Rac1 recruitment to the archipelago structure of the focal adhesion through the fluid membrane as revealed by single‐molecule analysis

Abstract

The focal adhesion (FA) is an integrin-based structure built in/on the plasma membrane (PM), linking the extracellular matrix to the actin stress-fibers, working as cell migration scaffolds. Previously, we proposed the archipelago architecture of the FA, in which FA largely consists of fluid membrane, dotted with small islands accumulating FA proteins: membrane molecules enter the inter-island channels in the FA zone rather freely, and the integrins in the FA-protein islands rapidly exchanges with those in the bulk membrane. Here, we examined how Rac1, a small G-protein regulating FA formation, and its activators αPIX and βPIX, are recruited to the FA zones. PIX molecules are recruited from the cytoplasm to the FA zones directly. In contrast, majorities of Rac1 molecules first arrive from the cytoplasm on the general inner PM surface, and then enter the FA zones via lateral diffusion on the PM, which is possible due to rapid Rac1 diffusion even within the FA zones, slowed only by a factor of two to four compared with that outside. The constitutively-active Rac1 mutant exhibited temporary and all-time immobilizations in the FA zone, suggesting that upon PIX-induced Rac1 activation at the FA-protein islands, Rac1 tends to be immobilized at the FA-protein islands. © 2013 Wiley Periodicals, Inc

Single-molecule tracking of small GTPase Rac1 uncovers spatial regulation of membrane translocation and mechanism for polarized signaling

“…This approach has been used to study interactions between Rac1 and focal adhesions (20). Consistent with the fast 2D diffusion of membrane-associated Rac1 (20), our results also indicate that the majority of Rac1 exists in a freely diffusing state. We further show that sptPALM can be successfully implemented for accurate quantification of Rac1 membrane translocation processes.…”

“…In cell-expressing Eos-caRac1, the majority of the recruitment occurs in focal adhesion-like structures at the cell edge, in accordance with previous studies reporting Rac1 immobilization on focal adhesions (20) (Fig. 2G).…”

“…The emergence of the sptPALM technique has made particle tracking in live cells relatively easy to implement (18,19,22). This approach has been used to study interactions between Rac1 and focal adhesions (20). Consistent with the fast 2D diffusion of membrane-associated Rac1 (20), our results also indicate that the majority of Rac1 exists in a freely diffusing state.…”

“…Thus, ensemble measurements of fluorescently labeled Rac1, even with the "membrane-selective" techniques such as total internal reflection fluorescence microscopy (TIRFM), can have significant contributions from the cytosolic pool. A second problem with TIRFM is that the distance between the plasma membrane to the substrate may vary within the same cell and thus contribute to intensity variations.To address these problems, we took a single-particle tracking Photoactivation Localization Microscopy (sptPALM) approach (18)(19)(20) to track the temporal trajectories of individual Rac1 molecules in cells. By confining the analysis to molecules that are undergoing 2D instead of 3D free diffusion (Fig.…”

“…To address these problems, we took a single-particle tracking Photoactivation Localization Microscopy (sptPALM) approach (18)(19)(20) to track the temporal trajectories of individual Rac1 molecules in cells. By confining the analysis to molecules that are undergoing 2D instead of 3D free diffusion (Fig.…”