Rab3A and Rab3D control the total granule number and the fraction of granules docked at the plasma membrane in PC12 cells

Rab proteins are Ras-like GTPases that regulate traffic along the secretory or endocytic pathways. Within the Rab family, Rab3 proteins are expressed at high levels in neurons and endocrine cells where they regulate release of dense core granules and synaptic vesicles. Immuno-electron microscopy shows that Rab3A and Rab3D can coexist on the same granule before and after docking. Using electron microscopy of transfected PC12 cells, we report that expression of wild-type Rab3A (or Rab3D) increases the total number of granules and the percentage that is docked at the plasma membrane. Mutated Rab3A N135I (or Rab3D N135I) decreases the total granule number and the fraction of granules docked to the plasma membrane. These data show that at least one of the functions of Rab3A and Rab3D proteins is to control the number of granules docked at the plasma membrane.     

The GAP activity of Msb3p and Msb4p for the Rab GTPase Sec4p is required for efficient exocytosis and actin organization

Polarized growth in Saccharomyces cerevisiae is thought to occur by the transport of post-Golgi vesicles along actin cables to the daughter cell, and the subsequent fusion of the vesicles with the plasma membrane. Previously, we have shown that Msb3p and Msb4p genetically interact with Cdc42p and display a GTPase-activating protein (GAP) activity toward a number of Rab GTPases in vitro. We show here that Msb3p and Msb4p regulate exocytosis by functioning as GAPs for Sec4p in vivo. Cells lacking the GAP activity of Msb3p and Msb4p displayed secretory defects, including the accumulation of vesicles of 80-100 nm in diameter. Interestingly, the GAP activity of Msb3p and Msb4p was also required for efficient polarization of the actin patches and for the suppression of the actin-organization defects in cdc42 mutants. Using a strain defective in polarized secretion and actin-patch organization, we showed that a change in actin-patch organization could be a consequence of the fusion of mistargeted vesicles with the plasma membrane.     

Phosphatidic acid production,required for cholecystokinin octapeptide-stimulated amylase secretion from pancreatic acinar AR42J cells,is regulated by a wortmannin-sensitive process

To investigate the role of phospholipids in exocytotic secretory events, we utilized rat pancreatic acinar AR42J cells that secreted amylase in response to cholecystokinin octapeptide (CCK-8). Wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), was found to inhibit the secretion in a dose-dependent manner. When changes in cell membrane phospholipids were investigated before and after CCK-8 stimulation using [32P]orthophosphoric acid-labeled AR42J cells, we observed a rapid increase in phosphatidic acid (PtdOH) levels right after stimulation, which was not observed in non-stimulated cells. The increase, however, was suppressed by wortmannin pre-treatment, which also inhibited amylase secretion. Changes in other major phospholipids were not significant. These results indicate that CCK-8 induces amylase secretion through PI3K-regulated production of PtdOH in cell membranes.     

Transcriptional cascade from Math1 to Mbh1 and Mbh2 is required for cerebellar granule cell differentiation

Cerebellar granule cells (CGCs) are the most abundant neuronal type in the mammalian brain, and their differentiation is regulated by the basic helix-loop-helix gene, Math1. However, little is known about downstream genes of Math1 and their functions in the cerebellum. To investigate them, we have here established an electroporation-based in vivo gene transfer method in the developing mouse cerebellum. Misexpression of Math1 ectopically induced expression of Bar-class homeobox genes, Mbh1 and Mbh2, which are expressed by CGCs. Conversely, their expression was repressed in CGCs by knockdown of Math1. These findings, taken together with chromatin immunoprecipitation assays, suggest that Math1 directly regulates the Mbh genes in CGCs. Furthermore, a dominant-negative form of the Mbh proteins disrupted proper formation of the external granule layer and differentiation of CGCs, whereas misexpression of the Mbh genes ectopically induced expression of a CGC marker in nonneuronal cells, indicating that the Mbh proteins are required for the differentiation of CGCs.     

Degranulation of eosinophilic granule cells in neurofibromas and gastrointestinal tract in the bicolor damselfish

Damselfish neurofibromatosis (DNF) is a neoplastic disease affecting bicolor damselfish (Stegastes partitus Poey) on Florida reefs. Previous studies have demonstrated high densities of eosinophilic granule containing cells (EGC), the proposed equivalent of mast cells in fishes, in neurofibromas and malignant peripheral nerve sheath tumors (mpnst) in DNF. These lesions are similar to those in the disease neurofibromatosis type 1 (NF1) in humans, which contain large numbers of mast cells. In the present study, experiments were conducted to measure the response of EGC in these tumors as well as in the submucosa of the digestive tract to the mast cell degranulating agent compound 48/80. Degranulation of these cells was visible by light microscopy and characterized by conspicuous swelling of granules and often by the presence of free granules adjacent to the EGC. Degranulation occurred by release of intact granules (diacytosis), as reported in other fishes, rather than by fusion of granules with the cell membrane (exocytosis) as reported in mast cells in mammals. Baseline levels of EGC exhibiting degranulation ranged from 20-26% in the submucosa to 30% in tumors. Within 1-2h of exposure to compound 48/80, significant increases in average levels of degranulation were observed, to 67% in the gut and 72% in tumors. Degranulation was significantly more extensive in the tumors than in the gut. The outermost edges of the tumors contained significantly higher densities of EGC but these cells exhibited lower rates of degranulation than those in the inner regions of tumors. These observations support the hypothesis that the EGC present in neurofibromas and mpnst in DNF are equivalent to the mast cell component in neurofibromas in NF1.     

The polybasic sequence in the C2B domain of rabphilin is required for the vesicle docking step in PC12 cells

Rabphilin is generally thought to be involved in the regulation of secretory vesicle exocytosis in neurons and neuroendocrine cells, and it has recently been hypothesized that the C2B domain of rabphilin promotes the docking of dense-core vesicles to the plasma membrane through simultaneous interaction with a vesicle protein, Rab3A/27A, and a plasma membrane protein, SNAP-25 (synaptosome-associated protein of 25 kDa). However, the physiological significance of the rabphilin-SNAP-25 interaction in the vesicle-docking step has never been elucidated. In this study we demonstrated by a mutation analysis that the polybasic sequence (587 KKAKHKTQIKKK 598) in the C2B domain of rabphilin is required for SNAP-25 binding, and that the Asp residues in the Ca(2+)-binding loop 3 (D628 and D630) of the C2B domain are not required. We also investigated the effect of Lys-->Gln (KQ) mutations in the polybasic sequence of the C2B domain on vesicle dynamics by total internal reflection fluorescence microscopy in individual PC12 cells. A rabphilin(KQ) mutant that completely lacks SNAP-25-binding activity significantly decreased the number of plasma-membrane-docked vesicles and strongly inhibited high-KCl-induced dense-core vesicle exocytosis. These results indicate that the polybasic sequence in the C2B domain functions as an effector domain for SNAP-25 and controls the number of 'releasable' vesicles docked to the plasma membrane.     

Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania.     

Cytoplasmic cleavage of IMPA1 3′ UTR is necessary for maintaining axon integrity

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Involvement of Rab3A in vesicle priming during exocytosis: interaction with Munc13-1 and Munc18-1

Rab3A is a small G-protein of the Rab family that is involved in the late steps of exocytosis. Here, we studied the role of Rab3A and its relationship with Munc13-1 and Munc18-1 during vesicle priming. Phorbol 12-myristate 13-acetate (PMA) is known to enhance the percentage of fusion-competent vesicles and this is mediated by protein kinase C (PKC)-independent Munc13-1 activation and PKC-dependent dissociation of Munc18-1 from syntaxin 1a. Our results show that the effects of PMA varied in cells overexpressing Rab3A or mutants of Rab3A and in cells with Rab3A knockdown. When Munc13-1 was overexpressed in Rab3A knockdown cells, secretion was completely inhibited. In cells overexpressing a Rab-interacting molecule (RIM)-binding deficient Munc13-1 mutant, 128-Munc13-1, the effects of Rab3A on PMA-induced secretion was abolished. The effect of PMA, which disappeared in cells overexpressing GTP-Rab3A (Q81L), could be reversed by co-expressing Munc18-1 but not its mutant R39C, which is unable to bind to syntaxin 1a. In cells overexpressing Munc18-1, manipulation of Rab3A activity had no effect on secretion. Finally, Munc18-1 enhanced the dissociation of Rab3A, and such enhancement correlated with exocytosis. In summary, our results support the hypothesis that the Rab3A cycle is coupled with the activation of Munc13-1 via RIM, which accounts for the regulation of secretion by Rab3A. Munc18-1 acts downstream of Munc13-1/RIM/Rab3A and interacts with syntaxin 1a allowing vesicle priming. Furthermore, Munc18-1 promotes Rab3A dissociation from vesicles, which then results in fusion.     

CtBP1/BARS is an activator of phospholipase D1 necessary for agonist-induced macropinocytosis

Vesicular trafficking such as macropinocytosis is a dynamic process that requires coordinated interactions between specialized proteins and lipids. A recent report suggests the involvement of CtBP1/BARS in epidermal growth factor (EGF)-induced macropinocytosis. Detailed mechanisms as to how lipid remodelling is regulated during macropinocytosis are still undefined. Here, we show that CtBP1/BARS is a physiological activator of PLD1 required in agonist-induced macropinocytosis. EGF-induced macropinocytosis was specifically blocked by 1-butanol but not by 2-butanol. In addition, stimulation of cells by serum or EGF resulted in the association of CtBP1/BARS with PLD1. Finally, CtBP1/BARS activated PLD1 in a synergistic manner with other PLD activators, including ADP-ribosylation factors as demonstrated by in vitro and intact cell systems. The present results shed light on the molecular basis of how the ‘fission protein' CtBP1/BARS controls vesicular trafficking events including macropinocytosis.     

A sulfated proteoglycan is necessary for storage of exocrine secretory proteins in the rat parotid gland

Sulfated proteoglycans have beenproposed to play a role in the sorting and storage of secretoryproteins in exocrine secretory granules. Rat parotid acinar cellsexpressed a 40- to 60-kDa proteoglycan that was stored in secretorygranules. Treatment of the tissue with the proteoglycan synthesisinhibitor paranitrophenyl xyloside resulted in the complete abrogationof the sulfated proteoglycan. Pulse-chase experiments in the presenceof the xyloside analog showed a significant reduction in the stimulatedsecretion and granule storage of the newly synthesized regulatedsecretory proteins amylase and parotid secretory protein. Inhibition ofproteoglycan sulfation by chlorate did not affect the sorting of theseproteins. The effect of proteoglycan synthesis inhibition on proteinsorting was completely reversed upon treatment with a weak acid. These results suggest that the sulfated proteoglycan is necessary for sortingand storage of regulated secretory proteins in the exocrine parotidgland. Preliminary evidence suggests that the mechanism involves themodulation of granule pH by the proteoglycan rather than a directinteraction with other granule components.

     

A platform for efficient establishment and drug-response profiling of high-grade serous ovarian cancer organoids

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Immunohistochemical identification and localization of pancreatic polypeptide cells in the pancreas and gastrointestinal tract of the human fetus and adult man

Summary Pancreatic polypeptide (PP)-containing cells were detected by using anti-bovine PP (BPP) serum in the pancreas and gastrointestinal tract of human fetuses, premature infants and in the pancreas, antrum and jejunum of adult man obtained by biopsy from patients with normal gastroduodenal endoscopy. The localization was established by studying the distribution of PP cells in comparison to the distribution of glucagon-, somatostatin- and insulin cells. The first PP cells are seen in the pancreas at 10 weeks of gestation. They are located preferentially in the lower part of the head of the pancreas. The specificity of immunocytological reaction was ascertained by the inhibition of the reaction by bovine pancreatic polypeptide, glucagon and insulin did not modify the immunocytological reaction.     

Rab11-FIP3 is critical for the structural integrity of the endosomal recycling compartment

Rab11-FIP3 is an endosomal recycling compartment (ERC) protein that is implicated in the process of membrane delivery from the ERC to sites of membrane insertion during cell division. Here we report that Rab11-FIP3 is critical for the structural integrity of the ERC during interphase. We demonstrate that knockdown of Rab11-FIP3 and expression of a mutant of Rab11-FIP3 that is Rab11-binding deficient cause loss of all ERC-marker protein staining from the pericentrosomal region of A431 cells. Furthermore, we find that fluorophore-labelled transferrin cannot access the pericentrosomal region of cells in which Rab11-FIP3 function has been perturbed. We find that this Rab11-FIP3 function appears to be specific because expression of the equivalent Rab11-binding deficient mutant of Rab-coupling protein does not perturb ERC morphology. In addition, we find that other organelles such as sorting and late endosomes are unaffected by loss of Rab11-FIP3 function. Finally, we demonstrate the presence of an extensive coiled-coil region between residues 463 and 692 of Rab11-FIP3, which exists as a dimer in solution and is critical to support its function on the ERC. Together, these data indicate that Rab11-FIP3 is necessary for the structural integrity of the pericentrosomal ERC.     

Expression of Rab27B-binding protein Slp1 in pancreatic acinar cells and its involvement in amylase secretion

Slp1 is a putative Rab27 effector protein and implicated in intracellular membrane transport; however, the precise tissue distribution and function of Slp1 protein remain largely unknown. In this study we investigated the tissue distribution of Slp1 in mice and found that Slp1 is abundantly expressed in the pancreas, especially in the apical region of pancreatic acinar cells. Slp1 interacted with Rab27B in vivo and both proteins were co-localized on zymogen granules. Morphological analysis of fasted Slp1 knockout mice showed an increased number of zymogen granules in the pancreatic acinar cells, indicating that Slp1 is part of the machinery of amylase secretion by the exocrine pancreas.     

Galpha(olf) is necessary for coupling D1 and A2a receptors to adenylyl cyclase in the striatum

In the brain, dopamine and adenosine stimulate cyclic AMP (cAMP) production through D1 and A2a receptors, respectively. Using mutant mice deficient in the olfactory isoform of the stimulatory GTP-binding protein alpha subunit, Galpha(olf), we demonstrate here the obligatory role of this protein in the adenylyl cyclase responses to dopamine and adenosine in the caudate putamen. Responses to dopamine were also dramatically decreased in the nucleus accumbens but remained unaffected in the prefrontal cortex. Moreover, in the caudate putamen of mice heterozygous for the mutation, the amounts of Galpha(olf) were half of the normal levels, and the efficacy of dopamine- and CGS 21680 A(2) agonist-stimulated cAMP production was decreased. Together, these results identify Galpha(olf) as a critical parameter in the responses to dopamine and adenosine in the basal ganglia.