[Show abstract][Hide abstract] ABSTRACT: NHERF1 is a multifunctional scaffolding protein that interacts with receptors and ion transporters in its PDZ domains, and with the Ezrin-Radixin-Moesin (ERM) family of proteins in its carboxy-terminus. The role of NHERF-1 in hepatocyte function remains largely unknown. Here we examine the distribution and physiological significance of NHERF1 and Mrp-2 in hepatocytes. A wild-type, radixin binding site mutant (F355R), and NHERF1 PDZ1- and PDZ2-domain adenoviral mutant constructs were tagged with yellow fluorescent protein (YFP) and expressed in polarized hepatocytes to study localization and function of NHERF1. Cellular distribution of NHERF1 and radixin was visualized by fluorescence microscopy. A CMFDA assay was used to characterize Mrp-2 function. Similar to Mrp-2, wild type NHERF-1 and the NHERF-1 PDZ2 deletion mutant were localized to the canalicular membrane. In contrast, the radixin binding site mutant (F355R) and the NHERF1 PDZ1 deletion mutant, which interacts poorly with Mrp-2, were rarely associated with the canalicular membrane. Knockdown of NHERF1 led to dramatically impaired CMFDA secretory response. The NHERF1 PDZ1 and F355R mutants were devoid of a secretory response using CMFDA, while wild type NHERF1 infected cells exhibited increased GSFM secretion. The data indicate that NHERF1 interacts with Mrp-2 via the PDZ1 domain of NHERF1. Further, the findings suggest that NHERF1 is essential for maintaining the localization and function of Mrp-2.
[Show abstract][Hide abstract] ABSTRACT: This study uses YFP-tagged Rab27b expression in rabbit lacrimal gland acinar cells, which are polarized secretory epithelial cells, to characterize early stages of secretory vesicle trafficking. Here we demonstrate the utility of YFP-Rab27b to delineate new perspectives on the mechanisms of early vesicle biogenesis in lacrimal gland acinar cells, where information is significantly limited. Protocols were developed to deplete the mature YFP-Rab27b-enriched secretory vesicle pool in the subapical region of the cell, and confocal fluorescence microscopy was used to track vesicle replenishment. This analysis revealed a basally-localized organelle, which we termed the "nascent vesicle site," from which nascent vesicles appeared to emerge. Subapical vesicular YFP-Rab27b was co-localized with p150(Glued), a component of the dynactin cofactor of cytoplasmic dynein. Treatment with the microtubule-targeted agent, nocodazole, did not affect release of mature secretory vesicles, although during vesicle repletion it significantly altered nascent YFP-Rab27b-enriched secretory vesicle localization. Instead of moving to the subapical region, these vesicles were trapped at the nascent vesicle site which was adjacent to, if not a sub-compartment of, the trans-Golgi network. Finally, YFP-Rab27b-enriched secretory vesicles which reached the subapical cytoplasm appeared to acquire the actin-based motor protein, Myosin 5C. Our findings show that Rab27b enrichment occurs early in secretory vesicle formation, that secretory vesicles bud from a visually discernable nascent vesicle site, and that transport from the nascent vesicle site to the subapical region requires intact microtubules.
PLoS ONE 01/2012; 7(2):e31789. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b(-/-) and Rab27(ash/ash)/Rab27b(-/-) mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.
[Show abstract][Hide abstract] ABSTRACT: Radixin, the dominant ezrin-radixin-moesin (ERM) protein in hepatocytes, has two important binding domains: an NH(2)-terminal region that binds to plasma membrane and a COOH-terminal region that binds to F-actin after a conformational activation by phosphorylation at Thr564. The present studies were undertaken to investigate the cellular changes in expression of radixin in WIF-B cells and to assess radixin distribution and its influence on cell polarity. We used a recombinant adenoviral expression system encoding radixin wild-type and Thr564 mutants fused to cyan fluorescent protein (CFP), as well as conventional immunostaining procedures. Functional analyses were characterized quantitatively. Similar to endogenous radixin, adenovirus-infected radixin-CFP-wild type and nonphosphorylatable radixin-CFP-T564A were found to be expressed heavily in the compartment of canalicular membrane vacuoles, typically colocalizing with multidrug resistance-associated protein 2 (Mrp-2). Expression of radixin-CFP-T564D, which mimics constant phosphorylation, was quite different, being rarely associated with canalicular membranes. The WIF-B cells were devoid of a secretory response, T567D radixin became predominantly redistributed to the basolateral membrane, usually in the form of dense, long spikes and fingerlike projections, and the altered cell polarity involved changes in apical membrane markers. Differences in polar distribution of radixin suggest a role for the linker protein in promoting formation and plasticity of membrane surface projections and also suggest that radixin might be an organizer and regulator of Mrp-2 and cell polarity in hepatocytes.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND & AIMS: Rabs are monomeric guan-osine triphosphatases that regulate membrane trafficking and acid secretion in gastric parietal cells. Using a pro-teomics approach, we identified a new Rab, Rab27b, in tubulovesicle membranes and determined its role in pa-rietal cell activation. METHODS: We used mass spec-trometry (MS) to identify Rab27b in purified tubulove-sicular membrane fractions and used immunoblot and immunofluorescence analyses to study its expression. Wild-type, constitutively active (Rab27bQ78L), and dom-inant negative (Rab27bN133I) forms of Rab27b were tagged with yellow fluorescent protein (YFP) and ex-pressed in parietal cells using adenoviral constructs to study localization and function. Localization was visual-ized by fluorescence microscopy in resting and stimu-lated cells. Acid secretion in primary cell cultures was measured by aminopyrine accumulation. RESULTS: A tandem MS peptide mass fingerprint was matched to 7 peptides of Rab27b. Rab27b localized to tubulovesicle membranes, based on immunoblot and immunocyto-chemical analyses. Endogenous Rab27b, YFP/wild-type Rab27b, Rab27bQ78L, and Rab27bN133I all distributed throughout the cytoplasm of resting parietal cells. After stimulation, wild-type Rab27b and YFP-Rab27bQ78L translocated to the apical membrane, but YFPR-ab27bN133I did not. Expression of wild-type YFP-Rab27b or YFP-Rab27bQ78L did not affect acid secre-tion, whereas expression of Rab27bN133I almost completely inhibited acid secretion. CONCLUSIONS: Rab27b is associated with tubulovesicle membranes in the parietal cell and Rab27b may play a role in stimulation-associated membrane recruitment and gastric acid secretion. A cid secretion by the gastric parietal cell in response to various secretagogues such as histamine, gastrin, and acetylcholine is associated with massive morphologic changes. 1,2 In resting parietal cells, H,K–adenosine triphosphatase (ATPase) is stored in an extensive system of tubulovesicles that have no physical continuity with the apical plasma membrane. Following stimulation, H,K-ATPase– containing, cytoplasmically localized tubu-lovesicles fuse with the apical (canalicular) membrane, resulting in the recruitment of H,K-ATPase to the apical surface for proton pumping. Membrane fusion and exo-cytosis have been dominated by the consideration of 2 types of proteins: small G proteins of the Rab family and soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins. 3,4 Studies of gastric parietal cells have largely focused on Rab11 and Rab25. Colocalization of Rab11a with H,K-ATPase in resting parietal cells was observed, and the distribution of Rab11a was shifted to the plasma mem-brane on stimulation of the cells, prompting speculation that Rab11a is responsible for stimulus-dependent mem-brane translocation and gastric acid secretion. 5,6 Rab27 consists of 2 isoforms: Rab27a and Rab27b. 7,8 These proteins share 71% amino acid sequence similarity. Rab27a is widely expressed. On the other hand, Rab27b is associated with various secretory vesicles or granules in platelets, the pituitary gland, salivary parotid acinar cells, pancreatic acinar cells, melanocytes, and urothelial blad-der cells and plays an important role in regulated exocy-tosis. 9 –17 Here we report the identification of Rab27b using a mass spectrometry (MS) approach and characterize its function in gastric parietal cells.
[Show abstract][Hide abstract] ABSTRACT: Rabs are monomeric guanosine triphosphatases that regulate membrane trafficking and acid secretion in gastric parietal cells. Using a proteomics approach, we identified a new Rab, Rab27b, in tubulovesicle membranes and determined its role in parietal cell activation.
We used mass spectrometry (MS) to identify Rab27b in purified tubulovesicular membrane fractions and used immunoblot and immunofluorescence analyses to study its expression. Wild-type, constitutively active (Rab27bQ78L), and dominant negative (Rab27bN133I) forms of Rab27b were tagged with yellow fluorescent protein (YFP) and expressed in parietal cells using adenoviral constructs to study localization and function. Localization was visualized by fluorescence microscopy in resting and stimulated cells. Acid secretion in primary cell cultures was measured by aminopyrine accumulation.
A tandem MS peptide mass fingerprint was matched to 7 peptides of Rab27b. Rab27b localized to tubulovesicle membranes, based on immunoblot and immunocytochemical analyses. Endogenous Rab27b, YFP/wild-type Rab27b, Rab27bQ78L, and Rab27bN133I all distributed throughout the cytoplasm of resting parietal cells. After stimulation, wild-type Rab27b and YFP-Rab27bQ78L translocated to the apical membrane, but YFPR-ab27bN133I did not. Expression of wild-type YFP-Rab27b or YFP-Rab27bQ78L did not affect acid secretion, whereas expression of Rab27bN133I almost completely inhibited acid secretion.
Rab27b is associated with tubulovesicle membranes in the parietal cell and Rab27b may play a role in stimulation-associated membrane recruitment and gastric acid secretion.
[Show abstract][Hide abstract] ABSTRACT: Syntaxins are differentially localized in polarized cells and play an important role in vesicle trafficking and membrane fusion. These soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are believed to be involved in tubulovesicle trafficking and membrane fusion during the secretory cycle of the gastric parietal cell. We examined the cellular localization and distribution of syntaxin-1 and syntaxin-3 in rabbit parietal cells. Fractionation of gastric epithelial cell membranes showed that syntaxin-1 was more abundant in a fraction enriched in apical plasma membranes, whereas syntaxin-3 was found predominantly in the H,K-ATPase-rich tubulovesicle fraction. We also examined the cellular localization of syntaxins in cultured parietal cells. Parietal cells were infected with CFP-syntaxin-1 and CFP-syntaxin-3 adenoviral constructs. Fluorescence microscopy of live and fixed cells demonstrated that syntaxin-1 was primarily on the apical membrane vacuoles of infected cells, but there was also the expression of syntaxin-1 in a subadjacent cytoplasmic compartment. In resting, non-secreting parietal cells, syntaxin-3 was distributed throughout the cytoplasmic compartment; after stimulation, syntaxin-3 translocated to the apical membrane vacuoles, there co-localizing with H,K-ATPase, syntaxin-1 and F-actin. The differential location of these syntaxin isoforms in gastric parietal cells suggests that these proteins may be critical for maintaining membrane compartment identity and that they may play important, but somewhat different, roles in the membrane recruitment processes associated with secretory activation.
[Show abstract][Hide abstract] ABSTRACT: The soluble N-ethylmaleimide-sensitive factor attachment protein of 25 kDa (SNAP-25) plays an important role in vesicle trafficking. Together with vesicle-associated membrane protein-2 (VAMP-2) and syntaxin, SNAP-25 forms a ternary complex implicated in docking and fusion of secretory vesicles with the plasma membrane during exocytosis. These so-called SNARE proteins are believed to regulate tubulovesicle trafficking and fusion during the secretory cycle of the gastric parietal cell. Here we examined the cellular localization and functional importance of SNAP-25 in parietal cell cultures. Adenoviral constructs were used to express SNAP-25 tagged with cyan fluorescent protein, VAMP-2 tagged with yellow fluorescent protein, and SNAP-25 in which the C-terminal 25 amino acids were deleted (SNAP-25 Delta181-206). Membrane fractionation experiments and fluorescent imaging showed that SNAP-25 is localized to the apical plasma membrane. The expression of the mutant SNAP-25 Delta181-226 inhibited the acid secretory response of parietal cells. Also, SNAP Delta181-226 bound poorly in vitro with recombinant syntaxin-1 compared with wild type SNAP-25, indicating that pairing between syntaxin-1 and SNAP-25 is required for parietal cell activation. Dual expression of SNAP-25 tagged with cyan fluorescent protein and VAMP-2 tagged with yellow fluorescent protein revealed a dynamic change in distribution associated with acid secretion. In resting cells, SNAP-25 is at the apical plasma membrane and VAMP-2 is associated with cytoplasmic H,K-ATPase-rich tubulovesicles. After stimulation, the two proteins co-localize on the apical plasma membrane. These data demonstrate the functional significance of SNAP-25 as a SNARE protein in the parietal cell and show the dynamic stimulation-associated redistribution of VAMP-2 from H,K-ATPase-rich tubulovesicles to co-localize with SNAP-25 on the apical plasma membrane.
Journal of Biological Chemistry 01/2003; 277(51):50030-5. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acid secretion by parietal cells involves secretagogue-dependent recycling of the H+-K+-ATPase. Proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have been implicated as participants in membrane trafficking, docking, and fusing processes. Here we studied the intracellular distribution and functional importance of one SNARE protein, vesicle associated membrane protein-2 (VAMP-2), in gastric parietal cells.
Using an adenoviral recombinant expression system encoding VAMP-2 (synaptobrevin-2) fused to the green fluorescent protein (GFP), we expressed the GFP-VAMP-2 protein in primary cultures of rabbit parietal cells, which enables us to visualize the dynamics of GFP-VAMP-2 in a variety of functional states by fluorescence microscopy. To ascertain the function of VAMP-2 in parietal cell activation, streptolysin-O permeabilized gastric glands were treated with tetanus toxin, a potent and preferential protease for VAMP-2, and acid secretion was measured.
In resting parietal cells GFP was detected throughout the cytoplasm in a pattern of distribution that was very similar to that of H+-K+-ATPase. After stimulation, we observed that the GFP-VAMP-2 translocated to the apical plasma membrane along with the H+-K+-ATPase. A relatively high degree of co-localization was detected between GFP-VAMP-2 and H+-K+-ATPase. Tetanus toxin inhibited cAMP/ATP-stimulated acid secretion by about 45% in permeabilized gastric glands with a concomitant reduction in the level of immunoreactive VAMP-2.
Adenovirus-based GFP reporter fusion proteins can be used to efficiently study the functional dynamics of SNAREs. VAMP-2 is associated with tubulovesicle membranes in the parietal cell and plays a role in stimulation-associated membrane recruitment and acid secretion.