Name: examining the dynamics of cellular adhesion and spreading of epithelial cells on fibronectin during oxidative stress
Uploaded: 2019-10-13T12:30:00
Description: Watch this Scientific Journal Video about Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress at JoVE.com

The authors thank Drs. Scott R. Hutton and Meghan S. Blackledge for the critical review of the manuscript.&#160;This work was funded by High Point University&#8217;s Research and Sponsored Programs (MCS) and the Biotechnology Program at North Carolina State University (MCS).

1. Preparations
NOTE: The protocol described below has been optimized for the use with REF52 cells and ATM+/+ or ATM-/- human fibroblasts. Other cell types may require further optimization as described in the notes and troubleshooting sections below.
<ol>
	<li>Make 500 mL of complete cell culture medium for REF52 cells. To 500 mL of high-glucose containing Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) add 10% FBS, 2 mM L-glutamine, and 100 units/mL penicillin-st...

<ol>
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D., Burridge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+on-off+relationship+of+Rho+and+Rac+during+integrin-mediated+adhesion+and+cell+migration.">The on-off relationship of Rho and Rac during integrin-mediated adhesion and cell migration.</a> Small GTPases. 5, e27958 (2014).</li><li>Heo, J., Campbell, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+redox-mediated+guanine+nucleotide+exchange+on+redox-active+Rho+GTPases.">Mechanism of redox-mediated guanine nucleotide exchange on redox-active Rho GTPases.</a> Journal of Biological Chemistry. 280 (35), 31003-31010 (2005).</li><li>Tolbert, C. E., Beck, M. V., Kilmer, C. E., Srougi, 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=Loss+of+ATM+positively+regulates+Rac1+activity+and+cellular+migration+through+oxidative+stress.">Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress.</a> Biochemical and Biophysical Research Communications. 508 (4), 1155-1161 (2019).</li><li>Hobbs, G. 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=Redox+regulation+of+Rac1+by+thiol+oxidation.">Redox regulation of Rac1 by thiol oxidation.</a> Free Radical Biology and Medicine. 79, 237-250 (2015).</li><li>Zhang, Y., 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=Mitochondrial+redox+sensing+by+the+kinase+ATM+maintains+cellular+antioxidant+capacity.">Mitochondrial redox sensing by the kinase ATM maintains cellular antioxidant capacity.</a> Science Signaling. 11 (538), (2018).</li><li>Hobbs, G. A., Zhou, B., Cox, A. D., Campbell, S. 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L., Martinez, R., Bokoch, G. M., LaFlamme, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+integrin+beta+tail+is+required+and+sufficient+to+regulate+adhesion+signaling+to+Rac1.">The integrin beta tail is required and sufficient to regulate adhesion signaling to Rac1.</a> Journal of Cell Science. 115 (Pt 22), 4285-4291 (2002).</li><li>Arthur, W. T., Petch, L. A., Burridge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+engagement+suppresses+RhoA+activity+via+a+c-Src-dependent+mechanism.">Integrin engagement suppresses RhoA activity via a c-Src-dependent mechanism.</a> Current Biology. 10 (12), 719-722 (2000).</li><li>Arthur, W. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+quantification+of+focal+adhesion+spatiotemporal+dynamics+in+living+cells.">High-resolution quantification of focal adhesion spatiotemporal dynamics in living cells.</a> PLoS One. 6 (7), e22025 (2011).</li><li>Horzum, U., Ozdil, B., Pesen-Okvur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Step-by-step+quantitative+analysis+of+focal+adhesions.">Step-by-step quantitative analysis of focal adhesions.</a> MethodsX. 1, 56-59 (2014).</li><li>Elosegui-Artola, 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=Image+analysis+for+the+quantitative+comparison+of+stress+fibers+and+focal+adhesions.">Image analysis for the quantitative comparison of stress fibers and focal adhesions.</a> PLoS One. 9 (9), e107393 (2014).</li><li>Meller, J., Vidali, L., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+RhoG+is+dispensable+for+integrin-mediated+cell+spreading+but+contributes+to+Rac-independent+migration.">Endogenous RhoG is dispensable for integrin-mediated cell spreading but contributes to Rac-independent migration.</a> Journal of Cell Science. 121 (Pt 12), 1981-1989 (2008).</li><li>Donaldson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+Staining.">Immunofluorescence Staining.</a> Current Protocols in Cell Biology. 69 (43), 1-7 (2015).</li><li>Burry, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+for+immunocytochemistry:+an+update.">Controls for immunocytochemistry: an update.</a> Journal of Histochemistry and Cytochemistry. 59 (1), 6-12 (2011).</li><li>Grashoff, C., 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=Measuring+mechanical+tension+across+vinculin+reveals+regulation+of+focal+adhesion+dynamics.">Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics.</a> Nature. 466 (7303), 263-266 (2010).</li><li>Kumar, 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=Correction:+Talin+tension+sensor+reveals+novel+features+of+focal+adhesion+force+transmission+and+mechanosensitivity.">Correction: Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity.</a> Journal of Cell Biology. 214 (2), 231 (2016).</li><li>Kumar, 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=Talin+tension+sensor+reveals+novel+features+of+focal+adhesion+force+transmission+and+mechanosensitivity.">Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity.</a> Journal of Cell Biology. 213 (3), 371-383 (2016).</li><li>Friedrichs, J., Helenius, J., Muller, D. 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The protocol described here is a versatile and economical way to rapidly screen a number of anchorage-dependent cell types for dynamic cytoskeleton remodeling during cell spreading. In particular, this method quantitatively examines stress fiber and focal adhesion formation during oxidative stress when cells adhere to FN (Figure 1A). Moreover, these cellular phenotypes may suggest a regulatory role for members of the Rho family of small GTPases since they have documented roles during cell at...

Cell-matrix adhesions (i.e., focal adhesions) are large and dynamic multimolecular protein complexes which mediate cell adhesion and spreading. These processes are critical for tissue development, maintenance, and physiological function. Focal adhesions are composed of membrane-bound receptors, such as integrins, as well as scaffolding proteins that link cytoskeletal actin to the extracellular matrix (ECM)1. These complexes are capable of responding to physiochemical cues present in the extracellular environment through the activation of various signaling transduction pathways. As such, focal adhesions serve as signaling centers to propagate extracellular mechanical cues into a number of cellular processes including directed migration, cell cycle regulation, differentiation, and survival1,2. One group of signaling molecules that regulate and interact with focal adhesions includes members of the Rho family of small GTPases. Rho GTPases are key proteins that regulate cell migration and adhesion dynamics through their specific spatiotemporal activation3. Not surprisingly, dysregulation of Rho protein function has been implicated in a number of human pathologies such as metastasis, angiogenesis, and others. Of particular interest, cellular redox status plays a predominant role in the modulation of cell migration and adhesion. Alterations in redox homeostasis, such as increases in reactive oxygen species (ROS), have been demonstrated to regulate Rho protein activity, as well as adhesion, in a number of cell types and human diseases4,5,6,7,8. For example, individuals suffering from the neurological disorder ataxia-telangiectasia (A-T), which is caused by a mutation in the DNA damage repair serine/threonine kinase A-T-mutated (ATM), have an increased risk of metastatic cancer9,10. Loss of ATM kinase activity in these patients and cell lines, either through genetic mutation or chemical inhibition, results in high levels of oxidative stress due to dysfunction of the pentose phosphate pathway7,11,12. Moreover, recent studies from the laboratory have highlighted a pathophysiological role for ROS in A-T by altering cytoskeletal dynamics (i.e. adhesion and spreading) as a direct result of activating Rho family GTPases in vitro5. Ultimately, these alterations in cytoskeletal dynamics caused by Rho family activation may lead to the increased risk of metastatic cancer noted in A-T patients5,13. Therefore, understanding the interplay between cell-matrix interactions during oxidative stress can provide insights into the regulation of adhesion and spreading. These studies can also set the stage for further investigations into a possible role for Rho family GTPases in these signaling processes.
Described herein is a protocol to study the early cellular dynamics of adhesion assembly and spreading during oxidative stress caused by inhibition of ATM kinase activity. This assay is based on the well-characterized mechanism of anchorage-dependent cells adhesion to the ECM protein fibronectin (FN). When cells maintained in suspension are plated onto FN, several Rho GTPases coordinate the control of the actin cytoskeletal remodeling3,14. Morphological changes are observed as cells shift from round and circular in appearance to flattened and expanded. Concomitant with these observations is the development of numerous matrix adhesions with the ECM. These changes are attributed to the biphasic activation of RhoA with Rac1 during the first hour as cells adhere and spread 15,16.
A variety of methods have been utilized to examine adhesion morphology and dynamics as well as cell spreading. However, these methods rely on sophisticated long-term, live-imaging total internal reflection fluorescence (TIRF) or confocal microscopy systems. Thus, users must have access to specialized equipment and software. Furthermore, the set-up time required by these bio-imaging systems makes capturing early adhesion events challenging, especially when testing multiple inhibitors or treatment conditions concurrently.
The methods detailed, herein, provide a straightforward, economical, yet quantitative way to assess parameters that govern the adhesion assembly and spreading in vitro. The protocol is performed using commonly available laboratory equipment, such as an epifluorescence microscope and CCD camera. This assay involves applying anchorage-dependent cells to an FN-coated surface after a period of oxidative stress caused by chemical inhibition of ATM kinase activity, which has been demonstrated previously5. Following plating, cells are allowed to attach and adhere for specified lengths of time. Unattached cells are washed away, while attached cells are fixed and labeled with fluorescence-based antibodies to markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin)2,5. These proteins are then visualized and recorded using an epifluorescence microscope. Subsequent data analysis is performed using freely available Fiji software. Moreover, this method can be adapted to examine adhesion dynamics under a wide range of conditions including different ECM proteins, treatment with various oxidants/cell culture conditions or a variety of anchorage-dependent cell lines to address a broad range of biological questions.

<table>
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA (1x)</td>
			<td>Gibco by Life Technologies</td>
			<td>25300-054</td>
			<td>cell dissociation</td>
		</tr>
		<tr>
			<td>10 cm2 dishes</td>
			<td>Cell Treat</td>
			<td>229620</td>
			<td>sterile, tissue culture treated</td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>05-539-5</td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>1X Phosphate Buffered Saline</td>
			<td>Corning Cellgro</td>
			<td>21-031-CV</td>
			<td>PBS, sterile, free of Mg2+ and Ca2+</td>
		</tr>
		<tr>
			<td>24-well cell culture treated plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-740</td>
			<td>sterile, tissue culture treated</td>
		</tr>
		<tr>
			<td>4&#176;C refrigerator</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG anti-paxillin primary antibody (clone 165)</td>
			<td>BD Transduction Laboratories</td>
			<td>610620</td>
			<td>marker of focal adhesions</td>
		</tr>
		<tr>
			<td>Aspirator</td>
			<td>Argos</td>
			<td>EV310</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet</td>
			<td>Nuair</td>
			<td>NU-477-400</td>
			<td>Class II, Type A, series 5</td>
		</tr>
		<tr>
			<td>Delipidated Bovine Serum Albumin (Fatty Acid Free) Powder</td>
			<td>Fisher Scientific</td>
			<td>BP9704-100</td>
			<td>dlBSA</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Fisher Scientific</td>
			<td>BP231-100</td>
			<td>organic solvent to dissolve Ku55933</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Media, High Glucose</td>
			<td>Fisher Scientific</td>
			<td>11965092</td>
			<td>REF52 base cell culture medium</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Fisher Scientific</td>
			<td>16000044</td>
			<td>certified, cell culture medium supplement</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>National Institutes of Health</td>
			<td>http://fiji.sc/</td>
			<td>image analysis program</td>
		</tr>
		<tr>
			<td>Filter syringe</td>
			<td>Fisher Scientific</td>
			<td>6900-2502</td>
			<td>0.2 &#181;M, sterile</td>
		</tr>
		<tr>
			<td>Glass coverslips (12-Cir-1.5)</td>
			<td>Fisher Scientific</td>
			<td>12-545-81</td>
			<td>autoclave in foil to sterilize</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG secondary antibody Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td>fluorescent secondary antibody, light sensitive</td>
		</tr>
		<tr>
			<td>Goat Serum</td>
			<td>Gibco by Life Technologies</td>
			<td>16210-064</td>
			<td>component of blocking solution for immunofluorescence</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher Scientific</td>
			<td>22-600-107</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>Human Plasma Fibronectin</td>
			<td>Gibco by Life Technologies</td>
			<td>33016-015</td>
			<td>FN</td>
		</tr>
		<tr>
			<td>IX73 Fluorescence Inverted Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>microscope to visualize fluorescence, cell morphology, counting and dissociation</td>
		</tr>
		<tr>
			<td>Ku55933</td>
			<td>Sigma-Aldrich</td>
			<td>SML1109-25MG</td>
			<td>ATM kinase inhibitor, inducer of reactive oxygen species</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Fisher Scientific</td>
			<td>25-030-081</td>
			<td>cell culture medium supplement</td>
		</tr>
		<tr>
			<td>Monochrome CMOS 16 bit camera</td>
			<td>Optimos</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148-500G</td>
			<td>PFA, fixative for immunofluorescence</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Fisher Scientific</td>
			<td>15-140-122</td>
			<td>P/S, antibiotic solution for culture medium</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 phalloidin (F-actin probe)</td>
			<td>Invitrogen</td>
			<td>A12381</td>
			<td>marker of F-actin, light sensitive</td>
		</tr>
		<tr>
			<td>ProLong Gold Anti-fade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36941</td>
			<td>cover slip mounting media including nuclear dye DAPI, light sensitive</td>
		</tr>
		<tr>
			<td>REF52 cells</td>
			<td></td>
			<td></td>
			<td>Graham, D.M. et. al. Journal of Cell Biology 2018</td>
		</tr>
		<tr>
			<td>Stir plate with heat control</td>
			<td>Corning Incorporated</td>
			<td>PC-420D</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD Biosciences</td>
			<td>309653</td>
			<td>60 mL syringe</td>
		</tr>
		<tr>
			<td>Tissue culture incubator</td>
			<td>Nuair</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td>detergent used to permeabilize cell membranes</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Fisher Scientific</td>
			<td>15-250-061</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>Trypsin Neutralizing Solution (1x)</td>
			<td>Gibco by Life Technologies</td>
			<td>R-002-100</td>
			<td>TNS, neutralizes trypsin instead of fetal bovine serum</td>
		</tr>
		<tr>
			<td>tube rotator</td>
			<td>Fisher Scientific</td>
			<td>11-676-341</td>
			<td></td>
		</tr>
		<tr>
			<td>water bath</td>
			<td>Fisher Scientific</td>
			<td>FSGPD02</td>
			<td></td>
		</tr>
	</tbody>
</table>

examining the dynamics of cellular adhesion and spreading of epithelial cells on fibronectin during oxidative stress

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

A general schema of the experimental set-up
Figure 1 represents the general schema for the cell adhesion and spreading protocol beginning with serum starvation of REF52 cells and ending with computational analysis of acquired fluorescence images. Key steps in the protocol are illustrated in the timeline. Of note, step 2 of the protocol describes the preparation of the FN-coated coverslips, which should be performed concurrently with step 3: serum starving REF52 cell...

Watch this Scientific Journal Video about Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress at JoVE.com

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

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