (-70 to +10 mV, 3 seconds) (B) activated I Ca (C) and increased [Ca 2+ ] c (A). At-70 mV (B), local photolysis of Ins(1,4,5)P 3 [F; concentrations producing maximum (red) or submaximum (blue) responses] increased [Ca 2+ ] c (A). Prior (7 minutes) introduction of BAPTA AM (50 M) to the bathing solution reduced the Ins(1,4,5)P 3-evoked [Ca 2+ ] c rise (A, unfilled bar) owing to increased cytoplasmic Ca 2+ buffering, as revealed by the reduced [Ca 2+ ] c rise for a similar Ca 2+ influx (A,C). Note the smaller rise in measured [Ca 2+ ] c (from the fluorescence measurements) for a given calculated [Ca 2+ ] c increase (from the Ca 2+ current) in BAPTA (D) compared with that of controls. Scaling the [Ca 2+ ] c transients obtained in BAPTA so that the depolarization-evoked transients in the presence and absence of the chelator are of comparable size allowed a compensation for the increased buffer capacity of the cell to be made (E; left-hand panel). Application of the same scaling factor to the Ins(1,4,5)P 3-evoked [Ca 2+ ] c increases revealed that the Ins(1,4,5)P 3-evoked [Ca 2+ ] c transients were substantially reduced in the presence of the chelator (E; middle and right panels). Significantly, when the cytoplasmic Ca 2+ buffer capacity had been increased (with BAPTA), the increase in velocity of the [Ca 2+ ] c rise (d[Ca 2+ ] c /dt) seen in control was substantially reduced (F). Thus the rate of Ca 2+ release was largely constant rather than increased (F) during the release process in the presence of the chelator-that is, the velocity increase arose as a result of a Ca 2+-dependent positive feedback acting at the cytoplasmic aspect of the Ins(1,4,5)P 3 R. BAPTA in its Ca 2+-free form (BAPTA Cafree ) might itself directly inhibit Ins(1,4,5)P 3 R (Richardson and Taylor, 1993). However, high concentrations of BAPTA are required-for example, increasing BAPTA Cafree from 90 M to 9 mM reduced Ins(1,4,5)P 3-mediated Ca 2+ release by 8% (Bootman et al., 1995). 
BAPTA in the membrane-permeable (AM) form prevents quantal Ca 2+ release. Depolarization (-70 to +10 mV, 3 seconds) (C) activated I Ca (D) and increased [Ca 2+ ] c (A). At-70 mV (C), CCh (50 M; B) produced a small, and CCh (500 M) a substantial, [Ca 2+ ] c increase (A). Approximately 7 minutes after the introduction of BAPTA AM (25 M; A, unfilled bar) to the bathing solution, the depolarization-evoked [Ca 2+ ] c rise was significantly reduced owing to increased cytoplasmic buffering, as revealed by the reduced [Ca 2+ ] c rise for a similar Ca 2+ influx (A,D), as was the CCh-evoked [Ca 2+ ] c rise (A,B). Scaling up the [Ca 2+ ] c transients obtained in BAPTA so that the depolarization-evoked transients in the presence and absence of the chelator are of comparable size allowed a compensation for the increased buffer capacity of the cell to be made (E). Application of the same scaling factor to the CCh-evoked [Ca 2+ ] c increases allowed comparison of the CCh-evoked [Ca 2+ ] c transients in the presence of the chelator (G-I). When the cytoplasmic Ca 2+ buffer capacity had been increased (with BAPTA), the lower concentration of CCh was affected to a smaller extent than the higher CCh concentration (inset shown on an expanded scale; note the colour coding). The change in noise in G (red trace) during CCh (500 M) occurred because of a decrease in data sampling rate (from 10 Hz to 1 Hz). 
Smooth muscle responds to activation of the inositol (1,4,5)-trisphosphate receptor [Ins(1,4,5)P(3)R] with a graded concentration-dependent ("quantal") Ca2+ release from the sarcoplasmic reticulum (SR) store. Graded release seems incompatible both with the finite capacity of the store and the Ca2+-induced Ca2+ release (CICR)-like facility, at Ins(1,4,5)P3Rs, that, once activated, should release the entire content of SR Ca2+. The structural organization of the SR and the regulation of Ins(1,4,5)P3R activity by inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] and Ca2+ have each been proposed to explain ;quantal' Ca2+ release. Here, we propose that regulation of Ins(1,4,5)P3R activity by lumenal Ca2+ acting at the cytoplasmic aspect of the receptor might explain ;quantal' Ca2+ release in smooth muscle. The entire SR store was found to be lumenally continuous and Ca2+ could diffuse freely throughout: peculiarities of SR structure are unlikely to account for ;quantal' release. While Ca2+ release was regulated by [Ca2+] within the SR, the velocity of release increased (accelerated) during the release process. The extent of acceleration of release determined the peak cytoplasmic [Ca2+] and was attenuated by a reduction in SR [Ca2+] or an increase in cytoplasmic Ca2+ buffering. Positive feedback by released Ca2+ acting at the cytoplasmic aspect of Ins(1,4,5)P3Rs (i.e. CICR-like) might (a) account for the acceleration, (b) provide the regulation of release by SR [Ca2+] and (c) explain the ;quantal' release process itself. During Ca2+ release, SR [Ca2+] and thus unitary Ins(1,4,5)P3R currents decline, CICR reduces and stops. With increasing [Ins(1,4,5)P3], coincidental activation of several neighbouring Ins(1,4,5)P3Rs offsets the reduced Ins(1,4,5)P3R current to renew CICR and Ca2+ release.
 
In skeletal remodeling, osteoclasts degrade bone, detach and move to new locations. Mechanical stretch and estrogen regulate osteoclast motility via nitric oxide (NO). We have found previously that NO stimulates guanylyl cyclase, activating the cGMP-dependent protein kinase 1 (PKG1), reversibly terminating osteoclast matrix degradation and attachment, and initiating motility. The PKG1 substrate vasodilator-stimulated protein (VASP), a membrane-attachment-related protein found in complexes with the integrin alphavbeta3 in adherent osteoclasts, was also required for motility. Here, we studied downstream mechanisms by which the NO-dependent pathway mediates osteoclast relocation. We found that NO-stimulated motility is dependent on activation of the Ca(2+)-activated proteinase mu-calpain. RNA interference (RNAi) showed that NO-dependent activation of mu-calpain also requires PKG1 and VASP. Inhibition of Src kinases, which are involved in the regulation of adhesion complexes, also abolished NO-stimulated calpain activity. Pharmacological inhibition and RNAi showed that calpain activation in this process is mediated by the inositol (1,4,5)-trisphosphate receptor 1 [Ins(1,4,5)P(3)R1] Ca(2+) channel. We conclude that NO-induced motility in osteoclasts requires regulated Ca(2+) release, which activates mu-calpain. This occurs via the Ins(1,4,5)P(3)R1.
 
Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] originating in the vascular smooth-muscle cells (VSMCs) has been shown to modulate the Ca(2+) stores in endothelial cells (ECs). However, the reverse is not found, suggesting that Ins(1,4,5)P(3) movement might be unidirectional across gap junctions at the myoendothelial junction (MEJ), or that distribution of the Ins(1,4,5)P(3) receptor [Ins(1,4,5)P(3)-R] is different between the two cell types. To study trans-junctional communication at the MEJ, we used a vascular-cell co-culture model system and selectively modified the connexin composition in gap junctions in the two cell types. We found no correlation between modification of connexin expression and Ins(1,4,5)P(3) signaling between ECs and VSMCs. We next explored the distribution of Ins(1,4,5)P(3)-R isoforms in the two cell types and found that Ins(1,4,5)P(3)-R1 was selectively localized to the EC side of the MEJ. Using siRNA, selective knockdown of Ins(1,4,5)P(3)-R1 in ECs eliminated the secondary Ins(1,4,5)P(3)-induced response in these cells. By contrast, siRNA knockdown of Ins(1,4,5)P(3)-R2 or Ins(1,4,5)P(3)-R3 in ECs did not alter the EC response to VSMC stimulation. The addition of 5-phosphatase inhibitor (5-PI) to ECs that were transfected with Ins(1,4,5)P(3)-R1 siRNA rescued the Ins(1,4,5)P(3) response, indicating that metabolic degradation of Ins(1,4,5)P(3) is an important part of EC-VSMC coupling. To test this concept, VSMCs were loaded with 5-PI and BAPTA-loaded ECs were stimulated, inducing an Ins(1,4,5)P(3)-mediated response in VSMCs; this indicated that Ins(1,4,5)P(3) is bidirectional across the gap junction at the MEJ. Therefore, localization of Ins(1,4,5)P(3)-R1 on the EC side of the MEJ allows the ECs to respond to Ins(1,4,5)P(3) from VSMCs, whereas Ins(1,4,5)P(3) moving from ECs to VSMCs is probably metabolized before binding to a receptor. This data implicates the MEJ as being a unique cell-signaling domain in the vasculature.
 
Inositol-(1,4,5)-triphosphate receptors (InsP(3)Rs) are ligand-gated Ca(2+) channels that control Ca(2+) release from intracellular stores and play a central role in a wide range of cellular responses. In most epithelial cells, InsP(3)Rs are not uniformly distributed within the endoplasmic reticulum (ER) membrane with the consequence that agonist stimulation results in compartmentalized Ca(2+) signals. Despite these observations, little is known about the mechanisms that regulate the intracellular localization of InsP(3)Rs. Here, we report that exogenously expressed InsP(3)R1-GFP and endogenous InsP(3)R3 interact with the K-Ras-induced actin-binding protein (KRAP) in both differentiated and undifferentiated Madin-Darby canine kidney (MDCK) cells. KRAP mediates InsP(3)R clustering in confluent MDCK cells and functions as an adapter, linking InsP(3)Rs to vimentin intermediate filaments (IF). Upon epithelial differentiation, KRAP and vimentin are both required for InsP(3)R accumulation at the periphery of MDCK cells. Finally, KRAP associates with vimentin in chicken B lymphocytes and with keratins in a breast cancer cell line devoid of vimentin. Collectively, our data suggest that IF in conjunction with KRAP may govern the localization of InsP(3)Rs in a large number of cell types (including epithelial cells) and in various physiological or pathological contexts.
 
ATP evoked [Ca 2+ ] c rises in only a minority of single voltage-clamped myocytes. (A,B) ATP (1 mM by pressure ejection; ii) evoked transient [Ca 2+ ] c increases in only ,10% of cells voltage-clamped at 270 mV (iii) as indicated by F/F 0 (Ai) and did not elicit any discernible current responses in these cells (iv). ATP failed to evoke Ca 2+ release in the remaining cells (Bi). (C) Although photolysed caged Ins(1,4,5)P 3 (m) increased [Ca 2+ ] c (i), ATP (1 mM by pressure ejection; ii), however, failed to evoke [Ca 2+ ] c rises as indicated by F/F 0 (i) in cells voltage clamped at 230 mV (iii). 
Adenosine 5'-triphosphate (ATP) mediates a variety of biological functions following nerve-evoked release, via activation of either G protein-coupled P2Y- or ligand-gated P2X-receptors. In smooth muscle, ATP, acting via P2Y receptors (P2YR), may act as an inhibitory neurotransmitter. The underlying mechanism(s) remain unclear, but have been proposed to involve the production of inositol 1,4,5-trisphosphate (IP(3)) by phospholipase C (PLC), to evoke Ca(2+) release from the internal store and stimulation of Ca(2+)-activated potassium (K(Ca)) channels to cause membrane hyperpolarization. This mechanism requires Ca(2+) release from the store. However, in the present study, ATP evoked transient Ca(2+) increases in only ∼10% of voltage-clamped single smooth muscle cells. These results do not support activation of K(Ca) as the major mechanism underlying inhibition of smooth muscle activity. Interestingly, ATP inhibited IP(3)-evoked Ca(2+) release in cells that did not show a Ca(2+) rise in response to purinergic activation. The reduction in IP(3)-evoked Ca(2+) release was not mimicked by adenosine and therefore, cannot be explained by hydrolysis of ATP to adenosine. The reduction in IP(3)-evoked Ca(2+) release was, however, also observed with its primary metabolite, ADP, and blocked by the P2Y(1)R antagonist, MRS2179, and the G protein inhibitor, GDPβS, but not by PLC inhibition. The present study demonstrates a novel inhibitory effect of P2Y(1)R activation on IP(3)-evoked Ca(2+) release, such that purinergic stimulation acts to prevent IP(3)-mediated increases in excitability in smooth muscle and promote relaxation.
 
Although ventricular cardiomyocytes express inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors, it is unclear how these Ca2+ channels contribute to the effects of Gq-coupled agonists. Endothelin-1 augmented the amplitude of pacing-evoked Ca2+ signals (positive inotropy), and caused an increasing frequency of spontaneous diastolic Ca2+-release transients. Both effects of endothelin-1 were blocked by an antagonist of phospholipase C, suggesting that Ins(1,4,5)P3 and/or diacylglycerol production was necessary. The endothelin-1-mediated spontaneous Ca2+ transients were abolished by application of 2-aminoethoxydiphenyl borate (2-APB), an antagonist of Ins(1,4,5)P3 receptors. Incubation of electrically-paced ventricular myocytes with a membrane-permeant Ins(1,4,5)P3 ester provoked the occurrence of spontaneous diastolic Ca2+ transients with the same characteristics and sensitivity to 2-APB as the events stimulated by endothelin-1. In addition to evoking spontaneous Ca2+ transients, stimulation of ventricular myocytes with the Ins(1,4,5)P3 ester caused a positive inotropic effect. The effects of endothelin-1 were compared with two other stimuli, isoproterenol and digoxin, which are known to induce inotropy and spontaneous Ca2+ transients by overloading intracellular Ca2+ stores. The events evoked by isoproterenol and digoxin were dissimilar from those triggered by endothelin-1 in several ways. We propose that Ins(1,4,5)P3 receptors support the development of both inotropy and spontaneous pro-arrhythmic Ca2+ signals in ventricular myocytes stimulated with a Gq-coupled agonist.
 
In the ciliate Paramecium, a variety of well characterized processes are regulated by Ca2+, e.g. exocytosis, endocytosis and ciliary beat. Therefore, among protozoa, Paramecium is considered a model organism for Ca2+ signaling, although the molecular identity of the channels responsible for the Ca2+ signals remains largely unknown. We have cloned - for the first time in a protozoan - the full sequence of the gene encoding a putative inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) receptor from Paramecium tetraurelia cells showing molecular characteristics of higher eukaryotic cells. The homologously expressed Ins(1,4,5)P3-binding domain binds [3H]Ins(1,4,5)P3, whereas antibodies unexpectedly localize this protein to the osmoregulatory system. The level of Ins(1,4,5)P3-receptor expression was reduced, as shown on a transcriptional level and by immuno-staining, by decreasing the concentration of extracellular Ca2+ (Paramecium cells rapidly adjust their Ca2+ level to that in the outside medium). Fluorochromes reveal spontaneous fluctuations in cytosolic Ca2+ levels along the osmoregulatory system and these signals change upon activation of caged Ins(1,4,5)P3. Considering the ongoing expulsion of substantial amounts of Ca2+ by the osmoregulatory system, we propose here that Ins(1,4,5)P3 receptors serve a new function, i.e. a latent, graded reflux of Ca2+ to fine-tune [Ca2+] homeostasis.
 
Nuclear Ca2+ plays a key role in the regulation of gene expression. Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3)] might be an important regulator of nuclear Ca2+ but its contribution to nuclear Ca2+ signalling in adult cardiomyocytes remains elusive. We tested the hypothesis that endothelin-1 enhances nuclear Ca2+ concentration transients (CaTs) in rabbit atrial myocytes through Ins(1,4,5)P3-induced Ca(2+) release from perinuclear stores. Cytoplasmic and nuclear CaTs were measured simultaneously in electrically stimulated atrial myocytes using confocal Ca2+ imaging. Nuclear CaTs were significantly slower than cytoplasmic CaTs, indicative of compartmentalisation of intracellular Ca2+ signalling. Endothelin-1 elicited a preferential (10 nM) or a selective (0.1 nM) increase in nuclear versus cytoplasmic CaTs. This effect was abolished by inhibition of endothelin-1 receptors, phospholipase C and Ins(1,4,5)P3 receptors. Fractional Ca2+ release from the sarcoplasmic reticulum and perinuclear stores was increased by endothelin-1 at an otherwise unaltered Ca2+ load. Comparable increases of cytoplasmic CaTs induced by beta-adrenoceptor stimulation or elevation of extracellular Ca2+ could not mimic the endothelin-1 effects on nuclear CaTs, suggesting that endothelin-1 specifically modulates nuclear Ca2+ signalling. Thus, endothelin-1 enhances nuclear CaTs in atrial myocytes by increasing fractional Ca2+ release from perinuclear stores. This effect is mediated by the coupling of endothelin receptor A to PLC-Ins(1,4,5)P3 signalling and might contribute to excitation-transcription coupling.
 
Cytosolic Ca(2+) controls a wide range of cellular events. The versatility of this second messenger depends on its ability to form diverse spatial and temporal patterns, including waves and oscillations. Ca(2+)-signaling patterns are thought to be determined in part by the subcellular distribution of inositol (1,4,5)-trisphosphate receptors [Ins(1,4,5)P(3)Rs] but little is currently known about how the localization of the Ins(1,4,5)P(3)R itself is regulated. Here, we report that the recruitment of GFP-tagged Ins(1,4,5)P(3)Rs in the vicinity of tight junctions in Madin-Darby canine kidney (MDCK) cells requires the N-terminal domain. Stable expression of this domain in polarized MDCK cells induced a flattened morphology, affected cytokinesis, accelerated cell migration in response to monolayer wounding and interfered with the cortical targeting of myosin IIA. In addition, downregulation of myosin IIA in polarized MDCK cells was found to mimic the effects of stable expression of the N-terminal part of Ins(1,4,5)P(3)R on cell shape and to alter localization of endogenous Ins(1,4,5)P(3)Rs. Taken together, these results support a model in which the recruitment of Ins(1,4,5)P(3)Rs at the apex of the lateral membrane in polarized MDCK cells, involves myosin IIA and might be important for the regulation of cortical actin dynamics.
 
The effect of chemoattractants such as cyclic AMP and folate on amoebae of the cellular slime mould Dictyostelium discoideum is to cause a series of rapid intracellular responses. One of the most rapid of these responses is the polymerization of actin associated with the cytoskeleton, an event correlated with pseudopodium formation, which occurs within 3-5 s of chemotactic receptor stimulation. We report that this response can be mimicked by addition of 5 microM-inositol 1,4,5-triphosphate (IP3) or by addition of 100 microM-Ca2+ to saponin-permeabilized amoebae. The data suggest that cytoskeletal actin polymerization occurs in normal cells as a result of IP3 formation in response to cell surface receptor stimulation and the consequent release of Ca2+ from internal stores.
 
Inositol 1,4,5-trisphosphate (InsP(3))-mediated increases in cytosolic Ca(2+) concentration ([Ca(2+)](c)) regulate activities which include division, contraction and cell death. InsP(3)-evoked Ca(2+) release often begins in a single site then regeneratively propagates through the cell as a Ca(2+) wave. The Ca(2+) wave consistently begins at the same site on successive activations. We addressed the mechanisms that determine the Ca(2+) wave initiation site in intestinal smooth muscle cells. Neither an increased sensitivity of InsP(3) receptors (InsP(3)R) to InsP(3) nor regional clustering of muscarinic receptors (mAChR3) or InsP(3)R1 explained the initiation site. However, examination of the overlap of mAChR3 and InsP(3)R1 by centre of mass analysis revealed a small percentage (∼10%) of sites which showed colocalisation. Indeed, the extent of colocalisation was greatest at Ca(2+) wave initiation site. The initiation site may arise from a selective delivery of InsP(3) from mAChR3 activity to particular InsP(3)R to generate faster local [Ca(2+)](c) increases at sites of co-localization. In support, a localized subthreshold 'priming' InsP(3) concentration applied rapidly but at regions distant from the initiation site shifted the wave to the site of priming InsP(3) release. Conversely, when the Ca(2+) rise at the initiation site was rapidly and selectively attenuated the Ca(2+) wave again shifted and initiated at a new site. These results indicate that Ca(2+) waves initiate where there is a structural and functional coupling of mAChR3 and InsP(3)R1 which generates junctions in which InsP(3) acts as a highly localized signal by being rapidly and selectively delivered to InsP(3)R.
 
Signal transduction in Dictyostelium for oriented movement and differentiation involves a fine tuning of the cytosolic Ca2+ concentration. We have previously shown that cAMP binding to the cell surface receptor elicits two cellular events: (i) to enhance Ca2+ entry across the plasma membrane; (ii) to increase Ca2+ uptake into Ca(2+)-sequestering organelles. Here we used permeabilised cells to show that cAMP-induced Ca2+ uptake in these cells was sensitive to the Ca2+ transport ATPase blocker 2,5-di-(tert-butyl)-1,4-hydroquinone (BHQ) and the vacuolar H(+)-ATPase inhibitor NBD-Cl. By contrast, bafilomycin A1 and vanadate, inhibitors of Ca2+ uptake into acidosomes in Dictyostelium, did not reduce the cAMP-induced Ca2+ uptake of permeabilised cells. GTP gamma S served as a tool to measure Ins(1,4,5)P3- (InsP3)-sensitive Ca2+ release. Following NBD-Cl or BHQ treatment Ca2+ release was reversibly inhibited. We conclude that the cAMP-controlled Ca2+ influx is directed into a NBD-Cl and BHQ-sensitive compartment, which comprises the InsP3-releasable pool. The acidosomal Ca2+ store seems to provide for additional Ca2+ if required.
 
Fructose-1,6-bisphosphatase (FBPase), an important enzyme in the gluconeogenic pathway in Saccharomyces cerevisiae, is expressed when cells are grown in media containing a poor carbon source. Following glucose replenishment, FBPase is targeted from the cytosol to intermediate Vid (vacuole import and degradation) vesicles and then to the vacuole for degradation. Recently, several vid mutants that are unable to degrade FBPase in response to glucose were identified. Here, we present VID22, a novel gene involved in FBPase degradation. VID22 encodes a glycosylated integral membrane protein that localizes to the plasma membrane. Newly synthesized Vid22p was found in the cytoplasm and then targeted to the plasma membrane independent of the classical secretory pathway. A null mutation of VID22 failed to degrade FBPase following a glucose shift and accumulated FBPase in the cytosol. Furthermore, the majority of FBPase remained in a proteinase K sensitive compartment in the Deltavid22 mutant, implying that VID22 is involved in FBPase transport from the cytosol to Vid vesicles. By contrast, starvation-induced autophagy and peroxisome degradation were not impaired in the Deltavid22 mutant. This strain also exhibited the proper processing of carboxypeptidase Y and aminopeptidase I in the vacuole. Therefore, Vid22p appears to play a specific role in the FBPase trafficking pathway.
 
Characterisation of invadopodia of MDA-MB- 231 breast cancer cells. Cells were grown for 24 hours on a planar matrix of Matrigel TM containing DQ-Gelatin H . 
Na V 1.5 colocalises with caveolin-1 in invadopodia of MDA-MB-231 breast cancer cells. (A-C) Immunofluorescence imaging performed on MDA-MB-231 breast cancer cells grown on Matrigel TM and showing focal areas of strong colocalisation (Coloc, white pixels) between (A) MT1-MMP (green) and Na V 1.5 (red), (B) caveolin-1 (green) and Na V 1.5 (red), and (C) spots of F-actin (phalloidin-Alexa594, red) and caveolin-1 (green). (D) In situ proximity ligation assays (DUOLINK In cell coIP) showing a strong proximity between Na V 1.5 and caveolin-1 (red dots) in areas of matrix degradation (green). (E) Invadopodia (I) entrapped into a 2% gelatin matrix were fractionated and separated from cytosol (C)-and membrane (M)-enriched fractions. The quality of the fractions was assessed using invadopodia (cortactin, FAK, MT1-MMP), cytosolic (HSC70, b-actin) and membrane (b-adaptin) markers.
Na V 1.5 interacts with, and allosterically modulates NHE-1 function. (A) SIM immunofluorescence imaging of NHE-1 (green) and Na V 1.5 (red) 
Na V 1.5 controls F-actin cytoskeleton, cell morphology and foci of matrix degradation in MDA-MB-231 cancer cells. (A) shCTL and shNa V 1.5 cells were transfected with LifeAct–GFP to visualize the striking differences in F-actin cytoskeleton and cell morphology between both MDA-MB-231-derived 
The degradation of the extracellular matrix by cancer cells represents an essential step in metastatic progression and this is performed by cancer cell structures called invadopodia. NaV1.5 sodium channels are overexpressed in breast tumours and associated with metastatic occurrence. NaV1.5 activity was shown to enhance breast cancer cell invasiveness through perimembrane acidification and subsequent degradation of the extracellular matrix by cysteine cathepsins. Here, we showed that NaV1.5 was co-localised with NHE-1, and caveolin-1 in MDA-MB-231 breast cancer cells invadopodia, at sites of matrix remodelling. NHE-1, NaV1.5 and caveolin-1 co-immunoprecipitated, which indicated a close association between these proteins. The expression of NaV1.5 was responsible for the allosteric modulation of NHE-1 rendering it more active at intracellular pH range 6.4 to 7, thus potentially extruding more protons in the extracellular space. Furthermore, NaV1.5 increased Src kinase activity and the phosphorylation (Y421) of the actin-nucleation-promoting factor cortactin, controlled F-actin polymerization and the acquisition of an invasive morphology. Taken together, our study suggests that NaV1.5 is a central regulator of invadopodia formation and activity in breast cancer cells.
 
Human trophoblast cells offer a unique in vitro model for the study of aspects of the dynamic processes occurring during cell fusion and syncytium formation. In the human placenta, mononuclear cytotrophoblasts aggregate and fuse to form a multinucleated syncytiotrophoblast. In vitro, the addition of cyclic AMP analogs, 8-bromo-cyclic-AMP or Sp-8-bromo-cyclic AMPS, promotes syncytiotrophoblast formation, as shown by the disappearance of immunostained E-cadherin and desmoplakin, and increased numbers of nuclei per syncytium. An antagonist of cyclic AMP, Rp-8-bromo-cyclic AMPS, and an inhibitor of the cyclic AMP-dependent protein kinase catalytic subunit, H-89, impair cell fusion. This led us to study the pattern of expression and subcellular localization of cyclic-AMP-dependent protein kinase subunits during syncytium formation. Cytotrophoblasts expressed the RIalpha and RIIalpha regulatory subunits and the Calpha and Cbeta catalytic subunits. RIalpha was down-regulated during syncytium formation. No change in RIIalpha protein levels was observed, but there was a drastic subcellular redistribution. RIIalpha located in the Golgi-centrosomal area of cytotrophoblasts was scattered throughout the cytoplasm of the syncytiotrophoblast. Interestingly, an accumulation of RIIalpha was observed underneath the apical membrane of syncytiotrophoblast in vitro and in situ. This suggests a key role of cyclic AMP-dependent protein kinase type IIalpha during cell fusion and microvilli formation, both of which are essential for the secretory and transfer functions of the syncytiotrophoblast.
 
Maintenance of appropriate cell adhesion is crucial for normal cellular and organismal homeostasis. Certain microRNAs have recently been found capable of regulating molecules that oversee the fundamental cell biological events that drive cellular adhesion. It is now apparent that microRNAs play crucial roles in the great majority of biochemical pathways that contribute to normal cell adhesion. In this Commentary, we describe the latest advances within this still-emerging field, and highlight connections between the deregulation of microRNAs that affect cell-adhesion-associated molecules and the pathogenesis of several human diseases. Current evidence suggests that the ability of certain microRNAs--notably miR-17, miR-29, miR-31, miR-124 and miR-200--to pleiotropically regulate multiple molecular components of the cell adhesion machinery endows these microRNAs with the capacity to function as key modulators of adhesion-associated processes. This, in turn, holds important implications for our understanding of both the basic biology of cell adhesion and the etiology of multiple pathological conditions.
 
The mAb RK7, previously shown to recognize keratin 19, was also found to cross-react with a biologically unrelated 102 kDa protein, which becomes associated with the poles of the mitotic apparatus. This newly identified protein, called cytocentrin, is a stable cellular component, may be at least in part phosphorylated, and displays a cell cycle-dependent cellular localization. In interphase cells, it is diffusely distributed in the cytosol and shows no affinity for cytoplasmic microtubules. It becomes localized to the centrosome in early prophase, prior to nuclear envelope breakdown, separation of replicated centrosomes, and nucleation of mitotic apparatus microtubules. During metaphase, cytocentrin is located predominately at the mitotic poles, often appearing as an aggregate of small globular sub-components; it also associates with some polar microtubules. In late anaphase/early telophase cytocentrin dissociates entirely from the mitotic apparatus and becomes temporarily localized with microtubules in the midbody, from which it disappears by late telophase. In taxol-treated cells cytocentrin was associated with the center of the miniasters but also showed affinity for some cytoplasmic microtubules. Studies employing G2-synchronized cells and nocodazole demonstrated that cytocentrin can become associated with mitotic centrosomes independently of tubulin polymerization and that microtubules regrow from antigen-containing foci. We interpret these results to suggest that cytocentrin is a cytoplasmic protein that becomes specifically activated or modified at the onset of mitosis so that it can affiliate with the mitotic poles where it may provide a link between the pericentriolar material and other components of the mitotic apparatus.
 
A polyclonal antiserum raised against a HeLa cell microtubule-associated protein of Mr 210,000 (210 kD MAP or MAP4), an abundant non-neuronal MAP, was used to isolate cDNA clones encoding MAP4 from a human fetal brain lambda gt11 cDNA expression library. The largest of these clones, pMAP4.245, contains an insert of 4.1 kb and encodes a 245 kD beta-galactosidase fusion protein. Evidence that pMAP4.245 encodes MAP4 sequences includes immunoabsorption of MAP4 antibodies with the pMAP4.245 fusion protein, as well as identity of protein sequences obtained from HeLa 210 kD MAP4 with amino acid sequences encoded by pMAP4.245. The MAP4.245 cDNA hybridizes to several large (approximately 6-9 kb) transcripts on Northern blots of HeLa cell RNA. DNA sequencing of overlapping MAP4 cDNA clones revealed a long open reading frame containing a C-terminal region with three imperfect 18-amino acid repeats; this region is homologous to a motif present in the microtubule (MT)-binding domain of two prominent neuronal MAPs, MAP2 and tau. The pMAP4.245 sequence also encoded a series of unrelated repeats, located in the MAP's projection domain, N-terminal to the MT-binding domain. MAP4.245 fusion proteins bound to MTs in vitro, while fusion proteins that contained only the projection domain repeats failed to bind specifically to MTs. Thus, the major human non-neuronal MAP resembles two neuronal MAPs in its MT-binding domain, while most of the molecule has sequences, and presumably functions, distinct from those of the neuronal MAPs.
 
Radixin is a barbed end-capping actin-modulating protein which was previously reported to be concentrated at cell-to-cell adherens junctions (AJ) and cleavage furrows. Recently, cDNA encoding mouse radixin was isolated, showing that radixin is highly homologous to but distinct from ezrin. From mouse teratocarcinoma cells we isolated and analyzed cDNA encoding another radixin-related protein. Sequence analysis has demonstrated that this protein is a mouse homologue of human moesin (98.3% identity) and that it shares 71.7% and 80.1% identity with ezrin and radixin, respectively. Translation experiments in vitro combined with immunoblot analyses led us to conclude that there is a gene family consisting of ezrin, radixin and moesin. These members are coexpressed in various types of cells. Then, by immunofluorescence microscopy, we closely analyzed their distribution using polyclonal and monoclonal antibodies, which could recognize all three members. In addition to cell-to-cell AJ and cleavage furrows, it was shown that they were concentrated at microvilli and ruffling membranes in various types of cells. Furthermore, the cell-to-substrate AJ (focal contacts) were clearly stained by anti-radixin pAb only after the apical/lateral membranes and cytoplasm were removed by the zinc method. We conclude that at least one of the members of the ezrin-radixin-moesin family is concentrated at specific regions where actin filaments are densely associated with plasma membranes.
 
I have examined the possible involvement of specific cytoskeletal and peripheral membrane proteins in the early stages of acetylcholine receptor (AChR) aggregation in rat myotubes in culture by immunofluorescence localization of these proteins on the cytoplasmic face of isolated plasma membranes. A culture procedure utilizing selective replating of myoblasts and subsequent treatment with cytosine arabinoside was devised to obtain large, multipolar myotubes with extensive upper surfaces that are free of fibroblasts. These cultures were exposed for 4-6 h to embryonic pig brain extract (EBX) to induce AChR aggregate formation on the upper cell surface, and the AChRs were labeled with TRITC-conjugated alpha-bungarotoxin. Large sheets of plasma membranes from the upper cell surface were isolated by adhesion to a coverslip coated with a polypeptide adhesive (Cell-Tak) that was pressed on top of the culture. The membranes were labeled by indirect immunofluorescence with monoclonal antibodies against the 43 x 10(3) Mr and 58 x 10(3) Mr proteins, originally identified in the AChR-enriched membranes of Torpedo electroplaques, and with monoclonal antibodies against isoforms of actin and beta-spectrin. The labeling patterns showed that all four of these proteins are concentrated in the punctate AChR-enriched domains within the aggregates, suggesting that they may be involved in the early stages of AChR aggregation. Immunofluorescence labeling with monoclonal antibodies against vinculin and clathrin, and with an antiserum to talin, showed that these proteins are also associated with AChR aggregates; however, their labeling patterns did not correspond closely to the AChR-enriched domains. Furthermore, vinculin and talin dissociated from most of the membrane during isolation. The concentration of beta-spectrin and actin isoforms on the cytoplasmic fact of the AChR-enriched domains is consistent with the formation, early in the aggregation process, of a membrane-cytoskeleton association similar to that of erythrocytes.
 
The adhesion complex, which plays an important role in cell-substratum attachment, consists of a cellular hemidesmosomal plaque, anchoring filaments, the basement membrane zone and anchoring fibrils. An analysis of the temporal sequence of assembly of the adhesion complex was undertaken in an in vitro model of epithelial cell wound healing by immunofluorescence and electron microscopy. A monoclonal antibody directed against a 125K (K = 10(3) Mr) polypeptide (mAbHD), bullous pemphigoid (BP) autoantibodies, antibodies directed against collagen type VII and laminin antibodies were used as markers for anchoring filaments, the hemidesmosome, anchoring fibrils and the laminin component of the basement membrane zone, respectively. Fluorescence labeling could be detected with mAbHD before labeling with BP autoantibodies or collagen type VII antibodies. Laminin fluorescence was detected at the same time as mAbHD. Furthermore, the 125K polypeptide and laminin were located extracellularly prior to the appearance of BP antigen and collagen type VII. The appearance of the hemidesmosomal plaque at the electron microscope level succeeded the localization of BP antigen in basal cells detected by immunofluorescence microscopy. No evidence for the coordinated appearance of BP antigen, collagen type VII and laminin was observed in this model. We discuss the possibility that the 125K protein and laminin may play roles in the initiation of complex formation. Furthermore, although basement membrane zone components were detected early in adhesion complex re-formation, formation of the lamina densa region of the basement membrane zone followed the appearance of the hemidesmosomal plaque, indicating a role for the hemidesmosomal plaque in organizing the structure of the lamina densa.
 
Monoclonal antibodies were raised against a complex of proteins that was purified following the crosslinking of tubulin to the centromeres of CHO chromosomes using Lomant's reagent. One of the clones, hybridoma 32-9, produced antibodies that reacted with a 40 x 10(3) Mr protein present in the crosslinked complex. Furthermore, immunoblot analysis demonstrated that the 40 x 10(3) Mr antigen was present in various mammalian cell types from several different species. Indirect immunofluorescence using the antibody produced by clone 32-9 demonstrated that the 40 x 10(3) Mr antigen was associated with both spindle and cytoplasmic microtubules. In addition, centromere/kinetochore staining was detected in metaphase-arrested cells, while staining of prekinetochores in interphase nuclei was not observed. Unlike microtubule-associated proteins and microtubule-dependent ATPases, the 40 x 10(3) Mr protein did not copurify with microtubules when tubules were assembled from cellular homogenates using taxol and either GTP or GTP and AMP-PNP. Instead, the 40 x 10(3) Mr protein remained associated with the insoluble cellular material. The 40 x 10(3) Mr antigen could be released from the insoluble pelleted material by extraction with 1 M NaCl. Once solubilized, the 40 x 10(3) Mr protein was able to copurify with microtubules in assembly assays in vitro. This monoclonal antibody should serve as a valuable probe for studies of centromere/kinetochore structure and function.
 
The subcellular localization of human acidic FGF (aFGF; FGF-1) expressed to high levels by using a bacteriophage T7 RNA polymerase-driven vaccinia virus expression system was studied in BHK21 and HeLa cells. Acidic FGF was detected by immunoblotting or immunofluorescence using an affinity-purified rabbit polyclonal antibody. The nuclei of most transfected cells, but not nuclei of control cells, were strongly immunoreactive. The nuclear accumulation of aFGF was confirmed by subcellular fractionation and immunoblotting, indicating that about 50% of the expressed protein was located in the nuclei at 12 h after transfection. It has previously been reported that a putative N-terminal nuclear localization sequence (NLS) in aFGF is required for full mitogenic activity (Imamura et al., Science 249, 1567-1570, 1990). We found that deletion of the first 27 residues including the putative NLS did not prevent the nuclear translocation of aFGF in either cell type. This observation suggests that the putative NLS sequence is not essential for targeting aFGF to the cell nucleus. To analyze further the mechanism of nuclear import, purified aFGF was microinjected into the cytoplasm of growing BHK21 cells under various conditions. In chilled (4 degrees C) or ATP-depleted cells, the injected aFGF entered the nucleus with similar efficiency to that in control cells at 37 degrees C. This suggests that aFGF, which has a molecular mass of only 16,500, enters the cell nucleus by free diffusion, and possibly becomes trapped by binding to some nuclear structures. When added exogenously to growing BHK21 cells, aFGF was not localized to the nucleus. Instead, a punctate staining pattern in the cytosol was observed, reminiscent of that in the endosomal-lysosomal compartments. In addition, a diffuse extracellular surface-staining was evident. This result demonstrates that receptor-mediated endocytosis of aFGF does not result in its translocation to the nucleus, as has been reported for basic FGF.
 
Expression of p97 in melanoma and intestinal epithelial cell lines. (a) Immunoprecipitation of metabolically-labeled cell lysates with anti-p97 mAb KF23. Negative controls are isotype matched mouse mAbs (mAb SV63 for SK-MEL-28 cells and mAb B5.2 for intestinal epithelial cell lines) run with samples of the same lysates as for KF23. The positions of protein markers (kDa) are indicated on the left. (b) Northern blot analysis of total cellular RNA with probe 1j1. 
Subcellular localization of p97 in Caco-2 cells using mAb KF23. (a) Streptavidin-peroxidase immunocytochemistry of monolayers (×400). (b, c) Electron microscopic immunocytochemistry with Protein Agold. (d) Isotype-matched control antibody (mAb B5.2). mv, microvilli; arrows, gold particles in subapical vesicular compartment; arrowheads, basal and lateral membranes. Bar (b), 1 µm. 
Domain-selective biotinylation of p97. Caco-2 monolayers were labeled with biotin either apically (AP) or basolaterally (BL), and subjected to immunoprecipitation with mAb KF23 (p97). Immune complexes were electrophoresed on SDSPAGE, transferred to nitrocellulose filters, and biotin-labeled proteins were detected with 125 Istreptavidin. 
Localization of p97 in human fetal duodenum, and sensitivity to PI-PLC. (a) Streptavidin-peroxidase detection of mAb KF23. (b) Control. (c) PI-PLC treatment followed by immunocytochemistry. (a) ×200; (b),(c) ×400. 
GPI membrane anchoring of p97. (a) Hydropathicity plot (Kyte and Doolittle, 1982) and sequence of the carboxy terminus of p97. (b) PI-PLC-induced release of p97 from SK-MEL-28 cells. Supernatants: immunoprecipitation of with mAB KF23 of supernatants resulting from PI-PLC treatment of surface-iodinated SK-MEL-28 cells (released fraction). Lysates: immunoprecipitation with mAb KF23 of surface-iodinated SK-MEL-28 lysates after PI-PLC treatment (cell membrane-associated fraction). MHC-I and MHC-II correspond to samples immunoprecipitated with mAb W6/32 and mAb GRB1, respectively. (c) [ 14 C]ethanolamine is incorporated to p97. Lysates of [ 14 C]ethanolamine-labeled SK-MEL-28 cells were incubated with anti-p97 mAb KF23 and Protein ASepharose. Immunoprecipitated molecules were revealed by autoradiography. The position of marker proteins (kDa) is indicated. 
Melanotransferrin (p97) is an iron-binding membrane glycoprotein with 40% homology to transferrin and lactoferrin. It was first identified on the basis of its high level of expression in melanoma cells, as compared to normal melanocytes. It is also present in many cultured cell types. In normal tissues, p97 is expressed in fetal intestine, umbilical cord, sweat gland ducts and liver sinusoidal lining cells. Kinetic studies in melanoma cells have suggested that p97 plays a role in iron metabolism. We have examined expression of p97 in cell lines derived from human colorectal carcinomas which express a differentiated phenotype. When polarized, these cells showed a preferred apical distribution of p97, as demonstrated by immunohistochemistry, immune electron microscopy and domain-selective biotinylation. Correspondingly, p97 was only found on the apical brush border of epithelial cells in the fetal intestine. p97 was shown to be anchored to the membrane through a glycosyl phosphatidylinositol moiety by treatment with phophatidylinositol-specific phospholipase C (PI-PLC) and labeling with [14C]ethanolamine. These observations provide a basis for the elucidation of the physiological role of p97 in iron metabolism and its possible role in cell proliferation and malignant cell transformation.
 
GPI-anchored proteins are partially resistant to TX-100 extraction in Caco-2 cells. Cells were broken by passing through a 23 G needle. Post-nuclear supernatant was obtained by centrifugation (1,000 g, 10 min, 4°C). After 5 min of extraction with 1% OG or 1% TX-100, soluble (s) and insoluble (l) materials were separated by centrifugation (100,000 g, 1 h, 4°C). Immunodetection of antigens was performed by SDS-PAGE (6% to 15%) and Western blotting with 125 I-Protein A. Black arrowhead indicates CEA180; white arrowhead indicates CEA110. GPI-anchored proteins (PLAP, CEA180) were fully solubilized in OG and only partially solubilized in TX-100.  
Insolubility of membrane proteins in OG and TX-100
Immunogold detection of apical and basolateral markers in TX-100 pellet: double labeling with PLAP (5 nm) and (a) Ag525 (10 nm), (b) DPPIV (10 nm), (c) GP97 (10 nm), (d) SI (10 nm) was performed with gold-conjugated antibodies on TX-100 pellets recovered after sucrose density gradient centrifugation. Bar, 0.1 µm. PLAP was detected on membrane structures, associated with GP97 and SI (c,d). No labeling was detected using DPPIV or Ag525 antibodies (a,b).
In this study, we have investigated the possibility that glycosyl-phosphatidylinositol (GPI)-anchored proteins form insoluble membrane complexes in Caco-2 cells and that transmembrane proteins are associated with these complexes. GPI-anchored proteins were mainly resistant to Triton X-100 (TX-100) extraction at 4 degrees C but fully soluble in n-octyl-glucoside. Resistance to Triton X-100 extraction was not observed in the endoplasmic reticulum but appeared during transport through the Golgi complex. It was not dependent upon N-glycosylation processing, or pH variation from 6.5 to 8.5, and was not affected by sterol-binding agents. Other apical or basolateral transmembrane proteins were well solubilized in TX-100, with the exception of sucrase-isomaltase, which was partly insoluble. We isolated a membrane fraction from Caco-2 cells that contained GPI-anchored proteins and sucrase-isomaltase but no antigen 525, a basolateral marker, or dipeptidylpeptidase IV, an apical one. These data suggest that GPI-anchored proteins cluster to form membrane microdomains together with an apical transmembrane protein, providing a possible apical sorting mechanism for intestinal cells in vitro that might be related to apical sorting in MDCK cells, and that other mechanisms might exist to sort proteins to the apical membrane.
 
Microphotograph of a Feulgen-stained B16F10 culture taken using confocal microscopy. Note the presence of a highploidy nucleus.
DNA-content histograms of the B16F10 (A) and 3T3A31M (B) lines in three different phases of the cell cycle. At least 600 nuclei were measured in each of the subpopulations considered. The filled histogram bars represent high-ploidy cells. The DNA content of interphasic cells has the classical bimodal profile delimited by the two Gaussian curves corresponding to cells in G0/G1 and G2 phases of the cell cycle, respectively. This histogram includes 2.5% (B16F10) and 9% (3T3A31M) of high-ploidy cells. The DNA content of the metaphasic subpopulation has been plotted on a single Gaussian curve. In this case there is also a small percentage of high-ploidy metaphases (2.4% in the B16F10 line and 2.5% in the 3T3A31M line). However, the telophasic subpopulation is highly homogeneous and the presence of high-ploidy telophasic daughter cells is indetectable in the sample analysed. 
The presence of high-ploidy cells in malignant tumours has long been documented. However, the biological significance of these cells is not known and there is a great deal of controversy over their proliferative potential. We have analysed the behaviour of these cells in two murine tumour lines, B16F10 melanoma and 3T3A31M angiosarcoma, determining their DNA content by microspectrophotometry and using time-lapse film studies. We have found a discrepancy between the presence of high-ploidy cells in metaphase and the absence of hyperploid telophases. High-ploidy metaphases may be aborted (mitotic polyploidization), prolonged in time or evolve in the form of multipolar, generally tripolar, mitoses. Our results suggest that high-ploidy cells are capable of proliferating, despite certain peculiarities in their cell cycle, and constitute a tumour subpopulation whose role in neoplasia merits further study.
 
The alpha6beta4 integrin is located at the basal surface of keratinocytes, in hemidesmosomal structures that mediate stable adhesion of epidermal cells to the underlying basement membrane component laminin-5. The absence of alpha6beta4 integrin causes junctional epidermolysis bullosa, a severe blistering disease of the skin leading to perinatal death, confirming its essential role in mediating strong keratinocyte adhesion. Several studies have suggested that alpha6beta4 integrin can also regulate signaling cascades that control cell proliferation, survival and migration through a mechanism independent of its adhesive function. We have generated a conditional knockout mouse strain, in which the gene encoding the beta4 integrin subunit (Itgb4) was inactivated only in small stretches of the skin. These mice were viable and permitted an accurate analysis of the consequences of the loss of beta4 on various biological processes by comparing beta4-positive and -negative parts of the skin in the same animal. Despite the complete loss of hemidesmosomes in regions lacking alpha6beta4 integrin, the distribution of a range of adhesion receptors and basement membrane proteins was unaltered. Moreover, loss of alpha6beta4 did not affect squamous differentiation, proliferation or survival, except for areas in which keratinocytes had detached from the basement membrane. These in vivo observations were confirmed in vitro by using immortalized keratinocytes - derived from beta4-subunit conditional knockout mice - from which the gene encoding beta4 had been deleted by Cre-mediated recombination. Consistent with the established role of alpha6beta4 in adhesion strengthening, its loss from cells was found to increase their motility. Our findings clearly demonstrate that, after birth, epidermal differentiation, proliferation and survival all proceed normally in the absence of alpha6beta4, provided that cell adhesion is not compromised.
 
C-CAM (Cell-CAM 105) is a cell surface glycoprotein that is involved in cell-cell adhesion of rat hepatocytes in vitro. To elucidate the adhesion mechanism the binding properties of purified C-CAM were investigated. Using proteins immobilized on nitrocellulose it was found that radiolabeled C-CAM bound to C-CAM but not to a variety of other proteins. Partitioning in Triton X-114 showed that C-CAM has hydrophobic properties. In accordance with this, C-CAM was effectively incorporated into phosphatidylcholine liposomes by dialysis from octylglucoside-containing solutions. The C-CAM-containing liposomes bound specifically to isolated hepatocytes. This binding was blocked by Fab fragments of anti-C-CAM antibodies. Furthermore, preincubation of hepatocytes with anti-C-CAM antibodies followed by washing of the cells blocked binding of C-CAM-containing liposomes. At increasing C-CAM contents in the reconstituted liposomes a marked self-aggregation of the liposomes occurred. This aggregation was blocked by Fab fragments of anti-C-CAM antibodies and by alkaline pH. After neutralization a rapid reaggregation occurred. Neither C-CAM binding to C-CAM immobilized on nitrocellulose nor C-CAM-liposome aggregation required calcium ions. Liposomes reconstituted with C-CAM-depleted membrane glycoproteins did not self-aggregate or bind to hepatocytes. Thus, it is concluded that C-CAM can bind specifically to C-CAM in a homophilic binding reaction that does not require calcium. Accordingly, C-CAM has the potential of directly mediating cell-cell adhesion via C-CAM-C-CAM binding between adjacent cells.
 
A monoclonal antibody, designated TeM 106, that recognizes an intrinsic protein from the vacuole membrane (tonoplast) of cauliflower (Brassica oleracea L. var. botrytis) is described. Mice were immunized with a tonoplast fraction that had been purified from differentiating meristematic cells from the cauliflower head. Hybridomas were generated and screened by means of Enzyme Linked Immuno Sorbent Assays for differential reactivity to tonoplast over non-related proteins (bovine serum albumin). One out of 14 reactive murine clones was selected on the basis of its stability, secretory efficiency, and high affinity of the secreted antibodies. TeM 106 is an IgM which was shown by indirect immunofluorescence microscopy of frozen thin sections to bind specifically to the tonoplast of highly vacuolated cells as well as to the tonoplast of small vacuoles in meristematic cells. The molecular specificities of TeM 106 were preliminarily determined using electrophoretic transfer procedures (immunoblotting). TeM 106 reacted with a single protein band of 106,000 M(r) from the tonoplast of cauliflower. Using two-dimensional gel electrophoresis, it was shown that the epitope is borne by a single polypeptide. The antigen is a glycopeptide containing mannose and/or glucose residues in the oligosaccharide side chain but the epitope, resistant to the metaperiodate oxidation, is contained in the polypeptide backbone. Salt elution experiments indicated that the antigen, unlike several proteins from the tonoplast, is not eluted from the membrane by KCl treatments and is, therefore, tentatively considered as a tonoplast intrinsic protein, designated TIP 106.
 
bFGF transcripts in SV40-transformed human mammary cell lines. Samples of RNA (10 μ g) from epithelial Huma 7 (lane 1) and myoepithelial-like Huma 62 (lane 2) cells were subjected to agarose gel electrophoresis, transferred to nylon filters, and incubated with the cDNA corresponding to human bFGF mRNA (A) or with the cDNA corresponding to the mRNA for rat non- muscle actin (B), as described in Materials and Methods. The washed filters were exposed against Kodak XAR-5 film for 12 hours at − 70°C with an intensifying screen (A) or against Fuji RX film for 30 minutes at − 70°C with an intensifying screen (B). The molecular sizes of RNA markers are shown in kb. 
bFGF transcripts in cell lines from a benign human breast lesion. Samples of RNA (10 μ g) from cell lines epithelial Huma 123 (lane 1); epithelial Huma 121 (lane 2); myoepithelial-like Huma 109 (lane 3); and the rat myoepithelial-like cell line Rama 29 (lane 4) were subjected to agarose gel electrophoresis, transferred to nylon filters, and incubated with the cDNA corresponding to human bFGF mRNA (A) or with the cDNA corresponding to the mRNA for rat non-muscle actin (B), as described in Materials and Methods. The washed filters were exposed against Kodak XAR-5 film for 70 hours at room temperature (A) or against Fuji RX film for 30 minutes at − 70°C with an intensifying screen (B). The molecular sizes of RNA markers are shown in kb. 
Northern hybridization of cell-line RNA to strand-specific bFGF probes. Samples of RNA (10 µg) from the human myoepithelial-like cell lines Huma 109 (lanes 1 and 2); Huma 62 (lane 3), and the rat myoepithelial-like cell line, Rama 29 (lane 4), were subjected to agarose gel electrophoresis, transferred to nitrocellulose filters and incubated with a single-stranded RNA probe complementary to bFGF mRNA (Materials and Methods). The washed filters were exposed against Fuji RX X-ray film for 24 hours at −70°C with an intensifying screen. The molecular sizes of RNA markers are shown in kb.
Detection of bFGF in human cell lines by immunoblotting. Immunoblots of 2 M NaCl eluates of cell extracts fractionated on columns of heparin-Sepharose from an uncloned epithelial cell line HMT 3522 (lane 2); myoepithelial-like Huma 62 (lane 3); myoepithelial-like Huma 109 (lane 4) were incubated with antibFGF serum and bound antibodies detected as described in Materials and Methods. 100 ng human recombinant bFGF (lane 1) is present as a marker. All these lanes were from a single immunoblot. Major immunoreactive bands are indicated by arrows. The apparent molecular sizes of protein markers are shown in kDa.
Dissociation constants and numbers of receptors for bFGF on human mammary cell lines
mRNA for basic Fibroblast Growth Factor (bFGF) was expressed in a series of SV40-transformed human mammary cell lines as molecules of 7.1, 3.6, 2.0 and 1.2 kb. This expression was much weaker in those lines of epithelial morphology than in myoepithelial-like cell lines derived from them. It was confirmed, using northern hybridization to single-stranded RNA probes, that the multiple mRNAs were transcribed from the coding strand for bFGF. bFGF activity was detected in extracts of the cells and the relative amounts of activity corresponded in general to the amounts of mRNA found. Similar results were obtained from spontaneously transformed cell lines derived from a human benign breast lesion. The presence of bFGF protein in the extracts was confirmed by western blotting, which showed a band of 18-19 kDa, migrating in the same position as authentic bFGF; in addition, the myoepithelial-like cells showed prominent bands of bFGF at 24 and 26 kDa. No FGF receptor was detectable by the binding of 125I-bFGF to the SV40-transformed cell lines or to the epithelial cell lines from the benign breast lesion, but both high- and low-affinity receptors were found on myoepithelial-like cells derived from the latter. The results indicate that differentiation to the human myoepithelial-like phenotype in culture is associated with the enhanced expression of bFGF, and it is suggested that bFGF, immunocytochemically detected in the basement membrane of the human breast, may arise, at least in part, from the myoepithelial cells of the mammary parenchyma.
 
Top-cited authors
Valerie M Weaver
  • University of California, San Francisco
Mathieu Laplante
  • Institut universitaire de cardiologie et de pneumologie de Québec
Martin J Humphries
  • The University of Manchester
Jonathan D Humphries
  • Manchester Metropolitan University
Adam Byron
  • The University of Manchester