ForC, a novel type of formin family protein lacking an FH1 ... aggregates behave as a multicellular

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  • Introduction Proper spatial and temporal regulation of cytoskeletal function is essential for such eukaryotic cell activities as mitosis, endocytosis, exocytosis, cell migration and morphogenesis. To better understand the molecular basis for cell motion and the underlying regulation of the cytoskeletal system, we are using the soil amoeba Dictyostelium discoideumas a model system.

    Dictyostelium discoideum has a relatively simple cytoskeleton; nevertheless, many of its movements appear similar to those observed in higher eukaryotes. In rich medium, they proliferate as a unicellular organism and carry out cytokinesis that looks morphologically very similar to that of vertebrate cells in culture. When starved, the cells aggregate to form multicellular structures called fruiting bodies, which consist of spores and stalks that hold sori above the substrate. During this process, the cells first migrate to an aggregation center in a fashion similar to leucocytes. The resultant aggregates behave as a multicellular entity and undergo programmed cell differentiation and morphogenesis to yield a fruiting body. In this way, Dictyosteliumprovides a model system with which to investigate how individual cells behave within a multicellular system and how multicellular morphogenesis is regulated. In addition, Dictyostelium is highly amenable to genetic manipulation, including gene disruption and introduction of exogenous genes. And since its genome is haploid, it is possible to see an effect of a mutation even when it is recessive.

    Formin family proteins are thought to play crucial roles in the regulation of cytoskeletal function (Tanaka, 2000; Wasserman, 1998). They are found in a wide variety of eukaryotic cells, from unicellular organisms and fungi to higher plant and animal cells. Many of the formin proteins were isolated genetically on the basis of mutations that affect cytoskeletal function. For example, budding yeast Bni1 (Kohno et al., 1996) and Bnr1 (Imamura et al., 1997), fission yeast Cdc12 (Imamura et al., 1997), Asperugius nidanasSepA (Harris et al., 1997), nematode Cyk-1 (Swan et al., 1998), and fruit fly diaphanous(Castrillon and Wasserman, 1994) and cappuccino (Emmons et al., 1995) were all discovered through mutations that affected cytokinesis. Of these, Bni1 (Jansen et al., 1996; Zahner et al., 1996), Bnr1 and cappuccino are also known to be involved in the establishment of cell polarity. In the fission yeast, however, establishment of cell polarity is mediated by another formin protein, For3 (Feierbach and Chang, 2001). In addition, mutation of mouse formin, the first formin isoform identified, results in limb deformity and renal agenesis (Jackson-Grusby et al., 1992; Woychik et al., 1990); mutation of DFNA1(hDia1), a human homologue of diaphanous, results in nonsyndromic deafness caused by a defect in actin organization in the hair cells of the inner ear (Lynch et al., 1997), and a mutation in DIA(hDia2), another human homologue of diaphanous,results in premature ovarian failure (Bione et al., 1998).

    Formin proteins are characterized by the presence of three

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    Formins are highly conserved regulators of cytoskeletal organization and share three regions of homology: the FH1, FH2 and FH3 domains. Of the nine known formin genes or pseudogenes carried byDictyostelium, forC is novel in that it lacks an FH1 domain. Mutant Dictyostelium lacking forC (∆forC) grew normally during the vegetative phase and, when starved, migrated normally and formed tight aggregates. Subsequently, however, ∆forC cells made aberrant fruiting bodies with short stalks and sori that remained unlifted. ∆forC aggregates were also unable to migrate as slugs, suggesting forC is involved in mediating cell movement during multicellular stages of Dictyostelium development. Consistent with this idea, expression of forC was increased significantly in aggregates of wild-type cells.

    GFP-ForC expressed in ∆forC cells was localized at the crowns, which are macropinocytotic structures rich in F- actin, suggesting that, like other formin isoforms, ForC functions in close relation with the actin cytoskeleton. Truncation analysis of GFP-ForC revealed that the FH3 domain is required for ForC localization; moreover, localization of a truncated GFP-ForC mutant at the site of contacts between cells on substrates and along the cortex of cells within a multicellular culminant suggests that ForC is involved in the local actin cytoskeletal reorganization mediating cell-cell adhesion.

    Key words: Cellular slime mold, Actin, Culmination, Slug, Morphogenesis, Profilin

    Summary

    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 and ‡Gene Function Research Laboratory, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8562, Japan §Author for correspondence (e-mail: c.kitayama@aist.go.jp)

    Accepted 11 November 2002 Journal of Cell Science 116, 711-723 © 2003 The Company of Biologists Ltd doi:10.1242/jcs.00265

    Research Article

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    FH (formin homology) domains (FH1, FH2 and FH3) (Tanaka, 2000; Wasserman, 1998). The FH1 domain consists of multiple poly-proline stretches and is located at the middle of the protein. Many formin proteins are known to interact with profilin, an actin-monomer-binding protein, via the FH1 domain (Evangelista et al., 1997; Holt and Koffer, 2001; Imamura et al., 1997; Wasserman, 1998; Watanabe et al., 1997). In addition, some formin proteins interact with the Src homology 3 (SH3) domain or WW domain through the FH1 domain (Holt and Koffer, 2001). The FH2 domain is a highly conserved region that spans about 130 amino acid residues, and is located near the C-terminus (Tanaka, 2000; Wasserman, 1998). Recent truncation analysis of Bni1 indicated that the FH2 domain alone is able to nucleate polymerization of actin filaments in vitro (Pruyne et al., 2002). The FH3 domain is less well conserved than the other two FH domains, is located near the N-terminus and is thought to be important for determining intracellular localization of formin family proteins (Kato et al., 2001; Petersen et al., 1998).

    These biochemical properties of the FH1 and FH2 domains, as well as the phenotypes related to formin mutations, implicate formin proteins in the regulation of the actin cytoskeleton. Consistent with this view, a variety of mutations affecting one or more formin proteins, or their overproduction, all result in actin cytoskeletal disorganization (Castrillon and Wasserman, 1994; Chang et al., 1997; Evangelista et al., 1997; Swan et al., 1998; Watanabe et al., 1997; Watanabe et al., 1999). In addition, a growing number of studies, including analyses of phenotype and protein localization, suggest that formin proteins are also involved in regulating microtubule function (Giansanti et al., 1998; Lee et al., 1999; Miller et al., 1999; Palazzo et al., 2001).

    Several formin proteins have been shown to bind Rho-type small GTPases. This places formin proteins at a critical position, where they can receive signals from Rho and organize the actin and/or microtubule cytoskeleton in response to that signal. This prompted us to examine the functions of formin proteins using Dictyostelium discoideumas a genetic model with which to study cell motility. Our aim was to establish a general model of cytoskeletal regulation in eukaryotic cells.

    Materials and Methods DNA manipulation Standard methods were used for DNA manipulation (Sambrook et al., 1989). The sequences of the entire coding regions of forA, forB and forC were determined mainly by inverse PCR using genomic DNA of wild-type DictyosteliumAx2 cells. For each PCR, the sequences of several clones were determined, and their consensus was taken as the sequence of each gene.

    Disruption construct of forC gene Entire genomic DNA of forC was obtained by PCR and cloned into the pGEM-T cloning vector (Promega). The 2.4 kb SalI-EcoRV fragment of the forC ORF was then replaced with the Blasticidin resistance gene cassette (Adachi et al., 1994). The resultant disruption construct was digested with SpeI and NcoI, and used to transform Ax2 cells. Successful disruption was determined with PCR using primers 5′-ATGAAAATTAGAGTTGAATTAATAAATGG-3 ′, and 5′-GCTC- GTTTTACCATATCATTTG-3′.

    Cells and media Wild-type Dictyostelium(strain Ax2) and ∆forC cells were cultured in HL5 medium (Sussman, 1987) supplemented with 60 µg/ml each of penicillin and streptomycin (+PS) at 20°C. Blasticidin selection was performed by adding 10 µg/ml Blasticidin to HL5+PS. Transformants with pBIG-based plasmids were maintained in HL5+PS supplemented with 15 µg/ml G418. For suspension cultures, cells were shaken in conical flasks at ~140 rpm. Dictyostelium development was carried out either on MES agar plates (Peterson et al., 1995) or on Klebsiella aerogeneson SM/5 agar plates (Sussman, 1987).

    RT-PCR Ax2 cells were allowed to develop on MES agar plates, during which cells were collected from each 100 mm plate every 4 hours. RNA was extracted from the cells using TriZol reagent (Gibco Invitrogen), and was used for synthesis of first strand cDNA using reverse transcriptase (ReverTra Ace; Toyobo) with Oligo dT primer (5′-CCAGTGA- GCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTT- TTT-3′), after which 1% of the first strand cDNA was used for standard PCR using primers specific for both sides of the intron of forC (5′-ACAACAATCTCAACAAACTCC-3′ and 5′-ACAAGCC- AACAGTACGGTATC-3′). The PCR products were subjected to agarose gel electrophoresis.

    Construction of plasmids expressing ForC or GFP-ForC Genomic DNA encoding ForC was amplified by