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

The
mechanical microenvironment serves as an important factor influencing
stem cell differentiation. Mechanobiological responses depend strongly
on actomyosin contractility and integrin-based cell–extracellular
matrix (ECM) interactions mediated by adhesive structures such as
focal adhesions (FAs). While the roles of FAs in mechanobiology have
been intensively studied in many mesenchymal and migratory cell types,
recently it has been recognized that certain pluripotent stem cells
(PSCs) exhibited significantly attenuated FA-mediated mechanobiological
responses. FAs in such PSCs are sparsely distributed and much less
prominent in comparison to “classical” FAs of typical
adherent cells. Despite these differences, insights into how FAs in
PSCs are structurally organized to perform their functions are still
elusive. Using mouse embryonic stem cells (mESCs) to study PSC-ECM
interactions, here we surveyed the molecular composition and nanostructural
organization of FAs. We found that, despite being small in size, mESC
FAs appeared to be compositionally mature, containing markers such
as vinculin, zyxin, and α-actinin, and dependent on myosin II
contractility. Using super-resolution microscopy, we revealed that
mESC FAs were organized into a conserved multilayer nanoscale architecture.
However, the nanodomain organization was compressed in mESCs, with
the force transduction layer spanning ∼10 nm, significantly
more compact than in FAs of other cell types. Furthermore, we found
that the position and orientation of vinculin, a key mechanotransduction
protein, were modulated in an ECM-dependent manner. Our analysis also
revealed that while most core FA genes were expressed, the expression
of LIM domain proteins was comparatively lower in PSCs. Altogether
our results suggest that while core structural and mechanosensitive
elements are operational in mESC FAs, their structural organization
and regulatory aspects may diverge significantly from “classical”
FAs, which may account for the attenuated mechanobiological responses
of these cell types.