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. 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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 ...

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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 ...