Ultrafast single-molecule imaging reveals focal adhesion nano-architecture and molecular dynamics

Abstract

Using our newly developed ultrafast camera described in the companion paper, we reduced the data acquisition periods required for photoactivation/photoconversion localization microscopy (PALM, using mEos3.2) and direct stochastic reconstruction microscopy (dSTORM, using HMSiR) by a factor of ≈30 compared with standard methods, for much greater view-fields, with localization precisions of 29 and 19 nm, respectively, thus opening up previously inaccessible spatiotemporal scales to cell biology research. Simultaneous two-color PALM-dSTORM and PALM-ultrafast (10 kHz) single fluorescent-molecule imaging-tracking has been realized. They revealed the dynamic nanoorganization of the focal adhesion (FA), leading to the compartmentalized archipelago FA model, consisting of FA-protein islands with broad diversities in size (13–100 nm; mean island diameter ≈30 nm), protein copy numbers, compositions, and stoichiometries, which dot the partitioned fluid membrane (74-nm compartments in the FA vs. 109-nm compartments outside the FA). Integrins are recruited to these islands by hop diffusion. The FA-protein islands form loose ≈320 nm clusters and function as units for recruiting FA proteins.

Nano-clusters of ligand-activated integrins organize immobile, signalling active, nano-clusters of phosphorylated FAK required for mechanosignaling in focal adhesions

“…S3). These also revealed the presence of clustered regions within focal adhesions similar to previous studies [46,[49][50][51][52].…”

“…1g) [31,48]. Taken together our results revealed that adhesions were composites of persistent regions with dense integrin β3 nanoclusters, as well as short-lived fast exchanging regions [31,32,46,48]. Since activation of integrins promotes their immobilization, which is further stabilized by their connections to ECM and actin-binding proteins [31,32], organisation of integrins into these long-lived immobile regions could provide a high spatial and temporal stability, which could function as robust platforms to organise signalling hubs of downstream signalling proteins within the focal adhesions.…”

Differential spatial regulation and activation of integrin nanoclusters inside focal adhesions

“…hierarchical fashion inside FAs, forming nanoclusters similar to the ones reported here, that further enrich into compartmentalized regions around 300 nm in size (39). To enquire whether such hierarchical organization is present in our data, we performed nearest-neighbor distance (NND) analysis between nanoclusters of the same proteins (Fig.…”

“…Considering the canonical view that integrins inside FAs are bound to paxillin and engaged to talin and vinculin (45,47), these results might be surprising at first sight, as one would expect a much higher fraction of EED-NN values below zero. Nevertheless, using super-resolution microscopy, others have also found negligible spatial colocalization of integrins with these adaptor proteins (38,39).…”

“…Indeed, it has been shown that integrins can rapidly transition between different conformational states that relate to their activation and binding capacity to their ligands (27,33,34). Moreover, in addition to the nanoscale axial segregation of different AC components inside FAs (35), it has been reported that integrins as well as different adaptor proteins also segregate laterally into small nanoclusters inside FAs (36)(37)(38)(39). Recent super-resolution studies have further shown that α5β1 segregates in both active and inactive nanoclusters inside FAs (38) and single-molecule tension measurements uncovered distinct sub-populations of load-bearing integrins and a high fraction of unengaged integrins (40,41).…”