NFIA regulates granule recruitment and exocytosis in the adult pancreas
Marissa A. ScavuzzoJolanta ChmielowiecJessica TeawDiane YangMatthew C. HillAndrea R. WaksmunskiLita DuraineJoan Camuñas-SolerXiao-Qing DaiJordon KingStephen R. QuakePatrick E. MacDonaldAndré CaticMalgorzata Borowiak
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Summary After food ingestion, pancreatic cells secrete zymogen and hormone-containing granules to precisely control digestion and blood glucose levels. Identifying regulators of this process is paramount to combatting multiple pancreatic diseases. Here we show that pancreatic deletion of the transcription factor nuclear factor IA (NFIA) leads to hyperglycemia, hypoinsulinemia, and hypolipidemia. Surprisingly, insulin and digestive enzymes are produced in the absence of NFIA, however, they are not secreted properly and instead accumulate inside pancreatic cells. In NFIA-deficient mice we saw a reduction of insulin granules in the ready releasable pool and the first-phase insulin response was impaired. We found that NFIA binds to and activates Rab39b, a Rab GTPase critical for exocytosis. Re-expression of Rab39b in NFIA knockout islets restored glucose-stimulated insulin secretion. In sum, the NFIA-Rab39b axis regulates pancreatic physiology through granule recruitment and docking , linking NFIA to a new process with potential effects in diabetes, pancreatitis, and lipid disorders.Keywords:
Rab
Multiple physiology-pertinent transmembrane proteins reach the cell surface via the Golgi-bypassing unconventional protein secretion (UcPS) pathway. By employing C. elegans–polarized intestine epithelia, we recently have revealed that the small GTPase RAB-8/Rab8 serves as an important player in the process. Nonetheless, its function and the relevant UcPS itinerary remain poorly understood. Here, we show that deregulated RAB-8 activity resulted in impaired apical UcPS, which increased sensitivity to infection and environmental stress. We also identified the SNARE VTI-1/Vti1a/b as a new RAB-8–interacting factor involved in the apical UcPS. Besides, RAB-11/Rab11 was capable of recruiting RABI-8/Rabin8 to reduce the guanine nucleotide exchange activity of SMGL-1/GEF toward RAB-8, indicating the necessity of a finely tuned RAB-8/RAB-11 network. Populations of RAB-8– and RAB-11–positive endosomal structures containing the apical UcPS cargo moved toward the apical side. In the absence of RAB-11 or its effectors, the cargo was retained in RAB-8– and RAB-11–positive endosomes, respectively, suggesting that these endosomes are utilized as intermediate carriers for the UcPS.
Rab
Transport protein
Vesicular Transport Proteins
Choroideremia
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Rab
Identification
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Rab
Organelle
Munc-18
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The idea that pancreatic digestive enzyme secretion can occur in a nonparallel manner has been controversial because of its presumed incompatibility with the exocytosis secretory mechanism. Correlation and regression analysis of enzyme output by the rabbit pancreas after it is stimulated with cholecystokinin and chymodenin revealed that digestive enzymes are secreted in a highly linked fashion, compatible with exocytosis and with nonparallel secretion. Thus, exocytosis and nonparallel secretion are not contradictory processes, but rather nonparallel secretion is due to exocytosis from heterogeneous sources within the pancreas.
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Apocrine
Secretory Vesicle
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Secretory Vesicle
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Three cases of chronic alcoholic pancreatitis associated with aberrant pancreas are reported. All three patients underwent laparotomy, and an aberrant pancreas was found in the jejunum of each of the three patients. Microscopic examination of the aberrant pancreas did not show any changes suggestive of chronic pancreatitis, despite severe chronic pancreatitis in the main pancreas.
Pancreatic Disease
Chronic alcoholic
Jejunum
Exocrine pancreas
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RAB-10/Rab10 is a master regulator of endocytic recycling in epithelial cells. To better understand the regulation of RAB-10 activity, we sought to identify RAB-10(GDP)–interacting proteins. One novel RAB-10(GDP)–binding partner that we identified, LET-413, is the Caenorhabditis elegans homologue of Scrib/Erbin. Here, we focus on the mechanistic role of LET-413 in the regulation of RAB-10 within the C. elegans intestine. We show that LET-413 is a RAB-5 effector and colocalizes with RAB-10 on endosomes, and the overlap of LET-413 with RAB-10 is RAB-5 dependent. Notably, LET-413 enhances the interaction of DENN-4 with RAB-10(GDP) and promotes DENN-4 guanine nucleotide exchange factor activity toward RAB-10. Loss of LET-413 leads to cytosolic dispersion of the RAB-10 effectors TBC-2 and CNT-1. Finally, we demonstrate that the loss of RAB-10 or LET-413 results in abnormal overextensions of lateral membrane. Hence, our studies indicate that LET-413 is required for DENN-4–mediated RAB-10 activation, and the LET-413–assisted RAB-5 to RAB-10 cascade contributes to the integrity of C. elegans intestinal epithelia.
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Vesicular Transport Proteins
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Here we describe two assays to measure dense core vesicle (DCV) exocytosis-mediated cargo secretion in neuroendocrine cells. To conduct siRNA screens for novel genes in regulated DCV exocytosis, we developed a plate reader-based secretion assay using DCV cargo, NPY-Venus, and an orthogonal
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Rab GTPases are master regulators of intracellular trafficking. They are involved in every aspect of membrane transport from vesicle budding to vesicle fusion. Their functions are regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). The mammalian genome encodes over 60 different Rab proteins. Each of these Rabs localizes to specific compartments. The actions of multiple Rabs are coordinated through Rab cascades where a single Rab recruits the machinery to activate or inactivate a secondary Rab. Considering the large Rab family size, very little is known about the majority of its members and how they work together. As opposed to the human system, the C. elegans RAB family encodes 28 members with normally just one isoform per Rab member. The smaller Rab family size, together with the genetic tractability of C. elegans allowed for a broad-scale analysis of each RAB protein. For this analysis it was decided to initially determine the expression pattern and sub-cellular localization of all C. elegans RABs. Expression pattern analyses revealed that C. elegans rabs are differentially expressed with a pattern of preference for the nervous system. Additionally, the sub-cellular localization analysis of the Rabs showed that they localize to specific sub-cellular compartments with many displaying partial staining to the Golgi apparatus. The availability of mutants for 90% of the rabs in C. elegans further provided the opportunity to elucidate novel RAB functions. Since most RABs were neuronally expressed, all rab mutants were tested for changes in a set of nervous system mediated behaviors: movement, defecation motor program, and egg laying. Analysis revealed that several RABs are important regulators of these behaviors. Additionally all rab mutants were also tested for defects in synaptic transmission through sensitivity to aldicarb. Several new RABs were identified to modulate aldicarb sensitivity. Mutant analysis showed that although several rab mutants displayed phenotypes of nervous system dysfunction, the majority of animals were healthy. This suggested that many RABs function redundantly together in C. elegans. To elucidate which RABs cooperate together, a synthetic RNAi screen was conducted where each rab mutant was co-depleted with the remaining 27 Rabs. Co-depletion of multiple rabs has provided novel insights into the higher order RAB network. In the second chapter of this study, we aimed to identify novel RABs involved in dense core vesicle (DCV) signaling. Interestingly rab-5 and rab-10 mutants showed defects in DCV secretion. Additionally, two TBC (Tre-2/Cdc16/Bub2) domain-containing molecules, TBC-2 and TBC-4, were identified to function as potential GAPs for RAB-5 and RAB-10 respectively. Lastly we have identified an interaction between a RAB-5 effector, Rabaptin-5 (RABN-5), and TBC-4, which provides a link between RAB-5 and RAB-10 function. Taken together, these results suggest the existence of a novel Rab exclusion cascade in the regulation of DCV release, where active RAB-5 recruits TBC-4 for the local inactivation of RAB-10.
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Vesicular Transport Proteins
Small GTPase
Transport protein
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