Phospholipids regulate localization and activity of mDia1 formin
Introduction
Formins are ubiquitous and highly conserved multi-domain proteins that nucleate and elongate linear actin filaments by insertional incorporation of monomers to the filament barbed ends (Faix and Grosse, 2006, Kovar and Pollard, 2004, Pollard, 2007). The proline-rich formin homology domain 1 (FH1) recruits profilin–actin complexes for filament elongation (Kovar et al., 2006, Paul and Pollard, 2008, Romero et al., 2004) which is accomplished by the adjacent FH2 domain (Higashida et al., 2004, Shimada et al., 2004, Xu et al., 2004). Members of the family of Diaphanous-related formins (DRF) fold on themselves and are thus intrinsically inactive by virtue of additional regulatory sequences located in the N- and C-terminal regions of these proteins (Alberts, 2001, Li and Higgs, 2005, Wallar et al., 2006). Binding of activated small Rho family GTPases such as RhoA to the GTPase-binding domain (GBD) releases this intra-molecular inhibition by disrupting the interaction between the C-terminal Diaphanous-auto-regulatory domain (DAD) and the N-terminal Diaphanous-inhibitory domain (DID) (Brandt et al., 2007, Nezami et al., 2006, Otomo et al., 2005, Rose et al., 2005, Wallar and Alberts, 2003). The dimerization domain (DD) is sufficient to dimerize the N-terminal region even without the adjacent coiled-coil (CC) region, while a short linker within the FH2 domain facilitates the dimerization of the C-terminus (Otomo et al., 2005, Xu et al., 2004).
Although the auto-inhibition and the GTPase signaling in mammalian DRF's regulation are well understood, other mechanisms that control e.g. their localization are largely unknown. Formins are often enriched at the plasma membrane (Seth et al., 2006) or in filopodial tips (Block et al., 2008, Schirenbeck et al., 2005). The molecular basis of this distribution is still not entirely understood and the interaction with a membrane-associated GTPase is apparently not the only mechanism (Copeland et al., 2007, Seth et al., 2006, Zaoui et al., 2008). IQGAP1 and CLIP170 have been described to recruit mDia1 to the phagocytic cup (Brandt et al., 2007, Lewkowicz et al., 2008), and FMNL1 inserts into membranes after being myristoylated at the N-terminus (Han et al., 2009). Additional types of formin regulation have been reported for yeast formins. Budding yeast Bud6 interacts directly with the DAD of Bni1 and stimulates its activity, whereas Bud14 inhibits the activity of the formin Bnr1 by displacing it from the growing filament barbed end (Chesarone et al., 2009, Moseley et al., 2004). Furthermore, the N-terminal region and the FH1FH2 domain of Cdc12p are obviously important for its localization to the contractile ring (Yonetani et al., 2008).
Here we report that the mouse DRF mDia1 can be anchored to the plasma membrane through an interaction of its N-terminal basic domain (BD) with phospholipids. Furthermore, the C-terminal region of mDia1 also binds PIP2 and this interaction inhibits mDia1-induced actin filament assembly. Thus our observations suggest that the activity and localization of mDia1 are two distinct phenomena.
Section snippets
Cell culture and transfection
NIH 3T3 fibroblasts were maintained in DMEM with 10% FBS and 2 mM glutamine. Cells were transfected with 2 μg plasmid DNA using LipofectAMINE 2000 (Invitrogen). Microscopy was performed essentially as described (Schirenbeck et al., 2005). Briefly, 10 h after transfection live cells expressing GFP-fusion proteins were imaged in phosphate buffer using a LSM 510 Meta (Zeiss, Germany) at 30 °C.
Plasmids
For cloning and expression of EGFP-mDia1, the entire gene and truncated fragments (ΔDAD-amino acids #1–1179
The N-terminal basic region of mDia1 interacts directly with liposomes
The first 60 residues of mDia1 referred to as the basic domain (BD) harbor three clusters of positively charged residues encompassed by the amino acids #12–21, #35–42, and #47–54 (Fig. 1A). A similar basic region is also present in mDia2 but not in mDia3. DAAM proteins contain only a single poly-basic cluster close to the N-terminus (Fig. 1B). To test whether the basic domain of mDia1 can interact with the negatively charged phospholipids such as PS and PIP2, several truncation and deletion
Discussion
Formins have been reported to target themselves to specific subcellular compartments by various mechanisms (Brandt et al., 2007, Copeland et al., 2007, Lewkowicz et al., 2008, Seth et al., 2006, Yonetani et al., 2008, Zaoui et al., 2008). In the present study we have demonstrated that the BD of mDia1 directly interacted with membrane phospholipids and this interaction is required for the targeting of mDia1 to the plasma membrane. Presumably this type of membrane recruitment is adapted by other
Acknowledgements
The authors thank Drs. A. Wittinghofer (MPI Dortmund) for the mDia1 cDNA clone, W. Tegge (Helmholtz Centre for Infection Research, Braunschweig) for providing mDia1 synthetic peptides, and D. Rieger, R. Allam and D. Kumar for excellent technical advice. We also thank S. Koehler and Dr. A. Bausch (TU Muenchen) for help with preparation of LUVs for in vitro actin polymerization assays. This work was supported by grants from the Elite Network Bavaria (to N.R.), the Deutsche Forschungsgemeinschaft
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2019, Current Opinion in Cell BiologyCitation Excerpt :In the case of N-WASP, which is an activator of actin-nucleating Arp2/3 complex, interactions with PI(4,5)P2 contribute to conversion of the protein to an active form at the plasma membrane [23,24]. In the case of Dia1 and Dia2 formins, which promote actin filament nucleation and polymerization, interactions with PI(4,5)P2 appear to be important for their targeting to the PI(4,5)P2-rich regions of the plasma membrane [25,26]. On the other hand, ADF/cofilin, and heterodimeric capping protein, which are important negative regulators of lamellipodial actin filament assembly, are inhibited through interactions with PI(4,5)P2.
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Current address: Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA.