We thank the members of the Gelfand lab who contributed to the development of these protocols. Research reported in this publication was supported by the National Institute of General Medical Science of the National Institutes of Health under award number R01GM052111 to VIG and by Basque Government Department of Education, Universities and Research under award number BFI-2011-295 to UdC.

1. Drosophila S2 cells
<ol>
	<li>Preparation of ConA coated coverslips
	<ol>
		<li>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].</li>
		<li>Rinse coverslips thoroughly with constantly running dH2O&#160;for 30 min until the acid is completely washed out.</li>
		<li>Let coverslips air dry and coat them with ConA (0.5 mg/ml solution in dH2O) f...

<ol>
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The authors declare that they have no competing financial interests.

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...

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 &#176;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 literature2,3. 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 microtubules4-10, 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 system10,12. Since several familial neurodegenerative diseases could be caused by or associated with cargo transport defects (e.g.&#160;Parkinson&#39;s disease13, Huntington&#39;s disease14, Alzheimer&#39;s Disease15,16, Amyotrophic Lateral Sclerosis/ALS17, HMN7B and Perry syndrome18,19, 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.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:124px;">Powder Insect cell medium (Schneider&#8217;s)</td>
			<td style="width:133px;">Sigma&#160;</td>
			<td style="width:179px;">S9895</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Insect &#8211;Xpress medium</td>
			<td style="width:133px;">Fisher</td>
			<td style="width:179px;">BW12730Q</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:124px;">Insulin</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:179px;">I6634</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:124px;">Penicillin</td>
			<td style="width:133px;">Research Products International</td>
			<td style="width:179px;">P93000</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:124px;">Streptomycin</td>
			<td style="width:133px;">Research Products International</td>
			<td style="width:179px;">S62000</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Tetracycline hydrochloride</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:179px;">T7660-5G</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Fetal bovine serum</td>
			<td style="width:133px;">Atlanta Biologicals</td>
			<td style="width:179px;">S11150</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:124px;">Concanavalin A</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:179px;">C2010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:124px;">Cytochalasin D</td>
			<td style="width:133px;">VWR</td>
			<td style="width:179px;">IC15077105</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">LysoTracker Red DND-99</td>
			<td style="width:133px;">Molecular Probes</td>
			<td style="width:179px;">L7528</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">100 &#181;m Cell strainer</td>
			<td style="width:133px;">Fisher Scientific</td>
			<td style="width:179px;">22363549</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">25 mm Circle cover glass</td>
			<td style="width:133px;">VWR</td>
			<td style="width:179px;">48380 080</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Drosophila Vials</td>
			<td style="width:133px;">VWR</td>
			<td style="width:179px;">89092-730</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:124px;">Disposable pellet pestles with matching microtubes</td>
			<td style="width:133px;">Kimble Chase</td>
			<td style="width:179px;">749520-0000</td>
		</tr>
	</tbody>
</table>

organelle transport in cultured drosophila cells: s2 cell line and primary neurons.

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.

Drosophila S2 cells attached on the ConA-coated coverslip spread into a flat round &#8220;pancake&#8221; 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 ...

Watch this Scientific Journal Video about Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons. at JoVE.com

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

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.

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

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

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13.6K visualizações.  Feinberg School of Medicine, Northwestern University.  Células Drosophila S2 e neurônios cultivados são grandes sistemas para a imagem latente de transporte organela motorizado in vivo. Aqui nós descrevemos protocolos detalhados para o cultivo de ambos os tipos de células, a sua imagem e análise de transporte. 