Autoinhibition regulates cellular localization and actin assembly activity of the diaphanous-related formins FRLalpha and mDia1

Abstract

Diaphanous-related formins (DRFs) are key regulators of actin cytoskeletal dynamics whose in vitro actin assembly activities are thought to be regulated by autoinhibition. However, the in vivo consequences of autoinhibition and the involvement of DRFs in specific biological processes are not well understood. In this study, we show that in the DRFs FRLalpha (formin-related gene in leukocytes alpha) and mouse diaphanous 1, autoinhibition regulates a novel membrane localization activity in vivo as well as actin assembly activity in vitro. In FRLalpha, the Rho family guanosine triphosphatase Cdc42 relieves the autoinhibition of both membrane localization and biochemical actin assembly activities. FRLalpha is required for efficient Fc-gamma receptor-mediated phagocytosis and is recruited to the phagocytic cup by Cdc42. These results suggest that mutual autoinhibition of biochemical activity and cellular localization may be a general regulatory principle for DRFs and demonstrate a novel role for formins in immune function.

Figure 1.

The N terminus of FRLα negatively regulates actin assembly activity of the C-terminal FH2 domain. (A) Schematic diagrams of FRLα and mDia1 constructs used in this study. (B) Interaction between the N and C termini of FRLα. 3 μM GST-tagged N-terminal proteins were immobilized on glutathione–Sepharose beads and used to pull down 3 μM C-terminal constructs. Purified proteins are shown in lanes 2, 3, 6, and 9. Unbound proteins in the flow through (FT) and proteins remaining on the beads after five washes (B) are shown for each experiment. GST–N terminus was used to pull down wild-type C terminus (lanes 4 and 5) or C terminus L1062D containing a point mutation in the DAD domain (lanes 7 and 8). GST–N terminus T126D (Cdc42-binding mutant) was used to pull down wild-type C terminus (lanes 10 and 11). For each experiment, the volumes loaded on the SDS-PAGE gel were adjusted to equalize protein concentrations in the flow through and bead lanes. Gels are stained with Coomassie blue. (C) Similar to B except that the MBP-tagged wild-type C terminus (lane 2) or mutant C terminus (lane 6) was immobilized on amylose resin and used to pull down GST–N terminus (lane 3). Flow through and bead samples from the wild-type C-terminal pull down (lanes 4 and 5) or the mutant C-terminal pull down (lanes 7 and 8) are shown. (D) Dose-dependent inhibition of the actin assembly activity of 200 nM FRLα C terminus in the presence of the N terminus monitored by the increase in fluorescence of 4 μM pyrene-actin (5% labeled) upon incorporation into filaments. (E) Maximum actin assembly rate versus the concentration of N terminus. (F) Effect of the L1062D DAD motif mutation on the ability of 400 nM N terminus to inhibit 4 μM actin assembly (5% pyrene labeled) by 400 nM wild-type or mutant C terminus. Note that mutant C terminus alone or mutant C terminus + N terminus curves overlap significantly.

Figure 2.

Autoinhibition regulates FRLα localization. Confocal images of RAW cells coexpressing GFP fusion proteins and mRFP. The mRFP serves as an evenly distributed control fluorophore. For these and all subsequent confocal images, the intensities for each channel have been normalized against the maximum value in the cell depicted in each image. As illustrated in F, the degree of membrane localization is not dependent on the total expression level of FRLα proteins. GFP fusion proteins are as follows: (A) full-length FRLα-GFP; (B) FRLα N terminus–GFP; (C) full-length FRLα-GFP containing an L1062D mutation in the DAD; (D) full-length FRLα-GFP containing a V281E mutation in the DID; and (E) mini-FRLα–GFP consisting of the N terminus of FRLα fused directly to the DAD domain with an artificial (Gly-Gly-Ser)₂ linker. (F) Membrane enrichment versus expression level for the indicated constructs. Each data point represents one transfected cell. As described in Materials and methods, for each cell expressing a given GFP fusion protein, ratios were obtained for the GFP intensity at the plasma membrane against the GFP intensity in the cytosol. The ratios are plotted against the total GFP expression level in each cell (expressed as the fluorescence intensity per unit area). (G) Membrane enrichment versus expression level for the indicated constructs. Each data point represents one transfected cell. (H) Confocal images of cells coexpressing mRFP and the indicated mutant mini-FRLα–GFP fusion protein. The schematic protein diagram refers to the mini-FRLα construct depicted in Fig. 1 A and contains an asterisk signifying that the point mutations are all contained within the DAD region.

Figure 3.

Active Cdc42 relieves the autoinhibition of FRLα actin assembly activity. Actin assembly assays were performed with 4 μM actin (5% pyrene-labeled). (A) Effect on the inhibition of actin assembly by 200 nM C terminus in the presence of 400 nM N terminus by the addition of the indicated amount of Cdc42-GMPPNP. (B) Similar to A except using Cdc42-GDP. (C) Similar to A except using Rac1-GMPPNP. (D) Actin assembly rate versus the concentration of Cdc42 based on curves in A and B. (E) Actin assembly by 200 nM C terminus in the presence of 1 μM wild-type N terminus or 1 μM T126D N terminus with or without 200 μM Cdc42-GMPPNP.

Figure 4.

Active Cdc42 relieves the autoinhibition of FRLα localization. (A and B) Confocal images of RAW cells coexpressing mRFP, constitutively active Cdc42, and full-length FRLα-GFP (A) or mini-FRLα–GFP (B). (C–E) Confocal images of RAW cells expressing mRFP, full-length FRLα-GFP, and dominant-negative Cdc42 (C), constitutively active Rac1 (D), or constitutively active RhoA (E). (F) Confocal images of a cell expressing mRFP and the FRLα N terminus T126D-GFP mutant. (G and H) Confocal images of cells coexpressing N terminus–GFP and an mRFP fusion of WASP-GBD (G) or a mutant WASP-GBD (H) that does not bind Cdc42. For each transfection, 1 μg FRL construct was cotransfected with 4 μg GBD construct. (I) Membrane enrichment versus expression level for the indicated constructs. Each data point represents one transfected cell. (J) Coimmunoprecipitation experiments using lysates from 293T cells coexpressing myc-tagged Rho GTPase mutants and different FRLα-GFP constructs. Lysates were immunoprecipitated using anti-GFP or control IgG antibodies and blotted with anti-GFP or anti-myc antibodies. For each experiment (lanes a–c, d–f, g–i, j–l, m–o, and p–r), the lysate (starting material) is shown in the first lane followed by the material bound to control IgG beads or anti-GFP beads in the next two lanes. MW, molecular weight; SM, starting material.

Figure 5.

Autoinhibition controls localization of the Rho-regulated DRF mDia1. (A–D) Confocal images of RAW cells coexpressing mRFP and mDia1 N terminus–GFP (A), full-length mDia1-GFP (B), mini-mDia1–GFP (C), or mDia1 V161D N terminus–GFP (D). Three different cells are shown to illustrate the different patterns observed for this construct. The GFP fusion is localized at the plasma membrane in the first cell (top) and in the cytosol of the third cell (bottom). The second cell (middle) has an intermediate distribution between membrane and cytosol. (E) mDia1 ΔG–N terminus–GFP. Three different cells are shown to illustrate the different localization patterns observed with this construct. (F) Membrane enrichment versus expression level for the indicated constructs. Each data point represents one transfected cell.

Figure 6.

Cdc42 recruits FRLα to the phagocytic cup during Fc-γ receptor–mediated phagocytosis. (A) Western blot of lysates prepared from cells transfected with siRNAs directed against FRLα or GFP (control) and probed with anti-FRLα or anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control) antibodies. (B) Phagocytic index (number of RBCs/100 macrophages) of siRNA-transfected RAW macrophages. Experiments were performed in duplicate and repeated three times. Data are the means based on counting at least 300 macrophages per experiment. (C) FRLα-GFP–expressing cell undergoing Fc-γ receptor–mediated phagocytosis. The zero time point is a reference for when the phagosome closes around the RBC being engulfed. The top panels are pseudocolored to represent the GFP/mRFP ratio at each pixel in the cell. Low ratios are represented by blue or cool colors, whereas higher ratios are represented by increasingly red or warmer colors. Bottom panels are the corresponding DIC images for each time point. The ingested RBCs are indicated by arrows. The insets depict additional magnification of the phagocytic cup. (D) Time course of formin accumulation during Fc-γ receptor–mediated phagocytosis. n indicates the number of phagocytic events analyzed for each construct. For each time point, the GFP/mRFP ratio at the cell surface in contact with the RBC (R_p) was divided by the ratio in the cell cytoplasm (R_c). Error bars represent SEM.

Figure 7.

Recruitment of active Cdc42 to the phagocytic cup during Fc-γ receptor–mediated phagocytosis. (A) GBD-GFP–expressing cell undergoing Fc-γ receptor–mediated phagocytosis. Cells were transfected with plasmids encoding mRFP and the indicated GBD-GFP construct. 2.5 μg of each plasmid (5 μg of total DNA) was used for each transfection. As in Fig. 6 C, the GFP/mRFP ratio is pseudocolored in the top panel to reflect the relative accumulation of GBD-GFP over mRFP. Localization of the GBD-GFP protein reflects the distribution of Cdc42-GTP during phagocytosis. Arrows in the bottom panels of DIC images indicate the ingested RBCs. Insets depict additional magnifications of the phagocytic cup. (B) Time course of active Cdc42 accumulation during Fc-γ receptor–mediated phagocytosis. Multiple phagocytic events were analyzed as in Fig. 6 B in cells expressing mRFP and GBD-GFP or the mutant GBD-GFP (as a negative control). Error bars represent SEM.