Name: live cell imaging to assess the dynamics of metaphase timing and cell fate following mitotic spindle perturbations
Uploaded: 2019-09-20T15:00:00
Description: Watch this Scientific Journal Video about Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations at JoVE.com

DLM is supported by an NSF GRFP. ALM is supported by funding from the Smith Family Award for Excellence in Biomedical Research.

1. Generation of hTERT-RPE-1 cells stably expressing RFP-Histone 2B (RFP-H2B) and &#945;-tubulin-EGFP (tub-EGFP)
NOTE: All steps follow aseptic techniques and take place in a biosafety level II+ (BSL2+) safety cabinet.
<ol>
	<li>Generate retrovirus carrying the genes of interest (&#945;-tubulin-EGFP and RFP-H2B) by the transfection of 293T cells with the appropriate lentiviral plasmids according to the manufacturer&#39;s instructions of the lipid-based transfection delivery system.
	<ol>
		<...

<ol>
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D., Degrassi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merotelic+kinetochore+orientation+versus+chromosome+mono-orientation+in+the+origin+of+lagging+chromosomes+in+human+primary+cells.">Merotelic kinetochore orientation versus chromosome mono-orientation in the origin of lagging chromosomes in human primary cells.</a> Journal of Cell Science. 115, 507-515 (2002).</li><li>Kwon, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+to+suppress+multipolar+divisions+in+cancer+cells+with+extra+centrosomes.">Mechanisms to suppress multipolar divisions in cancer cells with extra centrosomes.</a> Genes &amp; Development. 22, 2189-2203 (2008).</li><li>Khodjakov, A., Rieder, C. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimitotic+drugs+in+the+treatment+of+cancer.">Antimitotic drugs in the treatment of cancer.</a> Cancer Chemotherapy and Pharmacology. 76, 1101-1112 (2015).</li><li>Chan, K. S., Koh, C. G., Li, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis-targeted+anti-cancer+therapies:+where+they+stand.">Mitosis-targeted anti-cancer therapies: where they stand.</a> Cell Death and Diseases. 3, 411 (2012).</li><li>Martin, M., Akhmanova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coming+into+Focus:+Mechanisms+of+Microtubule+Minus-End+Organization.">Coming into Focus: Mechanisms of Microtubule Minus-End Organization.</a> Trends in Cell Biology. 28, 574-588 (2018).</li><li>Maiato, H., Logarinho, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+spindle+multipolarity+without+centrosome+amplification.">Mitotic spindle multipolarity without centrosome amplification.</a> Nature Cell Biology. 16, 386-394 (2014).</li><li>Vitre, B. D., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosomes,+chromosome+instability+(CIN)+and+aneuploidy.">Centrosomes, chromosome instability (CIN) and aneuploidy.</a> Current Opinion in Cell Biology. 24, 809-815 (2012).</li><li>Godinho, S. A., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+and+consequences+of+centrosome+abnormalities+in+cancer.">Causes and consequences of centrosome abnormalities in cancer.</a> Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 369, (2014).</li><li>Navarro-Serer, B., Childers, E. P., Hermance, N. M., Mercadante, D., Manning, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aurora+A+inhibition+limits+centrosome+clustering+and+promotes+mitotic+catastrophe+in+cells+with+supernumerary+centrosomes.">Aurora A inhibition limits centrosome clustering and promotes mitotic catastrophe in cells with supernumerary centrosomes.</a> Oncotarget. 10, 1649-1659 (2019).</li><li>Conte, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TACC1-chTOG-Aurora+A+protein+complex+in+breast+cancer.">TACC1-chTOG-Aurora A protein complex in breast cancer.</a> Oncogene. 22, 8102-8116 (2003).</li><li>Schumacher, J. M., Ashcroft, N., Donovan, P. J., Golden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+conserved+centrosomal+kinase,+AIR-1,+is+required+for+accurate+cell+cycle+progression+and+segregation+of+developmental+factors+in+Caenorhabditis+elegans+embryos.">A highly conserved centrosomal kinase, AIR-1, is required for accurate cell cycle progression and segregation of developmental factors in Caenorhabditis elegans embryos.</a> Development. 125, 4391-4402 (1998).</li><li>Asteriti, I. A., Giubettini, M., Lavia, P., Guarguaglini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aurora-A+inactivation+causes+mitotic+spindle+pole+fragmentation+by+unbalancing+microtubule-generated+forces.">Aurora-A inactivation causes mitotic spindle pole fragmentation by unbalancing microtubule-generated forces.</a> Molecular Cancer. 10, 131 (2011).</li><li>Chan, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clinical+overview+of+centrosome+amplification+in+human+cancers.">A clinical overview of centrosome amplification in human cancers.</a> International Journal of Biological Sciences. 7, 1122-1144 (2011).</li><li>Kramer, A., Maier, B., Bartek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+clustering+and+chromosomal+(in)stability:+a+matter+of+life+and+death.">Centrosome clustering and chromosomal (in)stability: a matter of life and death.</a> Molecular Oncology. 5, 324-335 (2011).</li><li>Ganem, N. J., Godinho, S. A., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+linking+extra+centrosomes+to+chromosomal+instability.">A mechanism linking extra centrosomes to chromosomal instability.</a> Nature. 460, 278-282 (2009).</li><li>Silkworth, W. T., Nardi, I. K., Scholl, L. M., Cimini, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipolar+spindle+pole+coalescence+is+a+major+source+of+kinetochore+mis-attachment+and+chromosome+mis-segregation+in+cancer+cells.">Multipolar spindle pole coalescence is a major source of kinetochore mis-attachment and chromosome mis-segregation in cancer cells.</a> PLoS One. 4, e6564 (2009).</li><li>Nigg, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+aberrations:+cause+or+consequence+of+cancer+progression?.">Centrosome aberrations: cause or consequence of cancer progression?.</a> Nature reviews, Cancer. 2, 815-825 (2002).</li><li>Godinho, S. A., Kwon, M., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosomes+and+cancer:+how+cancer+cells+divide+with+too+many+centrosomes.">Centrosomes and cancer: how cancer cells divide with too many centrosomes.</a> Cancer Metastasis Reviews. 28, 85-98 (2009).</li><li>Quintyne, N. J., Reing, J. E., Hoffelder, D. R., Gollin, S. M., Saunders, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spindle+multipolarity+is+prevented+by+centrosomal+clustering.">Spindle multipolarity is prevented by centrosomal clustering.</a> Science. 307, 127-129 (2005).</li><li>Magidson, V., Khodjakov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circumventing+photodamage+in+live-cell+microscopy.">Circumventing photodamage in live-cell microscopy.</a> Methods in Cell Biology. 114, 545-560 (2013).</li></ol>

The temporal resolution provided by the time-lapse imaging allows for the visualization and assessment of sequential cellular events within single cells. Approaches that make use of cellular synchronization followed by the collection and fixation of cells at sequential time points are limited in that comparisons are ultimately made between populations of cells. In contexts where the cellular response to perturbations may be non-uniform, or where the process being visualized is dynamic, live cell time-lapse imaging is bet...

Image-based analysis of fixed cells is commonly used to assess the cell population level changes in response to various perturbations. When combined with cell synchronization, followed by the collection and imaging of serial time points, such approaches can be used to suggest a cellular sequence of events. Nevertheless, fixed cell imaging is limited in that temporal relationships are implied for a population and not demonstrated at the level of individual cells. In this way, while fixed cell imaging and analysis is sufficient to observe robust phenotypes and steady-state changes, the ability to detect transient changes over time and changes that impact only a subpopulation of the cells is imperfect. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular and subcellular processes within a single cell, or cellular population, over time and without the aid of synchronization approaches that may themselves impact cellular behavior1,2,3,4,5,6.
The formation of a bipolar mitotic spindle is essential for the proper chromosome segregation during cell division, resulting in two genetically identical daughter cells. Defects in mitotic spindle structure that corrupt mitotic progression and compromise the fidelity of chromosome segregation can result in catastrophic cell divisions and reduced cell viability. For this reason, mitotic poisons that alter spindle formation are promising therapeutics to limit the rapid proliferation of cancer cells7,8,9. Nevertheless, fixed cell analysis of spindle structure following the addition of mitotic poisons is limited in its ability to assess the dynamic process of spindle formation and may not indicate whether observed changes in spindle structure are permanent or are instead transient and may be overcome to permit successful cell division.
In this protocol, we describe an approach to assess the dynamics of mitosis following spindle perturbations by live cell imaging. Using the hTERT immortalized RPE-1 cell line engineered to express an RFP-tagged Histone 2B to visualize chromatin, together with an EGFP-tagged &#945;-tubulin to visualize microtubules, the timing of metaphase chromosome alignment, anaphase onset, and ultimately mitotic cell fate are assessed using visual cues of chromosome movement, compaction, and nuclear morphology.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin</td>
			<td>Gibo-Life sciences</td>
			<td>25-510</td>
			<td>A serine protease used to release adherent cells from culture dishes</td>
		</tr>
		<tr>
			<td>15ml centrifuge tubes</td>
			<td>Olympus Plastics</td>
			<td>28-101</td>
			<td></td>
		</tr>
		<tr>
			<td>20x CFI Plan Fluor objective&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td>For use in Live-cell imaging to visualize both bright field and fluorescence</td>
		</tr>
		<tr>
			<td>293T Cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td>For use in retroviral transfection; used in step 1.1</td>
		</tr>
		<tr>
			<td>a-tubulin-EGFP&#160;</td>
			<td>Addgene</td>
			<td>various numbers</td>
			<td>Expression vector for alpha tubulin fused to a green fluorescent protein tag; for use in the visualization of tubulin in live-cell imaging: commercially available through addgene and other vendors</td>
		</tr>
		<tr>
			<td>Alisertib</td>
			<td>Selleckchem</td>
			<td>S1133</td>
			<td>Small molecule inhibitor of the mitotic kinase Aurora A. Stock concentration is prepared at 10mM in DMSO, and used at a final concentration of 100nM.</td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Invitrogen</td>
			<td>A11139-03</td>
			<td>Antibiotic selection agent; used to select for a-tub-EGFP expressing cells</td>
		</tr>
		<tr>
			<td>C02</td>
			<td>Airgas</td>
			<td></td>
			<td>For use in cell culture and live cell imaging</td>
		</tr>
		<tr>
			<td>Chroma ET-DS Red (TRITC/Cy3)</td>
			<td>Chroma</td>
			<td>49005</td>
			<td>Single band filter set; excitation wavelength 545nm with 25nm bandwidth and emission at 605nm wavelength with 70nm bandwidth; for visualization of H2B-RFP</td>
		</tr>
		<tr>
			<td>Chroma ET-EGFP (FITC/Cy2)</td>
			<td>Chroma</td>
			<td>49002</td>
			<td>Single band filter set; excitation wavelength 470nm with 40nm bandwidth and emission at 525nm wavelength with 50nm bandwidth; for visualization of GFP-tubulin</td>
		</tr>
		<tr>
			<td>disposable glass Pastuer pipets, sterilized&#160;</td>
			<td>Fisher Scientific</td>
			<td>13-678-6A</td>
			<td>For use in aspirating cells&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Gibo-Life sciences</td>
			<td>11965-084</td>
			<td>Cell culture medium for growth of RPE-1 and 293T cells</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibo-Life sciences</td>
			<td>10438-026</td>
			<td>Cell culture medium supplement</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 and p3000</td>
			<td>Invitrogen</td>
			<td>L3000-015</td>
			<td>Lipid based transfection reagent for transfection of plasmids; used in 1.1.4</td>
		</tr>
		<tr>
			<td>Multi well Tissue Culture dishes</td>
			<td>Corning</td>
			<td>various</td>
			<td>for use in cell culture, transfection/infection, and live cell imaging</td>
		</tr>
		<tr>
			<td>Nikon Ti-E microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Inverted epifluorescence microscope for use in live-cell imaging</td>
		</tr>
		<tr>
			<td>NIS Elements HC&#160;</td>
			<td>Nikon</td>
			<td>Version 4.51</td>
			<td>Image acquisition and analysis software; used in sections 3 &#38; 4</td>
		</tr>
		<tr>
			<td>OPTI-MEM</td>
			<td>Gibo-Life sciences</td>
			<td>31985-070</td>
			<td>Reduced serum medium for cell transfection; used in step 1.1.3</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin&#160;</td>
			<td>Gibo-Life sciences</td>
			<td>15140-122</td>
			<td>antibiotic used in cell culture medium</td>
		</tr>
		<tr>
			<td>phosphate bufferred saline (PBS)</td>
			<td>Caisson labs</td>
			<td>PBP06-10X1LT</td>
			<td>sterile saline solution for use with cell culture</td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td>Lentiviral VSV-G envelope expression construct; used in step 1.1.4</td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma-Aldrich</td>
			<td>H9268</td>
			<td>Cationic polymer used to enhane viral infection efficiency; used in step 1.1.10</td>
		</tr>
		<tr>
			<td>psPAX</td>
			<td>Addgene</td>
			<td>12260</td>
			<td>2nd generation lentiviral packaging plasmid; used in step 1.1.4</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Invitrogen</td>
			<td>ant-pr-1</td>
			<td>Antibiotic selection agent; used to select for RFP-H2B expressing cells</td>
		</tr>
		<tr>
			<td>RFP- Histone 2B (H2B)</td>
			<td>Addgene</td>
			<td>various numbers</td>
			<td>Expression vector for red fluorescent protein-tagged histone 2B; for use in the visualization of chromatin in live-cell imaging: commercially available through Addgene and other vendors</td>
		</tr>
		<tr>
			<td>RNAi Max</td>
			<td>Invitrogen</td>
			<td>13778-150</td>
			<td>Lipid based transfection reagent for transfection of siRNA constructs</td>
		</tr>
		<tr>
			<td>RPE-1 cells</td>
			<td>ATCC</td>
			<td>CRL-4000</td>
			<td>Human retinal pigment epithelial cell line</td>
		</tr>
		<tr>
			<td>Tissue culture dish 100x20mm</td>
			<td>Corning&#160;</td>
			<td>353003</td>
			<td>for use in culturing adherent cells</td>
		</tr>
		<tr>
			<td>Zyla sCMOS camera&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td>Camera attached to the micrscope, used for capturing images of cells</td>
		</tr>
	</tbody>
</table>

live cell imaging to assess the dynamics of metaphase timing and cell fate following mitotic spindle perturbations

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identification&#160;and tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Assessment of mitotic progression in the presence of spindle perturbations 
The regulation of spindle pole focusing is an essential step in proper bipolar spindle formation. Disruption in this process through protein depletions, drug inhibition, or alterations in centrosome number corrupt spindle structure and delay or halt mitotic progression10,11,12,<sup class="...

Watch this Scientific Journal Video about Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations at JoVE.com

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, ...

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