Initiation of Attachment and Generation of Mature Focal Adhesions by Integrin-containing Filopodia in Cell Spreading

Abstract

Integrin receptors, and associated cytoplasmic proteins mediate adhesion, cell signaling and connections to the cytoskeleton. Using fluorescent protein chimeras, we analyzed initial integrin adhesion in spreading fibroblasts with Total Internal Reflection Fluorescence (TIRF) microscopy. Surprisingly, sequential radial projection of integrin and actin containing filopodia formed the initial cell-matrix contacts. These Cdc42-dependent, integrin-containing projections recruited cytoplasmic focal adhesion (FA) proteins in a hierarchical manner; initially talin with integrin and subsequently FAK and paxillin. Radial FA structures then anchored cortical actin bridges between them and subsequently cells reorganized their actin, a process promoted by Src, and characterized by lateral FA reorientation to provide anchor points for actin stress fibers. Finally, the nascent adhesions coalesced until they formed mature FAs.

The key feature for early migratory processes

“…Interestingly, depending on the analyzed cell type filopodial, sensing does not necessarily have to end up in cell matrix adhesions as described, for example, 3T3 fibroblasts during cell spreading. 8 Instead, filopodia can also mediate the interaction between macrophages and pathogens 36 or between two cells as shown for embryonic epithelial cells 37 or the formation of functional neuronal interactions. 38 These examples show the overall importance of filopodial sensing.…”

“…5,6 Furthermore, for many cell types the lamellipodium is described as the origin of adhesion site formation. 7 In addition, for cell types forming filopodia latest research has shown an important role of these finger-like extensions in cell spreading 8 and adhesion assembly during migration. 9,10 Here, nearly all lamellipodial focal adhesions (FAs) evolve from small focal complexes (FXs) originally assembled within filopodia and just increased in size when overgrown by the lamellipodial leading edge.…”

Quantification of the Cell-Substratum Contact and Cell Lift-off Under Different Intra/Extracellular Conditions

“…Therefore, it can be argued that the variation of the contact area and its increasing rate can be used to quantify cell viability. In a previous study (Partridge and Marcantonio 2006), the two-dimensional area that was spread by cells that are transfected by different plasmids was measured by using phase-contrast microscopy and TIRF microscopy. The findings of Partrige and Marcantonio (2006) that the increasing rate of the spread area varied with the transfecting material and that the area range was 100Â400 mm 2 are quite comparable to the present study.…”

“…Nutrition is supplied during the experiment from the incubator through the chip system. In the literature (Lanni et al 1985;Bretschneider et al 2004;Weisswange et al 2005;Partridge and Marcantonio 2006;Manneville 2006), there are many examples of the use of EF in combination with TIRF for measuring global and membrane-specific events. However, in the present study, EF setup was used only for illuminating the whole field to locate the cells before and/or after the TIRF images were recorded.…”

“…However, those studies did not consider the effect of different cellular conditions on cell adhesion. By using TIRF, Partridge and Marcantonio (2006) analyzed the initiation of attachment and the generation of focal adhesion. Using confocal and TIRF microscopy, Vilarino et al (2006) characterized the effect of a marine toxin (AZA-1) on the cytoskeleton of adherent cells and cell growth.…”