Entamoeba histolytica Encodes Unique Formins, a Subset of Which Regulates DNA Content and Cell Division

Abstract

The formin family of proteins mediates dynamic changes in actin assembly in eukaryotes, and therefore it is important to understand the function of these proteins in Entamoeba histolytica, where actin forms the major cytoskeletal network. In this study we have identified the formin homologs encoded in the E. histolytica genome based on sequence analysis. Using multiple tools, we have analyzed the primary sequences of the eight E. histolytica formins and discovered three subsets: (i) E. histolytica formin-1 to -3 (Ehformin-1 to -3), (ii) Ehformin-4, and (iii) Ehformin-5 to -8. Two of these subsets (Ehformin-1 to -3 and Ehformin-4) showed significant sequence differences from their closest homologs, while Ehformin-5 to -8 were unique among all known formins. Since Ehformin-1 to -3 showed important sequence differences from Diaphanous-related formins (DRFs), we have studied the functions of Ehformin-1 and -2 in E. histolytica transformants. Like other DRFs, Ehformin-1 and -2 associated with F-actin in response to serum factors, in pseudopodia, in pinocytic and phagocytic vesicles, and at cell division sites. Ehformin-1 and -2 also localized with the microtubular assembly in the nucleus, indicating their involvement in genome segregation. While increased expression of Ehformin-1 and -2 did not affect phagocytosis or motility, it clearly showed an increase in the number of binucleated cells, the number of nuclei in multinucleated cells, and the average DNA content of each nucleus, suggesting that these proteins regulate both mitosis and cytokinesis in E. histolytica.Polymerization of actin into helical filaments is nucleated by different groups of actin-binding proteins, which in turn are controlled by specific signaling molecules in eukaryotic cells (10, 66). Currently, the three known groups of F-actin nucleating factors are the Arp2/3 complex, formins, and spire (10, 21). Formin homology proteins promote rapid assembly of unbranched actin filaments and local reorganization of higherorder cellular structures to execute specified cytoskeletal functions (reviewed in reference 21). These multidomain proteins have many isoforms that are ubiquitously expressed and perform diverse cellular functions in almost all eukaryotic cells, from vertebrates and plants to unicellular protists (25). The defining feature of formin proteins is the formin homology 2 (FH2) domain, which is required for the nucleation and elongation of nascent actin filaments (24,43,46). The FH1 domain, preceding the FH2 domain, is another key region found in most formins and is made up of tandem repeats of proline and other amino acids (24). Polyproline residues in the FH1 domain interact with profilin to recruit assembly-competent actin monomers in the vicinity of the FH2 domain (50, 52, 61). The FH1 domain also interacts with Src homology 3 (SH3) domainor WW domain-containing proteins (17, 59). Thus, the core FH1-FH2 region nucleates unbranched actin filaments, unlike the Arp2/3 complex, which creates branched filaments (21). Other regulatory domains,...

Unique Structural and Nucleotide Exchange Features of the Rho1 GTPase of Entamoeba histolytica

“…EhFormin1 also co-localizes with microtubules during mitosis, and its overexpression leads to delays in cell cycle progression (20). It will be interesting to determine whether EhRho1 mediates actin and/or microtubule dynamics in response to extracellular cues in E. histolytica trophozoites.…”

“…EhRho1 Interacts with an mDia Homolog, EhFormin1-The E. histolytica genome encodes a family of eight Formins (20). EhFormins 1-3 contain a Rho GBD in tandem with a formin homology 3 (FH3) domain, homologous to mammalian mDia and Drosophila melanogaster Diaphenous (12).…”

“…EhFormins 1-3 contain a Rho GBD in tandem with a formin homology 3 (FH3) domain, homologous to mammalian mDia and Drosophila melanogaster Diaphenous (12). EhFormin1, also known as EhDia, is known to regulate actin polymerization and cell cycle progress in E. histolytica trophozoites (20). We cloned and purified the GBD-FH3 tandem of EhFormin1 (Fig.…”

“…Not surprisingly, Rho-activating GEFs have also emerged as critical players in E. histolytica pathogenesis (17,18). Similarly, E. histolytica possesses homologs to key Rho GTPase effectors, such as p21-activated kinase and Diaphanous-like formins (19,20). Although none of the formin family members have been shown to interact with Rho GTPases from E. histolytica, three of them (EhFormin1-3) contain Rho GTPase-binding domains (GBDs), suggesting that they serve as a link between Rho GTPase activation and actin polymerization.…”

“…Although none of the formin family members have been shown to interact with Rho GTPases from E. histolytica, three of them (EhFormin1-3) contain Rho GTPase-binding domains (GBDs), suggesting that they serve as a link between Rho GTPase activation and actin polymerization. Interestingly, overexpression of EhFormin1 in E. histolytica trophozoites revealed its co-localization with actin assemblies promoted by serum factors, association with microtubules during mitosis, and aberration of cell division (20).…”