Superresolution architecture of pluripotency guarding adhesions

Abstract

Human pluripotent stem cells (hPSC) can generate almost all adult cell lineages. While it is clear that key transcriptional programmes are important elements for maintaining pluripotency, the equally essential requirement for cell adhesion to specific extracellular matrix components remains poorly defined. Our recent observation that hPSC colonies form unusually large "cornerstone" focal adhesions (FA), distinct from parental somatic cells, that are lost following differentiation, emphasises the potential of these atypical FA as gatekeepers of pluripotency. Here, using nanopatterns, we further demonstrate that physical restriction of adhesion size, in hPSC colonies, is sufficient to trigger differentiation. Using superresolution two-colour interferometric photoactivated localization microscopy (iPALM), we examined the three-dimensional architecture of these cornerstone adhesions and report vertical lamination of FA proteins with three main structural peculiarities: 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 expected with respect to the substrata, and 3) surprisingly, actin and α-actinin are present in two discrete layers, a previously undescribed localisation for these proteins. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a key role for kanks in hPSC colony architecture.

Molecular Organization of Integrin-Based Adhesion Complexes in Mouse Embryonic Stem Cells

“…We note that a recent preprint has also reported a study of FA nanoscale architecture in human induced PSCs (hiPSCs). 71 In that study, multilayer stratification of FA proteins into ISL/ FTL/ARL was observed, supporting our notion that the multilayer architecture may be a conserved architecture of cell adhesion complexes. 28 Interestingly, on vitronectin ECM used in that study, a relatively high position of vinculin was also observed similar to our observation for mESC FAs on fibronectin and gelatin, while the inverted position of vinculin is similar to our mESC results on laminin.…”

“…We note that a recent preprint has also reported a study of FA nanoscale architecture in human induced PSCs (hiPSCs) . In that study, multilayer stratification of FA proteins into ISL/FTL/ARL was observed, supporting our notion that the multilayer architecture may be a conserved architecture of cell adhesion complexes .…”