粒線體是一個動態的胞器，會透過融合、分裂、移動來改變粒線體型態，先前研究指出細胞骨架對於粒線體移動扮演重要角色。但是肌動蛋白對於粒線體動態平衡調控並不清楚。本實驗室致力於尋找與粒線體動態平衡調控相關因子，我們發現CAP2有潛力調控粒線體動態平衡，CAP2的主要功能之一為，其會與肌動蛋白單體結合並調控肌動蛋白聚合，然而CAP2對於粒線體動態平衡調控仍有待釐清，本研究乃利用CRISPR/Cas9基因編輯技術剔除人類細胞中CAP2基因，探討CAP2是否會調控粒線體動態平衡，並探究剔除CAP2基因對細胞造成之影響。本實驗結果顯示剔除CAP2的細胞其粒線體型態呈現網絡狀，且缺乏肌動蛋白微絲結構，進一步將肌動蛋白微絲以藥物抑制使其無法聚合，結果顯示粒線體分裂需要肌動蛋白微絲參與，本研究結果指出CAP2藉調控肌動蛋白聚合，參與粒線體動態平衡調控。

Mitochondria are highly dynamic organelles; mitochondria fusion, fission, and movement determine mitochondrial network morphology. Cytoskeleton are important for mitochondrial dynamics due to the role ofmitochondrial movement track,constrictionadjuvant, and mitochondrialanchor.However, the effects of actin on mitochondria dynamics remain to be further clarified. CAP2 is an actin monomer binding protein, which is known to regulate actin dynamics. We identified SRV2/CAP2 as a potentiallyfactorinvolved in mitochondrial dynamics regulationin yeast in previous work.To clarify whetherCAP2 involve inmitochondrialdynamicsin mammalian cells, we generate CAP2 deletion cells through CRISPR-Cas 9 systemto clarify the role of CAP2 in mammalian cells.The results demonstratedthatCAP2 deletion causes mitochondria hyperfused and disrupts actin filaments formation. Pharmacological inhibition indicated that actin polymerization is indispensable for mitochondrial fission. Our study implies that CAP2 may involves in mitochondrial dynamics as a pro-fission factorthrough regulating actin. Further understanding of CAP2 may contribute to clarify the mutual effects of mitochondria and actin dynamics.

Anesti, V., and L. Scorrano. 2006. The relationship between mitochondrial shape and function and the cytoskeleton. Biochim Biophys Acta. 1757:692-699.Borowicz, S., M. Van Scoyk, S. Avasarala, M.K. Karuppusamy Rathinam, J. Tauler, R.K. Bikkavilli, and R.A.Winn. 2014. The Soft Agar Colony Formation Assay. Journal of Visualized Experiments : JoVE:51998.Cecchini, M.J., M. Amiri, and F.A. Dick. 2012. Analysis of Cell Cycle Position in Mammalian Cells. Journal of Visualized Experiments : JoVE:3491.Chen, H., and D.C. Chan. 2009. Mitochondrial dynamics--fusion, fission, movement, and mitophagy--in neurodegenerative diseases. Hum Mol Genet. 18:R169-176.Chen, H., S.A. Detmer, A.J. Ewald, E.E. Griffin, S.E. Fraser, and D.C. Chan. 2003. Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development. J Cell Biol. 160:189-200.Dominguez, R., and K.C. Holmes. 2011. Actin structure and function. Annual review of biophysics. 40:169-186.Farkas, D.L., M.D. Wei, P. Febbroriello, J.H. Carson, and L.M. Loew. 1989. Simultaneous imaging of cell and mitochondrial membrane potentials. Biophys J. 56:1053-1069.Fedor-Chaiken, M., R.J. Deschenes, and J.R. Broach. 1990. SRV2, a gene required for RAS activation of adenylate cyclase in yeast. Cell. 61:329-340.Frederick, R.L., and J.M. Shaw. 2007. Moving mitochondria: establishing distribution of an essential organelle. Traffic (Copenhagen, Denmark). 8:1668-1675.Friedman, J.R., and J. Nunnari. 2014. Mitochondrial form and function. Nature. 505:335-343.Gautheron, D.C. 1984. Mitochondrial oxidative phosphorylation and respiratory chain: review. Journal of inherited metabolic disease. 7 Suppl 1:57-61.Gellerich, F.N., S. Trumbeckaite, J.R. Opalka, E. Seppet, H.N. Rasmussen, C. Neuhoff, and S. Zierz. 2000. Function of the mitochondrial outer membrane as a diffusion barrier in health and diseases. Biochemical Society transactions. 28:164-169.Hara, K.Y., and H. Mori. 2006. An efficient method for quantitative determination of cellular ATP synthetic activity. Journal of biomolecular screening. 11:310-317.Hatch, A.L., W.K. Ji, R.A. Merrill, S. Strack, and H.N. Higgs. 2016. Actin filaments
27as dynamic reservoirs for Drp1 recruitment. Mol Biol Cell. 27:3109-3121.Hatefi, Y. 1985. The mitochondrial electron transport and oxidative phosphorylation system. Annual review of biochemistry. 54:1015-1069.Heng, Y.W., and C.G. Koh. 2010. Actin cytoskeleton dynamics and the cell division cycle. The international journal of biochemistry & cell biology. 42:1622-1633.Horbay, R., and R. Bilyy. 2016. Mitochondrial dynamics during cell cycling. Apoptosis : an international journal on programmed cell death. 21:1327-1335.Hubberstey, A.V., and E.P. Mottillo. 2002. Cyclase-associated proteins: CAPacity for linking signal transduction and actin polymerization. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 16:487-499.Itoh, K., K. Nakamura, M. Iijima, and H. Sesaki. 2013. Mitochondrial dynamics in neurodegeneration. Trends Cell Biol. 23:64-71.Jastroch, M., A.S. Divakaruni, S. Mookerjee, J.R. Treberg, and M.D. Brand. 2010. Mitochondrial proton and electron leaks. Essays in biochemistry. 47:53-67.Ji, W.K., A.L. Hatch, R.A. Merrill, S. Strack, and H.N. Higgs. 2015. Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites. Elife. 4:e11553.Knowles, G.C., and C.A. McCulloch. 1992. Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods. Journal of Histochemistry and Cytochemistry.40:1605-1612.Korobova, F., V. Ramabhadran, and H.N. Higgs. 2013. An actin-dependent step in mitochondrial fission mediated by the ER-associated formin INF2. Science. 339:464-467.Li, N., K. Ragheb, G. Lawler, J. Sturgis, B. Rajwa, J.A. Melendez, and J.P. Robinson. 2003. Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J Biol Chem. 278:8516-8525.Ma, X., M. Jin, Y. Cai, H. Xia, K. Long, J. Liu, Q. Yu, and J. Yuan. 2011. Mitochondrial Electron Transport Chain Complex III Is Required for Antimycin A to Inhibit Autophagy. Chemistry & biology. 18:1474-1481.Makrecka-Kuka, M., G. Krumschnabel, and E. Gnaiger. 2015. High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide
28fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules. 5:1319-1338.Mannella, C.A. 2006. Structure and dynamics of the mitochondrial inner membrane cristae. Biochim Biophys Acta. 1763:542-548.Mashal, R.D., J. Koontz, and J. Sklar. 1995. Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases. Nature Genetics. 9:177-183.Meyer, J.N. 2010. QPCR: a tool for analysis of mitochondrial and nuclear DNA damage in ecotoxicology. Ecotoxicology. 19:804-811.Moriyama, K., and I. Yahara. 2002. Human CAP1 is a key factor in the recycling of cofilin and actin for rapid actin turnover. J Cell Sci. 115:1591-1601.Morton, W.M., K.R. Ayscough, and P.J. McLaughlin. 2000. Latrunculin alters the actin-monomer subunit interface to prevent polymerization. Nature cell biology. 2:376-378.Ni, H.M., J.A. Williams, and W.X. Ding. 2015. Mitochondrial dynamics and mitochondrial quality control. Redox Biol. 4:6-13.Nomura, K., and S. Ono. 2013. ATP-dependent regulation of actin monomer-filament equilibrium by cyclase-associated protein and ADF/cofilin. Biochem J. 453:249-259.Park, K.S., I. Jo, K. Pak, S.W. Bae, H. Rhim, S.H. Suh, J. Park, H. Zhu, I. So, and K.W. Kim. 2002. FCCP depolarizes plasma membrane potential by activating proton and Na+ currents in bovine aortic endothelial cells. Pflugers Archiv : European journal of physiology. 443:344-352.Peche, V., S. Shekar, M. Leichter, H. Korte, R. Schroder, M. Schleicher, T.A. Holak, C.S. Clemen, Y.B. Ramanath, G. Pfitzer, I. Karakesisoglou, and A.A. Noegel. 2007. CAP2, cyclase-associated protein 2, is a dual compartment protein. Cell Mol Life Sci. 64:2702-2715.Penefsky, H.S. 1985. Mechanism of inhibition of mitochondrial adenosine triphosphatase by dicyclohexylcarbodiimide and oligomycin: relationship to ATP synthesis. Proceedings of the National Academy of Sciences of the United States of America. 82:1589-1593.Pernas, L., and L. Scorrano. 2016. Mito-morphosis: mitochondrial fusion, fission, and cristae remodeling as key mediatorsof cellular function. Annu Rev Physiol. 78:505-531.Perry, S.W., J.P. Norman, J. Barbieri, E.B. Brown, and H.A. Gelbard. 2011.
29Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. BioTechniques. 50:98-115.Ran, F.A., P.D. Hsu, J. Wright, V. Agarwala, D.A. Scott, and F. Zhang. 2013. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 8:2281-2308.Rose, S., R.E. Frye, J. Slattery, R. Wynne, M. Tippett, O. Pavliv, S. Melnyk, and S.J. James. 2014. Oxidative stress induces mitochondrial dysfunction in a subset of autism lymphoblastoid cell lines in a well-matched case control cohort. PloS one. 9:e85436.Rosner, M., K. Schipany, and M. Hengstschläger. 2013. Merging high-quality biochemical fractionation with a refinedflow cytometry approach to monitor nucleocytoplasmic protein expression throughout the unperturbed mammalian cell cycle. Nat. Protocols. 8:602-626.Rowland, A.A., and G.K. Voeltz. 2012. Endoplasmic reticulum-mitochondria contacts: function of the junction. Nat Rev Mol Cell Biol. 13:607-625.Saxton, W.M., and P.J. Hollenbeck. 2012. The axonal transport of mitochondria. J Cell Sci. 125:2095-2104.Scalettar, B.A., J.R. Abney, and C.R. Hackenbrock. 1991. Dynamics, structure, and function are coupled in the mitochondrial matrix. Proceedings of the National Academy of Sciences of the United States of America. 88:8057-8061.Schieber, M., and N.S. Chandel. 2014. ROS function in redox signaling and oxidative stress. Current biology : CB. 24:R453-R462.Senior, A.E., S. Nadanaciva, and J. Weber. 2002. The molecular mechanism of ATP synthesis by F1F0-ATP synthase. Biochimica et Biophysica Acta (BBA) -Bioenergetics. 1553:188-211.Seo, A.Y., A.M. Joseph, D. Dutta, J.C. Hwang, J.P. Aris, and C. Leeuwenburgh. 2010. New insights into the role of mitochondria in aging: mitochondrial dynamics and more. J Cell Sci. 123:2533-2542.Smirnova, E., L. Griparic, D.-L. Shurland, and A.M. van der Bliek. 2001. Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells. Molecular Biology of the Cell. 12:2245-2256.Varanita, T., MariaE. Soriano, V. Romanello, T. Zaglia, R. Quintana-Cabrera, M. Semenzato, R. Menabò, V. Costa, G. Civiletto, P. Pesce, C. Viscomi, M. Zeviani, F. DiLisa, M. Mongillo, M. Sandri, andL. Scorrano. The Opa1-dependent mitochondrial cristae remodeling pathway controls atrophic, apoptotic, and ischemic tissue damage. Cell Metabolism. 21:834-844.
30Vojtek, A., B. Haarer, J. Field, J. Gerst, T.D. Pollard, S. Brown, and M. Wigler. 1991. Evidence for a functional link between profilin and CAP in the yeast S. cerevisiae. Cell. 66:497-505.Westermann, B. 2014. Mitochondrial inheritance in yeast. Biochimica et Biophysica Acta (BBA) -Bioenergetics. 1837:1039-1046.Woods, L.C., G.W. Berbusse, andK. Naylor. 2016. Microtubules are essential for mitochondrial dynamics-fission, fusion, and motility-in dictyostelium discoideum. Front Cell Dev Biol. 4:19.Xeros, N. 1962. Deoxyriboside control and synchronization of mitosis. Nature. 194:682-683.Zelicof, A., V. Protopopov, D. David, X.Y. Lin, V. Lustgarten, and J.E. Gerst. 1996. Two separate functions are encoded by the carboxyl-terminal domains of the yeast cyclase-associated protein and its mammalian homologs. Dimerization and actin binding. J Biol Chem. 271:18243-18252.