α-Actinin and Filamin Cooperatively Enhance the Stiffness of Actin Filament Networks

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, display synergistic mechanical functions. METHODOLOGY/PRINCIPAL FINDINGS: Using quantitative rheology, we find that combining alpha-actinin and filamin is much more effective at producing elastic, solid-like actin filament networks than alpha-actinin and filamin separately. Moreover, F-actin networks assembled in the presence of alpha-actinin and filamin strain-harden more readily than networks in the presence of either alpha-actinin or filamin. SIGNIFICANCE: These results suggest that cells combine auxiliary proteins with similar ability to crosslink filaments to generate stiff cytoskeletal structures, which are required for the production of internal propulsive forces for cell migration, and that these proteins do not have redundant mechanical functions

Rho-Regulatory Proteins in Breast Cancer Cell Motility and Invasion

The importance of the Rho-GTPases in cancer progression, particularly in the area of metastasis, is becoming increasingly evident. This review will provide an overview of the role of the Rho-regulatory proteins in breast cancer metastatis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44220/1/10549_2004_Article_5264599.pd

Microinjection of fluorescently labeled alpha-actinin into living fibroblasts

alpha-Actinin from chicken gizzard labeled with tetramethylrhodamine isothiocyanate has been incorporated into living fibroblast cells by microinjection. Fluorescent labeling of alpha-actinin was carried out such that the conjugated protein was functional in vitro as shown by its ability to bind to F-actin. Within 1-2 hr after injection, diffuse fluorescence was observed throughout the cytoplasm and only faint fluorescence was apparently associated with the stress fibers. During the ensuing 2-15 hr, however, most of the fluorescence was seen as periodicities along the stress fibers and as foci of the microfilament polygonal networks. This distribution of alpha-actinin in the living cells was strikingly similar to that found by indirect immunofluorescence localization of endogenous alpha-actinin in fixed samples of the same cell type. Control studies in which heat-treated (100 degrees C, 2 min) fluorescent alpha-actinin or tetramethylrhodamine isothiocyanate alone was injected into the cells indicated that the stress fiber and polygonal network labeling was specific for "native" fluorescently labeled alpha-actinin. These results suggest that the dynamic properties of proteins and structures in cultured mammalian cells can be studied with the use of microinjection and fluorescence microscopic techniques