SIGNALING AND MECHANICAL REGULATION OF PODOSOME-TYPE ADHESIONS

Abstract

Podosomes represent a special class of integrin-mediated cell-matrix adhesions formed by migrating and matrix degrading cells. Here, I demonstrated that assembly of podosomes induced in macrophage-like THP1 cells and fibroblasts by different treatments were regulated by the ARF1 GTPase and its GEF ARNO. Down-regulation of ARNO and ARF1 by siRNA, and by pharmacological inhibitors led to a striking reduction in the numbers of podosome-forming cells. ARNO was found to co-localize with the adhesive ring components of podosomes while ARF1 localized to vesicular structures that transiently contact podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells, whilst the suppression of myosin-IIA rescued podosome formation regardless of ARF1 inhibition. Interestingly, overexpression of constitutively active ARF1 in fibroblasts lacking podosomes was sufficient to induce bona fide podosomes lacking adhesion ring components, suggesting that ARF1 can crosstalk and modulate Rho GTPase activity in controlling actin polymerization events.

Secondly, I provide a mechanism to explain the effect of microtubule (MT) disruption on podosome disassembly. Using structured illumination microscopy (SIM), I observed that addition of nocodazole, a microtubule depolymerizing drug, induced an outburst of myosin IIA filament assembly as a consequence of increased RhoA activity in macrophage-like THP1 cells. siRNA knockdown of MT-associated GEF-H1 or inhibition of ROCK by Y-27632 prevented the nocodazole-induced disassembly of podosomes and assembly of myosin II filaments. Prolonged treatment with nocodazole induced formation of sarcomeric-like actomyosin structures and larger focal adhesions typically observed in fibroblast-type cells. I propose a switch mechanism between focal adhesions and podosomes governed by an actomyosin-dependent force generation.

Finally, I showed using high-resolution microscopy that existing podosomes undergo dissolution upon artificial increase in membrane tension by exposure to hypo-osmotic medium and mechanical stretching, while expansion of the membrane to decrease membrane tension by deoxycholate promoted “clustering” of podosomes with shorter connecting actin links than podosomes of control cells. Intriguingly, podosomes exposed to 50% reduction in osmolarity but not deoxycholate-treated ones exhibit reduction in size and fluorescence intensity, but with increased “roundedness” indicated by actin markers. Based on these findings, I hypothesize that podosomes mimic the lamellipodia in response to changes in membrane tension.