Volume 8, Issue 2, December 2020, Page: 22-26
Mutation of Spastin Affects Microtubule Dynamics Through Differential Distribution
Liang Zhi, 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
Zhang Guowei, Department of Orthopedics, the First Affiliated Hospital of Jinan University, Guangzhou, China
Tan Minghui, 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
Received: Jul. 21, 2020;       Accepted: Jul. 29, 2020;       Published: Aug. 5, 2020
DOI: 10.11648/j.cb.20200802.11      View  78      Downloads  92
Abstract
SPG4 gene encodes Spastin, and its mutations are the main cause of hereditary spastic paraplegia (HSP). There are more than 50 gene mutations in HSP, and mutations of Spastin account for more than 40%, so SPG4 is the key gene that causes the disease. In order to clarify the effect on microtubule stability after inactivation of AAA functional domain caused by mutations of Spastin, we carried out PCR amplification and C413Y site-directed mutation of the target gene according to the sequence of SPG4 in PUBMED gene bank and constructed GFP-Spastin and GFP-Spastin C413Y recombinant plasmids. The recombinant plasmids were introduced into COS7 cells, and the expression of recombinant plasmids in COS7 cells and changes of microtubule stability were observed. The results of colony PCR and gene identification showed that recombinant plasmids were successfully constructed. Western blotting showed that GFP-Spastin and GFP-Spastin C413Y could be expressed normally in COS7 cells. Immunofluorescence assay showed that the distribution of GFP-Spastin was punctate in the cells and GFP-Spastin had strong microtubule cleavage ability, while GFP-Spastin C413Y was almost distributing in the nucleus, and the ability of microtubule cleavage was weakened and the microtubule was in a stable state. Therefore, we concluded that Spastin C413Y changed the distribution of Spastin in cells and weakened the ability of microtubule cleavage. Spastin C413Y accumulated in the nucleus which could not cut microtubules effectively.
Keywords
Spastin, Mutation, Microtubule Dynamics, Protein Distribution
To cite this article
Liang Zhi, Cai Zhenbin, Zhang Guowei, Tan Minghui, Lin Hongsheng, Mutation of Spastin Affects Microtubule Dynamics Through Differential Distribution, Cell Biology. Vol. 8, No. 2, 2020, pp. 22-26. doi: 10.11648/j.cb.20200802.11
Copyright
Copyright © 2020 Authors retain the copyright of this article.
This article is an open access article distributed under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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