SEIX 17-20 octobre 2005 - Atelier Calcium

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Figure 1 : Set-up used for ratiometric imaging of intracellular Ca 2+ using fluorescent dye Fura-2. SILENCING ENDOGENOUS CALCIUM SIGNALLING COMPONENTS USING RNA INTERFERENCE We have generated a number of S2 cell lines expressing metabotropic and ionotropic receptors (for review see [2]). Figure 2: Calcium imaging in Drosophila S2 cells A: S2-DM1 cells (an S2 cell line stably expressing a Drosophila muscarinic acetylcholine receptor (DM1)) in normal saline stained with fura-2 AM. B: The same cells following a 20 s application of 100 M carbamylcholine, a muscarinic receptor agonist. The Drosophila S2 cell line stably expressing a cloned muscarinic acetylcholine receptor (mAChR, DM1) [Fig 2] has been used to explore the utility of gene silencing by double- 92
stranded RNA interference (RNAi) in the study of mAChR activated calcium signalling [4]. Knockdown by RNAi of either the inositol 1,4,5-trisphosphate receptor and SERCA resulted in a reduction, in all cells tested, of carbamylcholine-evoked calcium transients to 10% or less of control values. This knockdown was achieved with soaking in dsRNA without the need for transfection agents, and only 10 microgrammes of dsRNA per million cells was needed. Although RNAi knockdown of the DM1 receptor was much less effective in reducing carbamylcholine responses, we attribute this to the high levels of DM1 expression. [Fig. 3] Figure 3: Application of carbamylcholine (CCh, 100 M) induces an increase of intracellular Ca 2+ concentration [Ca 2+ ] i (A). Intracellular Ca 2+ response from cells loaded with Fura-2/AM are presented in the panels. Timing of drug application is indicated by a horizontal bar. Application of CCh elicits an increase of [Ca 2+ ]i in S2-DM1 cells (A) but also in S2-DM1 cells treated with DM1 dsRNA (B). However, no response to CCh is observed when the cells are treated with either Ins(1,4,5)P3R (C) or SERCA (D) dsRNA. COMBINING REVERSE GENETICS WITH CALCIUM MEASUREMENTS TO IDENTIFY NOVEL CALCIUM SIGNALLING COMPONENTS in DROSOPHILA S2 CELLS The Drosophila genome contains approximately 14,000 genes and, of these, many remain to have a function assigned to them. Thus, it is likely that there will be many hitherto unknown genes that contribute to functions such as calcium signalling. Genome-wide RNAi screens have been fruitful in finding novel genes involved in the growth and viability of Drosophila cells [Boutros et al 2004][6]. Our own studies (Raymond et al 2004) [5] and those of Roos et al (2005) [7] have demonstrated the potential of using RNAi in conjunction with calcium 93
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SEIX 17-20 octobre 2005 - Atelier Calcium

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