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Quantitative Assessment of the Relationship Between Cellular Morphodynamics and Signaling Events by Stochastic Analysis of Fluorescent Images

Published online by Cambridge University Press:  10 June 2014

Giuseppe Maulucci Open the ORCID record for Giuseppe Maulucci [Opens in a new window] ,
Alessandro Maiorana ,
Massimiliano Papi ,
Giovambattista Pani  and
Marco De Spirito
Giuseppe Maulucci
Affiliation:
Istituto di Fisica, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00169, Roma
Alessandro Maiorana
Affiliation:
Istituto di Fisica, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00169, Roma
Massimiliano Papi
Affiliation:
Istituto di Fisica, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00169, Roma
Giovambattista Pani
Affiliation:
Istituto di Patologia Generale, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00169, Roma
Marco De Spirito*
Affiliation:
Istituto di Fisica, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00169, Roma
*
*Corresponding author. [email protected]
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Abstract

Cell motility involves a number of strategies that cells use in order to seek nutrients, escape danger, and fulfill morphogenetic roles. Here we present a methodology to quantify morphological changes and their relationship with signaling events from time-lapse imaging microscopy experiments, in order to characterize physiological and pathological processes. To this aim, the stationary spatial pattern of signaling events is determined through an intracellular fluorescent probe, and it is related with the frequency and entity of morphodynamic events, which are in turn quantified through a stochastic approach: two pseudoimages are obtained from a time series of moving cells that describe the probability that a pixel belongs to the cell, and the probability that a pixel is subject to a dynamic event. The simultaneous construction of these maps permits visualization of hot spots of dynamic events, i.e., zones of formation of membrane protrusions and retractions and their relationship with the signaling events reported by the specific probe employed. The method is tested on spontaneous movement of cells, trasfected with redox-sensitive yellow fluorescent protein, in which the distribution of the hot spots and its change upon expression of constitutively active Rac (V12-Rac), is related to the distribution of oxidized spots.

Keywords

cell trackingcellular morphodynamicsRacV12-Racconfocal microscopyreactive oxygen species
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 20 , Issue 4 , August 2014 , pp. 1198 - 1207
Copyright
© Microscopy Society of America 2014 

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References

Angelucci, C., Maulucci, G., Lama, G., Proietti, G., Colabianchi, A., Papi, M., Maiorana, A., De Spirito, M., Micera, A., Balzamino, O.B., Di Leone, A., Masetti, R. & Sica, G. (2012). Epithelial-stromal interactions in human breast cancer: Effects on adhesion, plasma membrane fluidity and migration speed and directness. PloS One 7, e50804. Available at http://www.plosone.org/article/related/info:doi/10.1371/journal.pone.0050804;jsessionid=DF961FF1D04EE9F72EA663A9974D6380 (retrieved May 15, 2013).Google Scholar
Arosio, D., Ricci, F., Marchetti, L., Gualdani, R., Albertazzi, L. & Beltram, F. (2010). Simultaneous intracellular chloride and pH measurements using a GFP-based sensor. Nat Met 7, 516–518. Available at http://dx.doi.org/10.1038/nmeth.1471 (retrieved August 11, 2013).Google Scholar
Balogh, G., Maulucci, G., Gombos, I., Horváth, I., Török, Z., Péter, M., Fodor, E., Páli, T., Benkő, S., Parasassi, T., De Spirito, M., Harwood, J.L. & Vígh, L. (2011). Heat stress causes spatially-distinct membrane re-modelling in K562 leukemia cells. PloS One 6, e21182. Available at http://www.ncbi.nlm.nih.gov/pubmed/21698159 (retrieved June 26, 2011).CrossRefGoogle ScholarPubMed
Banyard, J., Anand-Apte, B., Symons, M. & Zetter, B.R. (2000). Motility and invasion are differentially modulated by Rho family GTPases. Oncogene 19, 580–591. Available at http://www.nature.com/onc/journal/v19/n4/full/1203338a.html (retrieved May 15, 2013).Google Scholar
Diaz, B., Shani, G., Pass, I., Anderson, D., Quintavalle, M. & Courtneidge, S.A. (2009). Tks5-dependent, nox-mediated generation of reactive oxygen species is necessary for invadopodia formation. Sci Signal 2, ra53. Available at http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2810640&tool=pmcentrez&rendertype=abstract (retrieved May 29, 2013).CrossRefGoogle ScholarPubMed
Dormann, D., Libotte, T., Weijer, C.J. & Bretschneider, T. (2002). Simultaneous quantification of cell motility and protein-membrane-association using active contours. Cell Motil Cytoskeleton 52, 221–230. Available at http://www.ncbi.nlm.nih.gov/pubmed/12112136.CrossRefGoogle ScholarPubMed
Dubinthaler, B., Giannone, G., Dobereiner, H. & Sheetz, M. (2004). Nanometer analysis of cell spreading on matrix-coated surfaces reveals two distinct cell states and steps. Biophys J 86, 1794–1806. Available at http://linkinghub.elsevier.com/retrieve/pii/S0006349504742460.Google Scholar
Dzyubachyk, O., van Cappellen, W.A., Essers, J., Niessen, W.J. & Meijering, E. (2010). Advanced level-set-based cell tracking in time-lapse fluorescence microscopy. IEEE Trans Med Imag 29, 852–867. Available at http://ieeexplore.ieee.org/articleDetails.jsp?arnumber=5423293 (retrieved September 6, 2013).Google Scholar
Etienne-Manneville, S. & Hall, A. (2002). Rho GTPases in cell biology. Nature 420, 629–635. Available at http://www.ncbi.nlm.nih.gov/pubmed/18460342.Google Scholar
Ferraro, D., Corso, S., Fasano, E., Panieri, E., Santangelo, R., Borrello, S., Giordano, S., Pani, G. & Galeotti, T. (2006). Pro-metastatic signaling by c-Met through RAC-1 and reactive oxygen species (ROS). Oncogene 25, 3689–3698. Available at http://dx.doi.org/10.1038/sj.onc.1209409 (retrieved May 17, 2013).Google Scholar
Gianni, D., Diaz, B., Taulet, N., Fowler, B., Courtneidge, S.A. & Bokoch, G.M. (2009). Novel p47(phox)-related organizers regulate localized NADPH oxidase 1 (Nox1) activity. Sci Sig 2, ra54. Available at http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2850287&tool=pmcentrez&rendertype=abstract (retrieved May 29, 2013).Google Scholar
Giannone, G., Dubin-Thaler, B.J., Döbereiner, H.-G., Kieffer, N., Bresnick, A.R. & Sheetz, M.P. (2004). Periodic lamellipodial contractions correlate with rearward actin waves. Cell 116, 431–443. Available at http://www.ncbi.nlm.nih.gov/pubmed/15016377.Google Scholar
Kass, M., Witkin, A. & Terzopoulos, D. (1988). Snakes: Active contour models. Int J Comput Vis 1, 321–331. Available at http://link.springer.com/10.1007/BF00133570 (retrieved March 2, 2013).Google Scholar
Kiosses, W.B., Shattil, S.J., Pampori, N. & Schwartz, M.A. (2001). Rac recruits high-affinity integrin alphavbeta3 to lamellipodia in endothelial cell migration. Nat Cell Biol 3, 316–320. Available at http://www.nature.com/ncb/journal/v3/n3/pdf/ncb0301_316.pdf (retrieved May 15, 2013).Google Scholar
Koch, O.R., Fusco, S., Ranieri, S.C., Maulucci, G., Palozza, P., Larocca, L.M., Cravero, A.A, Farre’, S.M., De Spirito, M., Galeotti, T. & Pani, G. (2008). Role of the life span determinant P66(shcA) in ethanol-induced liver damage. Lab Invest 88, 750–760. Available at http://www.ncbi.nlm.nih.gov/pubmed/18490896 (retrieved May 14, 2011).Google Scholar
Machacek, M. & Danuser, G. (2006). Morphodynamic profiling of protrusion phenotypes. Biophys J 90, 1439–1452. Available at http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1367294&tool=pmcentrez&rendertype=abstract (retrieved July 21, 2010).CrossRefGoogle ScholarPubMed
Maulucci, G., Labate, V., Mele, M., Panieri, E., Arcovito, G., Galeotti, T., Østergaard, H., Winther, J.R., De Spirito, M. & Pani, G. (2008). High-resolution imaging of redox signaling in live cells through an oxidation-sensitive yellow fluorescent protein. Sci Signal 1, pl3. http://www.ncbi.nlm.nih.gov/pubmed/18957692.CrossRefGoogle ScholarPubMed
Maulucci, G., Pani, G., Fusco, S., Papi, M., Arcovito, G., Galeotti, T., Fraziano, M. & De Spirito, M. (2010). Compartmentalization of the redox environment in PC-12 neuronal cells. Eur Biophys J 39, 993–999. Available at http://www.ncbi.nlm.nih.gov/pubmed/19495741 (retrieved October 12, 2010).Google Scholar
Maulucci, G., Pani, G., Labate, V., Mele, M., Panieri, E., Papi, M., Arcovito, G., Galeotti, T. & De Spirito, M. (2009). Investigation of the spatial distribution of glutathione redox-balance in live cells by using Fluorescence Ratio Imaging Microscopy. Biosen Bioelectron 25, 682–687. Available at http://www.ncbi.nlm.nih.gov/pubmed/19748771 (retrieved March 2, 2011).Google Scholar
Meijering, E., Dzyubachyk, O. & Smal, I. (2012). Methods for cell and particle tracking. Met Enzymol 504, 183–200. Available at http://www.ncbi.nlm.nih.gov/pubmed/22264535 (retrieved August 9, 2013).Google Scholar
Nimnual, A.S., Taylor, L.J. & Bar-Sagi, D. (2003). Redox-dependent downregulation of Rho by Rac. Nat Cell Biol 5, 236–241. Available at http://dx.doi.org/10.1038/ncb938 (retrieved March 11, 2013).Google Scholar
Ostergaard, H., Henriksen, A., Hansen, F.G. & Winther, J.R. (2001). Shedding light on disulfide bond formation: Engineering a redox switch in green fluorescent protein. EMBO J 20, 5853–5862. Available at http://dx.doi.org/10.1093/emboj/20.21.5853 (retrieved May 16, 2013).Google Scholar
Padfield, D., Rittscher, J. & Roysam, B. (2011). Coupled minimum-cost flow cell tracking for high-throughput quantitative analysis. Med Image Anal 15, 650–668. Available at http://dx.doi.org/10.1016/j.media.2010.07.006 (retrieved August 13, 2013).Google Scholar
Pani, G., Bedogni, B., Colavitti, R., Anzevino, R., Borrello, S. & Galeotti, T. (2001). Cell compartmentalization in redox signaling. IUBMB Life 52, 7–16. Available at http://www.ncbi.nlm.nih.gov/pubmed/11795597 (retrieved May 15, 2013).Google Scholar
Pani, G., Galeotti, T. & Chiarugi, P. (2010). Metastasis: Cancer cell’s escape from oxidative stress. Cancer Metastasis Rev 29, 351–378. Available at http://www.ncbi.nlm.nih.gov/pubmed/20386957 (retrieved March 13, 2013).Google Scholar
Panieri, E., Toietta, G., Mele, M., Labate, V., Ranieri, S.C., Fusco, S., Tesori, V., Antonini, A., Maulucci, G., De Spirito, M., Galeotti, T. & Pani, G. (2010). Nutrient withdrawal rescues growth factor-deprived cells from mTOR-dependent damage. Aging 2, 487–503. Available at http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2954040&tool=pmcentrez&rendertype=abstract.CrossRefGoogle ScholarPubMed
Pollard, T.D. & Borisy, G.G. (2003). Cellular motility driven by assembly and disassembly of actin filaments. Cell 113, 549. Available at http://linkinghub.elsevier.com/retrieve/pii/S009286740300357X.Google Scholar
Ridley, A. & Peckham, M.C.P. (Eds.) (2004). Cell Motility: From Molecules to Organisms. The Atrium, Southern gate, Chirchester, West Sussex, England: John Wiley. Available at http://eu.wiley.com/WileyCDA/WileyTitle/productCd-0470848723.html.Google Scholar
Sahai, E. & Marshall, C.J. (2002). RHO-GTPases and cancer. Nat Rev Cancer 2, 133–142. Available at http://dx.doi.org/10.1038/nrc725 (retrieved February 27, 2013).Google Scholar
Taulet, N., Delorme-Walker, V.D. & DerMardirossian, C. (2012). Reactive oxygen species regulate protrusion efficiency by controlling actin dynamics. PloS One 7, e41342. Available at http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3410878&tool=pmcentrez&rendertype=abstract (retrieved May 29, 2013).Google Scholar
Woo, S. & Gomez, T.M. (2006). Rac1 and RhoA promote neurite outgrowth through formation and stabilization of growth cone point contacts. J Neurosci 26, 1418–1428. Available at http://www.ncbi.nlm.nih.gov/pubmed/16452665.Google Scholar
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