Volume 7, Issue 1, June 2019, Pages: 1-9
Received: Jan. 14, 2019;
Accepted: Feb. 25, 2019;
Published: Mar. 20, 2019
Views 665 Downloads 187
Yang Yuhao, Department of Orthopedics, the First Affiliated Hospital of Jinan University, Guangzhou, China
Zhang Guowei, Department of Orthopedics, the First Affiliated Hospital of Jinan University, Guangzhou, China
Ji Zhisheng, Department of Orthopedics, the First Affiliated Hospital of Jinan University, Guangzhou, China
Cai Zhenbin, Department of Orthopedics, the First Affiliated Hospital of Jinan University, Guangzhou, China
Liu Qiuling, Department of Orthopedics, the First Affiliated Hospital of Jinan University, Guangzhou, China
Li Shaojin, Department of Orthopedics, the First Affiliated Hospital of Jinan University, Guangzhou, China
Lin Hongsheng, Department of Orthopedics, the First Affiliated Hospital of Jinan University, Guangzhou, China
The research is aimed to explore the effect of different functional domains of SpastinM87V on microtubule cutting of Hela cells. First, the Spastin gene was extracted and its mutants were constructed and identified. Then, Spastin and its mutants were transfected into Hela cells and their cutting effects on microtubules were observed. The results showed that SpastinM87V and its truncated prokaryotic plasmids, including GST-SpastinN197, GST-Spastin△AAA, GST-Spastin△N1 and GST-Spastin△N2 were constructed, and the corresponding fusion proteins were expressed in vitro. In addition, SpastinM87V and its truncated eukaryotic plasmids including GFP-SpastinN197, GFP-Spastin△AAA, GFP-Spastin△N1 and GFP-Spastin△N2 were successfully constructed and introduced into Hela cells. At last, the microtubules of Hela cells could be cut into small fragments by SpastinM87V and Spastin△N1. Furthermore, the fluorescence intensity of the microtubules of Hela cells in the SpastinM87V and Spastin△N1 groups were significantly weaker than in the control group. In this way, Spastin is mainly involved in cell microtubule cutting, and the cutting function by SpastinM87V must contain complete microtubule binding- domain (MTBD) and cutting domain (AAA domain).
Different Functionals Domain of SpastinM87V Affect Cellular Microtubule Cutting, Cell Biology.
Vol. 7, No. 1,
2019, pp. 1-9.
Solowska, J. M., D'Rozario M, Jean D. C., Davidson M. W., Marenda D. R., Baas P. W. (2014). Pathogenic mutation of Spastin has gain-of-function effects on microtubule dynamics. J Neurosci 34 (5): 1856-1867.
Hazan, J., Fonknechten N., Mavel D., Paternotte C., Samson D., Artiguenave F., Davoine C. S., Cruaud C., Durr A., Wincker P., Brottier P., Cattolico L., Barbe V., Burgunder J. M., Prud'homme J. F., Brice A., Fontaine B., Heilig B., and Weissenbach J. (1999). Spastin, a new AAA protein, is altered in the most frequent form of autosomal dominant spastic paraplegia. Nature genetics; 23 (3): 296-303.
Fan, X., Lin Z., Fan G., Lu J., Hou Y., Habai G., Sun L., Yu P., Shen Y., Wen M., and Wang C. (2018). The AAA protein spastin possesses two levels of basal ATPase activity. FEBS letters; 592 (10): 1625-1633.
Reid, E., Connell J., Edwards T. L., Duley S., Brown S. E., Sanderson C. M. (2005). The hereditary spastic paraplegia protein spastin interacts with the ESCRT-III complex-associated endosomal protein CHMP1B. Human molecular genetics; 14 (1): 19-38.
Waclawek, E., Wloga D. (2016). Microtubule severing proteins - structure and activity regulation. Postepy biochemii; 62 (1): 46-51.
Tao, J., Feng C., and Rolls M. M. (2016). The microtubule-severing protein fidgetin acts after dendrite injury to promote their degeneration. journal of cell science; 129 (17): 3274-3281.
Bailey, M. E., Sackett D. L., Ross J. L. (2015). Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails. Biophysical journal; 109 (12): 2546-2561.
Taylor, J. L., White S. R., Lauring B., and Kull F. J. (2012). Crystal structure of the human spastin AAA domain. Journal of structural biology. J Struct Biol; 179 (2): 133-137.
Austin, T. O., Matamoros A. J., Friedman J. M., Friedman A. J., Nacharaju P., Yu W., Sharp D. J., and Baas P. W. (2017). Nanoparticle Delivery of Fidgetin siRNA as a Microtubule-based Therapy to Augment Nerve Regeneration. Scientific reports; 7 (1): 9675.
Leo, L., Yu W., Rozario M. D., Waddell E. A., Marenda D. R., Baird M. A., Davidson M. W., Zhou B., Wu B., Baker L., Sharp D. J., and Baas P. W. (2015). Vertebrate Fidgetin Restrains Axonal Growth by Severing Labile Domains of Microtubules. Cell reports; 12 (11): 1723-1730.
Sanderson, C. M., Connell J. W., Edwards T. L., Bright N. A., Duley S., Thompson A., Luzio J. P., and Reid E. (2006). Spastin and atlastin, two proteins mutated in autosomal-dominant hereditary spastic paraplegia, are binding partners. Human molecular genetics; 15 (2): 307-318.
Svenson, I. K., Kloos M. T., Jacon A., Gallione C., Horton A. C., Pericak-Vance M. A., Ehlers M. D., and Marchuk D. A. (2005). Subcellular localization of spastin: implications for the pathogenesis of hereditary spastic paraplegia. Neurogenetics; 6 (3): 135-141.
Claudiani, P., Riano E., Errico A., Andolfi G. and Rugarli E. I. (2005). Spastin subcellular localization is regulated through usage of different translation start sites and active export from the nucleus. Experimental cell research; 309 (2): 358-369.
Errico, A., Ballabio A. and Rugarli E. I. (2002). Spastin, the protein mutated in autosomal dominant hereditary spastic paraplegia, is involved in microtubule dynamics. Human molecular genetics; 11 (2): 153-163.
Wen, M. and Wang C. (2013). The nucleotide cycle of spastin correlates with its microtubule-binding properties. The FEBS journal; 280 (16): 3868-3877.
Zempel, H. and Mandelkow E. M. (2015). Tau missorting and spastin-induced microtubule disruption in neurodegeneration: Alzheimer Disease and Hereditary Spastic Paraplegia. Molecular neurodegeneration; 10: 68-80.
Taylor, J. L., White S. R., Lauring B. Kull F. J. (2012). Crystal structure of the hμman spastin AAA domain. J Struct Biol; 179 (2): 133-7.
Vietri, M., Schink K. O., Campsteijn C., Wegner C. S., Schultz S. W., Christ L., Thoresen S. B., Brech A., Raiborg C. and Stenmark H. (2015). Spastin and ESCRT-III coordinate mitotic spindle disassembly and nuclear envelope sealing. Nature; 522 (7555): 231-235.
Plaud, C., Joshi V., Marinello M., Pastre D., Galli T., Curmi P. A. and Burgo A. (2017). Spastin regulates VAMP7-containing vesicles trafficking in cortical neurons. Biochimica et biophysica acta Molecular basis of disease; 1863 (6): 1666-1677.
Lumb, J. H., Connell J. W., Allison R. and Reid E. (2012). The AAA ATPase spastin links microtubule severing to membrane modelling. Biochimica et biophysica acta; 1823 (1): 192-197.
Di Fabio, R., Tessa A., Marcotulli C., Leonardi L., Pierelli F., Santorelli F. M., and Casali C. (2014). 'When atlastin meets spastin'. Clinical genetics; 86 (5): 504-505.
Eckert, T., Le D. T., Link S., Friedmann L. and Woehlke G. (2012). Spastin's microtubule-binding properties and comparison to katanin. PloS one; 7 (12): e50161.
Byron, O. and Vestergaard B. (2015). Protein-protein interactions: a supra-structural phenomenon demanding trans-disciplinary biophysical approaches. Current opinion in structural biology; 35: 76-86.
Eckert, T., Link S., Le D. T., Sobczak J. P., Gieseke A., Richter K. and Woehlke G. (2012). Subunit Interactions and cooperativity in the microtubule-severing AAA ATPase spastin. The Journal of biological chemistry; 287 (31): 26278-26290.