This work was supported by NIH R01 EB014869.

1. Preparing Cells for Imaging
NOTE: The direct force probe (DFP) can be used for any adherent cell type. Here, NIH 3T3 mouse fibroblasts are used as the model cell line for this protocol.
<ol>
	<li>Culture NIH 3T3 fibroblast cells in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplemented with 10% donor bovine serum and 1% Penicillin-Streptomycin on a 35-mm glass bottom dish until desired confluency. Maintain cells at 37 &#176;C and 5% CO2.
	<ol>
		<li>Be sure to coat all ...

<ol>
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U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LINC+complexes+form+by+binding+of+three+KASH+peptides+to+domain+interfaces+of+trimeric+SUN+proteins.">LINC complexes form by binding of three KASH peptides to domain interfaces of trimeric SUN proteins.</a> Cell. 149 (5), 1035-1047 (2012).</li><li>Tapley, E. C., Starr, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connecting+the+nucleus+to+the+cytoskeleton+by+SUN-KASH+bridges+across+the+nuclear+envelope.">Connecting the nucleus to the cytoskeleton by SUN-KASH bridges across the nuclear envelope.</a> Current Opinion in Cell Biology. 25 (1), 57-62 (2013).</li><li>Arsenovic, P. T., 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=Nesprin-2G,+a+Component+of+the+Nuclear+LINC+Complex,+Is+Subject+to+Myosin-Dependent+Tension.">Nesprin-2G, a Component of the Nuclear LINC Complex, Is Subject to Myosin-Dependent Tension.</a> Biophysical Journal. 110 (1), 34-43 (2016).</li><li>Rowat, A. C., Lammerding, J., Ipsen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+the+cell+nucleus+and+the+effect+of+emerin+deficiency.">Mechanical properties of the cell nucleus and the effect of emerin deficiency.</a> Biophysical Journal. 91 (12), 4649-4664 (2006).</li><li>Rowat, A. C., Foster, L. J., Nielsen, M. M., Weiss, M., Ipsen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+elastic+properties+of+the+nuclear+envelope.">Characterization of the elastic properties of the nuclear envelope.</a> Journal of the Royal Society Interface. 2 (2), 63-69 (2005).</li><li>Pagliara, 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=Auxetic+nuclei+in+embryonic+stem+cells+exiting+pluripotency.">Auxetic nuclei in embryonic stem cells exiting pluripotency.</a> Nature Materials. 13 (6), 638-644 (2014).</li><li>Liu, 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=In+situ+mechanical+characterization+of+the+cell+nucleus+by+atomic+force+microscopy.">In situ mechanical characterization of the cell nucleus by atomic force microscopy.</a> ACS Nanotechnology. 8 (4), 3821-3828 (2014).</li><li>Krause, M., Te Riet, J., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+compressibility+of+tumor+cell+nuclei+by+combined+atomic+force-confocal+microscopy.">Probing the compressibility of tumor cell nuclei by combined atomic force-confocal microscopy.</a> Physical Biology. 10 (6), 065002 (2013).</li><li>Neelam, 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=Direct+force+probe+reveals+the+mechanics+of+nuclear+homeostasis+in+the+mammalian+cell.">Direct force probe reveals the mechanics of nuclear homeostasis in the mammalian cell.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (18), 5720-5725 (2015).</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. 104 (40), 15619-15624 (2007).</li><li>Lammerding, 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=Lamin+A/C+deficiency+causes+defective+nuclear+mechanics+and+mechanotransduction.">Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction.</a> Journal of Clinical Investigation. 113 (3), 370-378 (2004).</li><li>Chancellor, T. J., Lee, J., Thodeti, C. K., Lele, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actomyosin+tension+exerted+on+the+nucleus+through+nesprin-1+connections+influences+endothelial+cell+adhesion,+migration,+and+cyclic+strain-induced+reorientation.">Actomyosin tension exerted on the nucleus through nesprin-1 connections influences endothelial cell adhesion, migration, and cyclic strain-induced reorientation.</a> Biophysical Journal. 99 (1), 115-123 (2010).</li><li>Neelam, S., Dickinson, R. B., Lele, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+for+understanding+the+nuclear+force+balance+in+living,+adherent+cells.">New approaches for understanding the nuclear force balance in living, adherent cells.</a> Methods. 94, 27-32 (2016).</li></ol>

Measuring the mechanical integration of the nucleus with the cytoskeleton is a challenge for most current methods, such as micropipette aspiration16, because they require either isolated nuclei (where the nucleus is decoupled from the cytoskeleton) or nuclei in suspended cells (where extracellular forces, such as traction forces, are absent). Force has been applied to the nucleus by applying biaxial strain to cells adherent to a membrane17,18</s...

Pathologies such as cancer involve alterations to nuclear shape and structure1,2, which are generally accompanied by a &#39;softening&#39; of the nucleus3,4. Nuclear resistance to mechanical deformation has been generally characterized by applying a force to isolated nuclei5.
The nucleus in cells is molecularly connected to the cytoskeleton by the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex6,7,8,9. As a result, the nucleus is mechanically integrated with the cytoskeleton and, through cell-substratum adhesions, the extracellular matrix. Mechanically probing the nucleus inside adherent cells can provide insight into this mechanical integration. Methods to manipulate nuclei in living cells include micropipette aspiration10,11, and atomic force microscopy12,13,14. We recently described a direct force probe (DFP) that applies mechanical forces directly on the nucleus in a living adherent cell15.
Here, we outline the procedure for using a microinjection system that is commonly available in microscopy facilities to apply a known, nano-scale mechanical force directly to the nucleus in an adherent cell. A femtotip (0.5 &#181;m diameter micropipette tip) is mounted and connected to the microinjection system by a tube. The tip, positioned at a 45&#176; angle relative to the surface of the culture dish, is lowered until adjacent to the nuclear surface. The tube is then disconnected and opened to the atmosphere, which creates a negative suction pressure on the nuclear surface and seals the micropipette tip against the nuclear surface. Through translation of the micropipette tip, the nucleus is deformed and eventually (depending on the magnitude of force applied), detached from the micropipette. This detachment occurs when the restoring (resisting) forces, exerted by the nucleus and cell, equal the suction force applied by the micropipette. Analysis can be performed by measuring the displacement of the nucleus, the length strain (Equation 1), or the area strain (Figure 1A).

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">FluoroDish</td>
			<td style="width:71px;">WPI</td>
			<td style="width:119px;">FD35</td>
			<td style="width:355px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:299px;">SYTO 59</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">S11341</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Femtotips&#160;</td>
			<td style="width:71px;">Eppendorf</td>
			<td align="right">930000043</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">InjectMan NI2</td>
			<td style="width:71px;">Eppendorf</td>
			<td style="width:119px;">NA</td>
			<td>discontinued, current equivalent model: InjectMan 4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">FemtoJet</td>
			<td style="width:71px;">Eppendorf</td>
			<td style="width:119px;">NA</td>
			<td>Current model FemtoJet 4i</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Plan Fluor oil immersion 40x</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Apo TIRF oil immersion 60x</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:299px;">Donor Bovine Serum (DBS)</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">16030074</td>
			<td>NIH 3T3 serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dulbecco&#39;s Modification of Eagle&#39;s (DMEM)</td>
			<td style="width:71px;">Mediatech cellgro</td>
			<td style="width:119px;">MT10013CVRF</td>
			<td>NIH 3T3 medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:71px;">Mediatech</td>
			<td style="width:119px;">MT30004CIRF</td>
			<td>NIH 3T3 medium supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Immersion Oil Type LDF Non-Fluorescing</td>
			<td style="width:71px;">Nikon</td>
			<td align="right" style="width:119px;">77007</td>
			<td>Immersion oil for objective lens&#160;</td>
		</tr>
	</tbody>
</table>

a direct force probe for measuring mechanical integration between the nucleus and the cytoskeleton

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous with the cytoskeleton, methods are needed to probe its mechanical behavior in adherent cells. Here, we discuss the direct force probe (DFP) as a tool to apply force directly to the nucleus in a living adherent cell. We attach a narrow micropipette to the nuclear surface with suction. The micropipette is translated away from the nucleus, which causes the nucleus to deform and translate. When the restoring force is equal to the suction force, the nucleus detaches and elastically relaxes. Because the suction pressure is precisely known, the force on the nuclear surface is known. This method has revealed that nano-scale forces are sufficient to deform and translate the nucleus in adherent cells, and identified cytoskeletal elements that enable the nucleus to resist forces. The DFP can be used to dissect the contributions of cellular and nuclear components to nuclear mechanical properties in living cells.

Figure 2A shows the forcing of an NIH 3T3 mouse fibroblast nucleus. As the micropipette tip is translated to the right, the nucleus deforms and eventually detaches from the micropipette tip. The length strain of the nucleus is seen to increase with increasing suction force (Figure 2B). The front edge of the nucleus (micropipette pulling edge) forms a nuclear protrusion and the trailing edge is displaced from its original position...

Watch this Scientific Journal Video about A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton at JoVE.com

A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

In this protocol, we describe a micropipette method to directly apply a controlled force to the nucleus in a living cell. This assay allows interrogation of nuclear mechanical properties in the living, adherent cell.

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous ...

This method can help answer key questions in the nuclear mechanics field. For example, what is the force required to deform the nucleus in a cell? What are the contributions of cellular and nuclear components to nuclear resistance to deformation?Or, does nuclear coupling with cellular structures vary with cell type? The main advantage of this technique is that it allows us to apply probing force to deform the integrating nucleo-cytoskeleton in a living adherent cell. Though this method has been used to provide insight into the nuclear mechanical properties in NIH-3T3 cells, it can also be applied to any adherent cell type, such as probing nuclear mechanical properties in Hutchinson-Gilford progerial cells.To begin this procedure, coat a 35 millimeter glass bottom dish with 5 micrograms per milliliter of fibronectin. Then, culture the NIH 3T3 fibroblast cells in DMEM supplemented with 10%donor bovine serum and 1%Penicillin-Streptomycin on the coated dish at 37 degrees Celsius until desired confluency. Immediately prior to the experiment, wash the cells twice with PBS, followed by a single wash with complete growth medium.Afterward, add three milliliters of complete growth medium to a glass bottom dish. In this procedure, turn on the microinjector. Using an immersion oil dropper, apply a drop of immersion oil on the objective lens.Then, clamp the dish tightly to the dish holder and load the dish holder onto the stage. Please note that the cells should be maintained at 37 degrees Celsius and 5%carbon dioxide throughout the experiment. Next, adjust the height of the objective to bring the cells into focus.Move the microscope stage to find a cell of interest. Using the micromanipulator, move the pipette holder to the highest position. Subsequently, load the micropipette with a 0.5 micrometer diameter tip onto the pipette holder.Then raise the objective focal plane above plane A and the top of the cell to plane B by adjusting the fine control. Next, set the micromanipulator to coarse control position. Slowly bring the micropipette down to plane B by watching for the silhouette of the micropipette until the micropipette comes fully into focus.Once the micropipette tip is in focus, set the micromanipulator to fine control. Subsequently, lower the objective to the equatorial plane of the cell and lower the micropipette to around 15 micrometers above that. Set the compensation pressure on the microinjector to the desired pressure.Wait several seconds for the pressure to stabilize. It is important to check if the pipette tip is broken or clogged, otherwise the force measurement would be inaccurate. Insure that the micropipette is not clogged by using the clean setting on the micromanipulator panel and make sure that the air bubbles emerge from the micropipette tip.Then, insert the micropipette tip into the cell until it lightly touches the nuclear surface. Create a seal between the micropipette tip and the nuclear membrane by disconnecting the pressure supply tube from the microinjection system, thereby opening the end of the micropipette to the atmosphere. This step creates a negative pressure equal to the compensation pressure on the nuclear surface.Next, open the image collection software. Set up an AVI acquisition for video or ND acquisition for images in the image collection software. Then, toggle to the corresponding fluorescent imaging channel and begin imaging.Move the micropipette tip away from the body of the cell until the nucleus detaches from the micropipette. This figure shows the forcing of an NIH 3T3 mouse fibroblast nucleus. As the micropipette tip moves to the right, the nucleus deforms and eventually detaches from the micropipette tip.The length strain of the nucleus is seen to increase with increasing suction force. The front edge of the nucleus forms a nuclear protrusion and the trailing edge is displaced from its original position. The length of the protrusion is much greater than the trailing edge displacement, suggesting a tight integration between the nucleus and the surrounding cytoplasm.The time scales are short for relaxation of the nuclear front edge and the nuclear back edge. Direct force probe was used to determine the contribution of intranuclear and cytoskeletal structures to the resistance of the nucleus to deformation. Fluorescent images showed the overlay of the nucleus before and after at the indicated condition.While no significant differences in nuclear deformation or translation were found after F-actin disruption or microtubule disruption, reducing the menten expression through siRNA-based knockdown resulted in significantly greater nuclear translation and deformation. This suggests that the menten intermediate filaments in fibroblasts are the main cytoskeletal element that helped the nucleus resist local force. After watching this video, you should have a good understanding of how to apply a controlled and known force to the nucleus in a living adherent cell.

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Biochemistry

14.1K visualizações.  University of Florida. This method can help answer key questions in the nuclear mechanics field. For example, what is the force required to deform the nucleus in a cell? What are the contributions of cellular and nuclear components to nuclear resistance to deformation?Or, does nuclear coupling with cellular structures vary with cell type? The main advantage of this technique is that it allows us to apply probing force to deform the integrating nucleo-cytoskeleton in a living adherent cell. Though this method...