We would like to thank Patricia Wadsworth for the gift of the kinesin-5 construct. This work was supported by an NIH grant (5 R01 GM107026) to T.J.M. and by Research Grant No. 5-FY13-205 from the March of Dimes Foundation to T.J.M., as well as support from the Charles H. Hood Foundation, Inc., Boston, MA. to T.J.M.

1. Transfecting S2 Cells
<ol>
	<li>From a previously cultured 25 cm2 flask, add GFP-&#945;-tubulin-expressing S2 cells10 to a final volume of 2 ml at 100% confluency, and allow them to semi-adhere to the bottom of a 35 mm tissue culture dish for approximately 1 hr.</li>
	<li>Remove the media from the dish. Transfect 2 &#181;g of the Eg5-mCherry construct11 with 1 ml of Schneider&#39;s media supplemented with 10% FBS (referred to hereafter as Schneider&#39;s media) and Transfection Reagent, following the manufacturer&#39;s protocol. Seal the dish with Parafilm and incubate cells at 25 &#176;C for 16-18 hr.</li>
	<li>Add 1 ml of Schneider&#39;s media. Return cells to 25 &#176;C incubator.</li>
	<li>On day 3 post-transfection, transfer 500 &#181;l of transfected cells into a new 35 mm tissue culture dish containing 1.5 ml of Schneider&#39;s media for a test induction. Induce the expression of Eg5-mCherry by adding CuSO4 to a final concentration of 500 &#181;M. Seal the dish in Parafilm and incubate at 25 &#176;C O/N.</li>
	<li>To make concanavalin A-coated coverslips, pipette enough volume of concanavalin A solution (0.5 mg/ml dissolved in H2O) to coat an acid washed coverslip, remove the excess, and allow the coverslip to air dry.</li>
	<li>Place a 22 mm x 22 mm concanavalin A-coated coverslip in a clean 35 mm tissue culture dish, then seed 500 &#181;l at 100% confluency of the induced cells onto the coverslip and allow the cells to adhere for 1 hr at RT.</li>
	<li>To check for Eg5-mCherry expression, assemble the coverslip into a rose chamber (or appropriate imaging vessel) with media for imaging at RT on a fluorescence microscope. Use appropriate light sources and filter sets to visualize the cells. For example, the microscope used in this study has an LED white light source and EGFP (FITC/Cy2) and DSRed (TRITC/Cy3) filter cubes. Eg5-mCherry localizes to microtubules and is enriched near spindle poles.</li>
	<li>After ensuring that the cells are expressing both Eg5-mCherry and GFP-&#945;-tubulin, transfer the remainder of the transfected, uninduced cells to a 25 cm2 tissue culture flask containing 4 ml of Schneider&#39;s media.</li>
	<li>Add Blasticidin S HCL at a final concentration of 25 &#956;g/ml to the flask of transfected cells and continue splitting in the presence of 25 &#956;g/ml Blasticidin S HCl until cell death ceases being sure to check for expression of Eg5-mCherry periodically. Incubate cell lines in 25 &#176;C incubator. 
	NOTE: The percentage of cells expressing Eg5-mCherry increases over time.</li>
	<li>Once cells are stably transfected, continue to split in the absence of drug and freeze12 cells for future use.</li>
</ol>
2. RNA Interference
<ol>
	<li>PCR amplify a ~500 bp region of Klp61F from cDNA (LD15641) using primer with added T7 promoter sequence (sequence provided in Materials List) to be used as a template for dsRNA synthesis.
	<ol>
		<li>Set up four PCR reactions, 50 &#181;l each, containing 100 ng of template DNA, 25 &#181;M of each primer, appropriate buffer, and polymerase. Set the PCR protocol on the thermocycler according to the polymerase&#39;s manufacturer&#39;s protocol.</li>
		<li>Pool and clean four PCR reactions using a PCR clean up kit according to manufacturer&#39;s protocol. Store the clean template at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare double stranded RNA (dsRNA) from template using a T7 RNA transcription kit, following the manufacturer&#39;s instructions. Expect ~5-15 mg/ml concentration of dsRNA. Store dsRNA at -20 &#176;C.</li>
	<li>To Knockdown Klp61F with dsRNA, allow S2 cells expressing Eg5-mCherry at 25% confluency to semi-adhere to the bottom of a 35 mm tissue culture dish for 1 hr.</li>
	<li>Remove the media from the dishes and add 5 &#181;g of dsRNA against Klp61F (Drosophila kinesin-5) diluted into 1 ml of serum-free Schneider&#39;s media.</li>
	<li>Incubate at RT for 1 hr, then add 1 ml of Schneider&#39;s media with 10% FBS. Incubate cells at 25 &#176;C.</li>
	<li>24 hr after dsRNA treatment, induce expression of Eg5-mCherry by adding CuSO4 to a final concentration of 500 &#181;M. Incubate cells at 25 &#176;C for another 24 hr.&#160;</li>
</ol>
3. Live Cell Imaging
<ol>
	<li>Place a 22 mm x 22 mm concanavalin A coated coverslip into a clean 35 mm tissue culture dish.</li>
	<li>Seed 500 &#181;l of the cells that have been treated with dsRNA and induced O/N onto the concanavalin A coated coverslip and allow them to adhere for about 1 hr.</li>
	<li>Assemble the coverslip into a rose chamber (or appropriate vessel) for imaging.</li>
	<li>Find mitotic cells expressing both Eg5-mCherry and GFP-&#945;-tubulin. 
	NOTE: Mitotic cells expressing only GFP-&#945;-tubulin should have monopolar spindles since Klp61F, which is required for spindle bipolarity, is knocked down10,13.</li>
	<li>Collect time-lapse images of bipolar spindles (e.g., 1 frame per minute) in cells expressing both Eg5-mCherry and GFP-&#945;-tubulin.</li>
	<li>While imaging, remove the media from the rose chamber, and replace it with Schneider&#39;s media containing 1 &#181;M STLC over 3 consecutive media exchanges (~5 ml total) into the chamber to visualize spindle collapse.</li>
	<li>To washout the drug and reverse the spindle collapse, carefully remove the STLC-containing media from the rose chamber, and wash in Schneider&#39;s media 4x (6-8 ml total) before refilling the rose chamber one last time with fresh media. Continue imaging.</li>
</ol>
4. Error Correction Assay
<ol>
	<li>Place a 22 mm x 22 mm concanavalin A coated coverslips into clean 35 mm tissue culture dishes.</li>
	<li>Seed 500 &#181;l of cells that have been treated with Klp61F dsRNA onto the concanavalin A coated coverslip and allow them to adhere.</li>
	<li>After cells are adhered, add 1.5 ml of Schneider&#39;s media to each dish to bring the final volume up to 2 ml.</li>
	<li>To arrest cells in mitosis, add MG132 to a final concentration of 10 &#181;M to each of the dishes, and incubate for 1 hr.</li>
	<li>Add 1 &#181;M STLC and incubate for 1 hr to allow monopoles to form.</li>
	<li>Wash out the STLC by rinsing coverslips 3x with 2 ml of fresh Schneider&#39;s media each time.</li>
	<li>Incubate coverslips with Schneider&#39;s media in addition to any pharmacological drugs (e.g., Aurora kinase inhibitors) or DMSO as a control. 
	Note: Inhibitor concentrations vary and proper final concentrations must be determined by the experimenter. Stock solutions are typically made such that the inhibitor is diluted 1:1,000 into media. In this case a 1:1,000 dilution of DMSO (or appropriate solvent) serves as the vehicle control. We use the Aurora B inhibitor Binucleine 2 at a final concentration of 40 &#181;M.</li>
	<li>Fix cells at different time points to observe the progression of bipolar spindle formation and to assess kinetochore attachment states. To fix:
	<ol>
		<li>Quickly rinse the coverslips with 2 ml of 1x BRB-80.</li>
		<li>Fix cells by adding 2 ml of 10% paraformaldehyde diluted in 1x BRB-80 for 10 min. Pipet paraformaldehyde carefully in a chemical hood.</li>
		<li>Permeabilize cells with 2 ml of 1x PBS + 1% Triton-X for 8 min.</li>
		<li>Wash slides 3x with 2 ml of 1x PBS + 0.1% Triton-X.</li>
		<li>Transfer coverslips cell-side face up onto a sheet of Parafilm (use a marker to label Parafilm appropriately to keep track of the slides) in a 150 mm Petri dish.</li>
		<li>Cover the coverslips with 150 &#181;l boiled donkey serum (BDS) and incubate at RT for 1 hr to block non-specific antibody binding. 
		Note: Here, the fixation protocol can be stopped to be continued at a later point. Coverslips can be stored in a moisture chamber at 4 &#176;C for up to 24 hr. To make the moisture chamber, line the rim of a 150 mm dish with a wet laboratory wipe and cover with the Petri dish lid.</li>
		<li>Remove block, and incubate the coverslips for 1 hr at RT with 150 &#181;l of primary antibodies diluted appropriately in BDS to stain for kinetochores and microtubules to final concentrations of 2 &#181;g/ml (or according to manufacturer&#39;s recommendations) and 1 &#181;g/ml, respectively. 
		Note: Here, the fixation protocol can be stopped to be continued at a later point. Coverslips can be stored in a moisture chamber at 4 &#176;C for up to 24 hr.</li>
		<li>Wash coverslips 3x with 500 &#181;l of 1x PBS + 0.1% Triton-X.</li>
		<li>Incubate coverslips for 30-60 min at RT with the appropriate fluorophore-conjugated secondary antibodies diluted in BDS.</li>
		<li>Wash coverslips 3x with 500 &#181;l of 1x PBS + 0.1% Triton-X.</li>
		<li>Incubate coverslips with 150 &#181;l of BDS containing 1 &#181;g/ml DAPI final concentration for 5 min.</li>
		<li>Wash coverslips 2x with 1x PBS + 0.1% Triton-X, and mount coverslips cell side down on a 3x1&#34; slide with 8 &#181;l of mounting media. Paint the corners with nail polish to immobilize the coverslips onto slides.</li>
	</ol>
	</li>
	<li>Image cells with bipolar spindles that are expressing Eg5-mCherry. Take a Z series consisting of 30 planes at 0.2 &#181;m intervals in all channels using a 100X objective. Carefully analyze the kinetochores and microtubules to determine the attachment states (bioriented or syntelic attachments).</li>
</ol>

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S., Ohsugi, M., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+kinetochore+tracking+reveals+error-prone+homologous+chromosome+biorientation+in+mammalian+oocytes.">Complete kinetochore tracking reveals error-prone homologous chromosome biorientation in mammalian oocytes.</a> Cell. 146, 568-581 (2011).</li><li>Cimini, D., Wan, X., Hirel, C. B., Salmon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aurora+kinase+promotes+turnover+of+kinetochore+microtubules+to+reduce+chromosome+segregation+errors.">Aurora kinase promotes turnover of kinetochore microtubules to reduce chromosome segregation errors.</a> Curr Biol. 16, 1711-1718 (2006).</li><li>Lampson, M. A., Renduchitala, K., Khodjakov, A., Kapoor, T. 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The authors declare that they have no competing financial interests.

Visualizing error correction is a valuable technique to study the steps involved in this important and complex cellular process. To do so, erroneous attachments are generated using reversible inhibitors, and error correction is observed upon washout of the drug. This assay was originally developed using mammalian tissue culture cells5. However the presence of large number of kinetochores in many model mammalian cell types poses a challenge in observing individual error correction events. Drosophila S2 cells possess as few as 4 pairs of kinetochores, making them a more preferable cell line for observing error correction. However, a major drawback is that many inhibitors are ineffective in Drosophila S2 cells. Thus, the ability to generate a humanized Drosophila S2 cell line expressing human kinesin-5 provides a valuable tool to study error correction.
Although Drosophila S2 cells can be a better cell line to study error correction, the multiple steps involved in obtaining the cell line and knocking down essential genes poses some challenges to this technique. For instance, transfection efficiency can be quite low. If less than 20% of the cells are expressing the Eg5-mCherry, the transfection should be repeated as the selection process will take longer. Also, the percentage of cells expressing both fluorescent proteins may decrease over time. This can be overcome by splitting cells in the presence of Blasticidin S HCl and Hygromycin B to select for cells expressing Eg5-mCherry and GFP-&#945;-tubulin, respectively. It is also important to note that Drosophila S2 cells have orthologues to many human proteins and; therefore, it is critical to optimize the knockdown conditions for the endogenous proteins. Optimal knockdown conditions may vary in the amount of dsRNA and the length of treatment. Considering the emergence and rapid improvement of CRISPR-Cas9 technologies15-17, generation of a Drosophila cell line with the Klp61F gene replaced by human Eg5 presents a powerful alternative that would overcome the limitations of the transfection and knockdown approach. Our work demonstrates that fly-to-human gene replacement should be a viable option in this case although the necessary reagents to do so in Drosophila S2 cells are currently being developed.
This procedure is not limited to Eg5, but can be applied to study the function of other proteins of interest. If using inhibitors that have previously been established to be ineffective in Drosophila S2 cells, this protocol can be modified to study the direct effect of inhibitors in live cells without concerns about off target effects. The cell line produced using this protocol could also be used in high-throughput screening analyses to identify potential drugs targeting proteins involved in error correction.

Equal segregation of the genome during cell division requires correct interactions between the replicated DNA and spindle microtubules. Microtubules physically interact with chromosomes through an ensemble of proteins that assembles at centromeres known as the kinetochore1. Correct distribution of the chromosomes requires that sister kinetochores are bioriented, in which each sister is associated with microtubules originating from opposite spindle poles. Kinetochore microtubule (kt-MT) attachments that are not in the bioriented conformation are quickly and efficiently destabilized to provide the opportunity to establish biorientation in a process known as error correction. An error correction assay that was previously established in mammalian cells5 requires assembling monopolar spindles using reversible small molecule inhibitors against Eg5 (kinesin-5). The drug treatment generates a multitude of erroneous syntelic attachments in which both sister kinetochores attach to the same spindle pole. A subsequent washout of the drug allows for visualization of the error correction process. The error correction assay can be done in the presence of small molecule inhibitors or knockdowns to study the contribution of candidate proteins to correcting erroneous kt-MT attachments.
The ability to visualize error correction in living cells is a powerful tool to further understand the molecular mechanism involved in this complex process. However, the large number of chromosomes present in most cell lines poses a challenge in observing individual kt-MT attachments. Drosophila S2 cells would be ideal for applying the error correction assay as they contain as few as 4 chromosomes6, but small molecule inhibitors of kinesin 5 such as S-trityl-L-cysteine (STLC) and monastrol7-9 do not affect spindle assembly or kinesin-5 motor function in Drosophila cells. We therefore generated a Drosophila S2 cell line expressing human kinesin-5 under an inducible promoter that is sensitive to kinesin-5 inhibitors. This protocol describes how to knockdown the endogenous Drosophila kinesin-5 homologue, Klp61F, and use this cell line in the cell-based error correction assay.

<table><tbody><tr><td>Effectine Transfection Reagent</td><td>Qiagen</td><td>301425</td><td></td></tr><tr><td>Klp61F cDNA</td><td>Drosophila Genomic Resource Center</td><td>13690</td><td>Gene Name LD15641</td></tr><tr><td>Schneider&amp;rsquo;s Media</td><td>Invitrogen</td><td>21720-024</td><td></td></tr><tr><td>Fetal Bovine Serum, certified</td><td>Invitrogen</td><td>10082-147</td><td>Heat Inactivated, US origin</td></tr><tr><td>Copper(II) Sulfate (CuSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma</td><td>C8027-500G</td><td>500&amp;nbsp;mM stock</td></tr><tr><td>S-trityl-L-cysteine</td><td>Sigma</td><td>164739-5G</td><td>1&amp;nbsp;mM stock in DMSO</td></tr><tr><td>Blasticidin HCl</td><td>Invitrogen</td><td>R21001</td><td>5&amp;nbsp;mg/ml stock in 1x PBS</td></tr><tr><td>Hygromycin B</td><td>Invitrogen</td><td>10687010</td><td></td></tr><tr><td>T7 Large scale RNA Production System</td><td>Promega</td><td>P1320</td><td>The approximate dsRNA concentration from each reaction is about 5-15&amp;nbsp;&amp;micro;g/&amp;micro;l</td></tr><tr><td>Klp61F RNAi F primer</td><td>Invitrogen</td><td></td><td>TAATACGACTCACTATAGGGTA-TTTGCGCATTATTTTAAAA</td></tr><tr><td>Klp61F RNAi R primer</td><td>Invitrogen</td><td></td><td>TAATACGACTCACTATAGGGAT-ATTGATCAATTGAAAC</td></tr><tr><td>PCR clean-up kit</td><td>Mo Bio Laboratories, Inc</td><td>1250</td><td></td></tr><tr><td>Concanavalin A</td><td>Sigma</td><td>C5275</td><td>0.5&amp;nbsp;mg/ml solution made by dissolving in 1x PBS.</td></tr><tr><td>Boiled Donkey Serum</td><td>Jackson ImmunoResearch Labs</td><td>017-000-121</td><td>5% stock solution in 1x PHEM buffer, bring solution up to boil. Stored in 4&amp;nbsp;&amp;deg;C.</td></tr><tr><td>Mounting Media</td><td></td><td></td><td>20&amp;nbsp;mM Tris pH 8.0,&amp;nbsp; 0.5% &lt;em&gt;N&lt;/em&gt;-propyl gallate, 90% Glycerol. Stored in 4&amp;nbsp;&amp;deg;C.</td></tr><tr><td>1x BRB-80</td><td></td><td></td><td>80 mM PIPES pH 6.9; 1&amp;nbsp;mM EGTA; 1 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>White Light Source</td><td>Lumencor Inc</td><td></td><td></td></tr><tr><td>ET EGFP Filter Cube</td><td>Chroma</td><td>49002</td><td></td></tr><tr><td>DSRed Filter Cube</td><td>Chroma</td><td>49005</td><td></td></tr><tr><td>anti-NDC80 antibody&amp;nbsp;</td><td></td><td></td><td>Custom made by the Maresca Lab</td></tr><tr><td>DM1&amp;alpha; (anti-&amp;alpha;-tubulin)</td><td>Sigma</td><td>T6199</td><td></td></tr><tr><td>anti-CID antibody</td><td>AbCam</td><td>ab10887</td><td></td></tr></tbody></table>

generating a &#34;humanized&#34; drosophila s2 cell line sensitive to pharmacological inhibition of kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Klp61F is required to form bipolar spindles. The human kinesin-5, Eg5, can rescue the function of Klp61F in Drosophila S2 cells (Figure 1A and D). Upon addition of the kinesin-5 inhibitor (STLC), microtubule-associated levels of the motor drop (Figure 1B and E), and the spindle collapses, resulting in a monopole (Figure 1C and F) in cells lacking endogenous Klp61F upon successful RNAi (Supplemental movie 1). It is noteworthy that bipolar spindles collapse upon addition of STLC in the humanized S2 cells because kinesin-5 activity is not required to maintain spindle bipolarity in human cells. This interesting discrepancy may be a result of differences in interpolar microtubule overlap and/or stability in the two model systems14. Kinesin-5 inhibition (Figure 2A and E) can be reversed by washing out the drug and recovery of a bipolar spindle can be followed over time. Upon removal of the inhibitor (Figure 2B and F), Eg5-mCherry re-associates with the microtubules as a bipolar spindle assembles (Figure 2C and G), and cells can progress into a bipolar anaphase (Figure 2D and H, Supplemental movie 2).
<img alt="Figure 1" src="/files/ftp_upload/53594/53594fig1.jpg" /> 
Figure 1. Addition of 1 &#181;M STLC leads to monopolar spindles in Drosophila S2 cells. Representative images from time-lapse imaging of a Drosophila S2 cell expressing Eg5-mCherry (A-C) and GFP-&#945;-tubulin (D-F) during addition of 1 &#181;M STLC. The Eg5 inhibitor was added at 3 min. Scale bar = 5 &#181;m. Timestamp = min:sec. <a href="https://www.jove.com/files/ftp_upload/53594/53594fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53594/53594fig2.jpg" /> 
Figure 2. A bipolar spindle reforms upon removal of STLC. Representative images from time-lapse imaging of a Drosophila 2 cell expressing Eg5-mCherry (A-D) and GFP-&#945;-tubulin (E-H) during an STLC washout experiment. STLC was washed out at 5 min. A bipolar spindle reforms within 1 hr of removing the STLC. Scale bar = 5 &#181;m. Timestamp: min:sec. <a href="https://www.jove.com/files/ftp_upload/53594/53594fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/53594/53594mov1.jpg" /> 
<a href="https://www.jove.com/files/ftp_upload/53594/addin.mov" target="_blank">Supplemental Movie 1. (Right click to download).</a> Widefield fluorescence imaging of a representative example of a Drosophila S2 cell expressing Eg5-mCherry (left) and GFP-&#945;-tubulin (right). 1 &#181;M STLC was added at 2 min, and the spindle forms a monopole within 10 min&#160;after addition of the inhibitor. Images were acquired every 1 min&#160;and played at a rate of 10 frames per second. Scale bar = 5 &#181;m.
<img alt="Movie 2" src="/files/ftp_upload/53594/53594mov2.jpg" /> 
<a href="https://www.jove.com/files/ftp_upload/53594/washout.mov" target="_blank">Supplemental Movie 2. (Right click to download).</a> Widefield fluorescence imaging of a representative example of a Drosophila S2 cell expressing Eg5-mCherry (left) and GFP-&#945;-tubulin (right). Media containing 1 &#181;M STLC was removed and rinsed at 5 min. A bipolar spindle reforms within 1 hr of removing the inhibitor. Images were acquired every 1 min&#160;and played at a rate of 10 frames per second. Scale bar = 5 &#181;m.

Watch this Scientific Journal Video about Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5 at JoVE.com

Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

「ヒト化」を生成<em&gt;ショウジョウバエ</emキネシン5の薬理学的阻害に感受性&gt; S2細胞株

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

generating-humanized-drosophila-s2-cell-line-sensitive-to

Research

JoVE Journal

Biology

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

7.2K ビュー.  University of Massachusetts, Amherst. This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined. 

ビデオ: Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

ショウジョウバエ幼虫分節軸索内ではシナプス小胞の in vivo可視化で

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

神経科学

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:&#945;-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as &#8216;CID&#8217; gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Cell divisions can be visualized in real time using fluorescently tagged proteins and time-lapse microscopy. Using the protocol presented here, users can analyze cell division timing dynamics, mitotic spindle assembly, and chromosome congression and segregation. Defects in these events following RNA interference (RNAi)-mediated gene knockdown can be assessed and quantified.

細胞分裂のライブイメージングのためのショウジョウバエS2細胞の使用

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...

遺伝学

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

Drosophila is an excellent model organism that can be used to screen compounds that might be useful for cancer therapy. The method described here is a cost-effective in vivo method to identify heterochromatin-promoting compounds by using Drosophila. The Drosophila&#39;s DX1 strain, having a variegated eye color phenotype that reflects the extents of heterochromatin formation, thereby providing a tool for a heterochromatin-promoting drug screen. In this screening method, eye variegation is quantified based on the surface area of red pigmentation occupying parts of the eye and is scored on a scale from 1 to 5. The screening method is straightforward and sensitive and allows for testing compounds in vivo. Drug screening using this method provides a fast and inexpensive way for identifying heterochromatin-promoting drugs that could have beneficial effects in cancer therapeutics. Identifying compounds that promote the formation of heterochromatin could also lead to the discovery of epigenetic mechanisms of cancer development.

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

Drosophila is a widely used experimental model suitable for screening drugs with potential applications for cancer therapy. Here, we describe the use of Drosophila variegated eye color phenotypes as a method for screening small-molecule compounds that promote heterochromatin formation.

ショウジョウバエを用いたヘテロクロマチン促進薬の同定のためのスクリーニング方法

Drosophila is an excellent model organism that can be used to screen compounds that might be useful for cancer therapy. The method described here is a ...

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

全体の開発、 ショウジョウバエの脳のex vivoで培養

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted epithelial morphogen. In this assay, Fog is used as an agonist to activate Rho through a signaling cascade that includes a G-protein-coupled receptor (Mist), a G&#945;12/13 protein (Concertina/Cta), and a PDZ-domain-containing guanine nucleotide exchange factor (RhoGEF2). Fog signaling results in the rapid and dramatic reorganization of the actin cytoskeleton to form a contractile purse string. Soluble Fog is collected from a stable cell line and applied ectopically to S2R+ cells, leading to morphological changes like apical constriction, a process observed during developmental processes such as gastrulation. This assay is amenable to high-throughput screening and, using RNAi, can facilitate the identification of additional genes involved in this pathway.

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

Here we describe a contractility assay using Drosophila S2R+ cells. The application of an exogenous ligand, folded gastrulation (Fog), leads to the activation of the Fog signaling pathway and cellular contractility. This assay can be used to investigate the regulation of cellular contractility proteins in the Fog signaling pathway.

非筋ミオシン II 収縮を介して折り返されている陥シグナル伝達経路ショウジョウバエS2R + 細胞を調査する細胞ベースのアッセイ

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted ...

発生生物学

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

のライブ解剖ショウジョウバエ胚：抗体染色によってミュータントコレクションをスクリーニングするための合理化手法

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

生物学

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

の準備ショウジョウバエ</em光学顕微鏡用> S2細胞

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

ショウジョウバエにおけるRho1媒介性アクトミオシン収縮性の光遺伝学的阻害

Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos

Contractile forces generated by actin and non-muscle myosin II ("actomyosin contractility") are critical for morphological changes of cells and tissues at multiple length scales, such as cell division, cell migration, epithelial folding, and branching morphogenesis. An in-depth understanding of the role of actomyosin contractility in morphogenesis requires approaches that allow the rapid inactivation of actomyosin, which is difficult to achieve using conventional genetic or pharmacological approaches. The presented protocol demonstrates the use of a CRY2-CIBN based optogenetic dimerization system, Opto-Rho1DN, to inhibit actomyosin contractility in Drosophila embryos with precise temporal and spatial controls. In this system, CRY2 is fused to the dominant negative form of Rho1 (Rho1DN), whereas CIBN is anchored to the plasma membrane. Blue light-mediated dimerization of CRY2 and CIBN results in rapid translocation of Rho1DN from the cytoplasm to the plasma membrane, where it inactivates actomyosin by inhibiting endogenous Rho1. In addition, this article presents a detailed protocol for coupling Opto-Rho1DN-mediated inactivation of actomyosin with laser ablation to investigate the role of actomyosin in generating epithelial tension during Drosophila ventral furrow formation. This protocol can be applied to many other morphological processes that involve actomyosin contractility in Drosophila embryos with minimal modifications. Overall, this optogenetic tool is a powerful approach to dissect the function of actomyosin contractility in controlling tissue mechanics during dynamic tissue remodeling.

著者スポットライト:ショウジョウバエ胚におけるRho1を介したアクトミオシン収縮性の光遺伝学的阻害と上皮緊張の測定  

Actomyosin contractility plays an important role in cell and tissue morphogenesis. However, it is challenging to manipulate actomyosin contractility in vivo acutely. This protocol describes an optogenetic system that rapidly inhibits Rho1-mediated actomyosin contractility in Drosophila embryos, revealing the immediate loss of epithelial tension after the inactivation of actomyosin in vivo.

ショウ ジョウバエ 胚におけるRho1を介したアクトミオシン収縮性の光遺伝学的阻害と上皮圧の測定

Contractile forces generated by actin and non-muscle myosin II ("actomyosin contractility") are critical for morphological changes of cells and tissues at ...

「ヒト化」を生成

要約

さらに動画を探す

この動画の章

ショウジョウバエ幼虫分節軸索内ではシナプス小胞の in vivo可視化で

細胞分裂のライブイメージングのためのショウジョウバエS2細胞の使用

ショウジョウバエを用いたヘテロクロマチン促進薬の同定のためのスクリーニング方法

全体の開発、ショウジョウバエの脳のex vivoで培養

非筋ミオシン II 収縮を介して折り返されている陥シグナル伝達経路ショウジョウバエS2R + 細胞を調査する細胞ベースのアッセイ

のライブ解剖ショウジョウバエ胚：抗体染色によってミュータントコレクションをスクリーニングするための合理化手法

の準備ショウジョウバエ S2細胞

ショウジョウバエ胚におけるRho1を介したアクトミオシン収縮性の光遺伝学的阻害と上皮圧の測定

ショウジョウバエ胚におけるRho1を介したアクトミオシン収縮性の光遺伝学的阻害と上皮圧の測定

ショウジョウバエ胚におけるRho1を介したアクトミオシン収縮性の光遺伝学的阻害と上皮圧の測定

「ヒト化」を生成

要約

さらに動画を探す

この動画の章

ショウジョウバエ幼虫分節軸索内ではシナプス小胞の in vivo可視化で

細胞分裂のライブイメージングのためのショウジョウバエS2細胞の使用

ショウジョウバエを用いたヘテロクロマチン促進薬の同定のためのスクリーニング方法

全体の開発、 ショウジョウバエの脳のex vivoで培養

非筋ミオシン II 収縮を介して折り返されている陥シグナル伝達経路ショウジョウバエS2R + 細胞を調査する細胞ベースのアッセイ

のライブ解剖ショウジョウバエ胚：抗体染色によってミュータントコレクションをスクリーニングするための合理化手法

の準備ショウジョウバエ S2細胞

ショウ ジョウバエ 胚におけるRho1を介したアクトミオシン収縮性の光遺伝学的阻害と上皮圧の測定

ショウ ジョウバエ 胚におけるRho1を介したアクトミオシン収縮性の光遺伝学的阻害と上皮圧の測定

ショウ ジョウバエ 胚におけるRho1を介したアクトミオシン収縮性の光遺伝学的阻害と上皮圧の測定

全体の開発、ショウジョウバエの脳のex vivoで培養

ショウジョウバエ胚におけるRho1を介したアクトミオシン収縮性の光遺伝学的阻害と上皮圧の測定

ショウジョウバエ胚におけるRho1を介したアクトミオシン収縮性の光遺伝学的阻害と上皮圧の測定

ショウジョウバエ胚におけるRho1を介したアクトミオシン収縮性の光遺伝学的阻害と上皮圧の測定