title: Quantitative imaging of focal adhesion dynamics and their regulation by HGF and Rap1 signaling.
abstract: Cell migration is crucial in development, tissue repair and immunity and frequently aberrant in pathological processes including tumor metastasis. Focal adhesions (FAs) are integrin-based adhesion complexes that form the link between the cytoskeleton and the extracellular matrix and are thought to orchestrate cell migration. Understanding the regulation of FAs by (oncogenic) signaling pathways may identify strategies to target pathological cell migration. Here we describe the development of a robust FA tracker that enables the automatic, multi-parametric analysis of FA dynamics, morphology and composition from time-lapse image series generated by total internal reflection fluorescence (TIRF) microscopy. In control prostate carcinoma cells, this software recapitulates previous findings that relate morphological characteristics of FAs to their lifetime and their cellular location. We then investigated how FAs are altered when cell migration is induced by the metastasis-promoting hormone HGF and subsequently inhibited by activation of the small GTPase Rap1. We performed a detailed analysis of individual FA parameters, which identified FA size, sliding and intensity as primary targets of Rap1. HGF did not have strong effects on any of the FA parameters within the first hours of its addition. Subsequent Bayesian network inference (BNI), using all measured parameters as input, revealed little correlation between changes in cell migration and FA characteristics in this prostate carcinoma cell line. Instead BNI indicated a concerted coordination of cell size and FA parameters. Thus our results did not reveal a direct relation between the regulation of cell migration and the regulation of FA dynamics.
authors.affiliation: Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Emma
authors.initials: E
authors.lastname: Spanjaard
authors.affiliation: Departments of Medical Informatics and Radiology, Erasmus MC - University Medical Center Rotterdam, Rotterdam, The Netherlands.
authors.forename: Ihor
authors.initials: I
authors.lastname: Smal
authors.affiliation: Division of Molecular Carcinogenesis, Netherlands Cancer Institute, Amsterdam, The Netherlands.
authors.forename: Nicos
authors.initials: N
authors.lastname: Angelopoulos
authors.affiliation: Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Verlaan
authors.affiliation: Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Matov
authors.affiliation: Departments of Medical Informatics and Radiology, Erasmus MC - University Medical Center Rotterdam, Rotterdam, The Netherlands.
authors.forename: Erik
authors.initials: E
authors.lastname: Meijering
authors.affiliation: Division of Molecular Carcinogenesis, Netherlands Cancer Institute, Amsterdam, The Netherlands.
authors.forename: Lodewyk
authors.initials: L
authors.lastname: Wessels
authors.affiliation: Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Hans
authors.initials: H
authors.lastname: Bos
authors.affiliation: Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, The Netherlands. Electronic address: j.derooij-4@umcutrecht.nl.
authors.forename: Johan
authors.initials: J
authors.lastname: de Rooij
mesh_terms: D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002465:Cell Movement; D005109:Extracellular Matrix / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism*; D057809:HEK293 Cells; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008297:Male; D008856:Microscopy, Fluorescence; D009362:Neoplasm Metastasis; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology*; D015398:Signal Transduction; D012984:Software; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: C066855:HGF protein, human; D017228:Hepatocyte Growth Factor; D020827:rap1 GTP-Binding Proteins
keywords: Bayesian network inference; Cell migration; Computer Vision; Focal adhesion; HGF; Rap1