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.

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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 and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

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

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

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

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 the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

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

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

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

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



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

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

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

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

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 particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.

Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.

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 ...

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 depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

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

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

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 well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

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

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...