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.
Highlights
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
Results Human pluripotent stem cells (hPSC) cornerstone focal adhesions (FA) contribute to pluripotency maintenance. hPSC colonies plated on vitronectin (VTN), an extracellular matrix (ECM) ligand which supports pluripotency[4], assemble into tightly packed colonies with well-defined edges[20]
These colonies are encircled by paxillin-positive cornerstone FA that are themselves connected by prominent actin bundles termed actin fence (Fig. 1a, b19)
Summary
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. Even though integrins are key requirements for maintaining pluripotency in vitro[4,5,17], and adhesion signalling through focal adhesion kinase is implicated in pluripotent stem cell maintenance[18,19], hPSC FA composition and organisation remain poorly defined. We described that the hallmark sharp hPSC colony edge morphology is regulated by unusually large FA, termed cornerstone FA, connected by an actin fence defined by strong contractile ventral stress fibres[19]. These colony edge adhesions exert strong traction forces on the underlying. We include the FA scaffold partners kank[1] and kank[2] in our imaging, and describe a role for kanks in regulating hPSC adhesion and maintaining FA architecture in hPSCs
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