Name: all-optical mechanobiology interrogation of yes-associated protein in human cancer and normal cells using a multi-functional system
Uploaded: 2021-12-20T14:00:00
Duration: 9 min 55 s
Description: Watch this Scientific Journal Video about All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System at JoVE.com

This project is financially supported by the Cancer Pilot Award from UF Health Cancer Center (X. T. and D. S.) and the Gatorade Award Start-up Package (X. T.). The authors sincerely appreciate the intellectual discussions with and the technical supports from Dr. Jonathan Licht (UFHCC), Dr. Rolf Renne (UFHCC), Dr. Ji-Hyun Lee (Biostatistics, UF), Dr. Hugh Fan (MAE, UF), Dr. Warren Dixon (MAE, UF), Dr. Ghatu Subhash (MAE, UF), Dr. Mark Sheplak (MAE &#38; ECE, UF), Dr. Malisa Sarntinoranont (MAE, UF), Dr. Scott Banks (MAE, UF), Dr. Matthew Traum (MAE, UF), Dr. David Hahn (University of Arizona), Dr. Weihong Wang (Oracle Corporation), Dr. Youhua Tan (Hong Kong Polytechnic University), and the Support Team of Nikon (Drs. Jose Serrano-Velez, Larry Kordon, and Jon Ekman). The authors are deeply grateful for the generous and effective support from all members of Tang&#39;s, Siemann&#39;s, and Guan&#39;s research laboratories and all staff members of the MAE &#38; ECE &#38; Physics &#38; Radiation Oncology Departments, UF.

<ol>
	<li>Werley, C., Boccardo, S., Rigamonti, A., Hansson, E., Cohen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+optical+sensors+in+arrayed+islands+of+cells+for+multimodal+recordings+of+cellular+physiology.">Multiplexed optical sensors in arrayed islands of cells for multimodal recordings of cellular physiology.</a> Nature Communications. 11 (1), 3881 (2020).</li><li>Yang, B., 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=Epi-illumination+SPIM+for+volumetric+imaging+with+high+spatial-temporal+resolution.">Epi-illumination SPIM for volumetric imaging with high spatial-temporal resolution.</a> Nature Methods. 16 (6), 501-504 (2019).</li><li>Saraswathibhatla, A., Galles, E. E., Notbohm, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+force+and+motion+in+collective+cell+migration.">Spatiotemporal force and motion in collective cell migration.</a> Scientific Data. 7 (1), 197 (2020).</li><li>Saraswathibhatla, A., Henkes, S., Galles, E. E., Sknepnek, R., Notbohm, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+tractions+control+the+size+of+a+collectively+moving+pack+in+a+cell+monolayer.">Coordinated tractions control the size of a collectively moving pack in a cell monolayer.</a> Extreme Mechanics Letters. 48, 101438 (2021).</li><li>Wang, W., Kim, C. K., Ting, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+tools+for+imaging+and+recording+neuronal+activity.">Molecular tools for imaging and recording neuronal activity.</a> Nature Chemical Biology. 15 (2), 101-110 (2019).</li><li>Eliceiri, K. W., 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=Biological+imaging+software+tools.">Biological imaging software tools.</a> Nature Methods. 9 (7), 697-710 (2012).</li><li>Carpenter, A. E., Kamentsky, L., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+call+for+bioimaging+software+usability.">A call for bioimaging software usability.</a> Nature Methods. 9 (7), 666-670 (2012).</li><li>Skylaki, S., Hilsenbeck, O., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+long-term+imaging+and+quantification+of+single-cell+dynamics.">Challenges in long-term imaging and quantification of single-cell dynamics.</a> Nature Biotechnology. 34 (11), 1137-1144 (2016).</li><li>Lin, M. Z., Schnitzer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+indicators+of+neuronal+activity.">Genetically encoded indicators of neuronal activity.</a> Nature Neuroscience. 19 (9), 1142-1153 (2016).</li><li>Luo, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+multi-functional+integration+program+(AMFIP)+towards+all-optical+mechanobiology+interrogation.">Automatic multi-functional integration program (AMFIP) towards all-optical mechanobiology interrogation.</a> bioRxiv. , (2021).</li><li>Edelstein, A., Amodaj, N., Hoover, K., Vale, R., Stuurman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+control+of+microscopes+using+manager.">Computer control of microscopes using manager.</a> Current Protocols in Molecular Biology. 92 (1), 14-20 (2010).</li><li>Tulpule, 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=Kinase-mediated+RAS+signaling+via+membraneless+cytoplasmic+protein+granules.">Kinase-mediated RAS signaling via membraneless cytoplasmic protein granules.</a> Cell. 184 (10), 2649-2664 (2021).</li><li>Tang, X., Tofangchi, A., Anand, S. V., Saif, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+cell+traction+force+microscopy+to+study+multi-cellular+system.">A novel cell traction force microscopy to study multi-cellular system.</a> PLOS Computational Biology. 10 (6), 1003631 (2014).</li><li>Tang, X., 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+force+affects+expression+of+an+in+vitro+metastasis-like+phenotype+in+HCT-8+cells.">Mechanical force affects expression of an in vitro metastasis-like phenotype in HCT-8 cells.</a> Biophysical Journal. 99 (8), 2460-2469 (2010).</li><li>Guimar&#227;es, C. F., Gasperini, L., Marques, A. P., Reis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stiffness+of+living+tissues+and+its+implications+for+tissue+engineering.">The stiffness of living tissues and its implications for tissue engineering.</a> Nature Reviews Materials. 5, 351-370 (2020).</li><li>Phelps, E. 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=Maleimide+cross-linked+bioactive+PEG+hydrogel+exhibits+improved+reaction+kinetics+and+cross-linking+for+cell+encapsulation+and+in+situ+delivery.">Maleimide cross-linked bioactive PEG hydrogel exhibits improved reaction kinetics and cross-linking for cell encapsulation and in situ delivery.</a> Advanced Materials. 24 (1), 64-70 (2012).</li><li>Bajaj, P., Tang, X., Saif, T. A., Bashir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiffness+of+the+substrate+influences+the+phenotype+of+embryonic+chicken+cardiac+myocytes.">Stiffness of the substrate influences the phenotype of embryonic chicken cardiac myocytes.</a> Journal of Biomedical Materials Research. Part A. 95 (4), 1261-1269 (2010).</li><li>Temples, M. N., Adjei, I. M., Nimocks, P. M., Djeu, J., Sharma, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+three-dimensional+tumor+models+to+study+natural+killer+cell+suppression.">Engineered three-dimensional tumor models to study natural killer cell suppression.</a> ACS Biomaterials Science &amp; Engineering. 6 (7), 4179-4199 (2020).</li><li>Feng, 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=Improved+split+fluorescent+proteins+for+endogenous+protein+labeling.">Improved split fluorescent proteins for endogenous protein labeling.</a> Nature Communications. 8, 370 (2017).</li><li>Guan, J., Liu, H., Shi, X., Feng, S., Huang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+multiple+genomic+elements+using+correlative+CRISPR+imaging+and+sequential+DNA+FISH.">Tracking multiple genomic elements using correlative CRISPR imaging and sequential DNA FISH.</a> Biophysical Journal. 112 (6), 1077-1084 (2017).</li><li>. Micro-Manager Available from: <a target="_blank" href="https://micro-manager.org/wiki/NikonTi2">https://micro-manager.org/wiki/NikonTi2</a> (2021)</li><li>Schindelin, 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=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Martiel, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cell+traction+forces+with+ImageJ.">Measurement of cell traction forces with ImageJ.</a> Methods in Cell Biology. 125, 269-287 (2015).</li><li>Okumurai, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+generalization+of+Cerruti's+problem+in+an+elastic+half-space.">On the generalization of Cerruti's problem in an elastic half-space.</a> Doboku Gakkai Ronbunshu. 1995, 1-10 (1995).</li><li>Piccolo, S., Dupont, S., Cordenonsi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+YAP/TAZ:+hippo+signaling+and+beyond.">The biology of YAP/TAZ: hippo signaling and beyond.</a> Physiological Reviews. 94 (4), 1287-1312 (2014).</li><li>Hong, W. W., Guan, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+YAP+and+TAZ+transcription+co-activators:+Key+downstream+effectors+of+the+mammalian+Hippo+pathway.">The YAP and TAZ transcription co-activators: Key downstream effectors of the mammalian Hippo pathway.</a> Seminars in Cell and Developmental Biology. 23 (7), 785-793 (2012).</li><li>Zanconato, F., Cordenonsi, M., Piccolo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=YAP/TAZ+at+the+roots+of+cancer.">YAP/TAZ at the roots of cancer.</a> Cancer Cell. 29 (6), 783-803 (2016).</li><li>Wang, Y., 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=Overexpression+of+yes-associated+protein+contributes+to+progression+and+poor+prognosis+of+non-small-cell+lung+cancer.">Overexpression of yes-associated protein contributes to progression and poor prognosis of non-small-cell lung cancer.</a> Cancer Science. 101 (5), 1279-1285 (2010).</li><li>Li, 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=Inhibition+of+YAP+suppresses+CML+cell+proliferation+and+enhances+efficacy+of+imatinib+in+vitro+and+in+vivo.">Inhibition of YAP suppresses CML cell proliferation and enhances efficacy of imatinib in vitro and in vivo.</a> Journal of Experimental &amp; Clinical Cancer Research. 35 (1), 134 (2016).</li><li>Tang, X., 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+mechanically-induced+colon+cancer+cell+population+shows+increased+metastatic+potential.">A mechanically-induced colon cancer cell population shows increased metastatic potential.</a> Molecular Cancer. 13, 131 (2014).</li><li>Panciera, T., Azzolin, L., Cordenonsi, M., Piccolo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiology+of+YAP+and+TAZ+in+physiology+and+disease.">Mechanobiology of YAP and TAZ in physiology and disease.</a> Nature Reviews Molecular Cell Biology. 18 (12), 758-770 (2017).</li><li>Yuan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yes-associated+protein+(YAP)+functions+as+a+tumor+suppressor+in+breast.">Yes-associated protein (YAP) functions as a tumor suppressor in breast.</a> Cell Death and Differentiation. 15 (11), 1752-1759 (2008).</li><li>Koushki, N., 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+redistribution+mediated+by+nuclear+deformation+determines+dynamic+localization+of+YAP.">Lamin A redistribution mediated by nuclear deformation determines dynamic localization of YAP.</a> bioRxiv. , (2020).</li><li>Chang, S. S., Rape, A. D., Wong, S. A., Guo, W. H., 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=Migration+regulates+cellular+mechanical+states.">Migration regulates cellular mechanical states.</a> Molecular Biology of the Cell. 30 (26), 3104-3111 (2019).</li><li>Lee, J., Abdeen, A. A., Tang, X., Saif, T. A., Kilian, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+guidance+of+integrin+mediated+traction+stress+during+stem+cell+differentiation.">Geometric guidance of integrin mediated traction stress during stem cell differentiation.</a> Biomaterials. 69, 174-183 (2015).</li><li>Lee, J., Abdeen, A., Tang, X., Saif, T. A., Kilian, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+directed+adipogenesis+and+neurogenesis+of+mesenchymal+stem+cells+derived+from+adipose+tissue+and+bone+marrow.">Matrix directed adipogenesis and neurogenesis of mesenchymal stem cells derived from adipose tissue and bone marrow.</a> Acta Biomaterialia. 42, 46-55 (2016).</li><li>Tang, X., Bajaj, P., Bashir, R., Saif, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+far+cardiac+cells+can+see+each+other+mechanically.">How far cardiac cells can see each other mechanically.</a> Soft Matter. 7 (13), 6151-6158 (2011).</li><li>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=Stresses+at+the+cell-to-substrate+interface+during+locomotion+of+fibroblasts.">Stresses at the cell-to-substrate interface during locomotion of fibroblasts.</a> Biophysical Journal. 76 (4), 2307-2316 (1999).</li><li>Rape, A., Guo, W. H., 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=The+regulation+of+traction+force+in+relation+to+cell+shape+and+focal+adhesions.">The regulation of traction force in relation to cell shape and focal adhesions.</a> Biomaterials. 32 (8), 2043-2051 (2011).</li><li>Shiu, J. Y., Aires, L., Lin, Z., Vogel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopillar+force+measurements+reveal+actin-cap-mediated+YAP+mechanotransduction.">Nanopillar force measurements reveal actin-cap-mediated YAP mechanotransduction.</a> Nature Cell Biology. 20 (3), 262-271 (2018).</li><li>Fracchia, A., Asraf, T., Salmon-Divon, M., Gerlitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+lamin+B1+levels+promote+cell+migration+by+altering+perinuclear+actin+organization.">Increased lamin B1 levels promote cell migration by altering perinuclear actin organization.</a> Cells. 9 (10), 2161 (2020).</li><li>Ramdas, N. M., 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=Cytoskeletal+control+of+nuclear+morphology+and+chromatin+o1rganization.">Cytoskeletal control of nuclear morphology and chromatin o1rganization.</a> Journal of Molecular Biology. 427 (3), 695-706 (2015).</li><li>Khatau, S. B., 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+perinuclear+actin+cap+regulates+nuclear+shape.">A perinuclear actin cap regulates nuclear shape.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (45), 19017-19022 (2009).</li><li>Kim, D. H., Cho, S., Wirtz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+coupling+between+nucleus+and+cell+migration+through+the+perinuclear+actin+cap.">Tight coupling between nucleus and cell migration through the perinuclear actin cap.</a> Journal of Cell Science. 127 (11), 2528-2541 (2014).</li></ol>

There are no conflicts of interest to declare.

The imaging process (step 6.3) is critical to ensure that the fluorescence images are of sufficiently good quality to yield valid quantification results. The z-stack images of fluorescent protein or beads should have a z-range that is large enough to include the in-focus images for all the Z-positions that the sample spans. Another critical step is collecting the reference images of fluorescent beads after dissolving the cells (step 6.5). Because the reference images need to be taken at the same positions in step 6.3, no relative displacement should be induced between the Petri dish, environment chamber, and the microscope. The investigators performing the dissolving step must be careful to remove the lid of the Petri dish and ensure that the applied mechanical perturbation is not large enough to alter the location of the dish in the environment chamber.
Solutions are provided below to resolve some errors that might occur during experiments. If no macro is activated after clicking Enter in step 6.4, it is most likely because the left bottom area of the screen is occupied by a non-Element window. In such a case, the left bottom area of the window needs to be cleared so that macros can be activated in Elements. Another common error is that the bright-field images appear black. This issue is caused by an insufficient time interval between the acquisitions of fluorescence and bright-field images. Slight delays in the fluorescence imaging time counting can accumulate over time and cause considerable delays and interfere with the bright-field imaging. One solution is to adjust the duration of one imaging cycle of all the positions to be less than (not equal to) the time interval between the start of consecutive motions. This operation refreshes the time counting and eliminates the cumulative error at the beginning of each imaging cycle.
This all-optical interrogation technology supports (1) a wide range of hardware/software, including but not limited to Nikon, (2) diverse types of validated hydrogel systems, including gelatin, PEG, Matrigel, and collagen I gels, and (3) the programmable customization based on different needs of researchers. However, if any of the bottom-level control functions are not available from a commercial microscope, customization of the functions using AMFIP becomes challenging. Another limitation of this technique is the spatial drift of the sample in both the XY and focus (Z) planes. Although this limitation can be overcome during the post-processing of the images, it is essential to improve the auto-focus function to correct the real-time drift of the samples. This improvement will increase the throughput of the imaging process and reduce the potential error caused by the drift during experiments.
Mechanotransducers, such as YAP, may serve as new therapeutic targets for the development of promising cancer therapies25,26,27. Emerging data suggest that YAP promotes the proliferation and invasion of cancer cells. Mechanics-induced YAP translocation from the cytoplasm to the nucleus activates the transcription of genes related to cell migration, proliferation, invasion, and apoptosis, leading to aberrant cell behaviors28,29,30,31. This work aimed to explore the potential correlation of the YAP N/C ratio and cell mechanics in two typical human lung cancer and normal cell lines. During the 10 h cell spreading period, PC9 cells show similar YAP concentrations in the nucleus and cytoplasm (Figure 3D and Figure 5A). B2B cells show a higher YAP concentration in the nucleus than in the cytoplasm (Figure 3C and Figure 5A). This relationship found during the early spreading stage is different from the majority of published findings that compare YAP concentration in the nucleus between normal and cancer cells. Although not necessarily at the early spreading stage, most published findings show that YAP is more concentrated in the nucleus of cancer cells than in the nucleus of normal cells27,28. Only one study on breast cancer reported an exception32 that shows YAP is more concentrated in the cytoplasm, which agrees with our current observations made in lung cancer PC9 cells. To the best of the authors&#39; knowledge, this work is the first to show a lower YAP N/C ratio in a human lung cancer cell line. The authors hypothesize that the reason for a stable YAP N/C ratio in PC9 cells might be due to the low variation in the cell/nucleus spread area and traction in PC9 cells at the early spreading stage. The dissection of the underpinning molecular mechanisms of low YAP N/C ratio in PC9 and B2B cells is ongoing.
During the first 10 h of spreading, these two cell lines show a distinct relationship between the YAP N/C ratio, cell traction, and spread area (Figure 5). For B2B cells, a higher YAP N/C ratio is correlated with a higher cell and nucleus spread area (Figure 6A,B), which is consistent with the reported data of other normal cells33. Interestingly, although the developmental trend of this relationship is generally found in all B2B cells recorded, two different degrees (high and low) of this relationship are found. B2B cells that spread and migrate simultaneously show lower traction and higher cell and nucleus spread area with a higher YAP N/C ratio (2.05 &#177; 0.32). For B2B cells that spread and remain at the same location, they show higher traction and lower cell and nucleus spread area with a lower YAP N/C ratio (1.74 &#177; 0.21). These two degrees of relationships are demonstrated in the bifurcated scattered data groups (Figure 6C,D). As reported in the literature, stationary normal cells, such as embryonic fibroblast NIH 3T3 cells, have higher traction than migratory cells34. The data reported in this paper suggest that the spreading and non-migrating B2B cells applied higher traction than spreading and migrating B2B cells, likely suggesting that high traction is needed for non-migrating cells to stabilize on the substrate.
In addition, these data show that stationary normal B2B cells generate a higher peri-nuclear force, whereas previous research conducted by other researchers reported only higher cell traction generated at the periphery of stationary cells34,35,36,37. The authors think that the difference in the intrinsic tendency of migration in the experiments might cause these contradictory results. In the published experiments, square-shaped micropatterning had been used to confine single cells from spreading and inhibit migration; whether the cells had the tendency to migrate is unknown. As migratory cells often show high traction force at the periphery of the cells38, it is likely that cells with the tendency to migrate will still maintain high periphery traction even though their migration is restricted. In this present study, the stationary cells are not restricted by any micropattern but do not migrate, indicating that the cells tend to maintain their non-migrating state. Another possibility is that the cell shape defined by the micropattern may affect the distribution of focal adhesions and traction forces39. The results in this study were generated without any confining micropatterning and represent the force distribution of stationary cells in their original shape.
To the best of the authors&#39; knowledge, only one publication to date specifically reported the finding of peri-nuclear forces in normal cells (mouse embryonic fibroblasts), potentially caused by the actin cap spanning across the nucleus40. YAP cytoplasm-to-nucleus translocation is correlated with the increase in peri-nuclear force40. A thorough search of the relevant literature did not yield any publicationsthat report a peri-nuclear force or the actin cap in cancer cells. An indirect study on melanoma cancer cells demonstrated that the actin rim (another peri-nuclear actin organization located around but not covering the nucleus) reduces cell migration rates41, indirectly suggesting the existence of a peri-nuclear force. However, no direct experimental data are reported. In this study, the authors found that both PC9 and B2B cells show peri-nuclear displacement and traction. The mechanisms of the generation of the peri-nuclear forces and their effects remain controversial. In normal cells, the actin cap was reported to play a role in regulating nucleus morphology and chromatin organization42, transmitting mechanical signals from focal adhesions into the nucleus through linkers of nucleoskeleton and cytoskeleton (LINC) complex43, and regulating cell migration44. Lamin A/C is related to the formation and disruption of the actin cap40,41,42,43,44. However, the report that claimed that the actin cap generates a peri-nuclear force did not consider the potential role of the actin rim40. In cancer cells, overexpression of Lamin A facilitates the formation of an actin rim and restricts cancer cell migration. Overexpression of Lamin B reduces actin rim formation and promotes migration. The peri-nuclear force might be involved in this process due to the existence of peri-nuclear actin organization and the effect of Lamin A. However, the results of this study did not show any evidence of measured peri-nuclear forces or the behavior of the actin cap. Therefore, the discovery of peri-nuclear forces in PC9 cells in this present study is the first report showing peri-nuclear forces and displacements in lung cancer cells. The authors are currently investigating the molecular mechanisms and functions of peri-nuclear forces in CRISPR/Cas9-engineered PC9 and B2B cells.
Beyond the all-optical mechanobiology interrogation that is demonstrated in this paper, the integrated multi-functional system can be applied to optically probe a myriad of other essential physiological and pathobiological signals in living systems. For example, the authors&#39; laboratory has recently established multiple stably transduced human cancer cell lines that co-express three light-responsive membrane proteins: membrane voltage indicator QuasAr2 (excitation: 640 nm; emission: 660 nm-740 nm), membrane voltage depolarizer CheRiff (excitation: 488 nm), and membrane voltage hyperpolarizer eNpHR3 (excitation: 590 nm). These three functional proteins can be activated by spectrum-orthogonal laser lines in a crosstalk-free manner, enabling all-optical two-way signaling communications (readout and control) of membrane electrophysiology. Using an integrated opto-electronics system and a manual patch-clamp, the authors have validated the all-optical control and readout of the membrane voltage (Vm) in single human cancer cells and multicellular tumor spheroids. The all-optical electrophysiology interrogation opens the possibility for detailed explorations of previously inaccessible bioelectricity in cancer cells, which may help advance tumor biology from a new axis.

The overall goal of this method is to enable all-optical multi-functional imaging and analysis of living cells. An all-in-one imaging program that enables the automatic coordination of multi-functional optoelectronic devices will reduce labor-intensive and error-prone manual operations and is essential for researchers to conduct long-term live-cell imaging1,2,3,4. However, most existing public programs in the biomedical research community either only apply to limited optoelectronic devices or require additional hardware for the coordination of different equipment5,6,7,8,9. Recently, an open-source and software-based program has been developed, titled &#34;Automatic Multi-functional Integration Program (AMFIP),&#34; enabling multi-channel and time-lapse imaging. Based on Java language and the Application Programming Interface (API) of &#181;Manager11,12, AMFIP was developed as a plugin in &#181;Manager that executes customized Java scripts to accomplish software-based communications of multiple optoelectronic hardware and software platforms, including but not limited to those from Nikon. The establishment of AMFIP opens the possibility for programmable and multi-functional interrogation of cell behaviors. An integrated experimental and computational system is developed in this paper and combines AMFIP with digital imaging analysis and cell traction force microscopy. The system enables the elucidation of the distinct YAP mechanobiology in CRISPR/Cas9-engineered human normal B2B (Figure 1) and lung cancer PC9 (Figure 2) cell lines. The system provides the scientific community a comprehensive solution that avoids the demand of purchasing additional controlling devices that may not be available for and/or compatible with every imaging system.
The protocols presented in this paper introduce how to (1) apply AMFIP to conduct automatic long-term imaging for both CRISPR/Cas9-engineered cell lines that express mNEonGreen2-tagged YAP; and (2) combine Fiji ImageJ, MATLAB, and Origin for the quantitative analysis of YAP nuclear/cytoplasm (N/C) ratio based on their fluorescent intensity (Figure 3 and Figure 4), cellular displacement field (Figure 1C and Figure 2C), and cellular traction field (Figure 1D and Figure 2D). The results suggest that (1) during the first 10 h of cell spreading on the substrates that have physiologically relevant mechanical stiffness13,14,15,16,17,18, the YAP N/C ratio of single B2B cells shows more noticeable time-dependent variation and fluctuation compared to that of single PC9 cells (Figure 5 and Figure 6); and (2) PC9 cancer cells generate noticeable traction in their peri-nuclear regions (Figure 7). The integrated system and methodologies described in this protocol transcend the specific types of cells and optogenetic molecules. Researchers can apply the protocols to customize their specific live-cell interrogation experiments and elucidate multifaceted signaling dynamics in the context of cell physiology and pathology.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(3-Aminopropyl)triethoxysilane</td>
			<td>Sigma-aldrich</td>
			<td>440140</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05 % Trypsin</td>
			<td>Corning</td>
			<td>25-051-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 flask</td>
			<td>Corning</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>8 Benchtop Centrifuge</td>
			<td>Thermo</td>
			<td>75007210</td>
			<td></td>
		</tr>
		<tr>
			<td>A1R confocal system</td>
			<td>Nikon</td>
			<td>HD25</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-aldrich</td>
			<td>695092</td>
			<td>glacial, ACS reagent, &#8805;99.7%</td>
		</tr>
		<tr>
			<td>BEAS-2B (B2B) cells</td>
			<td>Sigma-aldrich</td>
			<td>95102433</td>
			<td>human epithelial cells from lung tissue</td>
		</tr>
		<tr>
			<td>Carboxylate-Modified Microspheres</td>
			<td>Invitrogen</td>
			<td>F8797</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture medium (RPMI-1640)</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop Computer</td>
			<td>Dell</td>
			<td>2018</td>
			<td>with Windows 10 operating system</td>
		</tr>
		<tr>
			<td>Environmental chamber TIZB</td>
			<td>Tokai Hit</td>
			<td>TIZB</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>26140</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin Human Protein, Plasma</td>
			<td>Gibco</td>
			<td>33016015</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji ImageJ</td>
			<td>National Institutes of Health and the Laboratory for Optical and Computational Instrumentation</td>
			<td>1.53k</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass-bottom petri dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffered saline</td>
			<td>Sigma-aldrich</td>
			<td>51558</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrazine hydrate solution</td>
			<td>Sigma-aldrich</td>
			<td>53847</td>
			<td></td>
		</tr>
		<tr>
			<td>IntelliJ IDEA</td>
			<td>JetBrains</td>
			<td>2020</td>
			<td>Java development platform</td>
		</tr>
		<tr>
			<td>Java Development Kit</td>
			<td>Oracle</td>
			<td>14.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Kimtech Science</td>
			<td>3066-05</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>2020b</td>
			<td></td>
		</tr>
		<tr>
			<td>Monochrome&#160;Camera</td>
			<td>FLIR</td>
			<td>BFS-U3-70S7M-C</td>
			<td></td>
		</tr>
		<tr>
			<td>MycoAlert Mycoplasma Detection Kit</td>
			<td>Lonza</td>
			<td>LT07-218</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#8242;-Methylenebisacrylamide solution</td>
			<td>Sigma-aldrich</td>
			<td>M1533</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements software platform</td>
			<td>Nikon</td>
			<td>4.50</td>
			<td>software platform</td>
		</tr>
		<tr>
			<td>Origin</td>
			<td>OriginLab</td>
			<td>OriginPro 2017 (Learning Edition)</td>
			<td>data analysis and graphing software</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PC9 cells</td>
			<td>Sigma-aldrich</td>
			<td>90071810</td>
			<td>human adenocarcinoma cells&#160; from lung tissue</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>&#160;F-553S</td>
			<td>high-fidelity DNA polymerase</td>
		</tr>
		<tr>
			<td>Scotch tape</td>
			<td>Scotch</td>
			<td></td>
			<td>adhesive tape</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate solution</td>
			<td>Sigma-aldrich</td>
			<td>05030</td>
			<td></td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>Gorilla</td>
			<td></td>
			<td>cyanoacrylate glue</td>
		</tr>
		<tr>
			<td>Ti2-E inverted microscope</td>
			<td>Nikon</td>
			<td>MEA54000</td>
			<td></td>
		</tr>
		<tr>
			<td>TI2-S-SE-E Motorized Stage with Encoder</td>
			<td>Nikon</td>
			<td>MEC56120</td>
			<td></td>
		</tr>
		<tr>
			<td>&#956;Manager</td>
			<td></td>
			<td>version 2.0 gamma</td>
			<td>open source microscopy software (https://micro-manager.org/)</td>
		</tr>
	</tbody>
</table>

all-optical mechanobiology interrogation of yes-associated protein in human cancer and normal cells using a multi-functional system

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. However, manual control of various equipment produced by different manufacturers is labor-intensive and time-consuming, potentially decreasing the accuracy, reproducibility, and quality of acquired data. Therefore, an all-in-one and user-programmable system that enables automatic, multi-functional, and long-term image acquisition and is compatible with most fluorescent microscopy platforms can benefit the scientific community. This paper introduces the complete operating protocols of utilizing a novel integrated software system that consists of (1) a home-built software program, titled "Automatic Multi-functional Integration Program (AMFIP)," which enables automatic multi-channel imaging acquisition, and (2) a suite of quantitative imaging analysis and cell traction computation packages. 
This integrated system is applied to reveal the previously unknown relationship between the spatial-temporal distribution of mechano-sensitive Yes-associated protein (YAP) and the cell mechanics, including cell spreading and traction, in CRISPR/Cas9-engineered human normal cells (B2B) and lung cancer cells (PC9). Leveraging this system's capability of multi-channel control and readout, the result shows: (1) B2B normal cells and PC9 cancer cells show a distinct relationship between YAP expression, traction, and cell dynamics during cell spreading and migration processes; and (2) PC9 cancer cells apply noticeable peri-nuclear forces on substrates. In summary, this paper presents a detailed stepwise protocol on how to utilize an integrated user-programmable system that enables automatic multi-functional imaging and analysis to elucidate YAP mechano-sensitivity. These tools open the possibility for detailed explorations of multifaceted signaling dynamics in the context of cell physiology and pathology.

Watch this Scientific Journal Video about All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System at JoVE.com

All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System

This paper presents a detailed stepwise protocol on how to utilize an integrated multi-functional and user-programmable system that enables automatic multi-channel imaging and mechanobiological analysis to elucidate the mechano-sensitivity of Yes-associated protein (YAP).

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. ...

Research

JoVE Journal

Bioengineering

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...