© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi:10.1111/tra.12182 Distinct Actions of Rab3 and Rab27 GTPases on Late Stages of Exocytosis of Insulin Victor A. Cazares1,2, Arasakumar Subramani1, Johnny J. Saldate1,2, Widmann Hoerauf1,2 and Edward L. Stuenkel1,2,∗ 1Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA 2Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI 48109, USA ∗Corresponding author: Edward L. Stuenkel, [email protected] Abstract Rab GTPases associated with insulin-containing secretory granules (SGs) readily releasable pool (RRP). By comparison, nucleotide cycling of Rab3 are key in targeting, docking and assembly of molecular complexes GTPases, but not of Rab27A, is essential for a kinetically rapid filling of governing pancreatic β-cell exocytosis. Four Rab3 isoforms along with the RRP with SGs. Aside from these distinct functions, Rab3 and Rab27A Rab27A are associated with insulin granules, yet elucidation of the GTPases demonstrate considerable functional overlap in building the distinct roles of these Rab families on exocytosis remains unclear. To readily releasable granule pool. Hence, while Rab3 and Rab27A coop- define specific actions of these Rab families we employ Rab3GAP erate to generate release-ready SGs in β-cells, they also direct unique and/or EPI64A GTPase-activating protein overexpression in β-cells from kinetic and functional properties of the exocytotic pathway. wild-type or Ashen mice to selectively transit the entire Rab3 fam- Keywords exocytosis, GTPase, insulin secretion, membrane fusion, Rab ily or Rab27A to a GDP-bound state. Ashen mice carry a sponta- proteins neous mutation that eliminates Rab27A expression. Using membrane capacitance measurements we find that GTP/GDP nucleotide cycling of Received 16 April 2014, revised and accepted for publication 4 June Rab27A is essential for generation of the functionally defined imme- 2014, uncorrected manuscript published online 6 June 2014, published diately releasable pool (IRP) and central to regulating the size of the online 26 June 2014 Insulin secretion from endocrine pancreatic β-cells is are less well understood (3–6). In addition, incretins essentialinregulatingbloodglucoselevels.Keytothis such as glucagon-like peptide-1 and glucose-dependent secretion is highly regulated membrane trafficking and insulinotropic polypeptide strongly potentiate insulin exocytotic pathways of insulin-containing secretory gran- secretion via cAMP signaling. Central to exocytosis of ules (SGs). Defects in the process of insulin production, insulin SGs is the assembly/disassembly of molecular trafficking and secretion can lead to profound metabolic complexes that direct SG membrane targeting, tethering disorders and diabetes mellitus (1). Physiologically, insulin and biochemical competency for undergoing membrane secretion is typically triggered from β-cells in response fusion (7–9). While evolutionarily conserved SNARE pro- to elevated blood glucose concentrations, which raises teins and regulators of SNARE complex assembly define the intracellular ATP/ADP concentration ratio and drives theprimingandfusionprocess,RabGTPasesarebelieved closure of KATP channels leading to a depolarization to be critical determinants of organelle identity, vesicle of the plasma membrane. Depolarization of the β-cell targeting, membrane tethering and SNARE complex 2+ membrane then triggers activation of voltage-gated Ca formation (10,11). channels, wherein Ca2+ influx results in an induction of exocytosis of the insulin SGs (2). Prolonged glucose Rab3A was the first Rab GTPase identified on activation of β-cells induces a second sustained phase of insulin-containing SGs (12). Genetic studies using Rab3A exocytotic insulin secretion, the mechanisms of which knockout mice (13,14) as well as investigations examining www.traffic.dk 997 Cazares et al. the effects of overexpression of Rab3A mutants (15–19) characteristic rates of GTP/GDP cycling and granule demonstrate that Rab3A is required for normal levels of residency. Notably, fluorescence imaging of Rab3A and insulin secretion and control of plasma glucose levels. Rab27A on SGs has demonstrated that they exhibit kinetic More recently, Rab27A, a close evolutionary relative of differences in granule membrane association (26,32). For Rab3, has also been found to be associated with these SGs example, activation of secretion is often accompanied by (20–22). Similarly, functional studies of Rab27A-deficient rapiddissociationofRab3Afromtheexocyticvesicles, mice (13,23,24) and overexpression of Rab27A mutants while Rab27A appears to be largely maintained on SGs (21,25) have determined that Rab27A exerts regulatory throughout fusion and early stages of endocytosis (33,34). activity over the exocytotic release of insulin. Although These results are consistent with studies showing that recent studies have confirmed that Rab3 and Rab27 are association between Rab3 and the SG membrane is rig- commonly colocalized on SGs and neuronal synaptic orously correlated with Rab3’s GTP-bound state, while vesicles (26), the precise mechanisms by which they reg- Rab27appearstobeabletoassociatewithgranulesinboth ulatelatestagesofexocytosisandthedegreetowhich GDP- and GTP-bound states (35,36). they exert functional overlap during vesicle priming and exocytosis remain poorly defined. Assigning specific roles Multiple effectors have been reported for the Rab3 and to these Rabs has been complicated by their high structural Rab27 GTPases, which fall largely within four groups homology and the promiscuity of effectors between the including, Rab-interacting molecules (RIMs and RIM-like Rab3/Rab27 families. Moreover, knockout mice where proteins); synaptotagmin-like proteins (Slp and rabphilin); three Rab3 isoforms are deleted do not survive when Slp-like proteins lacking C2 homologous regions (Slac and Rab3A is one of the deleted isoforms (14), further compli- Noc2); and a vesicle priming protein Munc13-4 (37,38). cating studies assigning specific actions of Rab3 proteins For these effector families, RIM proteins interact with all versus Rab27 within the exocytotic pathway. isoforms of Rab3 but apparently not Rab27. Conversely, Slp1, Slp2-a, Slp3-a, Slp5 and Slac2-a–c interact specifi- The subfamily of Rab3 consists of four isoforms cally with Rab27A/B but not Rab3. By comparison, Slp4A, (Rab3A–D), with each being present in pancreatic β-cells rabphilin and no C2 domain protein (Noc2) interact with and in part associated with SGs (27). By comparison, Rab27A/B, Rab3s and Rab8 in vitro;however,binding Rab27 consists of two isoforms (Rab27A,B), but only affinities suggest preference for interaction with Rab27s in Rab27A is expressed and targeted to insulin-containing vivo (38,39). Finally, Munc13-4, a member of the Munc13 SGs (24). The activity of Rab3 and Rab27 family pro- family that participates in exocytotic priming, has been teins, like other GTPases, is determined by their cycling reported to interact specifically with Rab27 (40). Impor- between a GDP-bound mostly inactive and cytosolic form tantly, Rab effectors may show cycling on/off membranes and a GTP-bound active and membrane-bound form. that differ kinetically from the Rabs themselves, suggest- Transitions between GTP/GDP states are mediated by ing that effector interactions with Rab GTPases may exhibit guanine nucleotide exchange factors (GEFs) that stimulate temporal and spatial differences and support distinct roles the binding of GTP, and by GTPase-activating proteins in the exocytic pathway (41). Although Rab3 and Rab27 are (GAPs) that accelerate the intrinsic rate of Rab-GTP important regulators of secretory activity their functional hydrolysis and return the Rab protein to an inactive state specificity remains poorly understood, in part as a result of (28). While greater than 60 Rab proteins have been iden- effector overlap. tified in mammalian cells, comparatively fewer regulatory Rab GEFs and Rab GAPs have been identified. Indeed, In this study, we identify distinct roles of Rab3 and Rab27 GTP binding of both Rab3 and Rab27 families is promoted family proteins in the final stages of the exocytotic path- by a single Rab GEF, termed Rab3GEP (29), suggesting a way for insulin secretion from pancreatic β-cells. The potential for commonality in their activation. Yet, return investigations are novel in employing selective overexpres- toaGDP-boundstatefortheseRabsisdrivenbydis- sion of the catalytic subunit of Rab3GAP and/or EPI64A tinct GAPs (Rab3GAP and EPI64, respectively) (30,31). to specifically activate GTP hydrolysis and respectively Differences in regulated GAP inactivation likely lead to deactivate Rab3 and/or Rab27 family proteins. Whole-cell 998 Traffic 2014; 15: 997–1015 Actions of Rab3 and Rab27 on Insulin Secretion capacitance measurements were used to evoke and mea- junctions of β-cells found in intact islet tissue; (ii) facili- sure exocytotic activity and to assign specific actions of the tate the maintenance of glucose responsiveness and elec- Rab families on functionally distinct vesicle pools of β-cells trical and secretory characteristics through organotypic in pancreatic islet slices. Furthermore, we employed total tissue culture, while allowing efficient viral infection; and internal reflection fluorescence (TIRF) microscopy of flu- (iii) promote a tissue platform allowing stable long-term orescently tagged insulin-containing SGs to examine GAP recording of membrane capacitance measurements