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Superresolution architecture of cornerstone focal adhesions in human pluripotent stem cells.

Nature communications (2019-10-20)
Aki Stubb, Camilo Guzmán, Elisa Närvä, Jesse Aaron, Teng-Leong Chew, Markku Saari, Mitro Miihkinen, Guillaume Jacquemet, Johanna Ivaska
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-β-Actin antibody, Mouse monoclonal, clone AC-15, purified from hybridoma cell culture
Sigma-Aldrich
Monoclonal Anti-Vinculin antibody produced in mouse, clone hVIN-1, ascites fluid
Sigma-Aldrich
MISSION® pLKO.1-puro Non-Mammalian shRNA Control Plasmid DNA, Targets no known mammalian genes
Sigma-Aldrich
Monoclonal Anti-Talin antibody produced in mouse, clone 8d4, ascites fluid
Sigma-Aldrich
Monoclonal Anti-α-Actinin antibody produced in mouse, clone BM-75.2, ascites fluid
Sigma-Aldrich
Anti-KANK2 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-Integrin αVβ5 Antibody, clone 15F11, Azide Free, clone 15F11, Chemicon®, from mouse