Formin 1-Isoform IV Deficient Cells Exhibit Defects in Cell Spreading and Focal Adhesion Formation

Leder P (2008) Formin 1-Isoform IV Deficient Cells Exhibit Defects in Cell Spreading and Focal Adhesion Formation. PLoS ONE 3(6): e2497. doi:10.1371/journal.pone.0002497 Formin 1-Isoform IV Deficient Cells Exhibit Defects in Cell Spreading and Focal Adhesion Formation Markus Dettenhofer 0 Fen Zhou 0 Philip Leder 0 Nils Cordes, Dresden University of Technology, Germany 0 Department of Genetics, Harvard Medical School , Boston, Massachusetts , United States of America Background: Regulation of the cytoskeleton is a central feature of cell migration. The formin family of proteins controls the rate of actin nucleation at its barbed end. Thus, formins are predicted to contribute to several important cell processes such as locomotion, membrane ruffling, vesicle endocytosis, and stress fiber formation and disassociation. Methodology/Principal Findings: In this study we investigated the functional role of Formin1-isoform4 (Fmn1-IV) by using genetically null primary cells that displayed augmented protrusive behaviour during wound healing and delayed cell spreading. Cells deficient of Fmn1-IV also showed reduced efficiency of focal adhesion formation. Additionally, we generated an enhanced green fluorescence protein (EGFP)-fused Fmn1-IV knock-in mouse to monitor the endogenous subcellular localization of Fmn1-IV. Its localization was found within the cytoplasm and along microtubules, yet it was largely excluded from adherens junctions. Conclusions/Significance: It was determined that Fmn1-IV, as an actin nucleator, contributes to protrusion of the cell's leading edge and focal adhesion formation, thus contributing to cell motility. - Eukaryotic cells utilize actin for a variety of specialized locomotor functions. As a cell senses its environment, filopodia and lamellipodia are projected forward and extracellular cues are monitored by cell surface receptors. Actin additionally participates in the trafficking of vesicles that transport membrane-associated cargo to and away from the cell surface. To date, three classes of actin nucleators control the rate of actin filament extension, the Arp2/3 complex, Spire, and the formins. The formin family of proteins consists of approximately 25 members, with the forming homology 2 (FH2) domain defining family membership. The formins are expressed ubiquitously in eukaryotic cells [13]. The FH2 of the yeast Formin, Bni1 [4,5] was originally shown to be sufficient for the nucleation of actin filaments at its barbed end, and subsequently FH2 domains derived from other formins have been shown to function similarly [68]. Crystal structure analyses have further suggested that FH2 domains function as dimers [9] and as nucleators of actin onto existing actin filaments [10,11]. Moreover, the FH2 domain acts by interfering with capping proteins gaining access to the growing actin filament, which leads to a model of formins acting as leaky cappers [12]. In addition to the FH2 domain, the majority of formins possess a forming homology 1 (FH1) domain that is highly proline-rich and functions in recruiting profilin to the emerging actin filament [1315]. Although the mechanism by which formins nucleate actin is becoming clearer, an understanding of their spatial and temporal regulation is just emerging. Several formins act in response to the Rho family of GTPases [16,17]. Dia1 functions through a dynamic self-folding mechanism that acts as an auto-inhibitory domain [18] to regulate stress fiber formation [19,20] and stabilize microtubules [21]. Moreover, recent knockdown studies convincingly demonstrated the role of Dia1 in stress fiber elongation [22]. While much has been learned about the Diaphanous-related formins (DRF), very little is known about the regulation of the founding member of the formin family, Formin1. Formin 1 consists of several splice variants that are differentially expressed in mammalian tissues [2326]. The most widely expressed variant is Fmn1-IV, which we have previously knocked-out in mice and found to result in weakly penetrant kidney aplasia [27]. Since they also possess an actin nucleation domain, functional regulation of the 6 different mRNA isoforms (Ia, Ib, II, III, IV, and V) of Fmn1 will in part be defined by the cell type in which they are expressed, as well as by the different peptide sequences they encode to determine their respective subcellular localizations. Formin1isoform1, which is highly expressed in the trigeminal and dorsal root ganglia [26,27] localizes to microtubules through a peptide encoded by exon 2 [28]. By examining primary cells derived from EGFP fused to Fmn1-IV knock-in mice we determined that the localization of Fmn1-IV was within the cytoplasm, and not significantly concentrated at adherens junctions. More specifically, Fmn1-IV also localizes along microtubules. Primary cells derived from Fmn1-IV knock-out mice demonstrated altered processive behavior at the cell periphery coupled with a delay in cell spreading and altered focal adhesion formation. Fmn1-IV localizes to the cytoplasm and along microtubules In this study, we examined the subcellular localization and functional role of Fmn1-IV, which has a broad pattern of tissue expression, including the limb bud, kidney, and gonad, and is distinguished from the other Formin1 isoforms by the inclusion of exon 6 within its coding region [25,26]. It has been shown that the FH1-FH2 domain of Fmn1 nucleates actin filaments in vitro [29]. Since the detection of endogenous Fmn1-IV has been difficult with the existing reagents, we generated an EGFP-Fmn1-IV fusion knock-in mouse in which the fusion protein is driven from the endogenous promoter (Fig. 1A). A targeting construct was generated by fusing exon 6 of Fmn1 in-frame with EGFP with the start of translation being from the first EGFP methionine. Using embryonic stem cell technology, mice were generated in which Fmn1-IV was replaced by the EGFP-Fmn1-IV fusion protein. Mouse tail DNA was used to screen for the stable insertion of EGFP fused to Fmn1-IV initially by Southern blot analysis and confirmed by PCR (Fig. 1B). Immunofluorescence analysis of primary epithelial cells derived from kidneys of EGFP-Fmn1-IV mice demonstrated its cytoplasmic localization, which was not significantly enriched in adherens junctions (Fig. 2). Fmn1-IV does not tend to colocalize with the major filamentous actin structures of cells, such as stress fibers or at the leading edge of lamellipodia. To gain a more thorough understanding of the nature of the cytoplasmic localization, immuno-gold electron microscopy was performed on primary MEFs derived from EGFP-Fmn1-IV knock-in and wild type control mice (Fig. 3A and B). These data show an association of EGFP-Fmn1-IV with microtubules. Fmn1-IV influences protrusive behavior at the leading edge of cells From an overall functional stand-point, it was important to determine what role Fmn1-IV plays in the cell. As an actin nucleator that localizes within the cytoplasm and can (...truncated)