Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Summary

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

Abstract

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Introduction

Drosophila S2 cells have gained increasing popularity for studying many cellular processes. These cells were originally obtained from trypsinized late stage embryos of OregonR Drosophila melanogaster and maintain macrophage-like features¹. Drosophila S2 cells offer many advantages over other cell lines. They are cultured at 25 °C without CO₂, and can be imaged for many hours at room temperature without the need of heating or gas exchange. More importantly, Drosophila S2 cells are very sensitive to RNAi treatment allowing high efficiency knockdown of proteins of interest. S2 cells are highly transf....

Protocol

1. Drosophila S2 cells

1. Preparation of ConA coated coverslips
   1. Place coverslips in a ceramic rack and wash them by chromic acid immersion for 1 hr. [CAUTION: chromic acid is corrosive and can cause irritation of eyes, nose, throat, and skin].
   2. Rinse coverslips thoroughly with constantly running dH₂O for 30 min until the acid is completely washed out.
   3. Let coverslips air dry and coat them with ConA (0.5 mg/ml solution in dH₂O) for 30 min.
   4. Rinse coverslips with dH₂O for 15 min and let them air dry. ConA coated coverslips can be stored up to 1 month.<....

Results

Drosophila S2 cells attached on the ConA-coated coverslip spread into a flat round “pancake” shape (Figures 1A and 1C). However, when S2 cells plated on coverslips in the presence of CytoD and allowed to spread for 2-3 hr, they form long thin processes filled by parallel microtubules (Figures 1B and 1D). These microtubules have uniform polarity with plus-ends out⁷. Time-lapse fluorescence imaging of Drosophila S2 stably expre.......

Discussion

Here we present detailed protocols for studying microtubule-dependent cargo transport in Drosophila S2 cells and primary neuron culture. Both S2 processes and neurites are structures filled by bundled microtubules arrays that serve as tracks for organelle transport.

Two strategies are typically used to label organelles: transfection of vectors encoding a fluorescent protein with an organelle-targeting sequence or staining with fluorescent dyes added directly to cultures. Common exampl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Powder Insect cell medium (Schneider’s)	Sigma	S9895
Insect –Xpress medium	Fisher	BW12730Q
Insulin	Sigma	I6634
Penicillin	Research Products International	P93000
Streptomycin	Research Products International	S62000
Tetracycline hydrochloride	Sigma	T7660-5G
Fetal bovine serum	Atlanta Biologicals	S11150
Concanavalin A	Sigma	C2010
Cytochalasin D	VWR	IC15077105
LysoTracker Red DND-99	Molecular Probes	L7528
100 µm Cell strainer	Fisher Scientific	22363549
25 mm Circle cover glass	VWR	48380 080
Drosophila Vials	VWR	89092-730
Disposable pellet pestles with matching microtubes	Kimble Chase	749520-0000