Dynamin 2 Mutants Linked to Centronuclear Myopathies Form Abnormally Stable Polymers

Abstract

Mutations in the dynamin 2 gene have been identified in patients with autosomal dominant forms of centronuclear myopathy (CNM). Dynamin 2 is a ubiquitously expressed ∼100-kDa GTPase that assembles around the necks of vesiculating membranes and promotes their constriction and scission. It has also been implicated in regulation of the actin and microtubule cytoskeletons. At present, the cellular functions of dynamin 2 that are affected by CNM-linked mutations are not well defined, and the effects of these mutations on the physical and enzymatic properties of dynamin have been not examined. Here, we report the expression, purification, and characterization of four CNM-associated dynamin mutants. All four mutants display higher than wild-type GTPase activities, and more importantly, the mutants form high order oligomers that are significantly more resistant than wild-type dynamin 2 to disassembly by guanine nucleotides or high ionic strength. These observations suggest that the corresponding wild-type residues serve to prevent excessive or prolonged dynamin assembly on cellular membranes or inappropriate self-assembly in the cytoplasm. To our knowledge, this report contains the first identification of point mutations that enhance the stability of dynamin polymers without impairing their ability to bind and/or hydrolyze GTP. We envision that the formation of abnormally large and stable complexes of these dynamin mutants in vivo contributes to their role in CNM pathogenesis. Mutations in the dynamin 2 gene have been identified in patients with autosomal dominant forms of centronuclear myopathy (CNM). Dynamin 2 is a ubiquitously expressed ∼100-kDa GTPase that assembles around the necks of vesiculating membranes and promotes their constriction and scission. It has also been implicated in regulation of the actin and microtubule cytoskeletons. At present, the cellular functions of dynamin 2 that are affected by CNM-linked mutations are not well defined, and the effects of these mutations on the physical and enzymatic properties of dynamin have been not examined. Here, we report the expression, purification, and characterization of four CNM-associated dynamin mutants. All four mutants display higher than wild-type GTPase activities, and more importantly, the mutants form high order oligomers that are significantly more resistant than wild-type dynamin 2 to disassembly by guanine nucleotides or high ionic strength. These observations suggest that the corresponding wild-type residues serve to prevent excessive or prolonged dynamin assembly on cellular membranes or inappropriate self-assembly in the cytoplasm. To our knowledge, this report contains the first identification of point mutations that enhance the stability of dynamin polymers without impairing their ability to bind and/or hydrolyze GTP. We envision that the formation of abnormally large and stable complexes of these dynamin mutants in vivo contributes to their role in CNM pathogenesis. IntroductionAutosomal dominant CNM 2The abbreviations used are: CNMcentronuclear myopathyPHpleckstrin homologyPI(4,5)P2l-α-phosphatidylinositol 4,5-bisphosphateGTPγSguanosine 5′-O-(thiotriphosphate). is a congenital disorder that commonly results in muscle weakness and wasting, ptosis, and ophthalmoplegia (reviewed in Ref. 1.Jungbluth H. Wallgren-Pettersson C. Laporte J. Orphanet J. Rare Dis. 2008; 3: 26Crossref PubMed Scopus (211) Google Scholar). As the name implies, the most evident histopathological feature is the presence of a large number of muscle fibers of centrally (rather than peripherally) located nuclei. Other characteristics include a relative increase in the number of type I fibers, hypertrophy of these fibers, and the presence of sarcoplasmic strands distributed radially around the central nuclei. In 2005, Bitoun et al. (2.Bitoun M. Maugenre S. Jeannet P.Y. Lacène E. Ferrer X. Laforêt P. Martin J.J. Laporte J. Lochmüller H. Beggs A.H. Fardeau M. Eymard B. Romero N.B. Guicheney P. Nat. Genet. 2005; 37: 1207-1209Crossref PubMed Scopus (330) Google Scholar) reported the identification of four mutations in the DNM2 (dynamin 2) gene in patients with autosomal dominant CNM, and an additional seven mutations have since been identified (3.Jungbluth H. Cullup T. Lillis S. Zhou H. Abbs S. Sewry C. Muntoni F. Neuromuscul. Disord. 2010; 20: 49-52Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 4.Durieux A.C. Prudhon B. Guicheney P. Bitoun M. J. Mol. Med. 2010; 88: 339-350Crossref PubMed Scopus (98) Google Scholar). DNM2 is a ubiquitously expressed ∼100-kDa GTPase that assembles into helical polymers around the necks of vesiculating membranes, thereby providing force for their constriction and scission (reviewed in Refs. 5.Hinshaw J.E. Annu. Rev. Cell Dev. Biol. 2000; 16: 483-519Crossref PubMed Scopus (580) Google Scholar, 6.Allan V.J. Thompson H.M. McNiven M.A. Nat. Cell Biol. 2002; 4: E236-E242Crossref PubMed Scopus (171) Google Scholar, 7.Song B.D. Schmid S.L. Biochemistry. 2003; 42: 1369-1376Crossref PubMed Scopus (102) Google Scholar, 8.Praefcke G.J. McMahon H.T. Nat. Rev. Mol. Cell Biol. 2004; l 5: 133-147Crossref Scopus (1099) Google Scholar). In addition to its well characterized roles in endocytosis and Golgi budding, DNM2 is also implicated in regulation of the actin (9.Schafer D.A. Traffic. 2004; 5: 463-469Crossref PubMed Scopus (127) Google Scholar, 10.Mooren O.L. Kotova T.I. Moore A.J. Schafer D.A. J. Biol. Chem. 2009; 284: 23995-24005Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) and microtubule (11.Thompson H.M. Cao H. Chen J. Euteneuer U. McNiven M.A. Nat. Cell Biol. 2004; 6: 335-342Crossref PubMed Scopus (137) Google Scholar, 12.Tanabe K. Takei K. J. Cell Biol. 2009; 185: 939-948Crossref PubMed Scopus (95) Google Scholar) cytoskeletons. The DNM2 molecule consists of five functional domains: an N-terminal catalytic domain; a so-called “middle domain” implicated in dynamin-dynamin interactions; a PH domain involved in phosphoinositide binding; a GTPase effector domain, which interacts with the catalytic domain and stimulates its GTPase activity; and a C-terminal proline/arginine-rich domain, which mediates interactions of dynamin with other proteins. Most of the currently known CNM-associated dynamin mutations are located in the middle and PH domains (4.Durieux A.C. Prudhon B. Guicheney P. Bitoun M. J. Mol. Med. 2010; 88: 339-350Crossref PubMed Scopus (98) Google Scholar, 13.Bitoun M. Bevilacqua J.A. Prudhon B. Maugenre S. Taratuto A.L. Monges S. Lubieniecki F. Cances C. Uro-Coste E. Mayer M. Fardeau M. Romero N.B. Guicheney P. Ann. Neurol. 2007; 62: 666-670Crossref PubMed Scopus (112) Google Scholar), but others have recently been identified in the GTPase effector domain (3.Jungbluth H. Cullup T. Lillis S. Zhou H. Abbs S. Sewry C. Muntoni F. Neuromuscul. Disord. 2010; 20: 49-52Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 14.Bitoun M. Durieux A.C. Prudhon B. Bevilacqua J.A. Herledan A. Sakanyan V. Urtizberea A. Cartier L. Romero N.B. Guicheney P. Hum. Mutat. 2009; 30: 1419-1427Crossref PubMed Scopus (64) Google Scholar).Besides the observation that expression of some mutants inhibits endocytosis in cultured cells (14.Bitoun M. Durieux A.C. Prudhon B. Bevilacqua J.A. Herledan A. Sakanyan V. Urtizberea A. Cartier L. Romero N.B. Guicheney P. Hum. Mutat. 2009; 30: 1419-1427Crossref PubMed Scopus (64) Google Scholar), there is no information regarding the consequences of these mutations on the physical and enzymatic properties of dynamin. To gain insight into the molecular basis of dynamin-dependent CNM pathogenesis, we have expressed, purified, and analyzed four CNM-linked DNM2 mutants, including three middle domain mutants that were identified in the original report (E368K, R369W, and R465W) (2.Bitoun M. Maugenre S. Jeannet P.Y. Lacène E. Ferrer X. Laforêt P. Martin J.J. Laporte J. Lochmüller H. Beggs A.H. Fardeau M. Eymard B. Romero N.B. Guicheney P. Nat. Genet. 2005; 37: 1207-1209Crossref PubMed Scopus (330) Google Scholar), and a PH domain mutant (A618T) identified more recently (14.Bitoun M. Durieux A.C. Prudhon B. Bevilacqua J.A. Herledan A. Sakanyan V. Urtizberea A. Cartier L. Romero N.B. Guicheney P. Hum. Mutat. 2009; 30: 1419-1427Crossref PubMed Scopus (64) Google Scholar). We found that all four mutants have a greater propensity to self-assemble than wild-type DNM2 and that the resulting structures are more resistant to depolymerization by guanine nucleotides or high ionic strength. The stability of these higher order oligomers in the presence of GTP is not due to defects in GTP binding and/or hydrolysis, as the mutants express higher than wild-type catalytic activities.DISCUSSIONThe goal of this study was to determine how CNM mutations alter the in vitro properties of purified DNM2 to gain insights into the molecular basis for the disease. Interestingly, none of the four mutants that we examined showed impaired basal or stimulated GTPase activities, despite the reported ability of one of the mutants (R465W) to inhibit endocytosis upon overexpression in COS cells (14.Bitoun M. Durieux A.C. Prudhon B. Bevilacqua J.A. Herledan A. Sakanyan V. Urtizberea A. Cartier L. Romero N.B. Guicheney P. Hum. Mutat. 2009; 30: 1419-1427Crossref PubMed Scopus (64) Google Scholar). However, they all shared one characteristic: stabilization of dynamin polymers in the presence of GTP, presumably reflecting abnormally strong dynamin-dynamin interactions. This observation suggests that the affected amino acid residues (Glu368, Arg369, Arg465, and Ala618) normally function to limit dynamin assembly, just as other residues (Arg361 and Arg399) were shown to be critical for promoting assembly (22.Ramachandran R. Surka M. Chappie J.S. Fowler D.M. Foss T.R. Song B.D. Schmid S.L. EMBO J. 2007; 26: 559-566Crossref PubMed Scopus (137) Google Scholar). Thus, the dynamin molecule may have evolved to maintain an energetic balance that ensures timely and efficient assembly and disassembly during the process of membrane vesiculation. Disruption of this energetic balance may contribute to the inherited neuropathies and myopathies associated with DNM2 mutations.Although three of the mutated residues (Glu368, Arg369, and Arg465) are located in the middle domain, which had previously been implicated in mediating dynamin assembly (21.Smirnova E. Shurland D.L. Newman-Smith E.D. Pishvaee B. van der Bliek A.M. J. Biol. Chem. 1999; 274: 14942-14947Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 22.Ramachandran R. Surka M. Chappie J.S. Fowler D.M. Foss T.R. Song B.D. Schmid S.L. EMBO J. 2007; 26: 559-566Crossref PubMed Scopus (137) Google Scholar), Ala618 is located in the PH domain, best characterized as the interaction site for phosphoinositides (30.Lemmon M.A. Biochem. Soc. Symp. 2007; 74: 81-93Crossref PubMed Scopus (248) Google Scholar). Interestingly, the A618T mutation does not impair PI(4,5)P2-activated GTPase activity (data not shown). A potential role for the PH domain as a suppressor of dynamin assembly was suggested by reports demonstrating that deletion of this domain enhances the ability of dynamin 1 to polymerize (31.Scaife R. Vénien-Bryan C. Margolis R.L. Biochemistry. 1998; 37: 17673-17679Crossref PubMed Scopus (14) Google Scholar, 32.Vallis Y. Wigge P. Marks B. Evans P.R. McMahon H.T. Curr. Biol. 1999; 9: 257-260Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 33.Muhlberg A.B. Schmid S.L. Methods. 2000; 20: 475-483Crossref PubMed Scopus (13) Google Scholar). However, the specific regions within the PH domain that regulate dynamin self-association are unknown, as previous mutational analyses have been directed only at identifying residues that participate in lipid binding (25.Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I. Driscoll P.C. Waterfield M.D. Panayotou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (488) Google Scholar, 32.Vallis Y. Wigge P. Marks B. Evans P.R. McMahon H.T. Curr. Biol. 1999; 9: 257-260Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 34.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar), membrane insertion (35.Ramachandran R. Pucadyil T.J. Liu Y.W. Acharya S. Leonard M. Lukiyanchuk V. Schmid S.L. Mol. Biol. Cell. 2009; 20: 4630-4639Crossref PubMed Scopus (81) Google Scholar), and binding to βγ-subunits of heterotrimeric G proteins (26.Lin H.C. Gilman A.G. J. Biol. Chem. 1996; 271: 27979-27982Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 36.Yang Z. Li H. Chai Z. Fullerton M.J. Cao Y. Toh B.H. Funder J.W. Liu J.P. J. Biol. Chem. 2001; 276: 4251-4260Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Our finding that the A618T mutation stabilizes dynamin 2 polymers suggests that the C-terminal α-helix contributes to the inhibition of assembly by the PH domain. IntroductionAutosomal dominant CNM 2The abbreviations used are: CNMcentronuclear myopathyPHpleckstrin homologyPI(4,5)P2l-α-phosphatidylinositol 4,5-bisphosphateGTPγSguanosine 5′-O-(thiotriphosphate). is a congenital disorder that commonly results in muscle weakness and wasting, ptosis, and ophthalmoplegia (reviewed in Ref. 1.Jungbluth H. Wallgren-Pettersson C. Laporte J. Orphanet J. Rare Dis. 2008; 3: 26Crossref PubMed Scopus (211) Google Scholar). As the name implies, the most evident histopathological feature is the presence of a large number of muscle fibers of centrally (rather than peripherally) located nuclei. Other characteristics include a relative increase in the number of type I fibers, hypertrophy of these fibers, and the presence of sarcoplasmic strands distributed radially around the central nuclei. In 2005, Bitoun et al. (2.Bitoun M. Maugenre S. Jeannet P.Y. Lacène E. Ferrer X. Laforêt P. Martin J.J. Laporte J. Lochmüller H. Beggs A.H. Fardeau M. Eymard B. Romero N.B. Guicheney P. Nat. Genet. 2005; 37: 1207-1209Crossref PubMed Scopus (330) Google Scholar) reported the identification of four mutations in the DNM2 (dynamin 2) gene in patients with autosomal dominant CNM, and an additional seven mutations have since been identified (3.Jungbluth H. Cullup T. Lillis S. Zhou H. Abbs S. Sewry C. Muntoni F. Neuromuscul. Disord. 2010; 20: 49-52Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 4.Durieux A.C. Prudhon B. Guicheney P. Bitoun M. J. Mol. Med. 2010; 88: 339-350Crossref PubMed Scopus (98) Google Scholar). DNM2 is a ubiquitously expressed ∼100-kDa GTPase that assembles into helical polymers around the necks of vesiculating membranes, thereby providing force for their constriction and scission (reviewed in Refs. 5.Hinshaw J.E. Annu. Rev. Cell Dev. Biol. 2000; 16: 483-519Crossref PubMed Scopus (580) Google Scholar, 6.Allan V.J. Thompson H.M. McNiven M.A. Nat. Cell Biol. 2002; 4: E236-E242Crossref PubMed Scopus (171) Google Scholar, 7.Song B.D. Schmid S.L. Biochemistry. 2003; 42: 1369-1376Crossref PubMed Scopus (102) Google Scholar, 8.Praefcke G.J. McMahon H.T. Nat. Rev. Mol. Cell Biol. 2004; l 5: 133-147Crossref Scopus (1099) Google Scholar). In addition to its well characterized roles in endocytosis and Golgi budding, DNM2 is also implicated in regulation of the actin (9.Schafer D.A. Traffic. 2004; 5: 463-469Crossref PubMed Scopus (127) Google Scholar, 10.Mooren O.L. Kotova T.I. Moore A.J. Schafer D.A. J. Biol. Chem. 2009; 284: 23995-24005Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) and microtubule (11.Thompson H.M. Cao H. Chen J. Euteneuer U. McNiven M.A. Nat. Cell Biol. 2004; 6: 335-342Crossref PubMed Scopus (137) Google Scholar, 12.Tanabe K. Takei K. J. Cell Biol. 2009; 185: 939-948Crossref PubMed Scopus (95) Google Scholar) cytoskeletons. The DNM2 molecule consists of five functional domains: an N-terminal catalytic domain; a so-called “middle domain” implicated in dynamin-dynamin interactions; a PH domain involved in phosphoinositide binding; a GTPase effector domain, which interacts with the catalytic domain and stimulates its GTPase activity; and a C-terminal proline/arginine-rich domain, which mediates interactions of dynamin with other proteins. Most of the currently known CNM-associated dynamin mutations are located in the middle and PH domains (4.Durieux A.C. Prudhon B. Guicheney P. Bitoun M. J. Mol. Med. 2010; 88: 339-350Crossref PubMed Scopus (98) Google Scholar, 13.Bitoun M. Bevilacqua J.A. Prudhon B. Maugenre S. Taratuto A.L. Monges S. Lubieniecki F. Cances C. Uro-Coste E. Mayer M. Fardeau M. Romero N.B. Guicheney P. Ann. Neurol. 2007; 62: 666-670Crossref PubMed Scopus (112) Google Scholar), but others have recently been identified in the GTPase effector domain (3.Jungbluth H. Cullup T. Lillis S. Zhou H. Abbs S. Sewry C. Muntoni F. Neuromuscul. Disord. 2010; 20: 49-52Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 14.Bitoun M. Durieux A.C. Prudhon B. Bevilacqua J.A. Herledan A. Sakanyan V. Urtizberea A. Cartier L. Romero N.B. Guicheney P. Hum. Mutat. 2009; 30: 1419-1427Crossref PubMed Scopus (64) Google Scholar).Besides the observation that expression of some mutants inhibits endocytosis in cultured cells (14.Bitoun M. Durieux A.C. Prudhon B. Bevilacqua J.A. Herledan A. Sakanyan V. Urtizberea A. Cartier L. Romero N.B. Guicheney P. Hum. Mutat. 2009; 30: 1419-1427Crossref PubMed Scopus (64) Google Scholar), there is no information regarding the consequences of these mutations on the physical and enzymatic properties of dynamin. To gain insight into the molecular basis of dynamin-dependent CNM pathogenesis, we have expressed, purified, and analyzed four CNM-linked DNM2 mutants, including three middle domain mutants that were identified in the original report (E368K, R369W, and R465W) (2.Bitoun M. Maugenre S. Jeannet P.Y. Lacène E. Ferrer X. Laforêt P. Martin J.J. Laporte J. Lochmüller H. Beggs A.H. Fardeau M. Eymard B. Romero N.B. Guicheney P. Nat. Genet. 2005; 37: 1207-1209Crossref PubMed Scopus (330) Google Scholar), and a PH domain mutant (A618T) identified more recently (14.Bitoun M. Durieux A.C. Prudhon B. Bevilacqua J.A. Herledan A. Sakanyan V. Urtizberea A. Cartier L. Romero N.B. Guicheney P. Hum. Mutat. 2009; 30: 1419-1427Crossref PubMed Scopus (64) Google Scholar). We found that all four mutants have a greater propensity to self-assemble than wild-type DNM2 and that the resulting structures are more resistant to depolymerization by guanine nucleotides or high ionic strength. The stability of these higher order oligomers in the presence of GTP is not due to defects in GTP binding and/or hydrolysis, as the mutants express higher than wild-type catalytic activities.