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Supervillin (p205): A novel membrane-associated, F-actin-binding protein in the villin/gelsolin superfamily.

Pestonjamasp KN, Pope RK, Wulfkuhle JD, Luna EJ - J. Cell Biol. (1997)

Bottom Line: The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin.The NH2 terminus is novel, but contains four potential nuclear targeting signals.Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

View Article: PubMed Central - PubMed

Affiliation: Worcester Foundation for Biomedical Research, University of Massachusetts Medical Center, Shrewsbury, Massachusetts 01545, USA.

ABSTRACT
Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE-purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell-cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions "rings." At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

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Distribution of p205  (A and C) and E-cadherin (B  and D) in MDBK cells grown  at low cell density. Cells were  double labeled with affinity-purified, rabbit anti-pepA  IgG and with monoclonal  anticadherin antibodies, as  described in Materials and  Methods. Regions of p205  and cadherin colocalization  at the plasma membrane  (white arrows), and regions  of anti-pepA staining alone  (hollow arrows) are indicated. Bar, 10 μm.
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Figure 8: Distribution of p205 (A and C) and E-cadherin (B and D) in MDBK cells grown at low cell density. Cells were double labeled with affinity-purified, rabbit anti-pepA IgG and with monoclonal anticadherin antibodies, as described in Materials and Methods. Regions of p205 and cadherin colocalization at the plasma membrane (white arrows), and regions of anti-pepA staining alone (hollow arrows) are indicated. Bar, 10 μm.

Mentions: As was the case in neutrophils (Fig. 1), p205 was associated with plasma membranes in HeLa (not shown) and MDBK (Figs. 8–10) cells. First, p205 was enriched ∼10-fold, about the same fold enrichment as a biotin cell surface marker, in crude plasma membrane fractions (4) from all three cell types (not shown). Second, immunofluorescence localization with affinity-purified, anti-pepA antibodies showed label at the plasma membrane in both low density (Fig. 8) and confluent (Fig. 9) MDBK cells. Low density cells (Fig. 8, A and C) and subconfluent cells (Fig. 9 A) also contained appreciable amounts of signal in punctate dots throughout the cytoplasm and within the nucleus. In nonjunctional regions of the plasma membrane, p205 staining was definite, but not pronounced, and did not necessarily colocalize with antibody against the cell–cell adhesion protein, E-cadherin (Figs. 8 and 9, hollow arrows). As the MDBK cells became confluent, the ratio between the plasma membrane and the internal p205 signal increased, as did the colocalization with E-cadherin at sites of both initial (Fig. 8, C and D) and established (Fig. 9, A and B) cell–cell contact (solid white arrows). In confluent cells, nearly all of the p205 staining was concentrated at lateral cell borders (Fig. 9 C), where it colocalized almost perfectly at the light level with E-cadherin (Fig. 9 D). Because p205 staining was not observed in apical microvilli, nor with actin meshworks at basal cell surfaces (not shown), we infer that the colocalization with cadherin-containing, adherens-type junctions is specific and may reflect a role for p205 in the formation or stabilization of these structures.


Supervillin (p205): A novel membrane-associated, F-actin-binding protein in the villin/gelsolin superfamily.

Pestonjamasp KN, Pope RK, Wulfkuhle JD, Luna EJ - J. Cell Biol. (1997)

Distribution of p205  (A and C) and E-cadherin (B  and D) in MDBK cells grown  at low cell density. Cells were  double labeled with affinity-purified, rabbit anti-pepA  IgG and with monoclonal  anticadherin antibodies, as  described in Materials and  Methods. Regions of p205  and cadherin colocalization  at the plasma membrane  (white arrows), and regions  of anti-pepA staining alone  (hollow arrows) are indicated. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 8: Distribution of p205 (A and C) and E-cadherin (B and D) in MDBK cells grown at low cell density. Cells were double labeled with affinity-purified, rabbit anti-pepA IgG and with monoclonal anticadherin antibodies, as described in Materials and Methods. Regions of p205 and cadherin colocalization at the plasma membrane (white arrows), and regions of anti-pepA staining alone (hollow arrows) are indicated. Bar, 10 μm.
Mentions: As was the case in neutrophils (Fig. 1), p205 was associated with plasma membranes in HeLa (not shown) and MDBK (Figs. 8–10) cells. First, p205 was enriched ∼10-fold, about the same fold enrichment as a biotin cell surface marker, in crude plasma membrane fractions (4) from all three cell types (not shown). Second, immunofluorescence localization with affinity-purified, anti-pepA antibodies showed label at the plasma membrane in both low density (Fig. 8) and confluent (Fig. 9) MDBK cells. Low density cells (Fig. 8, A and C) and subconfluent cells (Fig. 9 A) also contained appreciable amounts of signal in punctate dots throughout the cytoplasm and within the nucleus. In nonjunctional regions of the plasma membrane, p205 staining was definite, but not pronounced, and did not necessarily colocalize with antibody against the cell–cell adhesion protein, E-cadherin (Figs. 8 and 9, hollow arrows). As the MDBK cells became confluent, the ratio between the plasma membrane and the internal p205 signal increased, as did the colocalization with E-cadherin at sites of both initial (Fig. 8, C and D) and established (Fig. 9, A and B) cell–cell contact (solid white arrows). In confluent cells, nearly all of the p205 staining was concentrated at lateral cell borders (Fig. 9 C), where it colocalized almost perfectly at the light level with E-cadherin (Fig. 9 D). Because p205 staining was not observed in apical microvilli, nor with actin meshworks at basal cell surfaces (not shown), we infer that the colocalization with cadherin-containing, adherens-type junctions is specific and may reflect a role for p205 in the formation or stabilization of these structures.

Bottom Line: The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin.The NH2 terminus is novel, but contains four potential nuclear targeting signals.Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

View Article: PubMed Central - PubMed

Affiliation: Worcester Foundation for Biomedical Research, University of Massachusetts Medical Center, Shrewsbury, Massachusetts 01545, USA.

ABSTRACT
Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE-purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell-cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions "rings." At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

Show MeSH
Related in: MedlinePlus