Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells

The Arp2/3 complex and filamin A (FLNa) branch actin filaments. To define the role of these actin-binding proteins in cellular actin architecture, we compared the morphology of FLNa-deficient human melanoma (M2) cells and three stable derivatives of these cells expressing normal FLNa concentrations. All the cell lines contain similar amounts of the Arp2/3 complex. Serum addition causes serum-starved M2 cells to extend flat protrusions transiently; thereafter, the protrusions turn into spherical blebs and the cells do not crawl. The short-lived lamellae of M2 cells contain a dense mat of long actin filaments in contrast to a more three-dimensional orthogonal network of shorter actin filaments in lamellae of identically treated FLNa-expressing cells capable of translational locomotion. FLNa-specific antibodies localize throughout the leading lamellae of these cells at junctions between orthogonally intersecting actin filaments. Arp2/3 complex–specific antibodies stain diffusely and label a few, although not the same, actin filament overlap sites as FLNa antibody. We conclude that FLNa is essential in cells that express it for stabilizing orthogonal actin networks suitable for locomotion. Contrary to some proposals, Arp2/3 complex–mediated branching of actin alone is insufficient for establishing an orthogonal actin organization or maintaining mechanical stability at the leading edge

Phenotype.

<p>Phenotype.</p

Surface analysis of infected valves.

<p>For surface analysis using scanning electron microscopy (SEM), infected native (A-C; patient 3) and prosthetic (D-F; patient 7) heart valve tissues were cut into small pieces and fixed. After pretreatment and exposure to osmium tetroxide, tissues were dehydrated and mounted on standard SEM stubs with carbon tape. The cured samples were finally sputter-coated with a platinum layer and evaluated with a scanning electron microscope. (A-C) SEM images of the surface of an infected native valve. (A) Overview. (B) Epithelial cell boundaries are discernable. Inset: note that few bacteria are attached to the smooth surface and that these seem to be often damaged (arrow). (C) At higher magnification scattered bacteria showing apparently intact morphology (arrow) can also be found. (D-F) Ultrastructure images of infected biological prosthetic valve. (D) Overview. (E) The surface is characterized by deep holes and cracks where microbes may be concealed; the surface appears rough. Inset: note that the few bacteria attached to the outside often seem to be damaged (arrow). (F) At higher magnification a number of apparently intact bacteria (arrows) showing different morphologies can be found; note the fibrous structure of the substrate, providing ideal adhesion sites.</p

Clinical characteristics.

<p>Clinical characteristics.</p