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

Flanagan LA, Chou J, Falet H, Neujahr R, Hartwig JH, Stossel TP - J. Cell Biol. (2001)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Hematology Division, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.

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.

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Double labeling of FLNa and Arp3 in a sodium dodecyl sulfate–treated A7 cytoskeleton. A7 cells grown in serum were permeabilized, and then labeled with two separate monoclonal antibodies to FLNa (10-nm gold beads) and a polyclonal antibody against Arp3 (5-nm gold beads, highlighted by circles). Both antibodies label actin filament junctions at the cell periphery. Quantitation of the labeled junctions in A7 cells (shaded bars) and B3 cells (unshaded bars) reveals that the FLNa and Arp3 signals rarely overlap and are most often distinct (inset). The values are means ± SEM of measurements on four or more separate cells. Bar, 100 nm.
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fig4: Double labeling of FLNa and Arp3 in a sodium dodecyl sulfate–treated A7 cytoskeleton. A7 cells grown in serum were permeabilized, and then labeled with two separate monoclonal antibodies to FLNa (10-nm gold beads) and a polyclonal antibody against Arp3 (5-nm gold beads, highlighted by circles). Both antibodies label actin filament junctions at the cell periphery. Quantitation of the labeled junctions in A7 cells (shaded bars) and B3 cells (unshaded bars) reveals that the FLNa and Arp3 signals rarely overlap and are most often distinct (inset). The values are means ± SEM of measurements on four or more separate cells. Bar, 100 nm.

Mentions: The requirement for dodecyl sulfate denaturation of cytoskeletons to expose Arp3 epitopes reactive with available antibodies has hitherto hindered more direct comparisons of their location to those of FLNa, because the denaturation step interferes with binding of many FLNa antibodies, including those used for the experiments described in Fig. 3. We determined that a combination of two monoclonal anti-FLNa antibodies report FLNa in sodium dodecyl sulfate–treated A7 cells by immunofluorescence and immunogold staining and showed no staining in identically prepared M2 cells. These antibodies, generated against human platelet filamin antigen, are specific for FLNa (van der Ven et al., 2000). As expected from the results with separate staining protocols, FLNa and the Arp2/3 complex localize with actin filaments in the periphery of A7 cells (Fig. 4). However, by using gold beads of different sizes, unique aspects of staining are apparent. First, as observed with the polyclonal anti-FLNa antibodies, the monoclonal antibodies often reveal beads lining up more or less in rows at interfilament junctions, consistent with an array along FLNa subunits attached to the sides of filaments near the junctions to hold them in place. Second, Arp3 staining reveals more compact structures, sometimes at filament–filament junctions, but Arp3 and FLNa staining is infrequently visible at the same filament overlap sites. A quantitative analysis of the staining reveals that for both A7 and B3 cells, the majority of the immunolabeled junctions show staining for FLNa alone (inset). Some junctions contain only Arp2/3 complex, and a small minority of junctions are stained with both FLNa and Arp2/3.


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

Flanagan LA, Chou J, Falet H, Neujahr R, Hartwig JH, Stossel TP - J. Cell Biol. (2001)

Double labeling of FLNa and Arp3 in a sodium dodecyl sulfate–treated A7 cytoskeleton. A7 cells grown in serum were permeabilized, and then labeled with two separate monoclonal antibodies to FLNa (10-nm gold beads) and a polyclonal antibody against Arp3 (5-nm gold beads, highlighted by circles). Both antibodies label actin filament junctions at the cell periphery. Quantitation of the labeled junctions in A7 cells (shaded bars) and B3 cells (unshaded bars) reveals that the FLNa and Arp3 signals rarely overlap and are most often distinct (inset). The values are means ± SEM of measurements on four or more separate cells. Bar, 100 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198874&req=5

fig4: Double labeling of FLNa and Arp3 in a sodium dodecyl sulfate–treated A7 cytoskeleton. A7 cells grown in serum were permeabilized, and then labeled with two separate monoclonal antibodies to FLNa (10-nm gold beads) and a polyclonal antibody against Arp3 (5-nm gold beads, highlighted by circles). Both antibodies label actin filament junctions at the cell periphery. Quantitation of the labeled junctions in A7 cells (shaded bars) and B3 cells (unshaded bars) reveals that the FLNa and Arp3 signals rarely overlap and are most often distinct (inset). The values are means ± SEM of measurements on four or more separate cells. Bar, 100 nm.
Mentions: The requirement for dodecyl sulfate denaturation of cytoskeletons to expose Arp3 epitopes reactive with available antibodies has hitherto hindered more direct comparisons of their location to those of FLNa, because the denaturation step interferes with binding of many FLNa antibodies, including those used for the experiments described in Fig. 3. We determined that a combination of two monoclonal anti-FLNa antibodies report FLNa in sodium dodecyl sulfate–treated A7 cells by immunofluorescence and immunogold staining and showed no staining in identically prepared M2 cells. These antibodies, generated against human platelet filamin antigen, are specific for FLNa (van der Ven et al., 2000). As expected from the results with separate staining protocols, FLNa and the Arp2/3 complex localize with actin filaments in the periphery of A7 cells (Fig. 4). However, by using gold beads of different sizes, unique aspects of staining are apparent. First, as observed with the polyclonal anti-FLNa antibodies, the monoclonal antibodies often reveal beads lining up more or less in rows at interfilament junctions, consistent with an array along FLNa subunits attached to the sides of filaments near the junctions to hold them in place. Second, Arp3 staining reveals more compact structures, sometimes at filament–filament junctions, but Arp3 and FLNa staining is infrequently visible at the same filament overlap sites. A quantitative analysis of the staining reveals that for both A7 and B3 cells, the majority of the immunolabeled junctions show staining for FLNa alone (inset). Some junctions contain only Arp2/3 complex, and a small minority of junctions are stained with both FLNa and Arp2/3.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Hematology Division, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.

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.

Show MeSH
Related in: MedlinePlus