Name: high-resolution imaging of nuclear dynamics in live cells under uniaxial tensile strain
Uploaded: 2019-06-02T14:00:00
Description: Watch this Scientific Journal Video about High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain at JoVE.com

All authors gratefully acknowledge funding support from the National Research Foundation of Singapore through the Singapore-MIT Alliance for Research and Technology (SMART) BioSystems and Micromechanics (BioSyM) interdisciplinary research group. Authors Dr. Jagielska and Dr. Van Vliet also gratefully acknowledge funding and support from the Saks-Kavanaugh Foundation. The authors thank William Ong and Dr. Sing Yian Chew from Nanyang Technological University, Singapore, for providing rat oligodendrocyte progenitor cells for some experiments described in this work, and the authors thank Dr. G. V. Shivashankar from Mechanobiology Institute, National University of Singapore, Singapore, for discussions about nuclear area fluctuations.

1. Design of the single-well polydimethylsiloxane mold for high-resolution imaging
NOTE: The mold for manufacturing polydimethylsiloxane plates is designed with the following features to enable imaging with a 100x oil immersion lens and a correct fit in the custom-build strain device (Figure 1A,B).
<ol>
	<li>Keep the overall plate dimensions such that it fits in the clamps of the strain device. 
	NOTE: Here, they are 60 mm ...

<ol>
	<li>Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+tension+produced+by+nerve+cells+in+tissue+culture.">Mechanical tension produced by nerve cells in tissue culture.</a> Journal of Cell Science. 410, 391-410 (1979).</li><li>Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+growth+in+response+to+experimentally+applied+mechanical+tension.">Axonal growth in response to experimentally applied mechanical tension.</a> Developmental Biology. 389, (1983).</li><li>Van Essen, D. 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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=Strain+rate-dependent+induction+of+reactive+astrogliosis+and+cell+death+in+three-dimensional+neuronal-astrocytic+co-cultures.">Strain rate-dependent induction of reactive astrogliosis and cell death in three-dimensional neuronal-astrocytic co-cultures.</a> Brain Research. , 103-115 (2011).</li><li>Fisher, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+correlates+of+axonal+swelling+in+chronic+multiple+sclerosis+brains.">Imaging correlates of axonal swelling in chronic multiple sclerosis brains.</a> Annals of Neurology. 2, 219-228 (2007).</li><li>Niki&#263;, 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=A+reversible+form+of+axon+damage+in+experimental+autoimmune+encephalomyelitis+and+multiple+sclerosis.">A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis.</a> Nature Medicine. 17, 495-500 (2011).</li><li>Payne, S. C., Bartlett, C. A., Harvey, A. R., Dunlop, S. A., Fitzgerald, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+loss+during+chronic+secondary+degeneration+in+rat+optic+nerve.">Functional loss during chronic secondary degeneration in rat optic nerve.</a> Investigative Ophthalmology &amp; Visual Science. 53, (2018).</li><li>Zeiger, A. S., 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=Static+mechanical+strain+induces+capillary+endothelial+cell+cycle+re-entry+and+sprouting.">Static mechanical strain induces capillary endothelial cell cycle re-entry and sprouting.</a> Physical Biology. 13, 1-16 (2017).</li><li>Jagielska, 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=Mechanical+strain+promotes+oligodendrocyte+differentiation+by+global+changes+of+gene+expression.">Mechanical strain promotes oligodendrocyte differentiation by global changes of gene expression.</a> Frontiers in Cellular Neuroscience. 11, 93 (2017).</li><li>Pajerowski, J. D., Dahl, K. N., Zhong, F. L., Sammak, P. J., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+plasticity+of+the+nucleus+in+stem+cell+differentiation.">Physical plasticity of the nucleus in stem cell differentiation.</a> Proceedings of the National Academy of Sciences of the United States of America. 1, 0 (2007).</li><li>Talwar, S., Kumar, A., Rao, M., Menon, G. I., Shivashankar, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlated+spatio-temporal+fluctuations+in+chromatin+compaction+states+characterize+stem+cells.">Correlated spatio-temporal fluctuations in chromatin compaction states characterize stem cells.</a> Biophysical Journal. 104, 553-564 (2013).</li><li>Makhija, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+strain+alters+cellular+and+nuclear+dynamics+at+early+stages+of+oligodendrocyte+differentiation.">Mechanical strain alters cellular and nuclear dynamics at early stages of oligodendrocyte differentiation.</a> Frontiers in Cellular Neuroscience. 12, 59 (2018).</li></ol>

A device has previously1 been designed for the application of extracellular tensile strain on adherent cells. The design of the polydimethylsiloxane substratum in that work was sufficient for biochemical assays, as well as the low-resolution imaging of stretched cells. In this work, the substratum was redesigned, and a novel imaging configuration that facilitates high-resolution subcellular live cell imaging was introduced. The advantages of this system are numerous: it can be built in-lab using s...

Cells and tissues in the body are subjected to various mechanical cues, including tensile strains. However, the effects of these cues on the biology of neural cells have not yet been studied extensively and understood fully. In the central nervous system, sources of mechanical strain include developmental growth1,2,3,4, physiological processes such as spinal cord bending, blood and cerebrospinal fluid pulsation, and pathological conditions such as trauma, axon swelling, glial scarring, or tumor growth5,6,7,8. It is worth investigating how tensile strain affects the differentiation of oligodendrocytes and the subsequent myelination of axons, which is a critical process in the vertebrate central nervous system. Using a custom-designed strain device and elastomeric multiwell plates, previous works9,10 have shown that static uniaxial strain can increase oligodendrocyte differentiation via global changes in gene expression10. To gain further understanding of the mechanisms of strain mechanotransduction in these cells, the previous experimental apparatus must be redesigned as described here, to enable high-resolution fluorescence imaging of nuclear dynamics in living cells under strain. Specifically, a single-well polydimethylsiloxane plate is developed, and the imaging configuration is redesigned to allow for the time-lapse imaging of live cells under strain using a 100x oil immersion lens. To eliminate the negative optical effects of polydimethylsiloxane in the light pathway, cells are imaged not through the polydimethylsiloxane plate, but in the inverted position, through the cover glass covering the cell compartment. Using this new imaging design, hundreds of high-resolution time-lapse movies are recorded, of individual cell nuclei within intact adherent cells, where chromatin is labeled by tagging histone H2B to green fluorescent protein. These movies demonstrate that tensile strain induces changes in chromatin structure and dynamics that are consistent with the progression of oligodendrocyte differentiation.
Live cell imaging under applied strain is technically challenging and requires a device design that is compatible with the microscope system. The custom design described here presents an inexpensive alternative to commercial solutions. Its dimensions enable its installation on microscope stages and live cell imaging at high spatial resolution during applied strain. The imaging setup is designed to facilitate live cell imaging using a 100x oil immersion lens with the highest clarity, through the cover glass, not through the layer of polydimethylsiloxane plate which otherwise decreases the image quality and is common in most imaging setups under strain. The device, with a mounted plate containing cells, can also be stored easily in the incubator. This device is designed to apply uniaxial strain to substrata that facilitate adherent cell culture and maintain a stable and uniform strain over multiple days. The setup described here can be used for the high-resolution imaging of various adherent cell types under strain, making it applicable to mechanotransduction studies in many fields of cell mechanobiology.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Primary cells</td>
			<td></td>
			<td></td>
			<td>Primary Oligodendrocyte Progenitor Cells isolated from Neonatal Rats 
			were provided by Dr. S. Y. Chew&#39;s lab at Nanyang Technological University</td>
		</tr>
		<tr>
			<td>Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high&#160; glucose</td>
			<td>Gibco</td>
			<td>11996-065-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Gibco</td>
			<td>15070-063-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF</td>
			<td>Prospec</td>
			<td>CYT-608-50ug</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF</td>
			<td>Prospec</td>
			<td>CYT-776-10ug</td>
			<td></td>
		</tr>
		<tr>
			<td>Media Stock components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A9418-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P5780-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Selenite</td>
			<td>Sigma</td>
			<td>S5261-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-iodothyronine</td>
			<td>Sigma</td>
			<td>T6397-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Thyroxine</td>
			<td>Sigma</td>
			<td>T1775-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Holo-Transferrin</td>
			<td>Sigma</td>
			<td>T0665-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>Mold and PDMS plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS - Dow Corning Sylgard 184</td>
			<td>Ellsworth</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Mold - Liquid Plastic</td>
			<td>Smooth-On</td>
			<td>Smooth Cast 310 - Trail Size</td>
			<td></td>
		</tr>
		<tr>
			<td>Substrate Coatings</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma</td>
			<td>F1141-2MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Histone staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CellLigt H2B-GFP BacMam</td>
			<td>Molecular Probes</td>
			<td>C10594</td>
			<td></td>
		</tr>
		<tr>
			<td>Surface functionalization</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>APTES</td>
			<td>Sigma</td>
			<td>A3648-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>BS3</td>
			<td>Covachem</td>
			<td>13306-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer pH 8.0</td>
			<td>Alfa Aesar</td>
			<td>J63578-AK-250ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell detachment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Invitrogen</td>
			<td>A1110501-100ml</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-resolution imaging of nuclear dynamics in live cells under uniaxial tensile strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.&#160;

Recent work aimed at investigating the effect of tensile strain on oligodendrocytes10 showed that a 10% uniaxial tensile strain promotes the differentiation of oligodendrocyte progenitor cells by global changes in gene expression. The mechanism behind these changes in gene expression can be probed via the imaging of subcellular parameters, such as the cytoskeleton structure, transcription factor localization, nuclear dynamics, and chromatin organization. However, t...

Watch this Scientific Journal Video about High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain at JoVE.com

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...

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