Abstract
BackgroundCellular processes occur within dynamic and multi-molecular compartments whose characterization requires analysis at high spatio-temporal resolution. Notable examples for such complexes are cell-matrix adhesion sites, consisting of numerous cytoskeletal and signaling proteins. These adhesions are highly variable in their morphology, dynamics, and apparent function, yet their molecular diversity is poorly defined.Methodology/Principal FindingsWe present here a compositional imaging approach for the analysis and display of multi-component compositions. This methodology is based on microscopy-acquired multicolor data, multi-dimensional clustering of pixels according to their composition similarity and display of the cellular distribution of these composition clusters. We apply this approach for resolving the molecular complexes associated with focal-adhesions, and the time-dependent effects of Rho-kinase inhibition. We show here compositional variations between adhesion sites, as well as ordered variations along the axis of individual focal-adhesions. The multicolor clustering approach also reveals distinct sensitivities of different focal-adhesion-associated complexes to Rho-kinase inhibition.Conclusions/SignificanceMulticolor compositional imaging resolves “molecular signatures” characteristic to focal-adhesions and related structures, as well as sub-domains within these adhesion sites. This analysis enhances the spatial information with additional “contents-resolved” dimensions. We propose that compositional imaging can serve as a powerful tool for studying complex multi-molecular assemblies in cells and for mapping their distribution at sub-micron resolution.
Highlights
Molecular processes in cells involve multiple components that are dynamically interacting with each other
A fundamental feature of cell-matrix adhesions is the high diversity in their molecular composition and dynamics, which was studied by simultaneous two-component labeling of fixed cells, time-resolved experiments with GFP-tagged adhesion components and timelapse movies of cells fixed and labeled at the end-point[7,8,9,10,11,12]
8 molecules were visualized using 4 labeling sets (A–D, as defined in Fig. 1 and Table 1). This labeling was applied to well-spread, untreated fibroblasts, to cells treated with the Rho-kinase inhibitor Y-27632 and to cells at 3, 15 or 60 minutes after washout of the drug and recovery from this treatment
Summary
Molecular processes in cells involve multiple components that are dynamically interacting with each other. Based on morphological and molecular criteria, several types of cell-matrix adhesions were distinguished in cultured cells These include focal-complexes, which are small and short-lived contacts formed at the edge of the lamellipodium, focal-adhesions, which are associated with the ends of contractile actin stress-fibers, elongated fibrillar-adhesions, which play a role in extracellular matrix fibrillogenesis, and ‘‘3D matrix adhesions’’, which are formed with pre-assembled matrix fibrils. Cellular processes occur within dynamic and multi-molecular compartments whose characterization requires analysis at high spatio-temporal resolution Notable examples for such complexes are cell-matrix adhesion sites, consisting of numerous cytoskeletal and signaling proteins. These adhesions are highly variable in their morphology, dynamics, and apparent function, yet their molecular diversity is poorly defined
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
