Article

A Role for the Juxtamembrane Cytoplasm in the Molecular Dynamics of Focal Adhesions

Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
PLoS ONE (Impact Factor: 3.23). 02/2009; 4(1):e4304. DOI: 10.1371/journal.pone.0004304
Source: PubMed

ABSTRACT

Focal adhesions (FAs) are specialized membrane-associated multi-protein complexes that link the cell to the extracellular matrix and play crucial roles in cell-matrix sensing. Considerable information is available on the complex molecular composition of these sites, yet the regulation of FA dynamics is largely unknown. Based on a combination of FRAP studies in live cells, with in silico simulations and mathematical modeling, we show that the FA plaque proteins paxillin and vinculin exist in four dynamic states: an immobile FA-bound fraction, an FA-associated fraction undergoing exchange, a juxtamembrane fraction experiencing attenuated diffusion, and a fast-diffusing cytoplasmic pool. The juxtamembrane region surrounding FAs displays a gradient of FA plaque proteins with respect to both concentration and dynamics. Based on these findings, we propose a new model for the regulation of FA dynamics in which this juxtamembrane domain acts as an intermediary layer, enabling an efficient regulation of FA formation and reorganization.

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    • "Initial attempts to measure the internal dynamics of FAs relied on low resolution fluorescent techniques. FRAP measurements revealed that the exchange dynamics between FAs and the surrounding cytosol and plasma membrane is faster than the life-time of FAs as showed for integrin [27], FAK [65], paxillin/vinculin [66,67], and talin [68]. Moreover differential turnover of FAs components was shown to be dependent on acto-myosin contractile forces [69,70] . "
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    ABSTRACT: Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.
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    • "The intensity of the recovery curve was calculated according to Phair’s single normalization method [20]. To determine the τ1/2 of recovery, normalized recovery data were fitted to a single exponential equation [21], and the τ1/2 of recovery was calculated from the recovery curve. "
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    ABSTRACT: Focal adhesions (FAs), integrin-mediated macromolecular complexes located at the cell membrane extracellular interface, have been shown to regulate cell adhesion and migration. Our previous studies have indicated that HAb18G/CD147 (CD147) is involved in cytoskeleton reorganization and FA formation in human hepatocellular carcinoma (HCC) cells. However, the precise mechanisms underlying these processes remain unclear. In the current study, we determined that CD147 was involved in vinculin-mediated FA focal adhesion formation in HCC cells. We also found that deletion of CD147 led to reduced vinculin-mediated FA areas (P<0.0001), length/width ratios (P<0.0001), and mean intensities (P<0.0001). CD147 promoted lamellipodia formation by localizing Arp2/3 to the leading edge of the cell. Deletion of CD147 significantly reduced the fluorescence (t1/2) recovery times (22.7±3.3 s) of vinculin-mediated focal adhesions (P<0.0001). In cell-spreading assays, CD147 was found to be essential for dynamic focal adhesion enlargement and disassembly. Furthermore, the current data showed that CD147 reduced tyrosine phosphorylation in vinculin-mediated focal adhesions, and enhanced the accumulation of the acidic phospholipid phosphatidylinositol-4, 5-bisphosphate (PIP2). Together, these results revealed that CD147 is involved in vinculin-mediated focal adhesion formation, which subsequently promotes cytoskeleton reorganization to facilitate invasion and migration of human HCC cells.
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    • "Therefore, the cytosolic building blocks appear to be modules that are continuous along the functional axis of anchoring-mechanosensing-actin regulation and along the vertical axis across focal adhesions. Cell-matrix adhesion sites are dynamically maintained structures that constantly exchange their constituting material with the cytosol (Lele et al., 2008; Wolfenson et al., 2009). We pose here two alternative models for the mode of this exchange—symmetric and asymmetric (Figure 3A). "
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