PAK1 is involved in sensing the orientation of collagen stiffness gradients in mouse fibroblasts

Abstract

Migrating cells sense variations of stiffness in connective tissue matrices but how cells detect and respond to stiffness orientation is not defined. We examined cell extension formation on collagen with underlying support (vertical stiffness gradient) or on collagen laterally supported by nylon (lateral stiffness gradient). At 6 h after plating, cells plated on laterally-supported collagen exhibited >2-fold more abundant and ~2-fold longer cell extensions than cells plated on collagen with underlying support. We examined whether p21-activated kinase 1 (PAK1) influences extension formation that is dependent on the orientation of support. At 6 h after plating on collagen with underlying support, wild-type cell extensions were 40% shorter than PAK1 knockdown cells. In contrast, on laterally-supported collagen, wild-type cell extensions were 2-fold longer than PAK1 knockdown cells. In cells plated on laterally-supported collagen, there were ~2-fold reductions of collagen fiber alignment and compaction in PAK1 knockdown cells compared with wild-type cells. PAK1 knockdown did not affect collagen fiber alignment or compaction by cells plated on collagen with underlying support. Wild-type cells with lateral support of collagen exhibited 3-fold increases of phospho-myosin staining at 6h, which was 2-fold lower in PAK1 knockdown cells. In contrast, cells on collagen with underlying support showed no increase of phospho-myosin staining at any times. PAK1 knockdown did not affect α2 or β1 integrin expression or function. We conclude that PAK1 is involved in the ability of cells to sense the orientation of stiffness in collagen substrates and generate contractile forces that affect cell extension formation.

A Screening System for Evaluating Cell Extension Formation, Collagen Compaction, and Degradation in Drug Discovery

“…39 Our use of the floating collagen gel model to model cell extension formation, matrix remodeling, and collagen degradation provided a rapid and integrated system to screen a large number of compounds as potential inhibitors of cancer cell invasion. Notably, the alignment of collagen fibers is temporally and spatially correlated with the generation of cell extensions, 40 and our results here generally showed that those compounds that inhibited cell extension formation also affected collagen alignment. In summary, screening compounds with the use of the grid-supported floating collagen gel model may facilitate the linkage of cancer cell biology with the identification of new inhibitor of cancer cell invasion.…”

Vimentin tunes cell migration on collagen by controlling β1 integrin activation and clustering

“…Taken together, these processes contribute to increased cell extension formation. Our data presented here indicate that vimentin in focal adhesions plays a central role as an adaptor protein, which may contribute to mesenchymal cell migration and extracellular matrix remodeling (Pinto et al, 2015;Terriac et al, 2017). These processes, which are associated with EMT, are important for tissue regeneration and repair in wound healing (Stone et al, 2016).…”

Monocytes increase human cardiac myofibroblast-mediated extracellular matrix remodeling through TGF-β1

“…After 24 h, local ECM remodeling was quantified by measuring collagen fiber alignment (35) visualized by confocal reflectance microscopy, as we have previously described (29,24,25,31). In brief, a fast Fourier transform was employed to quantify the distribution of collagen fiber angles referenced to the frame of the acquired image measured as pixel intensity at any given angle.…”