Mechanosensing machinery for cells under low substratum rigidity

Abstract

Wei WC, Lin HH, Shen MR, Tang MJ. Mechanosensing machinery for cells under low substratum rigidity. Am J Physiol Cell Physiol 295: C1579 -C1589, 2008. First published October 15, 2008 doi:10.1152/ajpcell.00223.2008.-Mechanical stimuli are essential during development and tumorigenesis. However, how cells sense their physical environment under low rigidity is still unknown. Here we show that low rigidity of collagen gel downregulates ␤ 1 -integrin activation, clustering, and focal adhesion kinase (FAK) Y397 phosphorylation, which is mediated by delayed raft formation. Moreover, overexpression of autoclustered ␤ 1-integrin (V737N), but not constitutively active ␤ 1-integrin (G429N), rescues FAKY397 phosphorylation level suppressed by low substratum rigidity. Using fluorescence resonance energy transfer to assess ␤ 1-integrin clustering, we have found that substratum rigidity between 58 and 386 Pa triggers ␤ 1-integrin clustering in a dose-dependent manner, which is highly dependent on actin filaments but not microtubules. Furthermore, augmentation of ␤ 1-integrin clustering enhances the interaction between ␤ 1-integrin, FAK, and talin. Our results indicate that contact with collagen fibrils is not sufficient for integrin activation. However, substratum rigidity is required for integrin clustering and activation. Together, our findings provide new insight into the mechanosensing machinery and the mode of action for epithelial cells in response to their physical environment under low rigidity.focal adhesion kinase; ␤ 1-integrin activation; ␤1-integrin clustering; lipid raft; actin cytoskeleton EXTRACELLULAR MATRIX (ECM) has an active and complex role in regulating the behavior of the cells that contact it, influencing their survival, adhesion, spreading, migration, proliferation, apoptosis, and differentiation (32). The major receptors of ECM are integrins, which are transmembrane receptors composed of two subunits, ␣ and ␤, that form connections between the cytoskeleton and ECM. Each ␣␤ heterodimer has its own binding and signal specificity (25). The initiation of integrinmediated activities involves two steps, namely, integrin activation and clustering. Integrins are locked in the inactive form without stimulation of extracellular ligands. After binding to its ligand with the extracellular domain (outside-in) (19) or stimulation by intracellular signals (inside-out) (38), an integrin is activated and its extracellular domain undergoes conformational changes, which lead to exposure of several ligandinduced binding sites (LIBS) (49). In addition to conformation changes, ligand binding also induces elevation of lateral mobility and clustering of integrins in the plasma membrane (31). Both ligand binding and clustering of integrins are critically important to activate intracellular signals. However, the regulatory mechanisms of controlling integrin activation and clustering are still unclear.Activation of integrin leads to the recruitment and organization of a number of different cytoskeletal proteins (␣-actinin, tal...

Selective and uncoupled role of substrate elasticity in the regulation of replication and transcription in epithelial cells

“…Because only the rigidity changes between the different polyelectrolyte multilayers used in this study, the film stiffness is probably at the origin of the cell adhesion processes. This hypothesis agrees with a recent study, demonstrating that MDCK cells can sense the substratum rigidity to a sufficient extent to induce 1-integrin activation and clustering, allowing actin organization and downstream activation events to occur (Wei et al, 2008). In our present study, low rigidity above 50 kPa is sufficient for v-integrin engagement, allowing little cell spreading.…”

“…In addition, we could not exclude flow and creep phenomena of the film, and also a lateral cell pulling over several microns. It was previously reported that tyrosine phosphorylation increased at the adhesion sites of cells on stiff surfaces (Pelham and Wang, 1997;Giannone and Sheetz, 2006) and that the level of FAK-Y397 phosphorylation was progressively dependent on substratum rigidity (particularly at 390 Pa) on polyacrylamide gel for MDCK (Wei et al, 2008). In the present system, FAK-Y397 was only phosphorylated at around 200 kPa (E200) and again activated on the hard substratum (0/2, 0/5 and 0/12 and glass surfaces).…”

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“…40 The cell lysates were harvested, resolved on SDS-PAGE, and then electrophoretically blotted onto nitrocellular paper. The primary antibodies used in this study are listed as follows: Klf4, cyclin D1, glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology), Klf5 (Millipore, Temecula, CA), a-smooth muscle actin (Sigma-Aldrich), b1 integrin (BD Biosciences PharMingen), p-ERK, and ERK (Cell Signaling).…”

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