Name: adenofection: a method for studying the role of molecular chaperones in cellular morphodynamics by depletion-rescue experiments
Uploaded: 2016-09-16T15:00:00
Description: Watch this Scientific Journal Video about Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments at JoVE.com

This work was supported by the Canadian Institutes of Health Research (Grant no 7077), and by the Bellini Foundation and Roby Fondazione.

1. Preparation of Medium and Solutions (all sterile filtered)
<ol>
	<li>C2C12 mouse muscle cells (differentiation studies)
	<ol>
		<li>Prepare 500 ml of Growth Medium for C2C12 cell culture maintenance: DMEM High Glucose supplemented with 10 % FBS and 2mM L-Glutamine.</li>
		<li>Prepare 100 ml Differentiation Medium (DM) for C2C12 differentiation: DMEM High Glucose supplemented with 2% Horse Serum.</li>
	</ol>
	</li>
	<li>HeLa cells (studies in mitotic cells)
	<ol>
		<li>Prepare 500 ml...

<ol>
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A., 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=Caveolin-1+associated+adenovirus+entry+into+human+corneal+cells.">Caveolin-1 associated adenovirus entry into human corneal cells.</a> PLoS One. 8 (10), e77462 (2013).</li><li>Morton, P. E., Hicks, A., Nastos, T., Santis, G., Parsons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAR+regulates+epithelial+cell+junction+stability+through+control+of+E-cadherin+trafficking.">CAR regulates epithelial cell junction stability through control of E-cadherin trafficking.</a> Sci Rep. 3, 2889 (2013).</li><li>Carra, S., Seguin, S. J., Lambert, H., Landry, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HspB8+chaperone+activity+toward+poly(Q)-containing+proteins+depends+on+its+association+with+Bag3,+a+stimulator+of+macroautophagy.">HspB8 chaperone activity toward poly(Q)-containing proteins depends on its association with Bag3, a stimulator of macroautophagy.</a> J Biol Chem. 283 (3), 1437-1444 (2008).</li><li>Fuchs, 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=Identification+of+the+key+structural+motifs+involved+in+HspB8/HspB6-Bag3+interaction.">Identification of the key structural motifs involved in HspB8/HspB6-Bag3 interaction.</a> Biochem J. 425 (1), 245-255 (2010).</li><li>Rosati, A., Graziano, V., De Laurenzi, V., Pascale, M., Turco, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAG3:+a+multifaceted+protein+that+regulates+major+cell+pathways.">BAG3: a multifaceted protein that regulates major cell pathways.</a> Cell Death Dis. 2, e141 (2011).</li><li>Guilbert, S. M., Tanguay, R. M., Hightower, L. E., 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=.">.</a> The Big Book of Small Heat Shock Proteins. , 435-456 (2015).</li><li>Fasbender, A., 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=Complexes+of+adenovirus+with+polycationic+polymers+and+cationic+lipids+increase+the+efficiency+of+gene+transfer+in+vitro+and+in+vivo.">Complexes of adenovirus with polycationic polymers and cationic lipids increase the efficiency of gene transfer in vitro and in vivo.</a> J Biol Chem. 272 (10), 6479-6489 (1997).</li><li>Toyoda, K., 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=Cationic+polymer+and+lipids+enhance+adenovirus-mediated+gene+transfer+to+rabbit+carotid+artery.">Cationic polymer and lipids enhance adenovirus-mediated gene transfer to rabbit carotid artery.</a> Stroke. 29 (10), 2181-2188 (1998).</li><li>Fasbender, A., 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=Incorporation+of+adenovirus+in+calcium+phosphate+precipitates+enhances+gene+transfer+to+airway+epithelia+in+vitro+and+in+vivo.">Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo.</a> J Clin Invest. 102 (1), 184-193 (1998).</li><li>Fuchs, 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=A+Role+for+the+Chaperone+Complex+BAG3-HSPB8+in+Actin+Dynamics,+Spindle+Orientation+and+Proper+Chromosome+Segregation+during+Mitosis.">A Role for the Chaperone Complex BAG3-HSPB8 in Actin Dynamics, Spindle Orientation and Proper Chromosome Segregation during Mitosis.</a> PLoS Genetics. 11 (10), e1005582 (2015).</li><li>Champagne, C., Landry, M. C., Gingras, M. C., Lavoie, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+adenovirus+type+2+early+region+4+ORF4+cytoplasmic+death+function+by+direct+binding+to+Src+kinase+domain.">Activation of adenovirus type 2 early region 4 ORF4 cytoplasmic death function by direct binding to Src kinase domain.</a> J Biol Chem. 279 (24), 25905-25915 (2004).</li><li>Riedl, J., 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=Lifeact:+a+versatile+marker+to+visualize+F-actin.">Lifeact: a versatile marker to visualize F-actin.</a> Nat Methods. 5 (7), 605-607 (2008).</li><li>Takahashi, A., 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=Myogenic+Akt+signaling+regulates+blood+vessel+recruitment+during+myofiber+growth.">Myogenic Akt signaling regulates blood vessel recruitment during myofiber growth.</a> Mol Cell Biol. 22 (13), 4803-4814 (2002).</li><li>Murray, T. V., 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=A+non-apoptotic+role+for+caspase-9+in+muscle+differentiation.">A non-apoptotic role for caspase-9 in muscle differentiation.</a> J Cell Sci. 121 (Pt 22), 3786-3793 (2008).</li><li>Terada, K., Misao, S., Katase, N., Nishimatsu, S., Nohno, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+Wnt+Signaling+with+BMP/Smad+Signaling+during+the+Transition+from+Cell+Proliferation+to+Myogenic+Differentiation+in+Mouse+Myoblast-Derived+Cells).">Interaction of Wnt Signaling with BMP/Smad Signaling during the Transition from Cell Proliferation to Myogenic Differentiation in Mouse Myoblast-Derived Cells).</a> Int J Cell Biol. 2013, 616294 (2013).</li><li>Hindi, S. M., Tajrishi, M. M., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+mechanisms+in+mammalian+myoblast+fusion.">Signaling mechanisms in mammalian myoblast fusion.</a> Sci Signal. 6 (272), re2 (2013).</li><li>Klionsky, D. J., 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=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+autophagy+(3rd+edition).">Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).</a> Autophagy. 12 (1), 1-222 (2016).</li></ol>

Here, we described a method enabling depletion-rescue experiments to be performed, which is applicable to functional analyses of cell biological processes that are particularly sensitive to overexpression of proteins affecting the stoichiometry and dynamics of protein complexes and macromolecular structures. Mitotic cell division is an extreme example of finely tuned cell morphodynamics that involves the most dramatic and spectacular changes in the overall structure of a cell. Using adenofection combined with commerciall...

Functional inactivation of gene expression in mammalian cells is the gold standard to dissect protein functions. Newly developed technologies of genome editing based on the use of site-specific nucleases such as Zinc-finger nucleases and clustered regularly interspaced short palindromic repeats (CRISPR)/CAS9 now allow the generation of cell lines with targeted gene deletion and mutation1,2. These novel approaches should revolutionize the way we are studying protein function and our understanding of the genetics of human diseases. In some instances, however, long-term or complete gene knockout is not desirable and may provoke secondary cell compensation mechanisms. The generation of genetically modified cell lines can also be limiting when dealing with primary cell cultures with limited proliferation capacity, or when screening of a large set of mutations in various cell types is sought. This is often required for determining the dependence of a cell biological process on structural requirements of a protein. To that end, reversible knockdown by RNA interference that enables transient depletion-rescue experiments in various cellular backgrounds still remains a simple and powerful approach to perform structure-function analyses of a protein of interest3. However, a major drawback to this approach is the difficulty to achieve efficient silencing and to reintroduce the protein of interest or its variants at near physiological levels in a majority of the cell population. This is crucial to enable comprehensive studies that attempt to correlate functional effects seen at the level of single cells (hypomorphic phenotype) with those seen in cell population-based assays, for instance on protein-protein interactions.
Using classic transfection methods, one can hardly achieve homogenous and low expression of exogenous proteins in a large population of cells. Transduction of cells with recombinant viruses like adenoviruses often enables more normalized expression of exogenous proteins. Yet, adenovirus uptake is limited by the CAR receptor, which is absent in non-human cells or only weakly expressed in some human cell types. Furthermore, the cellular entry of adenoviruses activates signaling pathways that regulate cell shape and adhesion4-6. This is obviously not desirable when studying regulatory mechanisms of cell morphodynamics. We were facing this problematic when we undertook functional analyses of a chaperone complex, BAG3-HSPB8, in cell division and actin dynamics. Pioneering work had described a role for this chaperone complex in protein quality control and autophagy during stress7,8. Most of these studies, however, relied on protein overexpression, assuming that the chaperones are normally upregulated during stress. This has left open the question of whether BAG3, in complex with HSPB8, can contribute to the normal operation of dividing cells expressing these chaperones like many cancer cell types9. In particular, whether the chaperone complex contributes to the remodeling of actin-based structures that control mitotic progression was of great interest, given the emerging connections between HSPB chaperones and cytoskeletal dynamics10. To address this issue, we were seeking to develop an efficient method for depletion-rescue experiments that would not interfere with mitotic progression or cellular morphology, and which would preserve protein homeostasis to avoid secondary perturbation of the dynamics of macromolecular complexes regulating cell-shape changes. Thus ideally, depletion-add-back of the gene of interest should be performed simultaneously.
The use of complexes of adenovirus with a cationic polymer or lipids has been described to promote gene transfer in vitro and in vivo11,12. For instance, calcium phosphate (CaPi) appears to form a precipitate with adenovirus that enhance virus binding-entry via a CAR-independent pathway13. Indeed, we found that combining adenovirus-based cell transduction and transfection with cationic compounds could enhance the efficiency of the depletion-rescue experiments. This allowed us to lower the amounts of virus by 3- to 20-fold, depending on the cell line and the gene of interest, and benefit from a wider window in order to adjust the expression of exogenous proteins at near endogenous levels in the majority of a cell population of interest with minimum impact on cellular morphology. Under such conditions, we could also achieve high efficiency knockdown of endogenous protein expression (&#62;75%). We hereby describe the method step by step and provide evidence that protein homeostasis is not significantly perturbed as assessed by the unchanged levels of stress-induced chaperones of the Heat Shock Protein family, making the method suitable for functional analyses of the physiological role of molecular chaperones by time-lapse video microscopy. The protocol is amenable to cell synchronization procedures and to the use of commercially available baculoviruses for co-expression of low levels of fluorescent markers, with minimum interference with normal actin-based and spindle dynamics during mitotic progression. We further show the versatility of the method, which is applicable to &#34;hard to transduce&#34; mouse C2C12 cells, with no significant impact on myoblast differentiation into myotubes in vitro.

<table border="0" cellpadding="0" cellspacing="0" width="1349">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">C2C12 Mouse Myoblasts</td>
			<td style="width:331px;">ATCC</td>
			<td style="width:192px;">CRL-1772</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Adenovirus custom design</td>
			<td style="width:331px;">Welgen</td>
			<td style="width:192px;">Custom design</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">CellLight&#174; Actin-GFP, BacMam 2.0</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">C10582</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">CellLight&#174; Tubulin-RFP, BacMam 2.0</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">C10614</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM), High Glucose</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">EDTA</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">12483-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Fibronectin</td>
			<td style="width:331px;">Sigma</td>
			<td style="width:192px;">F1141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Glass bottom dishes, 35mm</td>
			<td style="width:331px;">MatTek Corperation</td>
			<td style="width:192px;">P35G-1.5-20-C Case</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">HeLa-RFP-H2B</td>
			<td style="width:331px;">Kind gift of Dr Sabine Elowe, Qu&#233;bec, Canada</td>
			<td style="width:192px;"></td>
			<td>Klebig C et al. 2009</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">HEPES</td>
			<td style="width:331px;">Fisher Scientific</td>
			<td>BP310-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Horse Serum, New Zealand</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">16050-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">KCl</td>
			<td style="width:331px;">Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">L-Glutamine</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine&#174; RNAiMAX Transfection Reagent</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">13778-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Minimal Essential Medium&#160; (MEM) Alpha&#160;</td>
			<td style="width:331px;">Wisent</td>
			<td style="width:192px;">310-101-CL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:523px;">Minimal Essential Medium&#160; (MEM) Alpha without Desoxyribonuleosides/Ribonucleosides</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">12000-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Minimal Essential Medium&#160; (MEM) Alpha without Phenol Red</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">41061-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td style="width:331px;">Biobasic</td>
			<td style="width:192px;">S0404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">OptiMEM</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td style="width:192px;">11058-021</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:523px;">rAVCMV-LifeAct-TagGFP2</td>
			<td style="width:331px;">IBIDI</td>
			<td style="width:192px;">60121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">siRNA duplexes</td>
			<td style="width:331px;">Dharmacon</td>
			<td style="width:192px;">Custom design</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Thymidine</td>
			<td style="width:331px;">Sigma</td>
			<td style="width:192px;">T9250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Trypsine 2.5%</td>
			<td style="width:331px;">Thermo Fisher</td>
			<td>15090-046</td>
			<td></td>
		</tr>
	</tbody>
</table>

adenofection: a method for studying the role of molecular chaperones in cellular morphodynamics by depletion-rescue experiments

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell cytoskeletal structures. This involves the assembly-disassembly of higher-order macromolecular structures at a given time and location, a process that is particularly sensitive to perturbations caused by overexpression of proteins. Methods that can preserve protein homeostasis and maintain near-to-normal cellular morphology are highly desirable to determine the functional contribution of a protein of interest in a wide range of cellular processes. Transient depletion-rescue experiments based on RNA interference are powerful approaches to analyze protein functions and structural requirements. However, reintroduction of the target protein with minimum deviation from its physiological level is a real challenge. Here we describe a method termed adenofection that was developed to study the role of molecular chaperones and partners in the normal operation of dividing cells and the relationship with actin remodeling. HeLa cells were depleted of BAG3 with siRNA duplexes targeting the 3&#39;UTR region. GFP-tagged BAG3 proteins were reintroduced simultaneously into &#62;75% of the cells using recombinant adenoviruses coupled to transfection reagents. Adenofection enabled to express BAG3-GFP proteins at near physiological levels in HeLa cells depleted of BAG3, in the absence of a stress response. No effect was observed on the levels of endogenous Heat Shock Protein chaperones, the main stress-inducible regulators of protein homeostasis. Furthermore, by adding baculoviruses driving the expression of fluorescent markers at the time of cell transduction-transfection, we could dissect mitotic cell dynamics by time-lapse microscopic analyses with minimum perturbation of normal mitotic progression. Adenofection is applicable also to hard-to-infect mouse cells, and suitable for functional analyses of myoblast differentiation into myotubes. Thus adenofection provides a versatile method to perform structure-function analyses of proteins involved in sensitive biological processes that rely on higher-order cytoskeletal dynamics.

Transfection of BAG3-GFP plasmid DNA using cationic lipids was associated with heterogeneous expression in HeLa cells, some cells showing barely detectable levels of the protein and others bearing very high BAG3 levels (Figure 2A). In these cells, loss of protein homeostasis was evidenced by accumulation of BAG3-GFP into perinuclear aggregates (Figure 2A, arrows). In contrast, cell transduction with adenoviruses carrying BAG3-GFP exhibited more homogenous...

Watch this Scientific Journal Video about Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments at JoVE.com

Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments

We describe a method for depletion-rescue experiments that preserves cellular integrity and protein homeostasis. Adenofection enables functional analyses of proteins within biological processes that rely on finely tuned actin-based dynamics, such as mitotic cell division and myogenesis, at the single-cell level.

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell ...

The overall goal of this method is to enable depletion rescue experiments to be performed under conditions that preserve cellular integrity and protein homeostasis. For functional analyses of biological processes at the single cell level. This method can help answer key questions regarding protein functions and structure requirements in cell dynamics such as mitosis and cell differentiation.The main advantage of this technique is to deplete a protein of interest and reintroduce at the same time variant of this protein at neo physiological level in a measurity of a cell population. Begin by coating glass bottom dishes with fribronectin. To do this, dilute a one milligram per milliliter firbronectin stock one to 100 with sterile PBS.Add one milliliter to each 35 milliliter dish to cover the whole surface. Incubate for one hour at 37 degrees celsius and 5%carbon dioxide. During the incubation, pre-warm alpha MEM supplemented with 10%FBS and an aliquot of 0.05%trypsin EDTA to 37 degrees celsius.After 45 minutes of incubation, start preparing the cell solution. Aspirate the medium from a 10 centimeter plate of HeLa RFP H2B cells. And gently rinse twice with 1.5 milliliters of 0.05%trypsin EDTA.Leave 0.5 milliliters of trypsin EDTA on the plate after the last aspiration. And incubate the cells for two to three minutes at 37 degrees celsius. And 5%carbon dioxide.Gently tap the plate. And observe the cell layer under the microscope to verify that all the cells have been detached. Then add 10 milliliters of medium and pipette gently several times to separate the cells and transfer the cell suspension in a 15 milliliter tube.Count a 10 microliter sample of the cell suspension using a hemocytometer. Next, aspirate the fibronectin solution from the glass bottom dishes. Do not allow the fibronectin to dry before cell plating.Prepare a cell suspension solution to plate 1.5 times ten to the fifth cells per 35 millimeter dish in two milliliters of medium. Plate one 35 millimeter plate additional to the number of conditions in order to count the cell number prior to virus transduction. Incubate for 30 to 45 minutes.After the incubation time has elapsed, check the cells under the microscope to ensure that the cells are well separated. As seen here. Return the plates to the incubator and grow the cells until the next day.On the day after plating, ensure that the cells have reached the density of at least 50%A cell density lower than 50%results in decreased virus transduction efficiency. Increased variation of protein expression per cell, and increased cell toxicity. Then harvest the cells from the additional plated dish as before.And count the number of cells. Calculate the volume of viruses required for efficient transduction of the cells. Label a 1.5 milliliter microcentrifuge tube for each condition.And add 400 microliters of warm alpha MEM minus. After slowly thawing aliquots of the viruses on ice, add the required volume of each virus to the tubes and mix gently by pipetting. Next, after aspirating the medium from the cells, gently add the virus mix drop wise.Incubate the cells at 37 degrees celsius with 5%carbon dioxide. Every 15 minutes for a period of one hour, agitate the plates carefully under the sterile hood to cover the cells with virus. Using a small amount of medium facilitates the contact of the virus with the cells.After the incubation, gently pipette 1.6 milliliters of alpha MEM minus to each plate to obtain a total volume of two milliliters. Then start cell synchronization by adding two millimolar thymidine before incubating the cells for a further two hours. Meanwhile, prepare the siRNA transfection mix by adding in a microtube 139 microliters of sterile water, 50 microliters of one molar calcium chloride, and 11 microliters of 20 micromolar siRNA.If no siRNA is necessary, replace the amount of siRNA by sterile water to perform an empty transfection. Add HBSS2X solution drop wise slowly on to the calcium chloride siRNA mixture. Gently mix two times by air injection using a 200 microliter pipette.As smaller precipitates result in better transfection efficiency. Then incubate the mix for 30 minutes at room temperature. After the incubation, slowly add 200 microliters drop wise to each plate and agitate to mix.Incubate the plates at 37 degrees celsius, 5%carbon dioxide for 16 hours. The next day, remove the dishes from the incubator and transport them to a tissue culture hood. After rinsing the cells twice with two milliliters of warm hipis, add two milliliters of alpha MEM minus before returning to the incubator.Seven hours later, add two millimolar thymidine to the plates before returning them to the incubator. Work with no more than four cell plates at a time as temperature variations effect the length of the cell cycle. The following day, 48 hours after siRNA transfection and virus infection, rinse the cells twice with two milliliters of warm PBS then add two milliliters of alpha MEM without phenol red for live cell imaging.Incubate for seven hours. Perform short term live cell imaging experiments on mitotic cells with an inverted microscope equipped with a humidified 5%carbon dioxide thermo regulated chamber. Prior to acquisition, verify that the chamber has reached the appropriate temperature of 37 degrees celsius.Place the cultured dishes in the microscope chamber one hour before acquisition to enable proper equilibrium of the medium and avoid focus drifting due to temperature changes. Examine the mitotic status of the cells. At this point.10 to 15%of the cells should be in the early stages of mitosis. During the equilibration time, set up the acquisition parameters as determined in prior experiments. It is important to achieve appropriate resolution without photo bleaching.As this causes cell damage and perturbs mitotic progression. Choose several fields containing cells that are at mitotic entry per condition to obtain a significant number of cells to analyze without exceeding the acquisition interval. Once all fields have been chosen, reset the focus and start the acquisition as fast as possible.Using a spinning disc can focus system. A typical set up would include four different conditions. Seven fields per plate.And two color channel with a 1.5 to two minute interval over 75 minute period. Determine well defined criteria to analyze mitotic pheno types in cells which will depend on the fluorescent markers being used. In contrast to transfection of BAG3 GFP, plasmic DNA, as seen on the left, adenofection with recompetent adenoviruses shown here on the right enables a more homogeneous and efficient expression of BAG3 GFP.In a majority of the cell population at low multiplicity of infection. This representative western blot shows that adenofection with BAG3 specific siRNAs and recompotent adenovirus BAG3 GFP allows efficient depletion of endogenous BAG3 and reintroduction of the BAG3 GFP protein at near endogenous levels. As shown here, adenofection can preserve protein homeostasis since there is no detectable increase in the levels of the stress induced heat shock proteins in HeLa cells that have been adenofected.In contrast to HeLa cells treated with the proteotoxic stress inducer, MG132. Here we see that the progression of cells through mitosis is not significantly perturbed by adenofection as shown by these con focal time lapse images from HeLa cells adenofected with control siRNA and BacMam GFP alpha Tubulin. And BacMam FRP Actin.The graph indicates the proportion of cells with abnormal mitosis. Cells adenofected with BAG3 specific siRNA alone exhibited an approximate two fold increase in the level of mitotic defects. As depicted by the red bar.Which was restored to near the control cell level by reintroduction of BAG3 GFP shown by the black bar. After watching this video, you should have a good understanding on how to perform depletion rescue experiments using adenofection. This method provides a fast and simple system to create hypomorphic knock downs for structure function analysis in multiple cellular backgrounds.Following this method, key structural requirements on the protein of interest for a given function can be identified. Other approaches of genome editing can then be used to deepen the mechanics involved.

Research

JoVE Journal

Biology

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

A System for ex vivo Culturing of Embryonic Pancreas

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...