Name: live cell imaging of chromosome segregation during mitosis
Uploaded: 2018-03-14T15:00:00
Description: Watch this Scientific Journal Video about Live Cell Imaging of Chromosome Segregation During Mitosis at JoVE.com

Nous tenons à remercier Dr Guo-Chiuan Hung et m. Bharatkumar Joshi de précieux commentaires qui ont amélioré le manuscrit.

Toutes les expériences menées dans ces études ont été effectuées conformément aux protocoles approuvés par le Comité de l’emploi et d’institutionnels animalier dans le centre de recherche de Food and Drug Administration (FDA).
1. isolement et Culture de fibroblastes embryonnaires de souris (MEFs)
<ol><li>Isoler les fibroblastes embryonnaires de souris (MEFs) d’une souche de PP2A-B56γ-souris et ceux de type sauvage par le protocole standard 7,</sup...

<ol>
	<li>Funk, L. C., Zasadil, L. M., Weaver, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+in+CIN:+Mitotic+Infidelity+and+Its+Consequences+for+Tumor+Promotion+and+Suppression.">Living in CIN: Mitotic Infidelity and Its Consequences for Tumor Promotion and Suppression.</a> Dev Cell. 39, 638-652 (2016).</li><li>Etemad, B., Kops, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+issues:+kinetochore+transformations+and+spindle+checkpoint+silencing.">Attachment issues: kinetochore transformations and spindle checkpoint silencing.</a> Curr Opin Cell Biol. 39, 101-108 (2016).</li><li>Lara-Gonzalez, P., Westhorpe, F. G., Taylor, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spindle+assembly+checkpoint.">The spindle assembly checkpoint.</a> Curr Biol. 22, 966-980 (2012).</li><li>Jallepalli, P. V., Lengauer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+segregation+and+cancer:+cutting+through+the+mystery.">Chromosome segregation and cancer: cutting through the mystery.</a> Nat Rev Cancer. (2), 109-117 (2001).</li><li>Zhu, L., 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=Mitotic+protein+CSPP1+interacts+with+CENP-H+protein+to+coordinate+accurate+chromosome+oscillation+in+mitosis.">Mitotic protein CSPP1 interacts with CENP-H protein to coordinate accurate chromosome oscillation in mitosis.</a> J Biol Chem. 290 (45), 27053-27066 (2015).</li><li>Pikor, L., Thu, K., Vucic, E., Lam, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+detection+and+implication+of+genome+instability+in+cancer.">The detection and implication of genome instability in cancer.</a> Cancer Metastasis Rev. 32 (3-4), 341-352 (2013).</li><li>Varadkar, P., Despres, D., Kraman, M., Lozier, J., Phadke, A., Nagaraju, K., McCright, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+phosphatase+2A+B56&#947;+regulatory+subunit+is+required+for+heart+development.">The protein phosphatase 2A B56&#947; regulatory subunit is required for heart development.</a> Dev Dyn. 243 (6), 778-790 (2014).</li><li>Jozefczuk, J., Drews, K., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Mouse+Embryonic+Fibroblast+Cells+Suitable+for+Culturing+Human+Embryonic+and+Induced+Pluripotent+Stem+Cells.">Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells.</a> J. Vis. Exp. (64), e3854 (2012).</li><li>Varadkar, P., Abbasi, F., Takeda, K., Dyson, J. J., McCright, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PP2A-B56&#947;+is+required+for+an+efficient+spindle+assembly+checkpoint.">PP2A-B56&#947; is required for an efficient spindle assembly checkpoint.</a> Cell Cycle. 18 (12), 1210-1219 (2017).</li><li>Rosner, M., Schipany, K., Hengstschl&#228;ger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merging+high-quality+biochemical+fractionation+with+a+refined+flow+cytometry+approach+to+monitor+nucleocytoplasmic+protein+expression+throughout+the+unperturbed+mammalian+cell+cycle.">Merging high-quality biochemical fractionation with a refined flow cytometry approach to monitor nucleocytoplasmic protein expression throughout the unperturbed mammalian cell cycle.</a> Nat Protoc. 8 (3), 602-626 (2013).</li><li>Jonkman, J., Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Any+Way+You+Slice+It-A+Comparison+of+Confocal+Microscopy+Techniques.">Any Way You Slice It-A Comparison of Confocal Microscopy Techniques.</a> J Biomol Tech. 26 (2), 54-65 (2015).</li><li>Versaevel, M., Braquenier, J. B., Riaz, M., Grevesse, T., Lantoine, J., Gabriele, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+reveals+LINC+complex+recruitment+at+nuclear+indentation+sites.">Super-resolution microscopy reveals LINC complex recruitment at nuclear indentation sites.</a> Sci Rep. 8 (4), 7362 (2014).</li><li>Wild, T., Larsen, M. S., Narita, T., Schou, J., Nilsson, J., Choudhary, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Spindle+Assembly+Checkpoint+Is+Not+Essential+for+Viability+of+Human+Cells+with+Genetically+Lowered+APC/C+Activity.">The Spindle Assembly Checkpoint Is Not Essential for Viability of Human Cells with Genetically Lowered APC/C Activity.</a> Cell Rep. 1 (8), 1829-1840 (2016).</li><li>Iuso, D., 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=Exogenous+Expression+of+Human+Protamine+1+(hPrm1)+Remodels+Fibroblast+Nuclei+into+Spermatid-like+Structures.">Exogenous Expression of Human Protamine 1 (hPrm1) Remodels Fibroblast Nuclei into Spermatid-like Structures.</a> Cell Rep. 1 (9), 1765-1771 (2015).</li><li>Ratcliffe, E., Glen, K. E., Naing, M. W., Williams, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+and+perspectives+on+stem+cell-based+therapies+undergoing+clinical+trials+for+regenerative+medicine:+case+studies.">Current status and perspectives on stem cell-based therapies undergoing clinical trials for regenerative medicine: case studies.</a> Br Med Bull. 108, 73-94 (2013).</li></ol>

Points de contrôle de contrôle cycle cellulaire qui assurent la ségrégation des chromosomes précis empêchent aneuploïdie et cell transformation 1,2,3. Dans la présente étude, nous avons trouvé que l’inactivation de PP2A-B56γ a donné lieu à un poste de contrôle de l’Assemblée broche affaibli. L’imagerie de cellules vivantes nous a permis d’observer la mis-ségrégation chromosomique au cours de la mitose ch...

Dans le protocole suivant, nous décrivons une méthode pratique pour visualiser la ségrégation chromosomique et la progression mitotique au cours du cycle cellulaire dans les fibroblastes embryonnaires de souris à l’aide d’Histone 2 b-GFP, marquage BacMam 2.0 et l’imagerie de cellules vivantes.
Points de contrôle de cycle cellulaire surveillent la ségrégation des chromosomes et jouent un rôle important dans le maintien de l’intégrité génétique de la cellule 1,2,3. Accumulation de mauvaise ségrégation chromosomes peut conduire à aneuploïdie, qui est une caractéristique de plus solides tumeurs 4. Par conséquent, détection des chromosomes traînards utilisable comme une méthode pour étudier l’instabilité chromosomique.
Fluorescent étiqueté protéines peuvent être utilisé pour visualiser la ségrégation des chromosomes vivants et chromosomiques en retard mais la génération de mCherry-tag ou protéine H2B-GFP Taggé nécessite des connaissances considérables de gène de biologie moléculaire et la livraison 5. Nous décrivons ici l’utilisation du réactif de CellLight Histone2B-GFP BacMam 2.0, ci-après appelé regent CL-HB, par souci de simplicité. Ce réactif peut être utilisé immédiatement et élimine donc les préoccupations concernant la qualité de vecteur et de l’intégrité. En outre, ce réactif ne nécessite pas l’utilisation des traitements potentiellement dangereux ou des lipides et des produits chimiques de colorant-chargement. À la différence des étiquettes fluorescentes conventionnelles, le régent de CL-HB taches indépendamment de fonction (i.e., potentiel membranaire). Le régent de CL-HB peut être simplement ajouté aux cellules et incubé durant une nuit à l’expression de la protéine. Le régent de CL-HB ne se réplique pas dans les cellules de mammifères et sont utilisables dans les paramètres de niveau 1 laboratoire (BSL) de prévention des risques biotechnologiques. Aussi, cette transfection transitoire peut être détectée après une nuit d’incubation jusqu'à 5 jours, suffisamment de temps pour procéder à des analyses cellulaires plus dynamiques.
Par ailleurs, les anomalies chromosomiques pourraient être étudiées par différentes techniques telles que la cytométrie en flux, immunohistochimie ou fluorescence in situ hybridation (poisson) 6. Cytométrie en flux peut être utilisée pour étudier l’aneuploïdie, qui peut être mesurée basée sur le contenu en ADN et la phase de cellules dans le cycle cellulaire. Bien que la cytométrie en flux peut être utilisée pour mesurer l’aneuploïdie, il ne fournit pas les informations sur la ségrégation chromosomique mauvaise. Techniques de poisson et l’immunohistochimie utilisent des sondes fluorescentes pour lier à l’ADN ou des chromosomes. Bien que ces techniques fournissent un instantané de l’état d’une population de cellules, ils ne permettent pas de cellules vivantes d’imagerie manquant ainsi toute information obtenue par le biais de visualisation spatio-temporelle de la cytokinèse dans certaines cellules suivies sur une période de temps.
Ce protocole a été utilisé pour étudier les chromosomes retardataires ou chromosomique mauvaise ségrégation dans la nocodazole traité fibroblastes embryonnaires de souris (MEFs) isolés de PP2A-B56γ-souris. Ce protocole prévoit, en plus de demande, un outil simple pour étiqueter et visualiser la ségrégation chromosomique dans divers types de cellules qui permet d’étudier la régulation du cycle cellulaire ou une instabilité chromosomique dans les cellules tumorales. En outre, il peut également être utilisé pour étudier l’instabilité chromosomique causée par différents traitements médicamenteux ou à étudier les effets du gène assommer résultant de la mis ségrégation chromosomique.

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			<td height="21" style="height:21px;width:191px;">DMEM/F12</td>
			<td style="width:135px;">Gibco</td>
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			<td style="width:191px;"></td>
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			<td height="21" style="height:21px;">L-Glutamine</td>
			<td>Gibco</td>
			<td align="right">25030081</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:191px;">MEM Non-Essential amino acids solution (100X)</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">11140050</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">15140122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Dulbecco&#8217;s Phosphate Buffered saline</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">14190144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Trypsin-EDTA</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Fetal bovine serum</td>
			<td style="width:135px;">Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td style="width:135px;">EMD Millipore</td>
			<td>MX 1458-6</td>
			<td></td>
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			<td height="21" style="height:21px;">Cryogenic vial storage boxes</td>
			<td style="width:135px;">Fisherbrand</td>
			<td style="width:128px;">10-500-28</td>
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			<td height="21" style="height:21px;width:191px;">Cryogenic vials</td>
			<td style="width:135px;">Corning/costar</td>
			<td align="right">431416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">T75 flasks</td>
			<td style="width:135px;">Cellstar</td>
			<td align="right" style="width:128px;">658175</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">2 well chambered cover glass</td>
			<td style="width:135px;">Nunc</td>
			<td style="width:128px;">155380PK</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Cellometer Vision Cell Profiler</td>
			<td style="width:135px;">Nexcelom Bioscience LLC</td>
			<td style="width:128px;">Cellometer Vision Trio</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Nocodazole</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:128px;">M1404</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">CellLight Histone 2B-GFP, BacMam 2.0</td>
			<td style="width:135px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:128px;">C10594</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:191px;">Zeiss Cell Observer Spinning Disk Confocal Microscope system</td>
			<td style="width:135px;">Carl Zeiss Microscopy</td>
			<td style="width:128px;">Zeiss Cell Observer SD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Water Bath</td>
			<td style="width:135px;">Thermoscientific</td>
			<td style="width:128px;">280 series</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Incubator</td>
			<td style="width:135px;">Sanyo commercial solutions</td>
			<td style="width:128px;">MCO-18AIC (UV)</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Class II Biological safety cabinet</td>
			<td style="width:135px;">The Baker Company</td>
			<td style="width:128px;">SterilGard</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Axiovision software</td>
			<td style="width:135px;">Zeiss</td>
			<td style="width:128px;">Ver.4.8.2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">ImageJ software</td>
			<td style="width:135px;">National Institute of Health</td>
			<td style="width:128px;">Ver. 1.51r</td>
			<td></td>
		</tr>
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live cell imaging of chromosome segregation during mitosis

Chromosomes doivent être fiable et uniforme divisés en cellules filles pendant la division cellulaire mitotique. Fidélité de la ségrégation chromosomique est contrôlée par les mécanismes multiples qui incluent le Checkpoint de montage broche (SAC). Le SAC fait partie d’un système de rétroaction complexe qui est responsable de la prévention d’un progrès de cellulaire par mitose, à moins que tous les kinétochores chromosomiques ont joint à la broche de microtubules. Calorifugeage chromosomique et chromosome anormal est un indicateur des postes de contrôle dysfonctionnel cycle cellulaire de la ségrégation et peut être utilisée pour mesurer la stabilité génomique des cellules en division. Déréglementation du SAC peut entraîner la transformation d’une cellule normale en une cellule maligne à travers l’accumulation des erreurs au cours de la ségrégation chromosomique. Mise en œuvre du SAC et la formation du kinétochore complexe sont étroitement contrôlée par les interactions entre les kinases et phosphatases tels que de la protéine Phosphatase 2 a (PP2A). Ce protocole décrit l’imagerie de cellules vivantes des chromosomes dans les fibroblastes embryonnaires de souris isolés de souris qui avaient un masquage de la sous-unité régulatrice PP2A-B56γ à la traîne. Cette méthode permet de surmonter les défauts des autres cell cycle control techniques d’imagerie comme cytométrie ou immunocytochimie seulement présentent un portrait de cellulaire cytocinèse, au lieu d’une visualisation dynamique spatio-temporelle des chromosomes au cours de la mitose.

MEFs de type sauvage et PP2A-B56γ-souris ont été ensemencées dans une chambre de 2 puits couvercle en verre et autorisés à fixer. Jour 2, MEFs ont été synchronisées à l’aide de 0,1 % FBS pendant 24 h. Jour 3, MEFs dans les médias, avec 200 ng/mL nocodazole et 30 CL de PPC-HB regentwere incubés pendant 18 h à 37° C et 5 % CO2. Jour 4, les cellules ont été projetés en utilisant un système de microscope confocal de disque de rotation (<strong class="...

Watch this Scientific Journal Video about Live Cell Imaging of Chromosome Segregation During Mitosis at JoVE.com

Live Cell Imaging of Chromosome Segregation During Mitosis

Ce protocole décrit une méthode simple et pratique pour étiqueter et visualiser direct chromosomes dans les cellules mitotiques en utilisant l’étiquette Histone2B-GFP BacMam 2.0 et un système de microscopie confocale tourne disque.

Imagerie de cellules vivantes de la ségrégation des chromosomes pendant la mitose

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

The overall goal of this method is to label and visualize live chromosomes in mitotic cells using Histone 2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system. This method can be used to visualize mitotic chromosomes in cultured cells to study the effects of genetic modifications or drug treatments on chromosome segregation. The main advantage of this technique is that it provides an easy and convenient method to label and visualize live chromosomes in mitotic cells.Demonstrating the live imaging procedure will be Doctor Kazuyo Takeda who maintains the Microscopy and Imaging Core Facility in the Division of Viral Products at the FDA Center for Biologics Evaluation and Research. The mouse embryonic fibroblasts, or MEFs, used in this experiment are grown in a two-well chambered cover glass at 37 degrees Celsius and 5%CO2 overnight, as described in the text protocol. On the following day, synchronize the MEFs in G0/G1 phase by incubating in DMEM/F12 growth medium containing 0.1%FBS.On the day of labeling, prepare a 1.5 milligram per milliliter stock solution of nocodazole in DMSO. A critical step is to calculate the right amount of CL-HB particles per cells to achieve enough labeling for chromosome visualization without toxicity to cells. We have previously determined, by titration experiments, that 30 PPC works best.Add to the MEFs, 200 microliters of growth medium with 10%FCS, 200 nanograms per milliliter of nocodazole, and CL-HB reagent at 30 particles per cells. Incubate the MEFs at 37 degrees Celsius and 5%CO2 for 18 hours. Visualization of the MEFs is accomplished using a spinning disk confocal microscope system equipped with an environmental chamber and an oil immersion, 63X objective lens.One day before imaging, turn the environmental chamber power on and warm up the entire chamber at 37 degrees Celsius overnight. On the following day, turn the power on for the microscope stand, camera, spinning disk unit, illuminator, argon laser, computer, and motorized stage. After letting the system warm up for three minutes, start the argon laser by turning on the ignition key.Switch the toggle switch for the argon laser from standby to laser run. Launch the data acquisition and processing software. Initiate the CO2 controller for the stage top incubator, and set the concentration of CO2 at 5%Remove the chambered cover glass from the incubator, place it on the microscope stage, and select the oil immersion 63X objective lens.View through the ocular lenses, focus the image, and identify a cell in the stage of nuclear envelope breakdown, or NEBD. Initiate the 488-nanometer argon laser to visualize Histone 2B-GFP. Open the acquisition control window, and set exposure time for the GFP channel.Identify a cell that is in NEBD. Then, manually determine the cell's top and bottom focal plane, and enter the XYZ optical sectioning settings. Observe the cell for 20 minutes.It is important to be aware that since not all cells will go through mitosis, mitotic progress must be actively monitored. If the cell does not proceed through cell division after 20 minutes, stop image acquisition and move on to the next cell that is in NEBD. To obtain data for a movie, take pictures every three minutes.Approximately one hour per cell is needed to image lagging chromosomes during mitosis in PP2A-B56-gamma cells that escaped from the spindle assembly checkpoint. Save images in ZVI file format for further analysis. Live cell imaging was used to visualize the fate of individual cells as they progressed from NEBD through mitosis.Post-nocodazole treatment, all 21 wild-type cells observed were arrested at metaphase. In contrast, from the 21 cells lacking PP2A-B56-gamma observed, 16 were able to go through mitosis after nocodazole treatment. In addition, abnormal chromosomal segregation was detected in 13 of those MEFs.An example of a PP2A-B56-gamma cell undergoing chromosomal mis-segregation is shown in this video clip. Once mastered, this technique will take three to four days depending on the cell treatment prior to the labeling protocol. It is important to remember that this is a transient transfection and signals can be detected only up to five days.This protocol can be used to answer additional questions related to the cell cycle regulation, genomic instability, and the quality of the cell therapy products. After its development, this technique could be used to study live imaging of chromosomes in a wide variety of cell lines, such as primary cell lines, stem cells, neurons, and immortalized T-cells. After watching this video, you should have a good understanding of how to easily label and visualize live chromosomes in mitotic cells using Histone 2B-GFP BacMam 2 label and a spinning disc confocal microscopy system.

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Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Live-imagerie de la<em&gt; Drosophila</em&gt; Nymphe yeux

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

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Biologie du développement

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

Analyse de la ségrégation chromosomique, de l&#39;acétone d&#39;histone et de la morphologie de la broche dans les ovocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

Culture des adultes Zebrafish Retinal explants Transgenic pour des cellules vivantes par imagerie multiphotonique Microscopie

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Bioingénierie des microenvironnements humanisés de la moelle osseuse dans la souris et leur visualisation par imagerie en direct

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In Vitro Culture de l’ovule pour vivre-cellule d’imagerie du Zygote polarisation et structuration d’embryon chez Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Imagerie en direct de la souris Fusion de palais secondaire

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Imagerie en temps réel de bioluminescence de la dynamique de signalisation d'encoche pendant la neurogenèse de Murine

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in molecular and imaging technologies allowed us to gain insights into how biochemical signals influence morphogenetic events. However, it is increasingly evident that apart from biochemical signals, mechanical cues also impact several aspects of cell and tissue growth. The Arabidopsis shoot apical meristem (SAM) is a dome-shaped structure responsible for the generation of all aboveground organs. The organization of the cortical microtubule cytoskeleton that mediates apoplastic cellulose deposition in plant cells is spatially distinct. Visualization and quantitative assessment of patterns of cortical microtubules are necessary for understanding the biophysical nature of cells at the SAM, as cellulose is the stiffest component of the plant cell wall. The stereotypical form of cortical microtubule organization is also a consequence of tissue-wide physical forces existing at the SAM. Perturbation of these physical forces and subsequent monitoring of cortical microtubule organization allows for the identification of candidate proteins involved in mediating mechano-perception and transduction. Here we describe a protocol that helps investigate such processes.

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Here we describe a protocol for live cell imaging of the cortical microtubule cytoskeleton at the shoot apical meristem and monitoring its response to changes in physical forces.

Imagerie cellulaire en direct du cytosquelette microtubule et manipulation micromécanique de l’Arabidopsis Shoot Apical Meristem

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in ...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actinRed) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actinRed polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actinRed into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

Imagerie Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos Imaging Intra

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

Dissection et imagerie en direct de la drosophile embryonnaire tardive Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...