F–actin-bundling sites are conserved in proteins with villin-type headpiece domains

Figures

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  FIGURE 1:. Identification of the F–actin-bundling site in recombinant human villin and human advillin proteins. (A) Schematic map comparing the amino-terminal core (S1–S6) and carboxyl-terminal headpiece (HP) of human villin and advillin proteins. S1–S6 represent six homologous domains conserved between villin and other proteins of its family including advillin and gelsolin. The actin-binding residues identified in the structure of isolated villin headpiece and isolated advillin headpiece domain are highlighted in red. The “KEK” residues in the actin-binding motif 821KKEK824 identified in FL villin are shown in green. (B) Human recombinant FL villin, point mutants of villin (W815A, K816A, K821A, F827A), human recombinant FL advillin, and point mutant of advillin (W807A, K8808A, K813A, F819A) were expressed as GST-tagged proteins. Proteins were purified using glutathione-Sepharose 4B beads, separated by SDS–PAGE and visualized by GelCode Blue staining. (C) FL and mutant villin proteins (1.0 μM) were incubated with 0.76 μM F-actin and centrifuged at 10,000 × g for 15 min. Actin distribution in the supernatant (S) and pellet (P) fractions was analyzed by SDS–PAGE and GelCode Blue staining.…

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  FIGURE 2:. The actin bundling site in villin headpiece is well conserved in other proteins that contain a villin-type headpiece domain. Sequence alignment of the headpiece domain of villin proteins from different species and unrelated actin-bundling proteins that contain a villin-type headpiece domain. The four F–actin-binding residues required for actin bundling by villin (highlighted in yellow) are well conserved in these proteins. Sequences shown include: chicken villin headpiece (CV HP), human villin headpiece (HV HP), human advillin headpiece (HA HP), Mus musculus advillin headpiece (MA HP), Danio rerio advillin headpiece (DrA HP), D. rerio villin headpiece (DrV HP), chicken supervillin headpiece (CS HP), human supervillin headpiece (HS HP), D. rerio supervillin headpiece (DrS HP), Drosophila melanogaster headpiece-containing protein (Dm protein HP), Anopheles gambiae headpiece-containing protein (Ag protein HP), Xenopus laevis villin headpiece (XlV HP), X. laevis supervillin headpiece (XlS HP), chicken villin homolog (CVh HP), Arabidopsis thaliana villin headpiece (AtV HP), A. thaliana villin 1 headpiece (AtV1 HP), A. thaliana villin 2 headpiece (AtV2 HP), A. thaliana villin 3…

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  FIGURE 3:. Villin and advillin self-associate. (A) Recombinant FL villin and advillin proteins (20 nM each) were incubated with different cross-linkers (50-fold M excess) for 90 min at room temperature. Western analysis shows chemically cross-linked proteins analyzed under nonreducing conditions. DMSO-treated proteins were used as negative controls. (B) The expression of EGFP-villin and EGFP-advillin proteins in MDCK cells. To compare protein expression, Western analysis was performed using tubulin as a loading control. (C) MDCK cells expressing EGFP-villin or EGFP-advillin were incubated with different cross-linkers (2.5 mM at room temperature for 30 min) and cell extracts were analyzed under reducing conditions. Self-association of proteins was analyzed by Western analysis using villin and advillin antibodies. DMSO-treated cells were used as a negative control. All data shown are representative of three independent experiments.

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  FIGURE 4:. Validation of human villin F–actin-bundling site in HeLa cells. (A) Western analysis of HeLa cells transiently transfected with EGFP-tagged FL or point mutants (W815A, K816A, K821A, F827A) of villin. Also shown is Western analysis of HeLa cells transiently transfected with EGFP-tagged villin mutant KAAA. Tubulin was used as a loading control. (B) The cell morphology and the distribution of EGFP-tagged FL or mutant villin proteins was assessed by confocal microscopy. F-Actin staining was monitored using Alexa Fluor 568 Phalloidin. Merged images show colocalization of villin and F-actin. Nuclei were imaged using DAPI. Right panels are higher magnifications of the boxed areas. Scale bar represents 10 μm. All data shown are representative of three independent experiments.

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  FIGURE 5:. Validation of human advillin F–actin-bundling site in HeLa cells. (A) Western analysis of HeLa cells transiently transfected with EGFP-tagged FL or point mutants of advillin. Tubulin was used as a loading control. (B) HeLa cells expressing EGFP-tagged FL or EGFP-tagged mutant advillin proteins were examined by confocal microscopy to assess filopodial assembly and intracellular distribution of advillin proteins and F-actin. F-Actin staining was monitored using Alexa Fluor 568 Phalloidin. Merged images show colocalization of advillin with F-actin. Nuclei were visualized using DAPI staining. Right panels are magnifications of the boxed areas. Scale bar represents 10 μm. All data shown are representative of three independent experiments.