1. Insulin Secretory Pathway 1.In response to elevated Ca 2+ levels, insulin granules fuse with plasma membrane in a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) dependent process 2.Rapid exocytosis occurs within first 5-10 mins = FIRST PHASE 3.Sustained exocytosis upto several hours = SECOND PHASE 4.Storage limited model: it is proposed that biphasic insulin secretion occurs by the release of the geographically or functionally distinct pools of insulin containing granules

2. Insulin Granules Readily releasable pool (RRP) - first phase of insulin secretion -pre-docked at the plasma membrane -small pool (50-200) of cell’s had greater than 10,000 mature insulin granules, estimated by EM -glucose, KCl, and non-nutrient secretagogues evoke first phase release -~ 1% of the total number of granules present release insulin Storage granule pool -second phase of insulin secretion -mobilized to the cell periphery to replenish RRP -replenishment of RRP involves granule mobilization, docking, and priming -5-40 granules per cell per minute -fuel secretagogues such as glucose can only yield sustained second release

3. Mobilization of Insulin Granules-Role of F-Actin 1. Perfused rat islets treated with Latrunculin (specific F-actin disrupting agent) increased insulin secretion in both phases 2.In response to the glucose F-actin remodeling was observed 3.Current model: only nutrient and glucose-induces F-actin reorganization and initiates RRP replenishment during the first phase of insulin release. Non- nutrients cannot elicit effects on F-actin remodeling

4. Mobilization of Insulin Granules-Role of Microtubule 1.Glucose induces microtubule formation in islets 2.Microtuble polymerization and depolymerization is important for insulin-granule mobilization 3. Microtubles transfers granules from the cell interior to the cortical actin near the cell periphery, where they dock with SNARE proteins and release insulin-SECOND PHASE

5. Role of Small GTPases in Insulin Secretion 1.Cdc42 is involved in targeting insulin-containing granules to the SNARE protein syntaxin 1A 2.Cdc42 binds to VAMP2 and forms Cdc42-VAMP2-Syntaxin1A heterodimer- for normal insulin secretion

6. Proteins Phase implicated Reference(s) SNARE proteins t-SNAREs Syntaxin 1 First (Ohara-Imaizumi et al., 2007) Syntaxin 2 N.D. (Jacobsson et al., 1994; Kang et al., 2002) Syntaxin 3 N.D. (Kang et al., 2002) Syntaxin 4 First and second (Spurlin and Thurmond, 2006) SNAP-23 N.D. (Sadoul et al., 1997) SNAP-25 N.D. (Sadoul et al., 1995) v-SNAREs VAMP2 N.D. (Jacobsson et al., 1994; Regazzi et al., 1995) VAMP3 N.D. (Jacobsson et al., 1994; Regazzi et al., 1995) Accessory factors Syntaxin-1 accessory factors Munc18a N.D. (Tomas et al., 2008; Zhang et al., 2000) Munc13 First and second (Kwan et al., 2007) Tomosyn N.D. (Cheviet et al., 2006; Zhang et al., 2006) Granuphilin First and second (Gomi et al., 2005) Munc18b N.D. (Zhang et al., 2000) Doc2β N.D. (Verhage et al., 1997) Syntaxin-4 accessory factors Munc18c Second phase (Oh and Thurmond, 2009; Wheeler et al., 1996) WNK1 N.D. (Oh et al., 2007) Doc2β N.D. (Ke et al., 2007) Synip N.D. (Saito et al., 2003) N.D., not determined SNARE proteins and SNARE accessory factors implicated in biphasic insulin secretion

7. Role of SNARE Proteins in Vesicle Fusion 1.SNARE proteins comprise a superfamily of small membrane proteins distinguished by the SNARE motif, a conserved coiled-coil stretch of 60–70 amino acids. 2. SNARE motifs spontaneously assemble into elongated four-helix bundles

8. The Syntaxin Family 1.Syntaxin 1A -/- mouse showed impaired first- phase insulin secretion and normal second phase 2.Syntaxin4 +/- mouse showed impaired first and second phase insulin secretion 3.Syntaxin4 but not syntaxin1A was shown to associate with the F-actin

9. Syntaxin Accessory Factors 1.Munc18c negatively regulates sustained phase of insulin secretion by ablating the glucose-stimulated VAMP2- syntaxin4 association 2.Munc18a inhibits SNARE complex assembly by sequestering syntaxin1A in a closed confirmation 3.Tomosyn1 inhibits insulin secretion by binding to both syntaxin1A and SNAP-25, and it prevents association of V- AMP to syntaxin1A

10. Tomosyn 1.Discovered as syntaxin1A-binding protein( tomo=friend; syn=syntaxin) 2.Tomosyn is a cytosolic protein, but has been found to be localized in synaptic vesicles and insulin granules 3.Two mammalian tomosyn genes have been identified; tomosyn1 and tomosyn2 4.Tomosyn 1 has 3 spliced variants: m-, b-, and s 5.m- and s-tomosyn1 are expressed in brain, whereas b is expressed is ubiquitously expressed 6.Tomosyn 2 has four spliced variants: b, xb, b, and s 7.Not much is know about Tomosyn-2, except that s-, and m- tomosyn-2 is expressed in brain and b-tomosyn is ubiquitously expressed

11. Physiological Role of Tomosyn1 1.Tomosyn1 was shown to form a four-protein complex consisting of syntaxin, SNAP-25 and synaptotagmin by displacing Munc18 from syntaxin 2.Tomosyn was shown to inhibit the priming step in both neuroendrocrine cells and neurons

12. Domain Structure of Tomosyn and its Homolog's N--C 1.The R or V-SNARE coiled-coil domain of tomosyn1 shows high homology to V-AMP2 coiled-coil domain. The V-SNARE domain of tomosyn1 binds SNAP-25 and syntaxin1A to inhibit vesicle fusion 2.Tomosyn1 mutant that lacks SNARE domain inhibits exocytosis to a lesser extent-suggesting that SNARE motif is not the sole mediator of inhibitory activity of tomosyn1 3.The N-terminal WD40 motifs and a HVR are also important for tomosyn1 mediated inhibition 4.In chromaffin cells, it is proposed that N-terminal domain of tomosyn1 can interact with syntaxin-myosin to inhibit exocytosis 5.Tomosyn1 is phosphorylated by PKA at Ser724, which reduces the interaction of tomosyn with syntaxin 2.

13. Model of Inhibitory Action of Tomosyn

14. Based on congenic datatomosyn-2 was identified to be a potential candidate gene, which may be responsible for the fasting glucose phenotype Goal: To investigate the mechanism by which tomosyn-2 affects insulin secretion from beta cells

15. Amino Acid Alignment of b-tomosyn1 and xb-tomosyn2

16. Amino Acid Similarity and Identity of Tomosyn1 and Tomosyn2

17. Overexpressing b-Tomosyn-2 Isoform in INSIE Cells Inhibits Insulin Secretion

18. Hypothesis: b-Tomosyn-2 inhibits GSIS by inhibiting the formation of SNARE complex, thereby interfering with the fusion of insulin secretory vesicles to the plasma membrane

19. Specific Aims 1. Interaction and localization of b-tomosyn-2 with the SNARE complex 2. Structural components of b–tomosyn-2 isoform that inhibits formation of SNARE complex 3. Perform the screen to identify novel interactions of b-tomosyn-2

20. Future Goals: 1.Write American heart fellowship 2.Repeat insulin secretion experiments looking at the effects of tomosyn-2 isoforms in MIN6 and INS1 cells 3.Validate the protein expression of b-tomosyn-2 in MIN6 and INS1 cells 4.Make tagged expression vector for tomosyn-2 isoforms 5.Make adenovirus of tomosyn-2 isoforms 6.Make BTBR version of tomosyn-2 7.Perform pull-down experiments in MIN6 cells using tagged b- tomosyn-2, syntaxin1, syntaxin4, and vAMP2 8.In vitro vesicle fusion experiments

21. Specific Aim 2: Structural components of b-tomosyn-2 isoform that inhibits formation of SNARE complex

22. Specific Aim 3: Perform the screen to identify novel interactions of b-tomosyn-2 Experiment: 1. Perform yeast-two hybrid screen to identify the novel b-tomosyn- 2 isoform