The mechanism and structural features that are responsible for the fast motility of Chara corallina myosin (CCM) have not been elucidated, so far. The low yields of native CCM that can be purified to homogeneity were the major reason for this. Here, we describe the expression of recombinant CCM motor domains, which support the fast movement of actin filaments in an in vitro motility assay. A CC...

We have developed an in vitro assay to study actin assembly at cadherin-enriched cell junctions. Using this assay, we demonstrate that cadherin-enriched junctions can polymerize new actin filaments but cannot capture preexisting filaments, suggesting a mechanism involving de novo synthesis. In agreement with this hypothesis, inhibition of Arp2/3-dependent nucleation abolished actin assembly at ...

We have investigated the exchangeability of alpha-actinin in various structures of cultured chick cardiac fibroblasts and muscle cells using fluorescent analogue cytochemistry in combination with fluorescence recovery after photobleaching. Living cells were microinjected with tetramethylrhodamine-labeled alpha-actinin, which became localized in cellular structures. Small areas of labeled struct...

BACKGROUND The close subcellular proximity of different actin filament crosslinking proteins suggests that these proteins may cooperate to organize F-actin structures to drive complex cellular functions during cell adhesion, motility and division. Here we hypothesize that alpha-actinin and filamin, two major F-actin crosslinking proteins that are both present in the lamella of adherent cells, d...

We have used a positively charged lipid monolayer to form two-dimensional bundles of F-actin cross-linked by ! -actinin to investigate the relative orientation of the actin filaments within them. This method prevents growth of the bundles perpendicular to the monolayer plane, thereby facilitating interpretation of the electron micrographs. Using ! -actinin isoforms isolated from the three types...

The distribution of fluorescently labeled a-actinin after microinjection into fibroblasts has been determined in both living and fixed cells. We have found that the distribution of the injected tetramethylrhodamine isothiocyanate-labeled protein (TMRITC-a-actinin) in living cells, which is in ruffling membranes, actin microfilament bundles, and polygonal microfilament networks (Feramisco, 1979,...

The actin filament severing protein, Acanthamoeba actophorin, decreases the viscosity of actin filaments, but increases the stiffness and viscosity of mixtures of actin filaments and the crosslinking protein alpha-actinin. The explanation of this paradox is that in the presence of both the severing protein and crosslinker the actin filaments aggregate into an interlocking meshwork of bundles la...

Protein-protein interactions are thought to modulate the efficiency and specificity of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling in specific subcellular compartments. Here we show that the F-actin-binding protein α-actinin targets CaMKIIα to F-actin in cells by binding to the CaMKII regulatory domain, mimicking CaM. The interaction with α-actinin is blocked by CaMKI...

The actin cytoskeleton plays a fundamental role in eukaryotic cells. Its reorganization is regulated by a plethora of actin-modulating proteins, such as a-actinin. In higher organisms, α-actinin is characterized by the presence of three distinct structural domains: an N-terminal actin-binding domain and a C-terminal region with EF-hand motif separated by a central rod domain with four spectrin ...