SEIX 17-20 octobre 2005 - Atelier Calcium

CALCIUM SIGNALLING IN DROSOPHILA CELL LINES AND PRIMARY
CULTURES: COMBINING PHYSIOLOGY, RNA INTERFERENCE AND
MICROARRAY STUDIES
David B Sattelle, Steven D Buckingham and Valérie Raymond-Delpech
MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of
Oxford, South Parks Road, Oxford, OX1 3QX, UK
SUMMARY
With the completion of the Drosophila genome project (www.flybase.org), the ready
application of forward and reverse genetics, as well as calcium imaging and electrophysiology,
this model organism offers exciting opportunities for the investigation of calcium signalling
pathways. In particular the utility of an ease to culture Drosophila cell line (Schneider S2 cells)
will be demonstrated. The cells can be conveniently deployed to study endogenous calcium
signalling pathways and lend themselves to transfection with additional receptor /channel
molecules. The application of RNA interference (RNAi) to Drosophila S2 cells and also to
Drosophila neuronal primary cultures has facilitated the identification of novel calcium
signalling components.
INTRODUCTION
Access to the genome of the fruitfly Drosophila melanogaster provides exciting opportunities
for rapid advances in the functional analysis of gene products [1]. Such developments are
further facilitated by the ease of expression of novel proteins in Drosophila cell lines, such as
the S2 cells [2]. Also, the recent advances in gene silencing by double-stranded RNA
interference (RNAi) permit rapid and direct investigation of protein function in cell lines [3].
For example, a Drosophila S2 line expressing a musarinic acetylcholine receptor [4] has
proved a particularly useful vehicle for studying components in the IP3 signalling pathway to
which the receptor is coupled [5]. Primary cultures of Drosophila neurons also offer a
suitable substrate for combined calcium imaging and RNAi experiments. Here we illustrate,
using our own studies, how this approach can be used to identify the nicotinic acetylcholine
receptor (nAChR) subunits that play a key role in the responses to nicotine of primary
cultured larval Drosophila cholinergic neurons.
FURA-2 BASED CALCIUM MEASUREMENTS IN DROSOPHILA S2 CELLS
S2 cells are readily loaded with 2-4 M Fura-2/AM and 0.002% pluronic acid F-127
dispersing agent in saline for 45 min at room temperature. The coverslip on which the cells
are dispersed forms the base of the experimental chamber which is mounted on the stage of an
inverted microscope equipped with a 40x, 1.3N.A. oil immersion objective. Using a filter
wheel, cells are illuminated at 340 and 380nm. Emitted light is passed through a dichroic
mirror set to 510nm and passed first to an image intensifier, then to a CCD camera [Fig 1]. In
our laboratory the image acquisition and subsequent processing is performed using a Concord
imaging system from Perkin-Elmer (UK). Imaging experiments are performed at room
temperature; a pump and suction pipette maintain a constant volume of saline in the
experimental chamber.
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