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First published online 8 January 2003
doi: 10.1242/jcs.00265

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ForC, a novel type of formin family protein lacking an FH1 domain, is involved in multicellular development in Dictyostelium discoideum

Chikako Kitayama^*,, and Taro Q. P. Uyeda

^* Japan Society for the Promotion of Science, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8562, Japan
Gene Function Research Laboratory, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8562, Japan

View larger version (48K): [in a new window]   Fig. 1. (A) Box diagram illustrating the primary structural features of formin family proteins in Dictyostelium discoideum. The deduced amino acid sequences of each gene are shown as open boxes. The gray boxes represent clusters of proline residues in each polyproline stretch within FH1 domains. The black boxes indicate the FH2 domains. forA, forB and forC were found as partial sequences encoded by cDNA clones in the Japanese cDNA database (FCL-AB11, SLB408 and SSC675, respectively). The accession numbers for full-length forA, forB and forC are AB082542, AB082543 and AB082544, respectively. forD, forE, forF, forG, forH and forI are found in contigs from the Dictyostelium genome database. The contig numbers are 16730, 16789, 17584, 16652, 15079 and 14500, respectively. (B) The predicted amino acid sequence of the forC gene product. Three highly conserved regions within the FH3 domain are shaded in gray. The FH2 domain is shown by white letters on a black background. (C,D) Amino acid sequence alignment of the FH2 (C) and FH3 (D) domains of various formin homologues. Multiple sequence alignments were performed using ClustalW 1.8 and colored with BOXSHADE. Residues identical to the column consensus are shown on black backgrounds; residues similar to the column consensus are shown on gray backgrounds. (C) Eleven proteins are compared: from top to bottom: Dictyostelium discoideum ForC, ForA and ForB; mouse p140mDia (mDIA1) (Watanabe et al., 1997); human hDia1 (DFNA1) (Lynch et al., 1997); hDia2 (Bione et al., 1998); Drosophila melanogaster Diaphanous (Castrillon and Wasserman, 1994); Caenorhabditis elegans Cyk-1 (Swan et al., 1998); mouse Formin (Chan et al., 1996; Woychik et al., 1990); Saccharomyces cerevisiae Bni1 (Jansen et al., 1996; Zahner et al., 1996); and Schizosaccharomyces pombe Cdc12 (Chang et al., 1997). (D) Twelve proteins are aligned: from top to bottom: Dictyostelium discoideum ForC, ForA and ForB; mouse p140mDia; human hDia2; Drosophila melanogaster Diaphanous and Cappuccino (Emmons et al., 1995); Caenorhabditis elegans Cyk-1; mouse Formin; Saccharomyces cerevisiae Bni1; Schizosaccharomyces pombe Cdc12; and human FHOS (Westendorf et al., 1999). The reported conserved regions (Petersen et al., 1998) are indicated by solid underlines. A newly found conserved region in the third domain is indicated by a dashed underline.

View larger version (19K): [in a new window]   Fig. 2. (A) The genomic structure of forC and the forC disruption construct. (B) Agarose gel electrophoreses of the forC locus obtained by genomic PCR from wild-type (Ax2) and forC cells. Amplification of wild-type genomic forC locus yielded a 3.6 kb product; amplification of the forC knocked-out locus yielded a 2.4 kb product.

View larger version (78K): [in a new window]   Fig. 3. Developmental morphology of wild-type and forC mutant cells. (A) Morphology of fruiting bodies of wild-type (left) and forC cells (right) on lawns of Klebisiella aerogenes. forC cells made aberrant fruiting bodies. (B) Time lapse recording of wild-type (upper row) and forC (lower row) development on MES plates. The times (hours) after the onset of starvation are indicated above the pictures. (C) Slug formation by wild-type (left) and forC (right) cells. When wild-type and forC cells were starved on unbuffered agar plates, wild-type cells formed slugs, while forC cells remained as tipped mounds. (D) Complementation of the forC phenotype by supplying a plasmid that expresses ForC or GFP-ForC. forC cells carrying each plasmid indicated above the pictures were allowed to develop on MES agar plates.

View larger version (44K): [in a new window]   Fig. 4. Expression of forC at each stage during development. Total RNA was prepared at several time points during development, and RT-PCR was carried out using primers designed to amplify a 983 bp fragment that included a site from which an intron was excised. The time after the onset of starvation is indicated below each picture, and the status at each developmental stage is illustrated above the picture. 330 bp H7 gene fragment was amplified as an internal control (Zinda and Singleton, 1998).

View larger version (52K): [in a new window]   Fig. 5. Development of mixtures of wild-type and forC cells combined at different ratios. forC cells and wild-type cells were mixed at the indicated ratios and allowed to develop on MES agar plates. The representative morphology of the fruiting bodies in each mixture is drawn schematically below each picture.

View larger version (52K): [in a new window]   Fig. 6. Intracellular localization of GFP-ForC. (A) Live observation of forC cells expressing GFP-ForC in MES buffer. GFP-ForC was diffusely distributed in the cytoplasm. (B) forC cells expressing GFP-ForC were fixed and stained with rhodamine-phalloidin. The fluorescent signals were recorded separately from the GFP and rhodamine channels by using a CCD camera, and then pseudocolored and merged. GFP-ForC localized at the crowns (a,b), which are rich in F-actin (a', b' and c'), while GFP alone had no distinct localization (c). GFP-ForC co-localizated with F-actin at crowns were depicted in yellow in merged pictures (a'',b''). No yellow region is seen in the merged images of cells expressing GFP alone (c''). (C) Localization of GFP-ForC at the crowns in live cells compressed by agarose overlay. Arrows indicate GFP-ForC fluorescence.

View larger version (59K): [in a new window]   Fig. 7. Intracellular localization of ForC truncation mutants fused to GFP. (A) Full-length ForC and the truncated ForC mutants. Gray boxes in the full-length ForC indicate the FH3 and FH2 domains. Thick lines indicate the regions encoded by each mutant. All ForC constructs were tagged with GFP at their N-termini. Crown localization of each mutant in either fixed or live cells is indicated by `-' and `+' on the right. (B) Fluorescence micrographs of forC cells expressing the various GFP-ForC mutants. Cells were fixed and stained with rhodamine-phalloidin. The full-length protein (a) and the 1-633 (c), 1-468 (d) and 1-323 (e) mutants all localized at the crowns (indicated by arrows), whereas GFP-FH3 did not (b, the position of a crown is indicated by an arrowhead). (C) Fluorescence micrographs of living forC cells expressing the GFP-ForC-1-323 mutant (left) and GFP-ForC (right). Arrows indicate the crown localization of GFP-ForC-1-323, which includes the region from the first methionine of ForC to the end of the FH3 domain. Crown localization of full-length GFP-ForC was not detected without fixation.

View larger version (129K): [in a new window]   Fig. 8. Intracellular localization of GFP-ForC-1-323 during macropinocytosis, phagocytosis, and when touching a neighboring cell. Images were taken every 6 seconds using confocal microscopy. (A) Arrows indicate a typical crown during macropinocytosis. GFP-ForC-1-323 stays at the leading edge of the ruffling membrane until it eventually disappears. (B) Arrows indicate a phagocytotic cup engulfing a yeast cell. The yeast cells being engulfed and those already taken up by the Dictyostelium cell are visible due to their autofluorescence. (C) Arrows indicate the site at which a cell touches a neighboring cell.

View larger version (116K): [in a new window]   Fig. 9. Intracellular localization of GFP-ForC-1-323 in multicellular structures. Wild-type cells expressing either GFP-ForC-1-323 (left two columns) or GFP alone (right) were mixed with those harboring the pBIG vector at a ratio of about 1:10 and allowed to develop on agar plates. Culminating fruiting bodies were picked with tweezers, placed on a coverslip and observed with a confocal microscope. Specific localization of GFP-ForC-1-323 at the edges of the cells is indicated by arrows (left).