Dissecting Requirements for Auto-inhibition of Actin Nucleation by the Formin, mDia1

The mammalian formin, mDia1, is an actin nucleation factor. Experiments in cells and in vitro show that the N-terminal region potently inhibits nucleation by the formin homology 2 (FH2) domain-containing C terminus and that RhoA binding to the N terminus partially relieves this inhibition. Cellular experiments suggest that potent inhibition depends upon the presence of the diaphanous auto-regulatory domain (DAD) C-terminal to FH2. In this study, we examine in detail the N-terminal and C-terminal regions required for this inhibition and for RhoA relief. Limited proteolysis of an N-terminal construct from residues 1–548 identifies two stable truncations: 129–548 and 129–369. Analytical ultracentrifugation suggests that 1–548 and 129–548 are dimers, whereas 129–369 is monomeric. All three N-terminal constructs inhibit nucleation by the full C terminus. Although inhibition by 1–548 is partially relieved by RhoA, inhibition by 129–548 or 129–369 is RhoA-resistant. At the C terminus, DAD deletion does not affect nucleation but decreases inhibitory potency of 1–548 by 20,000-fold. Synthetic DAD peptide binds both 1–548 and 129–548 with similar affinity and partially relieves nucleation inhibition. C-terminal constructs are stable dimers. Our conclusions are as follows: 1) DAD is an affinity-enhancing motif for auto-inhibition; 2) an N-terminal domain spanning residues 129–369 (called DID for diaphanous inhibitory domain) is sufficient for auto-inhibition; 3) a dimerization region C-terminal to DID increases the inhibitory ability of DID; and 4) DID alone is not sufficient for RhoA relief of auto-inhibition, suggesting that sequences N-terminal to DID are important to RhoA binding. An additional finding is that FH2 domain-containing constructs of mDia1 and mDia2 lose >75% nucleation activity upon freeze-thaw. The mammalian formin, mDia1, is an actin nucleation factor. Experiments in cells and in vitro show that the N-terminal region potently inhibits nucleation by the formin homology 2 (FH2) domain-containing C terminus and that RhoA binding to the N terminus partially relieves this inhibition. Cellular experiments suggest that potent inhibition depends upon the presence of the diaphanous auto-regulatory domain (DAD) C-terminal to FH2. In this study, we examine in detail the N-terminal and C-terminal regions required for this inhibition and for RhoA relief. Limited proteolysis of an N-terminal construct from residues 1–548 identifies two stable truncations: 129–548 and 129–369. Analytical ultracentrifugation suggests that 1–548 and 129–548 are dimers, whereas 129–369 is monomeric. All three N-terminal constructs inhibit nucleation by the full C terminus. Although inhibition by 1–548 is partially relieved by RhoA, inhibition by 129–548 or 129–369 is RhoA-resistant. At the C terminus, DAD deletion does not affect nucleation but decreases inhibitory potency of 1–548 by 20,000-fold. Synthetic DAD peptide binds both 1–548 and 129–548 with similar affinity and partially relieves nucleation inhibition. C-terminal constructs are stable dimers. Our conclusions are as follows: 1) DAD is an affinity-enhancing motif for auto-inhibition; 2) an N-terminal domain spanning residues 129–369 (called DID for diaphanous inhibitory domain) is sufficient for auto-inhibition; 3) a dimerization region C-terminal to DID increases the inhibitory ability of DID; and 4) DID alone is not sufficient for RhoA relief of auto-inhibition, suggesting that sequences N-terminal to DID are important to RhoA binding. An additional finding is that FH2 domain-containing constructs of mDia1 and mDia2 lose >75% nucleation activity upon freeze-thaw. Formin proteins are emerging as regulators of many cellular actin-based structures (1Wallar B.J. Alberts A.S. Trends Cell Biol. 2003; 13: 435-446Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 2Zigmond S.H. Curr. Opin. Cell Biol. 2004; 16: 99-105Crossref PubMed Scopus (204) Google Scholar). Biochemically, formins exert several effects on actin polymerization dynamics, including acceleration of filament nucleation from monomers, inhibition of barbed end elongation rate, inhibition of complete barbed end capping by heterodimeric capping protein, and filament severing (3Pruyne D. Evangelista M. Yang C. Bi E. Zigmond S. Bretscher A. Boone C. Science. 2002; 297: 612-615Crossref PubMed Scopus (570) Google Scholar, 4Sagot I. Rodal A.A. Moseley J. Goode B.L. Pellman D. Nat. Cell Biol. 2002; 8: 626-631Crossref Scopus (392) Google Scholar, 5Pring M. Evangelista M. Boone C. Yang C. Zigmond S.H. Biochemistry. 2003; 42: 486-496Crossref PubMed Scopus (187) Google Scholar, 6Zigmond S.H. Evangelista M. Boone C. Yang C. Dar A.C. Sicheri F. Forkey J. Pring M. Curr. Biol. 2003; 13: 1820-1823Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 7Moseley J.B. Sagot I. Manning A.L. Xu Y. Eck M.J. Pellman D. Goode B.L. Mol. Biol. Cell. 2004; 15: 896-907Crossref PubMed Scopus (222) Google Scholar, 8Kovar D.R. Kuhn J.R. Tichy A.L. Pollard T.D. J. Cell Biol. 2003; 161: 875-887Crossref PubMed Scopus (272) Google Scholar, 9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 10Harris E.S. Li F. Higgs H.N. J. Biol. Chem. 2004; 279: 20076-20087Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). These in vitro activities are generally considered to result from the ability of formins to bind at or near the filament barbed end and to move processively with the barbed end as it elongates (11Higashida C. Miyoshi T. Fujita A. Oceguera-Yanez F. Monypenny J. Andou Y. Narumiya S. Watanabe N. Science. 2004; 303: 2007-2010Crossref PubMed Scopus (249) Google Scholar, 12Kovar D.R. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14725-14730Crossref PubMed Scopus (354) Google Scholar). Essential to these properties is the formin homology 2 (FH2) 1The abbreviations used are: FH, formin homology; DAD, diaphanous auto-regulatory domain; DID, diaphanous inhibitory domain; DTT, dithiothreitol; FITC, fluorescein isothiocyanate; GMP-PNP, guanosine 5′-[β,γ-imido]triphosphate; GDP, guanosine 5′-diphosphate. 1The abbreviations used are: FH, formin homology; DAD, diaphanous auto-regulatory domain; DID, diaphanous inhibitory domain; DTT, dithiothreitol; FITC, fluorescein isothiocyanate; GMP-PNP, guanosine 5′-[β,γ-imido]triphosphate; GDP, guanosine 5′-diphosphate. domain, a 400-residue region generally found in the C-terminal half of the protein. Biochemical and structural studies show that the FH2 domain is dimeric for several formins (10Harris E.S. Li F. Higgs H.N. J. Biol. Chem. 2004; 279: 20076-20087Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 13Xu Y. Moseley J. Sagot I. Poy F. Pellman D. Goode B.L. Eck M.J. Cell. 2004; 116: 711-723Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 14Shimada A. Nyitrai M. Vetter I.R. Kuhlmann D. Bugyi B. Narumiya S. Geeves M.A. Wittinghofer A. Mol. Cell. 2004; 13: 511-522Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), although longer constructs of the budding yeast formin, Bni1p, can tetramerize (6Zigmond S.H. Evangelista M. Boone C. Yang C. Dar A.C. Sicheri F. Forkey J. Pring M. Curr. Biol. 2003; 13: 1820-1823Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Mammals possess 15 formin genes, in seven distinct phylogenetic groups (15Higgs H.N. Peterson K.J. Mol. Biol. Cell. 2004; 16: 1-13Crossref PubMed Scopus (201) Google Scholar). For one mammalian formin, mDia1, the mechanisms regulating effects on actin have begun to be elucidated. The in vitro nucleation activity of the FH2-containing C terminus of mDia1 is inhibited potently by inclusion of a separate polypeptide containing the mDia1 N terminus (9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar), suggesting an auto-inhibitory regulatory mechanism. Cellular experiments and two-hybrid interactions implicate a short sequence C-terminal to FH2, known as the diaphanous auto-regulatory domain (DAD), as a critical binding site for the N terminus in both mDia1 and the related protein, mDia2 (16Alberts A.S. J. Biol. Chem. 2001; 276: 2824-2830Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Direct effects of DAD on auto-inhibition of actin nucleation in vitro have not been examined. Cellular studies suggest that binding of the Rho family GTPase, RhoA, to the N terminus of mDia1 can relieve auto-inhibition (17Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat. Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (724) Google Scholar). Biochemical studies on actin nucleation support these findings (9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar), with the caveat that RhoA does not relieve completely the auto-inhibitory effect of the N terminus of mDia1. This incomplete relief by RhoA might imply that a second, non-RhoA dependent, auto-inhibitory interaction between the N and C terminus might exist. In this study, we examine mDia1 auto-inhibition in more detail. Deletion experiments show that auto-inhibition can be uncoupled from RhoA relief, suggesting that mDia1's N-terminal binding sites for RhoA and for the mDia1 C terminus are not identical. In addition, deletion of DAD from the C terminus does not affect nucleation but decreases the inhibitory potency of the N terminus 20,000-fold, suggesting that DAD mediates a high affinity interaction important for potent auto-inhibition. A synthetic DAD peptide binds the N terminus and partially disrupts auto-inhibition, supporting the role of DAD. DNA Constructs—Constructs of mouse mDia1 (accession number U96963) were generated by reverse transcription-PCR and cloned into pGEX-KT, as described previously (9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). Deletion constructs were generated by PCR from longer constructs, using Pfu DNA polymerase (Stratagene). The mouse mDia2 521–1171 construct in pGEX-KT was a kind gift from Dr. Arthur Alberts (Van Andel Research Institute). Protein Preparation and Purification—All proteins were expressed and purified through the thrombin cleavage step following the procedure described in detail in Refs. 9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar and 10Harris E.S. Li F. Higgs H.N. J. Biol. Chem. 2004; 279: 20076-20087Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar. After elution of thrombin-cleaved protein from glutathione-Sepharose, further purification varied as follows. N-terminal constructs (1–548, 129–548, and 129–369) were purified by fast protein liquid chromatography on a SourceS15 5/5 or 10/10 column (Amersham Biosciences) and then concentrated on Q Sepharose Fast Flow (Amersham Biosciences) and dialyzed into the following buffer: 2 mm NaPO4, pH 7.0, 50 mm NaCl, 0.1 mm MgCl2, 0.1 mm EGTA, 0.5 mm DTT. N-terminal constructs could be frozen in aliquots of <50 μl in liquid nitrogen and subsequently stored at -70 °C with no loss of inhibitory activity or detectable aggregation. C-terminal constructs (748–1255, 748–1203, 748–1175, and mDia2-(521–1171)) were purified by fast protein liquid chromatography on a SourceQ15 5/5 or 10/10 column (Amersham Biosciences), concentrated on SP Sepharose Fast Flow (Amersham Biosciences), and dialyzed into the following buffer: 2 mm NaPO4, pH 7.0, 150 mm NaCl, 0.1 mm EGTA, 0.5 mm DTT. C-terminal constructs lost >75% of their nucleation activity when frozen. C-terminal constructs were stored at concentrations <10 μm at 4 °C for 2 weeks or with 1 volume of glycerol at -20 °C for two months with no loss of activity. Protein concentrations were determined by two methods: from absorbance at 280 nm (extinction coefficient calculated by protein sequence using us.expasy.org/cgi-bin/protparam) and by Coomassie-stained SDS-PAGE using known amounts of actin as standards. Rabbit skeletal muscle actin was purified from acetone powder (18Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar) and labeled with pyrenyl iodoacetamide (19Pollard T.D. Cooper J.A. Biochemistry. 1984; 23: 6631-6641Crossref PubMed Scopus (235) Google Scholar). Both unlabeled and labeled actin were gel-filtered on S200 (20MacLean-Fletcher S. Pollard T.D. Cell. 1980; 20: 329-341Abstract Full Text PDF PubMed Scopus (536) Google Scholar), which was crucial to obtain reproducible polymerization kinetics. DAD Peptide Synthesis, Fluorescent Labeling, and Fluorescence Anisotropy—A peptide containing amino acids 1177–1200 of mouse mDia1 (DETGVMDSLLEALQSGAAFRRKRG) was synthesized by the W. M. Keck Small Peptide Synthesis Facility at Yale University, with the following modifications: N-terminal acetylation; N-terminal cysteine residue; C-terminal amidation. The crude peptide was dissolved in water to 14 mg/ml in 100 mm NaPO4, pH 7.0. Peptide was labeled with fluorescein-5′-maleimide (FITC-maleimide, Molecular Probes F150) by mixing 1.4 mg/ml peptide with 1 mm FITC-maleimide at 20 °C for 1 h. FITC-peptide was separated from unincorporated FITC-maleimide by Sephadex G-10 gel filtration (Amersham Biosciences) in 2 mm NaPO4, pH 7.0, 50 mm NaCl and then dialyzed for 48 h in two 4-liter changes of the same buffer, using SnakeSkin 3,500 molecular weight of the purified peptide a at the of and no was with complete and unincorporated FITC-maleimide was determined using the coefficient by Molecular Probes at nm in 50 mm pH Fluorescence were in an using nm in mm pH 7.0, 50 mm 1 mm MgCl2, 1 mm EGTA, mm 0.5 mm DTT. Limited of mDia1 N 1–548 was with nm or in mm mm NaCl, 0.5 mm 0.5 mm for to 1 h at 4 °C or or at 20 °C was by the of to 1 mm 50 mm of in and on for 15 of was by the of to mm from a 1 in water and further for 15 on The from was by fast protein liquid chromatography on a SourceQ15 5/5 column (Amersham Biosciences) and then dialyzed in the same used for mDia1 1–548 frozen in aliquots in liquid and stored at -70 of were determined by and N-terminal sequences were determined by both in the Protein filtration chromatography was using a column (Amersham Biosciences) with both high and molecular weight standards. of and were calculated following the filtration was in mm NaCl, 1 mm MgCl2, 1 mm EGTA, mm NaPO4, pH 7.0, 0.5 mm Analytical ultracentrifugation was using a and an In ultracentrifugation the proteins used were the from the gel and were used in the For protein was at and 20 and absorbance was 2 by at For protein was at with a absorbance of 0.5 absorbance For N-terminal this absorbance was to and 2 μm for mDia1 129–548, and For mDia1 C-terminal this absorbance was to μm in 748–1203, and and to μm for mDia2 For mDia1 129–548 at absorbance at 280 nm was with a of 0.5 absorbance Protein and were determined using by D. and T. were using For concentrations of protein, in buffer, were at and for and 15 protein concentrations were the concentrations and two of these at or 280 nm and were by D. was used to and by D. was used to the by D. was used to the concentrations at were for and at concentrations and were to a the of this D. and J. in the Analytical Scholar), the from and concentrations were to a dimerization using a to the by by D. and T. Protein were by by Fluorescence procedure is described in H.N. Pollard T.D. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar. and actin were in mm pH 0.5 mm DTT, mm 0.1 mm and to an actin of the actin This was to by a at °C in 1 mm EGTA, 0.1 mm to was by the of mm mm MgCl2, mm EGTA, and 100 mm pH to a of with the volume by proteins were for 1 to their to actin to the was in a or an The between the mixing of and of was for and between and of from polymerization were determined at the of polymerization with were to filament the of to barbed in 10Harris E.S. Li F. Higgs H.N. J. Biol. Chem. 2004; 279: 20076-20087Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) with a of T.D. J. Cell Biol. PubMed Scopus Google Scholar) to the following is filament in is the to and is μm at is calculated by the is in is μm of in and and are of and actin in of mDia1 N-terminal have previously that an N-terminal of mDia1 potently inhibits actin nucleation by FH2 domain-containing C-terminal constructs (9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). In this study, we further the N terminus. we two of a from residues and a from residues 129–369 For at 20 °C for to in no additional cleavage the of Biol. Chem. Scopus Google Scholar, D. S. S. PubMed Scopus Google Scholar), to these suggests that the region from to is For at 4 °C for 1 h the whereas at 20 °C an additional with similar SDS-PAGE to that of the similar to with the and C-terminal residues of these we constructs, with the that the construct at of The of was using these expressed generated proteins their expressed in properties All protein by SDS-PAGE the effects of these N-terminal on we their molecular to (9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar), 1–548 with of by gel filtration whereas 129–548 and 129–369 at and that protein is these suggest molecular of and for 129–548, and ultracentrifugation of the gel filtration at concentrations between 1 and 2 in of and for 129–548, and both 1–548 and 129–548 at the been gel-filtered to the suggest a of protein constructs used in this suggests of and nm for and suggests of and nm for and at μm In concentrations were All constructs were from suggests of and nm for and at μm In concentrations were All constructs were from mDia1 in a ultracentrifugation at protein concentrations and 2 and that 129–369 was monomeric. In and 1–548 to with of and and For 1–548 and 129–548, the between molecular from ultracentrifugation and gel as as the calculated for ultracentrifugation suggest that these proteins are experiments were at concentrations μm 129–548 properties from of a and These suggest that the region between residues and dimerization with dimerization in the of 100 of N-terminal on the effects of 129–548, and 129–369 on actin polymerization using the polymerization in the or presence of nm mDia1 This construct the FH2 domain and DAD and is a potent of the N-terminal constructs alone actin polymerization at concentrations to 20 μm F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar) and not In N-terminal constructs inhibited nucleation by The for construct were nm for 20 nm for 129–548, and nm for 129–369 was used as the nucleation were from using then the ability of RhoA to relieve inhibition by the N-terminal In to the inhibition of which is partially relieved by RhoA (9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar), the inhibitory activities of 129–548 and 129–369 are by high concentrations of RhoA 4 and not The result was with or of DAD to the in a construct containing the FH2 domain and DAD to a construct containing the C terminus (9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google in that it is a potent and nucleation ability is inhibited by N-terminal the role of DAD in auto-inhibition, we expressed a construct in which DAD was This construct was potent to and in actin nucleation the ability of to be inhibited by inhibition required 1–548 concentrations μm but complete inhibition was at the concentrations The of 1–548 for is an interaction between DAD and the N terminus, we synthesized a peptide containing the DAD sequence J.B. Pollard T.D. Higgs H.N. Nat. Cell Biol. 2001; PubMed Scopus Google Scholar), binds both 1–548 and 129–548 with a of The by 129–369 was to affinity with but the presence of 129–369 the of suggesting that it as was sufficient to the auto-inhibitory interaction between and 1–548 to RhoA relief of auto-inhibition, was to full of activity of on mDia1 have in which nucleation by mDia1 FH2 domain-containing constructs is J.B. Sagot I. Manning A.L. Xu Y. Eck M.J. Pellman D. Goode B.L. Mol. Biol. Cell. 2004; 15: 896-907Crossref PubMed Scopus (222) Google Scholar, 9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 14Shimada A. Nyitrai M. Vetter I.R. Kuhlmann D. Bugyi B. Narumiya S. Geeves M.A. Wittinghofer A. Mol. Cell. 2004; 13: 511-522Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, S. C. D. C. D. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), and the nucleation potency of mDia1 these this could we to that might these activity found that in the loss of >75% nucleation potency for mDia1 and mDia2 521–1171 FH2, DAD, and C-terminal as as for mDia1 and and not were frozen in aliquots in PCR by into liquid in which the was frozen in This loss of activity of the presence of In similar of mDia1 N-terminal constructs not affect their to inhibit nucleation In this study, we the auto-inhibitory properties of mDia1 with the following cellular findings of DAD (16Alberts A.S. J. Biol. Chem. 2001; 276: 2824-2830Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), suggesting that the role of DAD is to binding affinity to the N-terminal auto-inhibitory to be of the auto-inhibitory mechanism. we a inhibitory residues which we to as DID inhibitory we show that auto-inhibition can be uncoupled from RhoA relief of auto-inhibition inhibition by the DID or DID with a C-terminal is not by we a region between DID and the domain that mediates DID by proteolysis using The of residues 129–369 to suggests that this region a stable In addition, the ability of this region to inhibit nucleation by the mDia1 C terminus suggests that it mediates the auto-inhibitory that this region the that many including of the and as as budding yeast Bni1p, regions similar to DID (15Higgs H.N. Peterson K.J. Mol. Biol. Cell. 2004; 16: 1-13Crossref PubMed Scopus (201) Google Scholar). All of these proteins possess auto-inhibitory through binding be to many are proteins in the and groups (15Higgs H.N. Peterson K.J. Mol. Biol. Cell. 2004; 16: 1-13Crossref PubMed Scopus (201) Google Scholar), which we to be by In this region the 129–548 construct to with RhoA, important for RhoA binding N-terminal to two-hybrid studies found that RhoA an mDia1 construct containing residues N. T. Ishizaki T. Watanabe A. Y. Narumiya S. J. 16: PubMed Scopus Google Scholar). full affinity for RhoA residues both DID and N-terminal to of residues from to N-terminal to with affinity in the 100 nm a region is from to (15Higgs H.N. Peterson K.J. Mol. Biol. Cell. 2004; 16: 1-13Crossref PubMed Scopus (201) Google Scholar), we that this region mediates at two does this region in a or is the of mDia1, which both this dimerization region and the dimeric FH2 to these be for a complete of mDia1 Our experiments show that DAD is required for high affinity auto-inhibition. Deletion of DAD from the C terminus the of the N-terminal 1–548 construct from 2 nm to or 20,000-fold. Synthetic DAD peptide binds constructs and partially relieves inhibition. that DAD in cells DAD in N. and A. DAD is not required for and inhibition of the C terminus can be at high concentrations of N terminus, we that the role of DAD is to a high affinity interaction that a interaction between DID and the FH2 This interaction is inhibitory to The that RhoA does not relieve auto-inhibition suggests that a is required for full This the interaction or further the The nucleation activity of mDia1 and mDia2 FH2 domain-containing constructs is by This might to mDia1 nucleation by J.B. Sagot I. Manning A.L. Xu Y. Eck M.J. Pellman D. Goode B.L. Mol. Biol. Cell. 2004; 15: 896-907Crossref PubMed Scopus (222) Google Scholar, 9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 14Shimada A. Nyitrai M. Vetter I.R. Kuhlmann D. Bugyi B. Narumiya S. Geeves M.A. Wittinghofer A. Mol. Cell. 2004; 13: 511-522Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, S. C. D. C. D. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The to might imply that FH2 domain-containing constructs are to as that mDia1 FH2 domain-containing constructs partially in a of when stored at high concentrations at 4 °C for For these we mDia1 <10 μm at 4 in which full activity is for For longer the of glycerol to and at -20 °C full activity for several This to might not be for FH2 FH2 domain-containing constructs of (10Harris E.S. Li F. Higgs H.N. J. Biol. Chem. 2004; 279: 20076-20087Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) does not affect polymerization or severing Li and N. Dr. Arthur Alberts (Van Andel Research for the kind gift of the mDia2 construct and The of and in the Facility is