Superresolution architecture of cornerstone focal adhesions in human pluripotent stem cells

Abstract

While it is clear that key transcriptional programmes are important for maintaining pluripotency, the requirement for cell adhesion to the extracellular matrix remains poorly defined. Human pluripotent stem cells (hPSCs) form colonies encircled by an actin ring and large stable cornerstone focal adhesions (FA). Using superresolution two-colour interferometric photo-activated localisation microscopy, we examine the three-dimensional architecture of cornerstone adhesions and report vertical lamination of FA proteins with three main structural features distinct from previously studied focal adhesions: 1) integrin β5 and talin are present at high density, at the edges of cornerstone FA, adjacent to a vertical kank-rich protein wall, 2) vinculin localises higher than previously reported, displaying a head-above-tail orientation, and 3) surprisingly, actin and α-actinin are present in two discrete z-layers. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a role for kanks in hPSC colony architecture.

Growth factor dependent changes in nanoscale architecture of focal adhesions

“…A seminal study revealed the presence of three different layers: (1), the so-called integrin signalling layer (ISL) closest to the adherent membrane (within ~ 10–20 nm) which includes the cytoplasmic tails of the transmembrane integrin receptors, focal adhesion kinase and paxillin, (2), the actin-regulatory layer (ARL) at the top, where mainly directly actin-binding proteins such as zyxin, vasodilator-stimulated phosphoprotein (VASP) and α-actinin are found, and (3), the force transduction layer (FTL) in between (from ~ 10–20 to ~ 50–60 nm from the adherent membrane) of which talin is the most well-known protein and which is also where vinculin is found 7 . Later studies, using different techniques, confirmed the layered nanostructure of FAs along the z-axis 8 – 10 .…”

Definition of the fibroblast adhesome using multiplexed proximity biotinylation

“…as reported for vinculin, LPP, and zyxin) 26 . Furthermore, a relatively large number of the proteins identified by proximity biotinylation were identified in published adhesomes, with 24.5% (36) and 19.7% (23) of the 147 proteins in the proximity-dependent adhesome identified in the literature-curated (232 components) and consensus adhesomes (60 components), respectively (supp. 3B) 6,11 .…”

“…B2 interacts with other bait clusters separately in different structures, such as nascent adhesions and fibrillar adhesions). Network analysis revealed that paxillin had a particularly large number of proximal interactions with BioID baits (19), and a high betweenness centrality (0.57) ( fig. 3B, and supp.…”

“…Although KANK is known to bind talin, a direct interaction with any of the baits used in this study has yet to be described 47 . As KANK2 was identified in a number of proximity interactomes, it is possible that additional direct interactions exist 19 . In addition to KANK2 and LL5-a (Phldb1), three other CMSC components were identified in the proximity-dependent adhesome, LL5-b (Phldb2), liprin-a1 (Ppfia1) and liprin-b1 (Ppfibp1).…”

“…Microscopic analyses have enabled the close examination of the ultrastructure of IACs, revealing a high degree of lateral and vertical organisation [14][15][16][17] . For example, super-resolution light microscopy revealed the vertical stratification of a number of components within IACs at a 10-20 nm resolution, revealing three distinct, but overlapping, functional layers, albeit with some differences in the localisation of specific components 14,15,18,19 . However, the organisation of adhesome components on a larger scale has yet to be experimentally defined, and an empirical view of protein interactions within IACs is lacking.…”