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 transfectable and stable cell lines can be selected in less than four weeks to allow stable ectopic expression of proteins of interest. S2 cells normally grow either loosely attached but not spread on a flask or in suspension. They can be induced to spread on coverslips precoated with a plant lectin concanavalin A (ConA). The periphery of the spread cells is especially thin and therefore is ideal for live cell microscopy. Detailed protocols for S2 cells culture, RNAi treatment, live light imaging and immunostaining can be found in the literature^2,3. Our lab has adopted S2 cells as a model system to study microtubule-based cargo transport. Drosophila S2 cells treated with any drug that depolymerizes or severs F-actin, such as cytochalasin D (CytoD), grow long processes filled with uniformly polarized microtubules^4-10, which makes it an ideal system to study cargo movement along microtubule arrays. Different parameters of transport in these processes (i.e. trajectories lengths, velocities, directionality etc.) can be easily measured using automated particle-tracking software, DiaTrack (Semasopht: http://www.microscopy.info/Organization/Details/4423; Dr. Pascal Vallotton: http://www.csiro.au/en/Organisation-Structure/Divisions/Mathematics-Informatics-and-Statistics/PascalVallotton.aspx#a4). These properties make S2 cells an appealing system for studying cargo transport along microtubules in tissue culture cells.

Pilot experiments in S2 cells can be extended to a physiologically important model of cargo transport studies in Drosophila primary neurons. Similar to S2 cells, neurons cultured from dissociated embryos are permeable to small molecules such as dyes and inhibitors, and high-resolution live imaging of organelle transport can be performed in neurons at room temperature. Using Drosophila with different genetic backgrounds, we can examine gene function in primary neurons by knockout (genetic loss-of-function mutations), knockdown (dsRNA injection or expression) or ectopic expression (transgenic flies). Specifically, fragmentation of actin filaments using CytoD treatment does not prevent neurite outgrowth; instead they grow faster, and thus we can study microtubule-dependent organelle transport in CytoD-treated neurons without the influence of actin filaments¹¹. Therefore, primary neuronal cultures combine the advantages of tissue culture cells and fly genetics, which makes it a great system to study cargo transport in a physiological relevant system^10,12. Since several familial neurodegenerative diseases could be caused by or associated with cargo transport defects (e.g. Parkinson's disease¹³, Huntington's disease¹⁴, Alzheimer's Disease^15,16, Amyotrophic Lateral Sclerosis/ALS¹⁷, HMN7B and Perry syndrome^18,19, and Spinocerebellar ataxia type 5/SCA5²⁰), primary neuronal cultures can be useful in analysis of the effects of specific mutations causing these diseases on microtubule-dependent transport.

Finally, important properties of microtubule-dependent transport are well conserved between mammals and flies, but Drosophila genetic and cell culture is less expensive and time-consuming, justifying the use of cultured Drosophila cells for studying essential aspects of organelle transport in normal and pathological conditions.

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.
2. Plating the cells
   1. Allow S2 cells to exponentially grow in T25 or T75 cm² flasks depending on the number of cells required for the experiment.
   2. Count the cell density using a hemocytometer. Add 1 ml growth medium (for example Insect-Xpress) into a 35 mm tissue culture dish with ConA-coated coverslip, and gently transfer ~1 x 10⁵ cells to the dish. This cell density is optimal for imaging because processes from different cells do not overlap.
   3. In order to induce formation of processes, immediately after plating add 1ml medium with 5 µM CytoD to the dish (to the final concentration of 2.5 µM CytoD). Allow full development of processes by incubating the cells at least for 2 hr at 25 ºC before imaging.

2. Drosophila Primary Neuron Culture

1. Preparation for neuron culture
   1. Prepare Drosophila neuronal growth medium (Schneider's medium supplemented with: 20% fetal bovine serum, heat inactivated at 55 ºC for 30 min; 5 µg/ml insulin; 100 µg/ml penicillin, 100 µg/ml streptomycin; 10 µg/ml tetracycline). This supplemented Schneider's medium can be stored at 4 ºC for 6 months.
   2. Prepare apple juice agar vials for collecting Drosophila embryos (22.5 g agar, 12.0 g sucrose and 750 ml H₂O in a 2-liter flask; autoclave for 20-25 min; add 250 ml apple juice and pour ~10 ml into each Drosophila vial; after solidification, plug vials with cotton balls, and wrap vials with plastic food wrap to avoid over drying of the food). The apple juice in agar vials can be stored at 4 ºC in well-sealed plastic bags for 4 weeks.
   3. Coat acid-washed 25 mm circle coverslips with ConA (see details in the Protocol for Drosophila S2 cells) and sterilize them under UV light for 10-15 min. These coverslips can be stored for a month.
2. Drosophila embryonic neuron culture
   1. Keep young adult flies in apple juice agar vials supplemented with dry yeast for 1-2 days before collecting embryos for neuron preparation.
   2. On the collecting day, in order to synchronize the collected embryos, transfer adult flies to a fresh apple juice agar vial for 1 hr precollection in light and discard these embryos.
   3. Transfer flies to a new apple juice agar vial, collect embryos for 1 hr, and transfer the flies to another vial for another hour, both in dark to increase the embryo yield.
   4. Remove the adult flies from the apple juice agar vial and let the embryos develop another 4 hr to stages 9-11 at room temperature (4-6 hr-old embryos are between stage 9-11).
   5. Remove the embryos by squirting water into vials and loosening the embryos from the apple juice agar with a small paintbrush (at least 10 embryos are required for one good neuron prep).
   6. Transfer the embryos to a 100 µm nylon mesh cell strainer with a transfer pipette to drain water (prerinse the transfer pipette in Drosophila embryo wash buffer, 0.4% NaCl, 0.03% Triton X-100).
   7. Collect the embryos from the strainer using a small paintbrush, and transfer them into a 1.5 ml microtube filled with 1 ml Drosophila embryo wash (Note: clean any agar or cotton fibers using the paintbrush or pipette tips). In this protocol, the 1.5 ml microtube comes with a matching disposable pellet pestle.
   8. Wash embryos in Drosophila embryo wash 3x.
   9. Dechorionate the embryos in a fresh 1:1 solution of  commercial bleach and 95% ethanol for 10 min [CAUTION: bleach can cause irritation to eyes, nose, throat, and skin].
   10. Move to the tissue culture hood, and wash embryos 2x in supplemented Schneider's medium.
   11. Leave 200-300 µl medium in the tube with the embryos, and gently mechanically dissociate dechorionated embryos using Kimble Chase disposable pellet pestles that have been sterilized by 70% ethanol (grind 10-15x).
   12. Centrifuge the homogenized mixture at 20 x g for 2 min, and transfer the supernatant to a fresh tube. [This step is optional; it helps to remove embryo debris]
   13. Centrifuge the supernatant at 550 x g for 2 min to pellet cells.
   14. Resuspend the pellet in 500 µl supplemented Schneider's medium and spin cells down again at 550 x g for 2 min; repeat the resuspension-spinning cycle once.
   15. Finally resuspend the pellet in ~100 µl supplemented Schneider's medium and plate the cells onto a 25 mm ConA-coated coverslip in a sterile 35 mm Petri dish. Incubate for 10 min to allow cell attachment.
   16. Add 2 ml of supplemented Schneider's medium, with 5 µM CytoD to submerge the coverslip with attached cells.
   17. Incubate the dish in a humidified 25 ºC incubator overnight before imaging.

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