Name: a simplified system for evaluating cell mechanosensing and durotaxis in vitro
Uploaded: 2015-08-27T15:00:00
Description: Watch this Scientific Journal Video about A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro at JoVE.com

This work is supported by NIH R01 GM47607, CA163296 and NSF 1334493 to CET. We thank members of the Turner lab for critical reading of the manuscript. All data shown in this report were reproduced by permission from Wormer et al. 201411.

1. Preparation of Durotaxis Chambers
<ol>
	<li>To prepare one 6-well tissue culture plate, tare the balance with a 50-ml conical tube. Weigh out approximately 10 g of the PDMS base solution in the 50-ml tube (the solution is quite viscous).</li>
	<li>For a 90:1 substrate (Compliance of ~1 kPa), divide the measured weight of the PDMS base solution in the tube by 90 to determine the correct amount of crosslinker solution needed. Add the calculated amount of the PDMS crosslinker solution to the same tube. 
	Example: ...

<ol>
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Q., 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=Force+and+focal+adhesion+assembly:+a+close+relationship+studied+using+elastic+micropatterned+substrates.">Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates.</a> Nature Cell Biology. 3 (5), 466-472 (2001).</li><li>Provenzano, P. P., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+signaling+through+the+cytoskeleton+regulates+cell+proliferation+by+coordinated+focal+adhesion+and+Rho+GTPase+signaling.">Mechanical signaling through the cytoskeleton regulates cell proliferation by coordinated focal adhesion and Rho GTPase signaling.</a> Journal of Cell Science. 124 (8), 1195-1205 (2011).</li><li>Wang, Y. L., Pelham, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+a+flexible,+porous+polyacrylamide+substrate+for+mechanical+studies+of+cultured+cells.">Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells.</a> Methods in Enzymology. 298, 489-496 (1998).</li><li>Prager-Khoutorsky, 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=Fibroblast+polarization+is+a+matrix-rigidity-dependent+process+controlled+by+focal+adhesion+mechanosensing.">Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing.</a> Nature Cell Biology. 13 (12), 1457-1465 (2011).</li><li>Wormer, D. B., Davis, K. A., Henderson, J. H., Turner, C. 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+focal+adhesion-localized+CdGAP+regulates+matrix+rigidity+sensing+and+durotaxis.">The focal adhesion-localized CdGAP regulates matrix rigidity sensing and durotaxis.</a> PloS ONE. 9 (3), e91815 (2014).</li><li>Trichet, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+large-scale+mechanosensing+mechanism+for+cellular+adaptation+to+substrate+stiffness.">Evidence of a large-scale mechanosensing mechanism for cellular adaptation to substrate stiffness.</a> Proceedings of the National Academy of Sciences. 109 (18), 6933-6938 (2012).</li><li>Eyckmans, J., Boudou, T., Yu, X., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hitchhiker&#8217;s+guide+to+mechanobiology.">A hitchhiker&#8217;s guide to mechanobiology.</a> Developmental Cell. 21 (1), 35-47 (2011).</li><li>Martinac, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensitive+ion+channels:+molecules+of+mechanotransduction.">Mechanosensitive ion channels: molecules of mechanotransduction.</a> Journal of Cell Science. 117 (12), 2449-2460 (2004).</li><li>Jafar-Nejad, H., 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=Sec15,+a+component+of+the+exocyst,+promotes+notch+signaling+during+the+asymmetric+division+of+Drosophila+sensory+organ+precursors.">Sec15, a component of the exocyst, promotes notch signaling during the asymmetric division of Drosophila sensory organ precursors.</a> Developmental Cell. 9 (3), 351-363 (2005).</li></ol>

Herein we describe a simple assay to study durotaxis in migrating cells. A major strength of this assay is the ease of preparing the durotaxis chambers using PDMS. The rigidity of the substrates can be easily manipulated by changing the ratio of PDMS base solution to crosslinker to allow the study of various rigidities in the assay. However, one potential limitation of the system is that cells are only exposed to a single change in substrate rigidity as opposed to experiencing a rigidity gradient that is provided by more...

The extracellular matrix (ECM) is comprised of a complex array of structural and crosslinking proteins including collagen, fibronectin and laminin. Although it is well established that the ECM provides important structural support for cellular tissues, there is increasing evidence to indicate that cells actively respond to physical changes in their ECM environment to regulate diverse cellular processes including cell survival, differentiation and cell migration. For example, differences in the rigidity of the ECM can drive mesenchymal stem cells towards different lineages, with soft substrates (~1 kPa) promoting neurogenic lineages while stiff (~25 kPa) substrates promote osteogenic differentiation1. Similarly, an increase in the stromal matrix rigidity has been shown to promote mammary epithelial cell tumorigenesis and invasion into the surrounding tissue2,3.
A particularly interesting aspect of this mechanosignaling activity results in a process known as durotaxis, in which cells migrate preferentially towards a more rigid substrate4,5. Cells constantly sense the physical characteristics of their extracellular environment through integrin receptor binding to the ECM. This, in turn, promotes the accumulation of numerous structural and signaling proteins to their cytoplasmic domains to drive the formation of adhesive structures known as focal adhesions or focal contacts6,7. Since integrins have no inherent enzymatic activity, signals are relayed from the ECM through these accessory proteins to coordinate the cell&#8217;s response to their changing environment8. Accordingly, the identification and characterization of the key proteins involved in regulating mechanosignaling and durotaxis is an important area of investigation.
Various model systems have been developed to study durotaxis in vitro, but most have utilized collagen-coated polyacrylamide substrates4. However, the preparation of the polyacrylamide substrates can be technically challenging and the collagen used in these assays must be chemically crosslinked to the substrate9. Polydimethylsiloxane (PDMS) substrates have been shown to exhibit comparable mechanical properties to the polyacrylamide substrates10. However, PDMS substrates are prepared by simply mixing a ratio of the base to crosslinker and these substrates can be coated with ECM proteins without the need for chemical crosslinking, thus making PDMS an easier tool to study the effects of rigidity on cell behavior. Herein, we describe how to prepare a simple durotaxis chamber in which a soft PDMS substrate is integrated with a rigid glass coverslip.
The assay, as outlined below, provides a quick and simple method to study durotaxis. For this study we used human U2OS osteosarcoma cells combined with siRNA-mediated knockdown of cdGAP to study the role of this focal adhesion protein in durotaxis11. Importantly, this protocol may be readily adapted to individual requirements. Other cell types may be substituted for the U2OS cells and any protein may be knocked down or overexpressed to determine the effects on cell behavior during durotaxis. Furthermore, this protocol may be adapted to incorporate fluorescently tagged proteins to analyze their dynamics and behavior using FRAP or FRET approaches.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
		</tr>
		<tr>
			<td>#1 Cover glass 12mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-82</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Celltreat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>15-017-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Cellgro</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Fisher Scientific</td>
			<td>BP356-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Cellgro</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>610077</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>21600-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>Celltreat</td>
			<td>229456</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>U2OS cells</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simplified system for evaluating cell mechanosensing and durotaxis in vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A schematic of the durotaxis chamber is shown in Figure 1A. Soft PDMS substrate (a 90:1 mixture of PDMS base to crosslinker solutions) is spread in a 6 well dish and a glass coverslip is placed on top of the PDMS, which then partially covers the upper surface of the coverslip, thereby creating an interface between the two substrates of different compliance. The rigidity of the soft PDMS substrate is ~1 kPa, which is comparable to the typical compliance of brain tissue, while the rigidity of glass is arou...

Watch this Scientific Journal Video about A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro at JoVE.com

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

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