QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online October 22, 2003
doi: 10.1242/10.1242/jcs.00769

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Takeya, R. Articles by Sumimoto, H. Search for Related Content PubMed PubMed Citation Articles by Takeya, R. Articles by Sumimoto, H.

Fhos, a mammalian formin, directly binds to F-actin via a region N-terminal to the FH1 domain and forms a homotypic complex via the FH2 domain to promote actin fiber formation

Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

View larger version (31K): [in a new window]   Fig. 4. Localization of active Fhos to actin stress fibers is mediated via its N-terminal F-actin-binding region. (A) Binding of Fhos to F-actin in a cosedimentation assay. Polymerized F-actin and His-tagged Fhos-N (1-569) were mixed in F-buffer to a final concentration of 6.6 µM and 1.3 µM, respectively, and incubated for 60 minutes at 25°C. The mixture was centrifuged for 60 minutes at 100,000 g, and both supernatants (S) and pellets (P) were analyzed by SDS-PAGE, followed by staining with CBB. (B) Quantitative analysis for binding of Fhos-N to F-actin. Various amounts of His-tagged Fhos-N (1-20 µg) were incubated with 1 µg of polymerized actin in a total volume of 50 µl. After ultracentrifugation, the free and bound His–Fhos-N were subjected to SDS-PAGE followed by staining with CBB. The amounts of the protein on the gel were estimated by an image analyzer. (C) Direct binding of F-actin to GST–Fhos-N in an F-actin overlay assay. Lysates of E. coli expressing GST–Fhos-N, GST–Fhos-FH2 and GST alone were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was probed with F-actin, and bound F-actins were detected using an anti-actin antibody (left panel). Proteins in bacterial lysates were also analyzed by immunoblot with the anti-GST antibodies (middle panel) or CBB staining (right panel). (D) Localization of Myc–Fhos-N to actin stress fibers in HeLa cells. Myc–Fhos-N and Flag–Fhos-FH1FH2 were coexpressed in HeLa cells. The cells were fixed and stained with the anti-Myc antibody (left panel) and phalloidin (right panel). Bars, 20 µm. (E) Direct binding of F-actin to His–Fhos-NC in an F-actin overlay assay. His–Fhos-NC and His–Fhos-FH1FH2 proteins were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was probed with F-actin, and bound F-actins were detected using an anti-actin antibody (left panel). Proteins were also analyzed by immunoblot with the anti-His antibody (right panel).

View larger version (81K): [in a new window]   Fig. 3. Fhos in an active form localizes to actin stress fibers. (A) Confocal images of HeLa cells expressing the GFP–Fhos-NC (415-1053), a constitutively active mutant protein. Confocal images near the bottom, middle and top of the cells, as well as stacked images, are shown. For each pair of images, GFP fluorescence (left panel) and phalloidin staining (right panel) are shown. (B) Actin fibers in HeLa cells expressing the GFP-NC mutant. GFP fluorescence (left panels) or phalloidin staining (right panels) are shown. A magnified view of the insert is also shown in the lower panels. (C) COS-7 cells expressing the GFP-NC mutant were fixed and detected by GFP fluorescence (left panel) or phalloidin staining (right panel). Bar, 20 µm.

View larger version (44K): [in a new window]   Fig. 5. Fhos forms a homotypic complex via the FH2 domain. (A) HeLa cells co-expressing Myc-tagged Fhos-F and GFP-fused Fhos mutants were lysed, and proteins were immunoprecipitated with the anti-Myc antibody. The precipitants were analyzed by immunoblot with the anti-GFP antibodies. Proteins in cell lysates were also analyzed directly by immunoblot (lower panel). (B) HeLa cells co-expressing Flag-tagged Fhos-FH2 and Myc-tagged Fhos mutants (FH1FH2 or N) were lysed and immunoprecipitated with the anti-Flag antibody. The immunoprecipitates were analyzed by immunoblot with the anti-Myc or anti-Flag antibodies. Proteins in cell lysates were also analyzed directly by immunoblot. (C) HeLa cells co-expressing Myc-tagged Fhos-FH2 and GFP-fused Fhos-NC. Cells were fixed and detected by triple-fluorescence microscopy for GFP fluorescence, Myc immunostaining and phalloidin staining. Merged images are shown in the lowest panel. (D) The direct interaction of MBP-FH2 with GST-FH2. MBP-FH2 or MBP alone was incubated with GST-FH2 or GST-DAD. Proteins were pulled down with glutathione-Sepharose-4B and subjected to SDS-PAGE, and stained with CBB. (E) The direct interaction between His-tagged FH1FH2 and GST-FH2. His-FH1FH2 was incubated with GSTFH2 or GST alone. An in vitro pull-down binding assay was performed as in Fig. 5D.

View larger version (71K): [in a new window]   Fig. 1. Stress fiber formation induced by Fhos mutant proteins. (A) HeLa cells were transfected with vectors encoding the indicated mutant proteins of Fhos or RhoAG14V. Cells were fixed 16 hours after the addition of DNA-lipofectamine, and expressed proteins were detected by GFP fluorescence or immunostaining for the Myc epitope (left panel). F-actin was detected by phalloidin staining (right panel). Bar, 20 µm. (B) The structure of Fhos and its truncation mutants. Numbers denote amino acid positions in Fhos. Although the FH2 domain is originally defined as a conserved sequence of approximately 100 residues, the analysis of additional formins revealed that the similarity extends over about 500 amino acids (Frazier and Field, 1997; Zeller et al., 1999). The FH2 domain of Fhos (amino acids 613-1053) is also delineated by the extended definition. All constructs were tagged with GFP at the N-termini. The effect of each mutant on the induction of actin stress fibers is summarized on the right.

View larger version (50K): [in a new window]   Fig. 2. The DAD of Fhos in the C-terminus appears to regulate the Fhos activity by interacting with the N-terminus. (A) HeLa cells were transfected with vectors encoding the indicated GFP-fused C-terminally truncation mutants of Fhos. Cells were fixed and then detected by GFP fluorescence (upper panels) or phalloidin staining (lower panels). Bar, 20 µm. (B) The C-terminally truncated mutant proteins are illustrated on the left, and the effects of each mutant on the induction of actin stress fibers (shown in Fig. 2A) are summarized on the right (indicated by plus and minus signs). Asterisks in the DAD indicate the polybasic region (amino acid sequences are shown in Fig. 2C). (C) Sequence alignment of the DAD and the polybasic region of Fhos, Diaphanous, p140mDia, Bni1 and SepA. Identical residues are shown on a black background and similar residues are shown on a gray background. The polybasic region is shaded in dark gray and basic residues are indicated by white letters. (D) The direct interaction between the N-terminus and DAD of Fhos. His–Fhos-N (1-569) or His–Fhos-FH1FH2 (533-1053) was incubated with GST–Fhos-DAD (1081-1145). Proteins were pulled down with glutathione-Sepharose-4B, subjected to SDS-PAGE, and stained with CBB. (E) Role for the polybasic region of the DAD in the intramolecular interaction. His–Fhos-N (1-569) binds directly to GST–Fhos-DAD (1081-1145), whereas it failed to bind to GST-Fhos (1081-1120), which lacks the polybasic region, or GST alone. An in vitro pull-down binding assay was performed as in Fig. 2D.

View larger version (60K): [in a new window]   Fig. 6. Both FH1 and FH2 domains of Fhos are required for promoting actin fiber formation, but the FH1 domain is dispensable for homotypic complex formation. (A) The structure of Fhos and its FH1- or FH2-truncated mutant proteins. (B) HeLa cells were transfected with vectors encoding the indicated Fhos truncation mutants. Cells were fixed and then detected by GFP fluorescence (left panels) or phalloidin staining (right panels). Bar, 20 µm. (C) HeLa cells expressing Myc-tagged Fhos mutants were lysed and immunoprecipitated with the anti-Myc antibody. The immunoprecipitates were blotted with anti-Fhos or anti-Myc antibodies. Proteins in cell lysates were also analyzed directly by immunoblot.