Name: single cell durotaxis assay for assessing mechanical control of cellular movement and related signaling events
Uploaded: 2019-08-27T14:45:00
Description: Watch this Scientific Journal Video about Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events at JoVE.com

1. Fabrication of Deformable Polyacrylamide Hydrogels with Embedded Fluorescent Microspheres
NOTE: Directions describe polymerization of a 25 kPa hydrogel that is 22 &#956;m in diameter and approximately 66 &#956;m thick. Each or all of these parameters can be modified and directions to do so can be found in Table 1 and in the notes17.
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	<li>Activation of glass-bottom dishes or coverslips
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		<li>Prepare the b...

<ol>
	<li>Acerbi, I., 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=Human+breast+cancer+invasion+and+aggression+correlates+with+ECM+stiffening+and+immune+cell+infiltration.">Human breast cancer invasion and aggression correlates with ECM stiffening and immune cell infiltration.</a> Integrative Biology (Camb. 7 (10), 1120-1134 (2015).</li><li>Mekhdjian, A. 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=Integrin-mediated+traction+force+enhances+paxillin+molecular+associations+and+adhesion+dynamics+that+increase+the+invasiveness+of+tumor+cells+into+a+three-dimensional+extracellular+matrix.">Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix.</a> Molecular Biology of the Cell. 28 (11), 1467-1488 (2017).</li><li>Paszek, M. 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=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8 (3), 241-254 (2005).</li><li>Lange, J. R., Fabry, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+tissue+mechanics+in+cell+migration.">Cell and tissue mechanics in cell migration.</a> Experimental Cell Research. 319 (16), 2418-2423 (2013).</li><li>Davidson, L. A., Oster, G. F., Keller, R. E., Koehl, 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=Measurements+of+mechanical+properties+of+the+blastula+wall+reveal+which+hypothesized+mechanisms+of+primary+invagination+are+physically+plausible+in+the+sea+urchin+Strongylocentrotus+purpuratus.">Measurements of mechanical properties of the blastula wall reveal which hypothesized mechanisms of primary invagination are physically plausible in the sea urchin Strongylocentrotus purpuratus.</a> Developmental Biology. 209 (2), 221-238 (1999).</li><li>Li, D., 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=Role+of+mechanical+factors+in+fate+decisions+of+stem+cells.">Role of mechanical factors in fate decisions of stem cells.</a> Regenerative Medicine. 6 (2), 229-240 (2011).</li><li>Handorf, A. M., Zhou, Y., Halanski, M. A., Li, W. 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D., Franze, K., Sivaniah, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+stiffness+gradient+substrates+for+studying+mechanotactic+cell+migration.">Complex stiffness gradient substrates for studying mechanotactic cell migration.</a> Advanced Materials. 24 (45), 6059-6064 (2012).</li><li>Breckenridge, M. T., Desai, R. A., Yang, M. T., Fu, J., 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=Substrates+with+engineered+step+changes+in+rigidity+induce+traction+force+polarity+and+durotaxis.">Substrates with engineered step changes in rigidity induce traction force polarity and durotaxis.</a> Cell and Molecular Bioengineering. 7 (1), 26-34 (2014).</li><li>Goreczny, G. J., Wormer, D. B., Turner, C. 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Demonstrated here is a repeatable, single-cell durotaxis assay that allows assessment of a cell&#8217;s ability to alter its migration behavior in response to acute mechanical cues. This technique can also be used in combination with fluorescence microscopy and appropriate fusion proteins or biosensors to examine subcellular signaling and cytoskeletal events within seconds of mechanical stimulation or over a longer timescale during durotactic movement. Understanding a cell&#8217;s relationship to its environment involves...

Over the past few decades, the importance of the mechanical properties of a cell&#8217;s environment has garnered increasing recognition in cell biology. Different tissues and extracellular matrices have different relative stiffnesses and, as cells migrate throughout the body, they navigate these changes, using these mechanical properties to guide them1,2,3,4,5,6,7. Cells use the stiffness of a given tissue to inform their motile behavior during processes such as development, wound healing, and cancer metastasis. However, the molecular mechanisms that allow sensation of and response to these mechanical inputs remain largely unknown1,2,3,4,5,6,7.
In order to study the mechanisms through which cells respond to physical environmental cues, the rigidity or stiffness of the substrate underlying adherent cells must be manipulated. In 2000, Chun-Min Lo, Yu-Li Wang and colleagues developed an assay8 whereby an individual cell&#8217;s motile response to changing mechanical cues could be directly tested by stretching deformable extracellular matrix (ECM)-coated polyacrylamide hydrogels on which the cells were plated. Cells exhibits a significant preference for migrating towards stiffer substrates, a phenomenon they dubbed &#8220;durotaxis.&#8221;
Since the original report in 2000, many other techniques have been employed for the study of durotaxis. Steep stiffness gradients have been fabricated by casting gels over rigid features such as polystyrene beads9 or stiff polymer posts10 or by polymerizing the substrate around the edges of a glass coverslips11 to create mechanical &#8216;step-boundaries&#8217;. Alternatively, hydrogels with shallower but fixed stiffness gradients have been fabricated by a variety of methods such as gradients of crosslinker created by microfluidic devices12,13 or side-by-side hydrogel solution droplets of differing stiffness8, or hydrogels with photoreactive crosslinker treated with graded UV light exposure to create a linear stiffness gradient14,15. These techniques have been used to great effect to investigate durotactic cellular movement en masse over time. However, typically these features are fabricated in advance of cell plating and their properties remain consistent over the course of the experiment, relying on random cell movement for sampling of mechanical gradients. None of these techniques are amenable to observation of rapid changes in cellular behavior in response to acute mechanical stimulus.
In order to observe cellular responses to acute changes in the mechanical environment, single cell durotaxis assays offer several advantages. In these assays, individual cells are given an acute, mechanical stimulus by pulling the underlying substrate away from the cell with a glass micropipette, thereby introducing a directional gradient of cell-matrix tension. Changes in the motile behavior, such as speed or direction of migration, are then observed by live-cell phase contrast microscopy. This approach facilitates direct observation of cause and effect relationships between mechanical stimuli and cell migration, as it allows rapid, iterative manipulation of the direction and magnitude of the tension gradient and assessment of consequent cellular responses in real time. Further, this method can also be used to mechanically stimulate cells expressing fluorescent fusion proteins or biosensors to visualize changes in the amount, activity, or subcellular localization of proteins suspected to be involved in mechanosensing and durotaxis.
This technique has been employed by groups who study durotaxis8,16 and is described here as it has been adapted by the Howe Laboratory to study the durotactic behavior of SKOV-3 ovarian cancer cells and the molecular mechanisms that underly durotaxis17. Additionally, a modified method is described for fabrication of hydrogels with a single, even layer of fluorescent microspheres near the cell culture surface; this facilitates visualization and optimization of micropipette-generated strain gradients and may allow assessment of cell contractility by traction force microscopy.

<table>
	<tbody>
		<tr>
			<td>Acrylamide 40 %&#160;</td>
			<td>National Diagnostic</td>
			<td>EC-810</td>
			<td></td>
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			<td>Ammonium Persulfate&#160;</td>
			<td>Fisher</td>
			<td>BP179-25</td>
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			<td>BD20A High frequency generator</td>
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			<td>12011A</td>
			<td>115 V -&#160;Handheld Corona Wand</td>
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			<td>Bind Silane (y-methacryloxypropyltrimethoxysilane) (</td>
			<td>Sigma Aldrich</td>
			<td>M6514</td>
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			<td>Bis-acrylamide 2%&#160;</td>
			<td>National Diagnostic</td>
			<td>EC-820</td>
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			<td>Borosilicate glass capillaries</td>
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			<td>1B100-4</td>
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			<td>Branson 2510 Ultrasonic Cleaner</td>
			<td>Bransonic</td>
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			<td>40 kHz frequency</td>
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			<td>Coarse Manipulator</td>
			<td>Narshige</td>
			<td>MC35A</td>
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			<td>DMEM</td>
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			<td>DMEM without phenol red</td>
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			<td>D5030</td>
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			<td>Dual-Stage Glass Micropipette Puller</td>
			<td>Narshige</td>
			<td>PC-10</td>
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			<td>Epidermal Growth Factor</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
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			<td>Ethanol</td>
			<td>Pharmco-aaper</td>
			<td>111000200</td>
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			<td>Fetal Bovine Serum (Qualified One Shot)</td>
			<td>Gibco</td>
			<td>A31606-02</td>
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			<td>Fibronectin&#160;</td>
			<td>EMD Millipore</td>
			<td>FC010</td>
			<td></td>
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			<td>Fluospheres Carboxylate 0.2 um&#160;</td>
			<td>Invitrogen</td>
			<td>F8810, F8807, F8811</td>
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			<td>Fugene 6</td>
			<td>Roche</td>
			<td>1815091</td>
			<td>1.5 ug DNA / 6uL fugene 6 per 35mm dish</td>
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			<td>Glacial Acetic Acid</td>
			<td>Fisher Chemical</td>
			<td>A38SI-212</td>
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			<td>Glass Bottom Dish</td>
			<td>CellVis</td>
			<td>D60-60-1.5-N</td>
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			<td>Glass Coverslip</td>
			<td>Electron Microscopy Sciences</td>
			<td>72224-01</td>
			<td>22 mm, #1.5</td>
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			<td>HCl</td>
			<td>JT Baker</td>
			<td>9535-03</td>
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			<td>Hellmanex III&#160;Special cleaning concentrate</td>
			<td>Sigma Aldrich</td>
			<td>Z805939</td>
			<td>Used at 2% in ddH2O for cleaning coverslips</td>
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		<tr>
			<td>HEPES powder</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td>Make 50mM HEPES buffer, pH 8.5</td>
		</tr>
		<tr>
			<td>Intelli-Ray 400 Shuttered UV Flood Light</td>
			<td>Uviton International</td>
			<td>UV0338</td>
			<td></td>
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		<tr>
			<td>Isopropanol</td>
			<td>Fisher Chemical</td>
			<td>A417-4</td>
			<td></td>
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			<td>Microforge</td>
			<td>Narshige</td>
			<td>MF900</td>
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			<td>Micromanipulator</td>
			<td>Narshige</td>
			<td>MHW3</td>
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			<td>Mineral Oil</td>
			<td>Sigma Aldrich</td>
			<td>M5904</td>
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			<td>Nanopure Life Science UV/UF System</td>
			<td>Barnstead</td>
			<td>D11931</td>
			<td>ddH2O</td>
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			<td>Nikon Eclipse Ti</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
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			<td>OptiMEM</td>
			<td>Invitrogen</td>
			<td>31985062</td>
			<td></td>
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			<td>Parafilm M</td>
			<td>Bemis Company, Inc</td>
			<td>PM-992</td>
			<td></td>
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			<td>PBS</td>
			<td></td>
			<td></td>
			<td>139 mM NaCl, 2.5 mM KCl, 28.6 mM Na2HPO4, 1.6 mM KH2PO4, pH 7.4</td>
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			<td>Platelet Derived Growth Factor-BB (PDGF-BB)</td>
			<td>Sigma Aldrich</td>
			<td>P4056</td>
			<td></td>
		</tr>
		<tr>
			<td>Ref52</td>
			<td></td>
			<td></td>
			<td>Rat embryonic fibroblast cell line; Culture in DMEM + 10% FBS</td>
		</tr>
		<tr>
			<td>Ringer&#39;s Buffer</td>
			<td></td>
			<td></td>
			<td>134 mM NaCl, 5.4 mM KCl, 1 mM MgSO4, 2.4 mM CaCl2, 20 mM HEPES, 5 mM D-Glucose, pH 7.4</td>
		</tr>
		<tr>
			<td>SKOV-3</td>
			<td>American Type Culture Collection</td>
			<td></td>
			<td>Culture in DMEM + 10% FBS</td>
		</tr>
		<tr>
			<td>Sulfo-SANPAH&#160;</td>
			<td>Covachem</td>
			<td>&#160;12414-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T9281-50</td>
			<td></td>
		</tr>
	</tbody>
</table>

single cell durotaxis assay for assessing mechanical control of cellular movement and related signaling events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

By preparing micropipettes (Figure 1) and normalizing the force generation of the pulls (Figure 2 and Figure 3) as described above, optimal durotactic conditions have been identified for multiple cell lines. Using this technique, as outlined in Figure 4, both SKOV-3 ovarian cancer cells17&#160;and Ref52 rat embryonic fibroblasts (Figure 5) move towar...

Watch this Scientific Journal Video about Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events at JoVE.com

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

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A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

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 ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...