SUMMARY The formation of epithelial tissues requires both the generation of apical-basal polarity and the co-ordination of this polarity between neighboring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to formation of a singular apical membrane, resulting in contribution of each cell to only a single lumen. Here, from a functional screen for genes required for 3D epithelial architecture we identify key roles for Synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PI(4,5)P2-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE Syntaxin-3. Together, Slp2-a/4-a co-ordinate the spatiotemporal organization of vectorial apical transport to ensure only a single apical surface, and thus formation of a single lumen, occurs per cell.

No abstract

Insulin secretion is essential for maintenance of glucose homeostasis, but the mechanism of insulin granule exocytosis, the final step of insulin secretion, is largely unknown. Here, we investigated the role of Rim2alpha in insulin granule exocytosis, including the docking, priming, and fusion steps. We found that interaction of Rim2alpha and Rab3A is required for docking, which is considered a brake on fusion events, and that docking is necessary for K(+)-induced exocytosis, but not for glucose-induced exocytosis. Furthermore, we found that dissociation of the Rim2alpha/Munc13-1 complex by glucose stimulation activates Syntaxin1 by Munc13-1, indicating that Rim2alpha primes insulin granules for fusion. Thus, Rim2alpha determines docking and priming states in insulin granule exocytosis depending on its interacting partner, Rab3A or Munc13-1, respectively. Because Rim2alpha(-/-) mice exhibit impaired secretion of various hormones stored as dense-core granules, including glucose-dependent insulinotropic polypeptide, growth hormone, and epinephrine, Rim2alpha plays a critical role in exocytosis of these dense-core granules.

The -conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The -conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The ␤ and ␣ 2 ␦-1 auxiliary subunits influence the expression and characteristics of the ␣ 1B subunit of N-type channels and are differentially regulated in disease states, including pain. In this study, we examined the influence of these auxiliary subunits on the ability of the -conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the ␤3 subunit had little influence on the on-and off-rates of -conotoxins, coexpression of ␣ 2 ␦ with ␣ 1B significantly reduced on-rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The The N-type (Ca v 2.2) voltage-gated calcium channels play an important role in the control of neurotransmitter release from nerve terminals (1, 2) and are important drug targets for the treatment of pain (3, 4) and ischemic brain injury (5). Native N-type Ca 2ϩ channels are hetero-oligomers that comprise a pore-forming ␣ 1 subunit (␣ 1B ) and at least two auxiliary subunits, ␤ and ␣ 2 ␦, which modulate the ␣ 1 subunit function (6, 7). Two splice variants of the ␣ 1B subunit have been identified that occur predominantly in the central (␣ 1B-d ) and peripheral (␣ 1B-b ) nervous systems (8, 9). Additional splice variants that lack large parts of the domain II-III linker region, including the synaptic protein interaction site, have also been isolated from human brain cDNA libraries (10). Multiple isoforms of the ␤ and ␣ 2 ␦ subunits also exist that can interact with the ␣ 1 subunit to produce N-type Ca 2ϩ channels with different gating properties, allowing the fine tuning of synaptic transmissions (11).A distinguishing feature of N-type channels is their high sensitivity to block by -conotoxins, which are relatively small (ϳ25 residue) polypeptides isolated from the venom of the marine snail of the genus Conus (12, 13). -Conotoxins have been used as research tools to help define the distribution and physiological roles of specific N-type calcium channels (14 -17) and have potential therapeutic value as intrathecal treatments for pain. MVIIA from Conus magus is being tested in clinical trials as a treatment for neuropathic pain, but dose-limiting side effects are a concern (18 -20). -Conotoxin CVID from Conus catus (21), which appears to have a wider therapeutic window (22), is also in clinical trials (23).The modulatory effects of auxiliary subunits on the biophysical properties of Ca 2ϩ channel ␣ 1 subunits have been well characterized. When expressed alone, ␣ 1 subunits produce functional channels with kinetic properties that differ substantially from those of the native channel (24, 25). The most commonly observed effect of the ␤ and ␣ 2 ␦ subunits when they are co-expressed with the ␣ 1 subunit in a heterologous expression system such as Xenopus oocytes or HE...

No abstract