α-Actinin1 and 4 tyrosine phosphorylation is critical for stress fiber establishment, maintenance and focal adhesion maturation

In polarized, migrating cells, stress fibers are a highly dynamic network of contractile actomyosin structures composed of bundles of actin filaments held together by actin cross-linking proteins such as alpha-actinins. As such, alpha-actinins influence actin cytoskeleton organization and dynamics and focal adhesion maturation. In response to environmental signals, alpha-actinins are tyrosine phosphorylated and this affects their binding to actin stress fibers; however, the cellular role of alpha-actinin tyrosine phosphorylation remains largely unknown. We found that non-muscle alpha-actinin1/4 are critical for the establishment of dorsal stress fibers and maintenance of transverse arc stress fibers. Analysis of cells genetically depleted of alpha-actinin1 and 4 reveals two distinct modes for focal adhesion maturation. An alpha-actinin1 or 4 dependent mode that uses dorsal stress fiber precursors as a template for establishing focal adhesions and their maturation, and an alpha-actinin-independent manner that uses transverse arc precursors to establish focal adhesions at both ends. Focal adhesions formed in the absence of alpha-actinins are delayed in their maturation, exhibit altered morphology, have decreased amounts of Zyxin and VASP, and reduced adhesiveness to extracellular matrix. Further rescue experiments demonstrate that the tyrosine phosphorylation of alpha-actinin1 at Y12 and alpha-actinin4 at Y265 is critical for dorsal stress fiber establishment, transverse arc maintenance and focal adhesion maturation. (c) 2013 Elsevier Inc. All rights reserved.