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

Abstract

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.

Comparison of Filamin A-induced Cross-linking and Arp2/3 Complex-mediated Branching on the Mechanics of Actin Filaments

“…Cells lacking FLNa expression have unstable surfaces and are unable to crawl, although they can transiently protrude flat lamellae (28). Hence, FLNa is required for locomotion and surface stability, but the findings do not rule out a requirement for the Arp2/3 complex in leading edge protrusion.…”

“…During each step, fractions containing recombinant FLNa protein were identified by immunoblotting with polyclonal antibodies against the hinge 1 region of FLNa (28) or by Coomassie Brilliant Blue staining. Either freshly prepared or thawed cell lysates were centrifuged at 20,000 ϫ g at 4°C and the supernatant fluid was loaded onto a HiTrap Q column (three connections of 5-ml columns, Amersham Biosciences, Inc.) preequilibrated with buffer solution QA8.5 (10 mM Tris-HCl, pH 8.5, 1 mM EDTA, 1 mM EGTA, 5 mM 2-mercaptoethanol, 0.02% Triton X-100).…”

Inhibiting the Arp2/3 Complex Limits Infection of Both Intracellular Mature Vaccinia Virus and Primate Lentiviruses

“…The Arp2/3 complex distributes throughout the cell but is enriched especially at the cortical layer underneath the plasma membrane through which viruses have to pass to infect cells (Flanagan et al, 2001). The Arp2/3 complex is regulated by both WiscottAldrich syndrome protein (WASP) family of proteins and cortactin (Weaver et al, 2003).…”

A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function

“…FlnA is composed of an N-terminal actin-binding domain followed by 24 Ig repeats, the C-terminal of which mediates their dimerization (Pudas et al, 2005). Human melanoma cells that lack FlnA have poor motility and continuous membrane blebbing (Cunningham et al, 1992;Flanagan et al, 2001). FLNA mutations have been associated with periventricular heterotopia, Ehlers-Danlos Syndrome, or familial cardiac valvular dystrophy (Fox et al, 1998;Robertson et al, 2003;Sheen et al, 2005;Kyndt et al, 2007;Unger et al, 2007).…”