Mitochondrial fusion protein MFN2 interacts with the mitostatin-related protein MNS1 required for mouse sperm flagellar structure and function

Cilia

George L Gerton¹  Angel M Lin¹  Melissa L Vadnais¹ 

[1] Center for Research on Reproduction and Women’s Health, Department of Obstetrics and Gynecology, Perelman School of Medicine, University of Pennsylvania, 421 Curie Blvd., 1309 BRB II/III, Philadelphia, PA 19104-6160, USA

关键词: Mitochondria;    Axoneme;    Flagella;    Sperm;    Mitostatin;    MNS1;    MFN2;   

Others  :  790558
DOI  :  10.1186/2046-2530-3-5

 received in 2014-03-07, accepted in 2014-05-13,  发布年份 2014

【 摘 要 】

Background

Cilia and the sperm flagellum share many structural properties. Meiosis-specific nuclear structural 1 (MNS1) is a recently characterized protein that is abundantly expressed in post-meiotic spermatids and is required for proper flagellar and motile cilia formation. To explore the possible functions of MNS1, we performed a BLAST search and determined it is homologous to the conserved domain pfam13868, exemplified by mitostatin. This protein interacts with mitofusin 2 (MFN2), a protein that participates in regulating mitochondrial associations to subcellular organelles. We hypothesized that an association between MFN2 and MNS1 in the sperm is involved in flagellar biogenesis and function.

Results

In the studies reported here, MFN2 was found in murine reproductive and somatic tissues high in ciliary content while MNS1 was present as two closely migrating bands in reproductive tissues. Interestingly, mitostatin was also present in reproductive tissues. Similar to Mns1 and mitostatin, Mfn2 was expressed in the testis as detected by RT-PCR. In addition, Mfn2 and Mns1 decreased in expression from pachytene spermatocytes to condensing spermatids as assessed by quantitative RT-PCR. Co-immunoprecipitation demonstrated an association between MFN2 and MNS1 in spermatogenic cells. Indirect immunofluorescence indicated that MFN2 and MNS1 co-localized to the sperm flagellum in freshly collected cauda epididymal sperm. MFN2 associated with the midpiece while MNS1 was present throughout the sperm tail in caput and cauda epididymal sperm. In spermatogenic cells, MFN2 was seen in the mitochondria, and MNS1 was present throughout the cell cytoplasm. MFN2 and MNS1 were present in detergent-resistant flagellar structures of the sperm.

Conclusions

These results demonstrate that MFN2 and MNS1 are present in spermatogenic cells and are an integral part of the sperm flagellum, indicating they play a role in flagellar biogenesis and/or function.

【 授权许可】

   
2014 Vadnais et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Zhou J, Yang F, Leu NA, Wang PJ: MNS1 is essential for spermiogenesis and motile ciliary functions in mice. PLoS Genet 2012, 8:e1002516.
• [2]Marchler-Bauer A, Zheng C, Chitsaz F, Derbyshire MK, Geer LY, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Lu S, Marchler GH, Song JS, Thanki N, Yamashita RA, Zhang D, Bryant SH: CDD: conserved domains and protein three-dimensional structure. Nucleic Acids Res 2013, 41:D348-D352.
• [3]Pugacheva EN, Jablonski SA, Hartman TR, Henske EP, Golemis EA: HEF1-dependent aurora a activation induces disassembly of the primary cilium. Cell 2007, 129:1351-1363.
• [4]Kinzel D, Boldt K, Davis EE, Burtscher I, Trümbach D, Diplas B, Attié-Bitach T, Wurst W, Katsanis N, Ueffing M, Lickert H: Pitchfork regulates primary cilia disassembly and left-right asymmetry. Dev Cell 2010, 19:66-77.
• [5]Inoko A, Matsuyama M, Goto H, Ohmuro-Matsuyama Y, Hayashi Y, Enomoto M, Ibi M, Urano T, Yonemura S, Kiyono T, Izawa I, Inagaki M: Trichoplein and aurora a block aberrant primary cilia assembly in proliferating cells. J Cell Biol 2012, 197:391-405.
• [6]Cerqua C, Anesti V, Pyakurel A, Liu D, Naon D, Wiche G, Baffa R, Dimmer KS, Scorrano L: Trichoplein/mitostatin regulates endoplasmic reticulum–mitochondria juxtaposition. EMBO Rep 2010, 11:854-860.
• [7]Bach D, Pich S, Soriano FX, Vega N, Baumgartner B, Oriola J, Daugaard JR, Lloberas J, Camps M, Zierath JR, Rabasa-Lhoret R, Wallberg-Henriksson H, Laville M, Palacı́n M, Vidal H, Rivera F, Brand M, Zorzano A: Mitofusin-2 determines mitochondrial network architecture and mitochondrial metabolism. J Biol Chem 2003, 278:17190-17197.
• [8]Ramió-Lluch L, Fernández-Novell JM, Peña A, Bucci D, Rigau T, Rodríguez-Gil JE: “In vitro” capacitation and subsequent acrosome reaction are related to changes in the expression and location of midpiece actin and mitofusin-2 in boar spermatozoa. Theriogenology 2012, 77:979-988.
• [9]Flores E, Fernández-Novell JM, Peña A, Rigau T, Rodríguez-Gil JE: Cryopreservation-induced alterations in boar spermatozoa mitochondrial function are related to changes in the expression and location of midpiece mitofusin-2 and actin network. Theriogenology 2010, 74:354-363.
• [10]Bellvé AR, Millette CF, Bhatnagar YM, O’Brien DA: Dissociation of the mouse testis and characterization of isolated spermatogenic cells. J Histo Cytochem 1977, 25:480-494.
• [11]Hasuwa H, Muro Y, Ikawa M, Kato N, Tsujimoto Y, Okabe M: Transgenic mouse sperm that have green acrosome and red mitochondria allow visualization of sperm and their acrosome reaction in vivo. Exp Anim 2010, 59:105-107.
• [12]Gerton GL, Millette CF: Generation of flagella by cultured mouse spermatids. J Cell Biol 1984, 98:619-628.
• [13]Romrell LJ, Bellvé AR, Fawcett DW: Separation of mouse spermatogenic cells by sedimentation velocity: a morphological characterization. Dev Biol 1976, 49:119-131.
• [14]Ruan YC, Shum WWC, Belleannée C, Da Silva N, Breton S: ATP secretion in the male reproductive tract: essential role of CFTR. J Physiol Lond 2012, 590:4209-4222.
• [15]Travis AJ, Jorgez CJ, Merdiushev T, Jones BH, Dess DM, Diaz-Cueto L, Storey BT, Kopf GS, Moss SB: Functional relationships between capacitation-dependent cell signaling and compartmentalized metabolic pathways in murine spermatozoa. J Biol Chem 2001, 276:7630-7636.
• [16]O’Brien DA, Bellvé AR: Protein constituents of the mouse spermatozoon. I. An electrophoretic characterization. Dev Biol 1980, 75:386-404.
• [17]Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227:680-685.
• [18]Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979, 76:4350-4354.
• [19]Kurien BT: Affinity purification of autoantibodies from an antigen strip excised from a nitrocellulose protein blot. Methods Mol Biol 2009, 536:201-211.
• [20]Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 2001, 25:402-408.
• [21]Jeong Y-J, Choi H-W, Shin H-S, Cui X-S, Kim N-H, Gerton GL, Jun JH: Optimization of real time RT-PCR methods for the analysis of gene expression in mouse eggs and preimplantation embryos. Mol Reprod Dev 2005, 71:284-289.
• [22]Karnik NS, Newman S, Kopf GS, Gerton GL: Developmental expression of G protein alpha subunits in mouse spermatogenic cells: evidence that G alpha i is associated with the developing acrosome. Dev Biol 1992, 152:393-402.
• [23]Carrera A, Gerton GL, Moss SB: The major fibrous sheath polypeptide of mouse sperm: structural and functional similarities to the A-kinase anchoring proteins. Dev Biol 1994, 165:272-284.
• [24]Hales KG, Fuller MT: Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase. Cell 1997, 90:121-129.
• [25]Santel A, Fuller MT: Control of mitochondrial morphology by a human mitofusin. J Cell Sci 2001, 114:867-874.
• [26]Rojo M, Legros F, Chateau D, Lombès A: Membrane topology and mitochondrial targeting of mitofusins, ubiquitous mammalian homologs of the transmembrane GTPase Fzo. J Cell Sci 2002, 115:1663-1674.
• [27]Ishihara N, Eura Y, Mihara K: Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity. J Cell Sci 2004, 117:6535-6546.
• [28]Cipolat S, Martins de Brito O, Dal Zilio B, Scorrano L: OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proc Natl Acad Sci U S A 2004, 101:15927-15932.
• [29]Chen H, Detmer SA, Ewald AJ, Griffin EE, Fraser SE, Chan DC: Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development. J Cell Biol 2003, 160:189-200.
• [30]Irons MJ, Clermont Y: Formation of the outer dense fibers during spermiogenesis in the rat. Anat Rec 1982, 202:463-471.