Abstract
While cell-substrate adhesions that form between the protruding edge of a spreading cell and flat surfaces have been studied extensively, processes that regulate the maturation of filopodia adhesions are far less characterized. Since little is known about how the kinetics of formation or disassembly of filopodia adhesions is regulated upon integration into the lamellum, a kinetic analysis of the formation and disassembly of filopodia adhesions was conducted at the leading edge of β3-integrin-EGFP-expressing rat embryonic fibroblasts spreading on fibronectin-coated glass or on soft polyacrylamide gels. Filopodia β3-integrin adhesions matured only if the lamellipodium in their immediate vicinity showed cyclic protrusions and retractions. Filopodia β3-integrin shaft adhesions elongated rapidly when they were overrun by the advancing lamellipodium. Subsequently and once the lamellipodium stopped its advancement at the distal end of the filopodia β3-integrin adhesion, these β3-integrin shaft adhesions started to grow sidewise and colocalize with the newly assembled circumferential actin stress fibers. In contrast, the suppression of the cyclic protrusions and retractions of the lamellipodium by blocking myosin light chain kinase suppressed the growth of filopodia adhesion and resulted in the premature disassembly of filopodia adhesions. The same failure to stabilize those adhesions was found for the advancing lamellipodium that rapidly overran filopodia shaft adhesions without pausing as seen often during fast cell spreading. In turn, plating cells on soft polyacrylamide gels resulted in a reduction of lamellipodia activity, which was partially restored locally by the presence of filopodia adhesions. Thus filopodia adhesions could also mature and be integrated into the lamellum for fibroblasts on soft polyacrylamide substrates.
Highlights
Cells use filopodia to explore the physical and biochemical characteristics of their environments [1,2,3,4,5,6], and the stabilization of filopodial contacts with the substrate directs cell movement [2,7,8]
Elongation and growth of b3-integrin-EGFP filopodia shaft adhesions is promoted in proximity to an advancing lamellipodium To capture and quantify the kinetics by which filopodia shaft adhesions form, mature and get incorporated in the lamellum, REF52 fibroblasts stably transfected with b3-integrin-EGFP (REF52-b3-integrin-EGFP) were allowed to adhere and spread on fibronectin coated glass slides
While filopodia adhesions were previously found to be integrated into the cell lamella of various cell types [20,21,22,32,58], no information was available about the integration process
Summary
Cells use filopodia to explore the physical and biochemical characteristics of their environments [1,2,3,4,5,6], and the stabilization of filopodial contacts with the substrate (matrix) directs cell movement [2,7,8]. Tensile forces will either immediately rupture or further stabilize filopodia adhesions: the temporal stability of a filopodium increases once a contact with the extracellular matrix has been formed, and is further maintained when cells pull on their substrates [2,3,5,8,22,23,24]. The presence of tensile forces acting on filopodia adhesions is reflected by the recruitment of certain force-regulated adaptor and scaffold proteins: the integrin containing cell-matrix adhesions within the filopodia shaft recruit talin and paxillin, VASP, and vinculin, tensin and even zyxin [16,20,21,22] This is important to note since vinculin recruitment to talin requires the stretching of talin [26,27]. Filopodia are known to contribute to the assembly of contractile bundles and substrate adhesions in the lamella [21], but how this process is influenced by the lamellipodia dynamic is not well understood
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