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Characterization of functional domains of mDia1, a link between the small GTPase Rho and the actin cytoskeleton

Anja Krebs*, Martin Rothkegel, Martin Klar and Brigitte M. Jockusch{ddagger}

Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38092 Braunschweig, Germany
* Present address: Medizinische Klinik I, University of Erlangen, D-91054 Erlangen



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  		Fig. 1. Molecular anatomy of wild type (wt) mDia1 and the deletion fragments used in this study. Location and extension of the domains and motifs within the polypeptide, as compiled from several studies, are indicated and colour-coded. For references see text. RBD, Rho-binding domain, comprising amino acid residues 63-260; FH1 (aa residues 571-735), FH2 (aa 945-1010), FH3 (aa 157-456), formin homology domains 1, 2 and 3, respectively. The two coiled coil regions are 1st CCD (aa 457-570) and 2nd CCD (aa 1011-1192). Within the C-terminal intramolecular interaction domain (CIID; aa 1116-1255), the highly conserved diaphanous-related formin autoregulatory domain (DAD; aa 1177-1207) has been identified.

 


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  		Fig. 2. Interaction of the mDia1 FH1 domain with profilins. (A) Autoradiograms of supernatants and pellets obtained after affinity precipitation of in vitro translated [35S]-labeled mDia1 protein and its fragments with mouse profilins I and IIa. Profilins were coupled to NHS-HiTrap material, incubated with in vitro translated [35S]-methione-labelled mDia1 polypeptides and subjected to centrifugation. Aliquots of supernatants (S) and pellets (P) were separated by SDS-PAGE, blotted and subjected to autoradiography. Control: BSA coupled to NHS-HiTrap material. Coprecipitation of the in vitro translated products depends on the presence of the FH1 domain, as in wt mDia1 and 456-761mDia1. (B) Immunoblots obtained by SDS-PAGE from immunoprecipitates of profilin I from extracts of HeLa cells transfected with BiPro-tagged mDia1 and two of its deletion fragments, containing (456-761mDia1) or lacking (1-448mDia1) the FH1 domain. HeLa cells were treated with the membrane permeant crosslinker DSP before lysis, mDia1 proteins were precipitated from the lysates with an antibody against the BiPro tag (4A6), centrifuged and subjected to SDS-PAGE and subsequent blotting. The presence of profilin I in the pellets was monitored with a monoclonal profilin antibody (2H11), which recognizes human profilin I. Note that profilin I only coprecipitates in the presence of the FH1.

 


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  		Fig. 3. Intramolecular interactions of the mDia1 domains. (A) Immunoblots obtained from BiPro-1-448mDia1-transfected HeLa and CHO cells. After transfection, cells were treated with the membrane permeant crosslinker DSP before lysis, centrifuged and subjected to SDS-PAGE and subsequent immunoblotting. The top panel shows the endogenous Dia1 (end. Dia1) in both cell lines, as detected with a monoclonal antibody against mDia1. The center panel shows the presence of the transfected fragment in both cell lines, as detected with the BiPro-tag antibody. The bottom panel shows an immunoblot analysis with the monoclonal mDia1 antibody of the precipitates obtained with the BiPro-tag. Note that the endogenous Dia1 protein is only precipitated from CHO cells, indicating species-restriction of recognition between the RBD and mDia1. (B) Immunoblots obtained from HeLa cells cotransfected with BiPro-tagged 1116-1255mDia1 and either the flag-tagged fragments 1-448mDia1 (lanes 1,1’) 1-260 mDia1 (lanes 2,2’), 63-448mDia1 (lanes 3,3') or 1-413mDia1 (lanes 4,4’). The top panel shows the expression of the N-terminal fragments, as obtained with the flag antibody, (lanes 1-4), the bottom panels show the expression of the C-terminal 1116-1255 fragment, as seen with the BiPro antibody. Note that all these fragments were expressed in the relevant cells. (C) Immunoblots obtained from immunoprecipitates of HeLa cells transfected as in (B). Cells were treated with the membrane permeant crosslinker DSP before lysis. Proteins were immunoprecipitated with the flag antibody, centrifuged and subjected to SDS-PAGE and blotting. 1116-1255mDia1 was monitored with the BiPro antibody. Note that coprecipitation of the CIID with the mDia1 fragments requires the presence of the RBD plus the FH3.

 


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  		Fig. 4. Actin reorganization and altered morphology of NIH 3T3 cells overexpressing the RBD. (A-C) Cells transfected with EGFP-63-448mDia1. (A,B) Double fluorescence analysis showing EGFP fluorescence (A) and F-actin (B). (C) Nomarski optics. The mDia1-fragment-expressing cells are flatter, lack stress fibers and display prominent ruffles (arrowheads). Bar: 10 µm. Insets are enlargements of the area marked by the arrowhead. (D) Catalogue of the fragments (expressed as fusion proteins with EGFP) tested with respect to loss of stress fibers and ruffling activity. Note that the exogenous, isolated RBD induces loss of stress fibers, while ruffling also requires the presence of either the N-terminal extension or the complete FH3 domain.

 


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  		Fig. 5. Spontaneous ruffling activity of stable CHO clones expressing EGFP-1-448 mDia1. (A) Immunoblots of control CHO cells (lane 1), CHO transfected with the EGFP-containing vector (lane 2), and two clones (termed 1A and 1B) stably transfected with EGFP-1-448mDia1 (lanes 3 and 4), as obtained with the polyclonal mDia1 antibody. Note that these clones express the fragment in amounts approximately equal to the endogenous mDia1. (B) Proportion of spontaneously ruffling cells in the mock-transfected (CHO/EGFP) and clone 1A cells. Cells were fixed and examined by Nomarski optics and for EGFP-fluorescence, and classified according to the presence of ruffles (black bars) or no ruffles (white bars). Approximately 300 cells were counted for each class, in each of 4 independent experiments.

 


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  		Fig. 6. Double fluorescence analysis of cells stably expressing EGFP-1-448mDia1. For each pair of images, the EGFP fluorescence is shown on the right, TRITC-phalloidin staining (indicative of F-actin) and immunostaining for ezrin and RhoA, respectively, are shown on the left. Note that these ruffles contain the cytoskeletal proteins actin and ezrin, which colocalize with the RBD (as contained in the 1-448mDia fragment) and its ligand RhoA. Bar, 20 µm.

 


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  		Fig. 7. Supertransfection of 1-448-mDia-expressing cells with dominant negative myc-tagged Rac1 (N17Rac1). (A,A’) Double fluorescence analysis with antibody against the myc-tag (A) and EGFP fluorescence (A’), to detect the N17Rac1-expressing cells among the 1-448mDia-expressing clone 1A cells (arrowheads). Bar, 20 µm. (B) Quantitation of ruffling activity of clone 1A cells coexpressing N17Rac1, identified from images as shown in (A). Note that N17Rac1 is effectively suppressing ruffling. At least 300 cells were counted, in four independent experiments.

 


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  		Fig. 8. Directed locomotion of CHO clones expressing EGFP-1-448mDia1 (clones, 1A and 1B) or EGFP only (control). Directed locomotory activity was recorded under phase-contrast as the velocity to close a wound set in the confluent culture, at the times indicated. Cells were kept and monitored either without (A) or with (B) colcemid in the medium. Note that wound closure is considerably faster in the control culture than in both RBD-expressing clones. Colcemid enhances this effect, possibly by inhibiting cell proliferation. Bar, 1 mm.

 

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