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>
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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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+stiffness+dictates+development,+homeostasis,+and+disease+progression.">Tissue stiffness dictates development, homeostasis, and disease progression.</a> Organogenesis. 11 (1), 1-15 (2015).</li><li>Lo, C. M., Wang, H. B., Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+movement+is+guided+by+the+rigidity+of+the+substrate.">Cell movement is guided by the rigidity of the substrate.</a> Biophysical Journal. 79 (1), 144-152 (2000).</li><li>Kuo, C. H., Xian, J., Brenton, J. 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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simplified+System+for+Evaluating+Cell+Mechanosensing+and+Durotaxis+In+Vitro.">A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro.</a> Journal of Visualized Experiments. (102), e52949 (2015).</li><li>Hartman, C. D., Isenberg, B. C., Chua, S. G., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+smooth+muscle+cell+durotaxis+depends+on+extracellular+matrix+composition.">Vascular smooth muscle cell durotaxis depends on extracellular matrix composition.</a> Proceedings of the National Acadademy of Sciences of the United States of America. 113 (40), 11190-11195 (2016).</li><li>Vincent, L. G., Choi, Y. S., Alonso-Latorre, B., del Alamo, J. C., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+durotaxis+depends+on+substrate+stiffness+gradient+strength.">Mesenchymal stem cell durotaxis depends on substrate stiffness gradient strength.</a> Biotechnology Journal. 8 (4), 472-484 (2013).</li><li>Sunyer, R., Jin, A. J., Nossal, R., Sackett, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+hydrogels+with+steep+stiffness+gradients+for+studying+cell+mechanical+response.">Fabrication of hydrogels with steep stiffness gradients for studying cell mechanical response.</a> PLoS One. 7 (10), e46107 (2012).</li><li>Martinez, J. S., Lehaf, A. M., Schlenoff, J. B., Keller, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+durotaxis+on+polyelectrolyte+multilayers+with+photogenerated+gradients+of+modulus.">Cell durotaxis on polyelectrolyte multilayers with photogenerated gradients of modulus.</a> Biomacromolecules. 14 (5), 1311-1320 (2013).</li><li>Plotnikov, S. V., Pasapera, A. M., Sabass, B., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+fluctuations+within+focal+adhesions+mediate+ECM-rigidity+sensing+to+guide+directed+cell+migration.">Force fluctuations within focal adhesions mediate ECM-rigidity sensing to guide directed cell migration.</a> Cell. 151 (7), 1513-1527 (2012).</li><li>McKenzie, A. 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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>
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		<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>
			<td>Electro Technic Products</td>
			<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>
			<td>World Precision Instruments</td>
			<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>
			<td>Corning</td>
			<td>10-013-CV</td>
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			<td>DMEM without phenol red</td>
			<td>Sigma Aldrich</td>
			<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>
			<td></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>
			<td></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>
			<td></td>
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			<td>Glass Bottom Dish</td>
			<td>CellVis</td>
			<td>D60-60-1.5-N</td>
			<td></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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		<tr>
			<td>HCl</td>
			<td>JT Baker</td>
			<td>9535-03</td>
			<td></td>
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		<tr>
			<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>
			<td></td>
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			<td>Micromanipulator</td>
			<td>Narshige</td>
			<td>MHW3</td>
			<td></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>
		</tr>
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			<td>OptiMEM</td>
			<td>Invitrogen</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis Company, Inc</td>
			<td>PM-992</td>
			<td></td>
		</tr>
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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>
		</tr>
		<tr>
			<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 ...

There's growing appreciation for the impact of the mechanical microenvironment on cell signaling and migration. Necessitating the use of unique, experimental approaches to deliver mechanical stimulation to individual cell. This technique allows the dynamic manipulation of the mechanical micro-environment underneath a cell in real time.Allowing the observation of mechanically regulated changes in cell migration behavior, and sub-cellular signaling events. Demonstrating the procedure with me will be Nyla Naim, a post-doc and Johnathan Patterson, a technician from our laboratory. Begin by using a Chorono-1 to activate the glass surface of each bottom cover slip for 20 seconds, before immediately over-laying 50 microliters of bind silane working solution onto each cover slip.Allow the solution to dry for 10 minutes before rinsing the cover slips two times with 95%ethanol and two times with isopropanol. Then, allow the cover slips to air dry for approximately 20 minutes. For fluorescent micro-bead deposition, first sonicate the working micro-bead solution for one hour.15 minutes before the end of the sonication, place the activated cover slips vertically in a ceramic cover slip holder and transfer the holder into a table-top plasma cleaner for room-air plasma treatment for three minutes. Next, place pieces of paraffin film in 60 milliliter Petri dish lids, and place the cover slips onto the films, lightly tapping each cover slip to ensure a good contact between the film and the coverslip. Then, add 150 microliters of the working bead solution to the top of each coverslip, and immediately aspirate the ethanol solution from the side of the coverslips, leaving the beads on the coverslip.To cast the Hydrogels immediately after their preparation, add a 25 microliter droplet of Hydrogel solution to the activated side of each bottom coverslip, and immediately flip the beaded top coverslips, bead-side down, onto the droplet. Allow the gels to polymerize for 30 minutes before using forceps to gently remove the coverslips. Then, rinse the gels with three, five minute washes in fresh 50 miliMoler Hepes per wash.To activate the Hydrogels surfaces, incubate the gels in fresh 50 miliMoler Hepes, supplemented with 0.4 miliMolar Sulfo Sanpah, and immediately expose the gels to an ultraviolet arch lamp in an enclosed area for 90 seconds. At the end of the activation, wash the gels with three, five minute washes in fresh Hepes, and treat the activated Hydrogels with 20 micrograms per milliliter of fibronectin for one hour at 37 degrees Celsius. At the end of the incubation, aspirate the excess fibronectin solution, and wash the gels three times in PBS for five minutes per wash.After the last wash, place the Hydrogels in a small volume of PBS and sterilize the gels for 15 minutes under ultra-violet light in a tissue culture hood. Then, wash the gels one time in fresh PBS. For cell plating, seed 2.1 times 10 the four of the cells of interest in three milliliters of medium per-Hydrogel and allow the cells to adhere at 37 degrees Celsius for four to 18 hours.While the cells are equilibrating, use a micro-pipette puller to pull a one millimeter diameter by 100 millimeter long borosilicate glass micro-pipette, to obtain a taper of over two millimeters that reduces to approximately 50 micrometers in diameter in the first millimeter, and extends to a long, parallel 10 micrometer diameter tube in the last millimeter. Next, load the pipette into a microforge and shape the pipette to have a 15 micrometer blunted tip that is enclosed at the very end of a 250 micrometer section, bent at approximately 35 degree angel from the rest of the pipette. The approximate diameter at the bend should be around 30 micrometers to lend strength to the tip.Next, place a Hydrogel onto the stage of an inverted microscope. Cover the medium with mineral oil and select the 10x objective. Sterilize the prepared micro-pipette with the 70%alcohol.Then, insert into the micro-pipette holder with the hook pointed toward the dish, and use the course manipulator to lower the pipette until the hook just touches the surface of the liquid. Focus the microscope on the cells adhered to the top of the gel, and taking care that the objective is in no danger of hitting the sample or stage, bring the focus above the gel to find the tip of the micro-pipette. Rotate the pipette until the tip is perpendicular to the focal plane, so that the blunted tip of the micro-pipette is pointing down, and refocus on the tip of the pipette as necessary.Focus back down to the cell layer to gauge how far the pipette is from the gel surface, and focus back up to a plane that is part-way between the gel and the tip of pipette. Then, slowly lower the pipette to reach the intermediate focal plane, and increase the magnification to that which will be used in the experiment, before lowering the pipette until it hovers just above the surface of the Hydrogel. For micro-manipulator calibration, image an area devoid of cells, an area with beads, but no manipulation, with the pipette engaging the gel, and with the engaged pipette pulling the gel.Then, use image J to compare the no bead fields to the bead fields without engagement, the bead fields with the gel engaged, and the pulled gel to calculate the relative bead displacements and force applied to the gels. To conduct a durotaxis assay, monitor a group of cells for 30 minutes to identify cells that are moving in a directed manner. Select a cell that is steadily moving in one direction, and monitor the cells of the desired frame-rate for an additional 30 minutes.Next, position the pipette about 50 micrometers in front of the near side of the leading edge of the selected cell, and move the micro-manipulator such that the gel is deformed orthogonally to the cell's direction of travel. Then, observe the cell over time as it responds to the acute, local gradient of Hydrogel stiffness. Using this technique as demonstrated, rat embryonic fibroblasts move towards the increased stiffness in gradients applied by a glass micro-pipette.Rat embryonic fibroblast cells, transiently expressing vinculin tension sensor on 125 kilopascal polyacrylamide gels also demonstrate the formation of focal adhesions in the directions of the stretch over a period of 40 minutes. Forster Resonance Energy Transfer analysis reveals that vinculin localized to focal adhesions experiences an immediate change in tension, when presented with an acute durotactic stimulation. Expanding the utility of this assay to the observation of sub-cellular signaling events, in response to durotactic stimulation.If you make multiple attempts to stretch a cell, or if the microneedle slips during the assay, start over with a new cell to avoid imparting multiple, variable, mechanical stimuli. Beyond its utility for assaying durotaxis, this technique can be combined with genetic approaches, such as biosensors, RNAI, or gene editing to help delineate the molecular mechanisms involved in mechanotransduction.

single-cell-durotaxis-assay-for-assessing-mechanical-control-cellular

Research

JoVE Journal

Biology

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

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 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.

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 development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

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

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

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

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...