SummaryMatrix-activated integrins can form different adhesion structures. We report that nontransformed fibroblasts develop podosome-like adhesions when spread on fluid Arg-Gly-Asp peptide (RGD)-lipid surfaces, whereas they habitually form focal adhesions on rigid RGD glass surfaces. Similar to classic macrophage podosomes, the podosome-like adhesions are protrusive and characterized by doughnut-shaped RGD rings that surround characteristic core components including F-actin, N-WASP, and Arp2/Arp3. Furthermore, there are 18 podosome markers in these adhesions, though they lack matrix metalloproteinases that characterize invadopodia and podosomes of Src-transformed cells. When nontransformed cells develop force on integrin-RGD clusters by pulling RGD lipids to prefabricated rigid barriers (metal lines spaced by 1–2 μm), these podosomes fail to form and instead form focal adhesions. The formation of podosomes on fluid surfaces is mediated by local activation of phosphoinositide 3-kinase (PI3K) and the production of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) in a FAK/PYK2-dependent manner. Enrichment of PIP3 precedes N-WASP activation and the recruitment of RhoA-GAP ARAP3. We propose that adhesion structures can be modulated by traction force development and that production of PIP3 stimulates podosome formation and subsequent RhoA downregulation in the absence of traction force.

A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions.

The turnover of integrin receptors is critical for cell migration and adhesion dynamics. Here we find that force development at integrins regulates adaptor protein recruitment and endocytosis. Using mobile RGD (Arg-Gly-Asp) ligands on supported lipid membranes (RGD membranes) and rigid RGD ligands on glass (RGD-glass), we find that matrix force-dependent integrin signals block endocytosis. Dab2, an adaptor protein of clathrin-mediated endocytosis, is not recruited to activated integrin-beta3 clusters on RGD-glass; however, it is recruited to integrin-mediated adhesions on RGD membranes. Further, when force generation is inhibited on RGD-glass, Dab2 binds to integrin-beta3 clusters. Dab2 binding to integrin-beta3 excludes other adhesion-related adaptor proteins, such as talin. The clathrin-mediated endocytic machinery combines with Dab2 to facilitate the endocytosis of RGD-integrin-beta3 clusters. From these observations, we propose that loss of traction force on ligand-bound integrin-beta3 causes recruitment of Dab2/clathrin, resulting in endocytosis of integrins.

Rafiq et al. demonstrate that the small G protein ARF1 and its activator, cytohesin 2 (ARNO), are required for podosome formation in macrophage-like cells and fibroblasts. Inhibition of ARNO-ARF1 signaling results in increased RhoA activity and disassembly of podosomes in a myosin-IIA–dependent fashion. In fibroblasts that normally do not form podosomes, constitutively active ARF1 induces actin-rich puncta associated with sites of matrix degradation, putative precursors of podosomes.

Podosomes are a singular category of integrin-mediated adhesions important in the processes of cell migration, matrix degradation and cancer cell invasion. Despite a wealth of biochemical studies, the effects of mechanical forces on podosome integrity and dynamics are poorly understood. Here, we show that podosomes are highly sensitive to two groups of physical factors. First, we describe the process of podosome disassembly induced by activation of myosin-IIA filament assembly. Next, we find that podosome integrity and dynamics depends upon membrane tension and can be experimentally perturbed by osmotic swelling and deoxycholate treatment. We have also found that podosomes can be disrupted in a reversible manner by single or cyclic radial stretching of the substratum. We show that disruption of podosomes induced by osmotic swelling is independent of myosin-II filaments. The inhibition of the membrane sculpting protein, dynamin-II, but not clathrin, resulted in activation of myosin-IIA filament formation and disruption of podosomes. The effect of dynamin-II inhibition on podosomes was, however, independent of myosin-II filaments. Moreover, formation of organized arrays of podosomes in response to microtopographic cues (the ridges with triangular profile) was not accompanied by reorganization of myosin-II filaments. Thus, mechanical elements such as myosin-II filaments and factors affecting membrane tension/sculpting independently modulate podosome formation and dynamics, underlying a versatile response of these adhesion structures to intracellular and extracellular cues. This article is part of a discussion meeting issue ‘Forces in cancer: interdisciplinary approaches in tumour mechanobiology’.

In the version of this Perspective originally published, Fig. 1 was missing the credit for the inset to panel a, and ref. 76 was missing from the reference list. The caption should have included the text: 'Reproduced from ref. 76 , American Chemical Society (a, inset). '; where ref. 76 is 'Gao, J., Loi, M. A., de Carvalho, E. J. F. & dos Santos, M. C. Selective wrapping and supramolecular structures of polyfluorene-carbon nanotube hybrids. ACS Nano 5, 3993-3999 (2011)'. These errors have now been amended in the online versions.

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The role of microtubules in the mechanoregulation of integrin--mediated adhesions is poorly understood. Here, we show that uncoupling of microtubules from integrin adhesions by depletion or displacement of KANK family proteins connecting the adhesion protein talin with microtubule tips led to disruption of podosomes and augmentation of focal adhesions, similarly to total disruption of microtubules. Using structured--illumination microscopy, we demonstrate that microtubule uncoupling from adhesions or total microtubule disruption brings about a massive assembly of myosin--IIA filaments, whilst a burst of microtubule polymerization led to a transient disassembly of myosin--IIA filaments. We showed that myosin--IIA filaments are indispensable for microtubule--dependent regulation of focal adhesions and podosomes. The microtubule--driven control of myosin--IIA filament formation is achieved through the regulation of Rho by microtubule--localized Rho guanine nucleotide exchange factor GEF--H1. Thus, a unifying mechanism of microtubule--mediated regulation of focal adhesions and podosomes operates via KANKs--and GEF--H1--dependent local reorganization of myosin--II filaments.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.