( Iezzi M Kouri G Fukuda M ...)Your Data: Use of pSilencer 1.0-U6 siRNA Expression Vector in the Analysis of Synaptotagmin Location and Function

Iezzi M, Kouri G, Fukuda M, Wollheim CB (2004) Synaptotagmin V and IX isoforms control Ca²⁺-dependent insulin exocytosis. J Cell Sci 117:3119-3127.

In a recently published paper in the Journal of Cell Science, Iezzi and colleagues used Ambions siRNA target finder and the pSilencer 1.0-U6 vector in an extensive study to investigate synaptotagmin location and function in rat pancreatic islets and in the clonal β cell line INS-1E. Synaptotagmins (Syts) are a family of proteins involved in Ca²⁺-triggered exocytosis (Chapman, 2002; Sdhof, 2002) of which vertebrates express at least 15 isoforms (Sdhof & Rizo, 1996; Rickman et al., 2004). Insulin exocytosis from pancreatic β cells is a Ca²⁺-dependent secretory process. However, previous studies have shown conflicting results as to which Syt isoform is present on the secretory granules in the native β cell. Iezzi and colleagues investigated Syt V and Syt IX as Syt V has been suggested to be one of the Ca²⁺ sensors for insulin exocytosis in the β cell (Gut et al., 2001). The involvement of Syt IX in insulin secretion was unclear at the time.

Western blotting and RT-PCR analysis revealed the presence of both Syt V and Syt IX in rat pancreatic islets and in INS-1E cells. The expression of Syt V was similar in the islets and in the cell lines, while Syt IX was expressed less in the rat pancreatic islets. Confocal microscopy showed that SytV and Syt IX co-localize with insulin-containing secretory granules which was confirmed by using a continuous sucrose density gradient. Interestingly, studies performed in primary pancreatic endocrine cells revealed that Syt V is present in glucagon-containing islet alpha-cells but is not expressed in β-cells whereas Syt IX is associated with insulin granules in β-cells.

To investigate the function of Syt V and Syt IX, the Syt isoforms were over-expressed or their levels reduced using RNA interference. Both proteins were produced at similar levels but the anti-Syt IX antibody recognised a smaller and a larger form of Syt IX. The two forms of Syt IX may be due to post-translational modification such as O-glycosylation and palmitoylation. Transient over-expression of Syt V and Syt IX did not alter exocytosis in INS-1E cells indicating that both proteins do not interfere with the stoichiometry of SNARE proteins whose over-expression has been shown to inhibit insulin exocytosis (Nagamatsu et al., 1999).

To investigate the hormone release, the pSilencer SytV and Syt IX siRNA expression plasmids were each transiently cotransfected into INS-1E cells with a plasmid encoding the human growth hormone hGH. hGH is targeted to insulin-containing granules and can be used to monitor exocytosis as an insulin substitute in a subpopulation of transfected cells (Iezzi et al., 2000). The pSilencer expression plasmids included GFP for identification of transfected cells. Silencing of SytV and Syt IX levels did not change basal secretion. However, glucose-induced insulin release was decreased suggesting that inhibition may be by impairment of the exocytotic process and that Syt V and Syt IX act as positive modulators of exocytosis. The addition of the phorbol ester PMA, which potentiates insulin exocytosis distal to the increase of cytosolic Ca²⁺ showed that both proteins are directly involved in the Ca²⁺-dependent stimulation of exocytosis. The association of Syt IX with insulin granules in β-cells suggests that Syt IX may be the Ca²⁺ sensor for insulin secretion in β-cells.

References

Chapman ER (2002) Synaptotagmin a Ca 2+ sensor that triggers exocytosis? Nat Rev Mol Cell Biol 3:498-508.

Gut A, Kiraly CE, Fukuda M, Mikoshiba K, Wollheim CB, Lang J (2001) Expression and localisation of synaptotagmin isoforms in endocrine beta-cells: their function in insulin exocytosis. J Cell Sci 114:1709-1716.

Iezzi M, Regazzi R, Wollheim, CB (2000) The Rab3-interacting molecule RIM is expressed in pancreatic beta-cells and is implicated in insulin exocytosis. FEBS Lett 26:66-70.

Nagamatsu S, Nakamichi Y, Yamamura C, Matsushima S, Watanabe T, Ozawa S, Furukawa H, Ishida H (1999) Decreased expression of t-SNARE, syntaxin 1, and SNAP-25 in pancreatic β -cells is involved in impaired insulin secretion from diabetic GK rat islets: restoration of decreased t- SNARE proteins improves impaired insulin secretion. Diabetes 48:2367-2373.

Rickman C, Craxton M, Osborne S, Davletov B (2004) Comparative analysis of tandem C2 domains from the mammalian synaptotagmin family. Biochem J (In press).

Sdhof TC (2002) Synaptotagmins: why so many? J Biol Chem 277:7629-7632.

Sdhof TC and Rizo J (1996) Synaptotagmins: C2-domain proteins that regulate membrane traffic. Neuron 17:379-388.

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Cat# Product Name Size 7208 pSilencer 1.0-U6 (linear) 20 rxns
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