Forces and constraints controlling podosome assembly and disassembly.

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'.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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

The interrelationship between microtubules and the actin cytoskeleton in mechanoregulation of integrin-mediated adhesions is poorly understood. Here, we show that the effects of microtubules on two major types of cell-matrix adhesion, focal adhesions and podosomes, are mediated by KANK family proteins connecting the adhesion protein talin with microtubule tips. Both total microtubule disruption and microtubule uncoupling from adhesions by manipulations with KANKs trigger a massive assembly of myosin IIA filaments, augmenting focal adhesions and disrupting podosomes. Myosin IIA filaments are indispensable effectors in the microtubule-driven regulation of integrin-mediated adhesions. Myosin IIA filament assembly depends on Rho activation by the RhoGEF GEF-H1, which is trapped by microtubules when they are connected with integrin-mediated adhesions via KANK proteins but released after their disconnection. Thus, microtubule capture by integrin-mediated adhesions modulates the GEF-H1-dependent effect of microtubules on the assembly of myosin IIA filaments. Subsequent actomyosin reorganization then remodels the focal adhesions and podosomes, closing the regulatory loop.

Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO.

Podosomes represent a class of integrin-mediated cell-matrix adhesions formed by migrating and matrix-degrading cells. We demonstrate that in macrophage-like THP1 cells and fibroblasts stimulated to produce podosomes, down-regulation of the G-protein ARF1 or the ARF1 guanine nucleotide exchange factor, ARNO, by small, interfering RNA or pharmacological inhibitors led to striking podosome elimination. Concomitantly, treatments inducing podosome formation increased the level of guanosine triphosphate (GTP)-bound ARF1. ARNO was found to colocalize with the adhesive rings of podosomes, whereas ARF1 was localized to vesicular structures transiently contacting podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells, whereas the suppression of myosin-IIA rescued podosome formation regardless of ARF1 inhibition. Finally, expression of constitutively active ARF1 in fibroblasts induced formation of putative podosome precursors: actin-rich puncta coinciding with matrix degradation sites and containing proteins of the podosome core but not of the adhesive ring. Thus, ARNO-ARF1 regulates formation of podosomes by inhibition of RhoA/myosin-II and promotion of actin core assembly.

Integrin-beta3 clusters recruit clathrin-mediated endocytic machinery in the absence of traction force.

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.

Integrin-matrix clusters form podosome-like adhesions in the absence of traction forces.

Matrix-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.

Branched disulfide-based polyamidoamines capable of mediating high gene transfection.

DNA condensation, endosomal escape of DNA/polymeric complexes, and unpacking of DNA are the key steps in the process of non-viral gene delivery. Amongst these steps, currently the unpacking of the DNA cargo from the DNA/polymeric nanocomplexes is the most challenging and arguably the most crucial if one wants to achieve high gene transfection with minimum cytotoxicity in the target cell. In this report we review current and past examples in the literature that demonstrate concerted efforts in designing and synthesizing various forms of cationic polymeric vectors having "built in" features. Such features can be certain types of chemical functional groups, such as amines and acids or other degradable bonds like esters, carbonates and disulfides, which allow for breakdown of polymeric vectors in certain cellular compartments. This may lead the DNA cargo to dissociate from the DNA/polymer complexes so as to maximize intracellular gene delivery. Furthermore, we provide further evidence that it is possible to achieve the goal of high gene transfection coupled with low cytotoxicity via rational design and formulation of branched polyamidoamines containing disulfide bonds. The DNA binding ability of these polymers and particle size as well as zeta potential of their DNA complexes were investigated. The cytotoxicity of pure polymer and polymer/DNA complexes at various polymer concentrations was studied in HEK293 human embryonic kidney, HepG2 human liver carcinoma, 4T1 mouse breast cancer and HeLa human cervical cancer cell lines. In vitro gene transfection efficiency induced by polymer/DNA complexes was explored in these cell lines by using luciferase and GFP reporter genes in comparison with PEI.