Mutation of Spastin Affects Microtubule Dynamics Through Differential Distribution

Liang Zhi; Cai Zhenbin; Zhang Guowei; Tan Minghui; Lin Hongsheng

doi:doi:10.11648/j.cb.20200802.11

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Mutation of Spastin Affects Microtubule Dynamics Through Differential Distribution

Liang Zhi, Cai Zhenbin, Zhang Guowei, Tan Minghui, Lin Hongsheng

Published in Cell Biology (Volume 8, Issue 2)

Received: 21 July 2020 Accepted: 29 July 2020 Published: 5 August 2020

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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.

Published in	Cell Biology (Volume 8, Issue 2)
DOI	10.11648/j.cb.20200802.11
Page(s)	22-26
Creative Commons	This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.
Copyright	Copyright © The Author(s), 2020. Published by Science Publishing Group

Keywords

Spastin, Mutation, Microtubule Dynamics, Protein Distribution

References

[1]	Hazan, J., et al., Spastin, a new AAA protein, is altered in the most frequent form of autosomal dominant spastic paraplegia. Nature genetics, 1999. 23 (3): p. 296-303.
[2]	McDermott, C. J., et al., Hereditary spastic paraparesis: a review of new developments. Journal Of Neurology Neurosurgery And Psychiatry, 2000. 69 (2): p. 150-160.
[3]	McDermott, C. J., et al., Clinical features of hereditary spastic paraplegia due to spastin mutation. Neurology, 2006. 67 (1): p. 45-51.
[4]	Fonknechten, N., et al., Spectrum of SPG4 mutations in autosomal dominant spastic paraplegia (vol 9, pg 637, 2000). Human Molecular Genetics, 2005. 14 (3): p. 461-461.
[5]	Sharp, D. J. and J. L. Ross, Microtubule-severing enzymes at the cutting edge. Journal Of Cell Science, 2012. 125 (11): p. 2561-2569.
[6]	Roll-Mecak, A. and R. D. Vale, Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin. Nature, 2008. 451 (7176): p. 363-U16.
[7]	Shoukier, M., et al., Expansion of mutation spectrum, determination of mutation cluster regions and predictive structural classification of SPAST mutations in hereditary spastic paraplegia (vol 17, pg 187, 2009). European Journal Of Human Genetics, 2009. 17 (3): p. 401-402.
[8]	Solowska, J. M., et al., Pathogenic Mutation of Spastin Has Gain-of-Function Effects on Microtubule Dynamics. Journal Of Neuroscience, 2014. 34 (5): p. 1856-1867.
[9]	Tadepalle, N., et al., Microtubule-dependent and independent roles of spastin in lipid droplet dispersion and biogenesis. Life science alliance, 2020. 3 (6).
[10]	Sandate, C. R., et al., An allosteric network in spastin couples multiple activities required for microtubule severing (vol 26, pg 671, 2019). Nature Structural & Molecular Biology, 2020. 27 (4): p. 400-400.
[11]	Connell, J. W., et al., ESCRT-III-associated proteins and spastin inhibit protrudin-dependent polarised membrane traffic. Cellular and molecular life sciences: CMLS, 2020. 77 (13): p. 2641-2658.
[12]	Goyal, U., et al., Spastin-Interacting Protein NA14/SSNA1 Functions in Cytokinesis and Axon Development. Plos One, 2014. 9 (11): p. 11.
[13]	Ji, Z. S., et al., Spastin Interacts with CRMP5 to Promote Neurite Outgrowth by Controlling the Microtubule Dynamics. Developmental Neurobiology, 2018. 78 (12): p. 1191-1205.
[14]	Jiang, T., et al., PlexinA3 Interacts with CRMP2 to Mediate Sema3A Signalling During Dendritic Growth in Cultured Cerebellar Granule Neurons. Neuroscience, 2020. 434: p. 83-92.
[15]	Leo, L., et al., Mutant spastin proteins promote deficits in axonal transport through an isoform-specific mechanism involving casein kinase 2 activation. Human Molecular Genetics, 2017. 26 (12): p. 2321-2334.
[16]	Evans, K., et al., Linking axonal degeneration to microtubule remodeling by Spastin-mediated microtubule severing. Molecular Biology Of the Cell, 2004. 15: p. 156A-156A.
[17]	Orso, G., et al., Disease-related phenotypes in a Drosphila model of hereditary spastic paraplegia are ameliorated by treatment with vinblastine. Journal Of Clinical Investigation, 2005. 115 (11): p. 3026-3034.
[18]	Mitchison, T. and M. Kirschner, Dynamic instability of microtubule growth. Nature, 1984. 312 (5991): p. 237-42.

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APA Style

Liang Zhi, Cai Zhenbin, Zhang Guowei, Tan Minghui, Lin Hongsheng. (2020). Mutation of Spastin Affects Microtubule Dynamics Through Differential Distribution. Cell Biology, 8(2), 22-26. https://doi.org/10.11648/j.cb.20200802.11

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ACS Style

Liang Zhi; Cai Zhenbin; Zhang Guowei; Tan Minghui; Lin Hongsheng. Mutation of Spastin Affects Microtubule Dynamics Through Differential Distribution. Cell Biol. 2020, 8(2), 22-26. doi: 10.11648/j.cb.20200802.11

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AMA Style

Liang Zhi, Cai Zhenbin, Zhang Guowei, Tan Minghui, Lin Hongsheng. Mutation of Spastin Affects Microtubule Dynamics Through Differential Distribution. Cell Biol. 2020;8(2):22-26. doi: 10.11648/j.cb.20200802.11

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@article{10.11648/j.cb.20200802.11,
  author = {Liang Zhi and Cai Zhenbin and Zhang Guowei and Tan Minghui and Lin Hongsheng},
  title = {Mutation of Spastin Affects Microtubule Dynamics Through Differential Distribution},
  journal = {Cell Biology},
  volume = {8},
  number = {2},
  pages = {22-26},
  doi = {10.11648/j.cb.20200802.11},
  url = {https://doi.org/10.11648/j.cb.20200802.11},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.cb.20200802.11},
  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.},
 year = {2020}
}

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TY  - JOUR
T1  - Mutation of Spastin Affects Microtubule Dynamics Through Differential Distribution
AU  - Liang Zhi
AU  - Cai Zhenbin
AU  - Zhang Guowei
AU  - Tan Minghui
AU  - Lin Hongsheng
Y1  - 2020/08/05
PY  - 2020
N1  - https://doi.org/10.11648/j.cb.20200802.11
DO  - 10.11648/j.cb.20200802.11
T2  - Cell Biology
JF  - Cell Biology
JO  - Cell Biology
SP  - 22
EP  - 26
PB  - Science Publishing Group
SN  - 2330-0183
UR  - https://doi.org/10.11648/j.cb.20200802.11
AB  - 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.
VL  - 8
IS  - 2
ER  -

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Author Information

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

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Plain Text BibTeX RIS

APA Style

Liang Zhi, Cai Zhenbin, Zhang Guowei, Tan Minghui, Lin Hongsheng. (2020). Mutation of Spastin Affects Microtubule Dynamics Through Differential Distribution. Cell Biology, 8(2), 22-26. https://doi.org/10.11648/j.cb.20200802.11

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ACS Style

Liang Zhi; Cai Zhenbin; Zhang Guowei; Tan Minghui; Lin Hongsheng. Mutation of Spastin Affects Microtubule Dynamics Through Differential Distribution. Cell Biol. 2020, 8(2), 22-26. doi: 10.11648/j.cb.20200802.11

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AMA Style

Liang Zhi, Cai Zhenbin, Zhang Guowei, Tan Minghui, Lin Hongsheng. Mutation of Spastin Affects Microtubule Dynamics Through Differential Distribution. Cell Biol. 2020;8(2):22-26. doi: 10.11648/j.cb.20200802.11

Copy | Download

@article{10.11648/j.cb.20200802.11,
  author = {Liang Zhi and Cai Zhenbin and Zhang Guowei and Tan Minghui and Lin Hongsheng},
  title = {Mutation of Spastin Affects Microtubule Dynamics Through Differential Distribution},
  journal = {Cell Biology},
  volume = {8},
  number = {2},
  pages = {22-26},
  doi = {10.11648/j.cb.20200802.11},
  url = {https://doi.org/10.11648/j.cb.20200802.11},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.cb.20200802.11},
  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.},
 year = {2020}
}

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TY  - JOUR
T1  - Mutation of Spastin Affects Microtubule Dynamics Through Differential Distribution
AU  - Liang Zhi
AU  - Cai Zhenbin
AU  - Zhang Guowei
AU  - Tan Minghui
AU  - Lin Hongsheng
Y1  - 2020/08/05
PY  - 2020
N1  - https://doi.org/10.11648/j.cb.20200802.11
DO  - 10.11648/j.cb.20200802.11
T2  - Cell Biology
JF  - Cell Biology
JO  - Cell Biology
SP  - 22
EP  - 26
PB  - Science Publishing Group
SN  - 2330-0183
UR  - https://doi.org/10.11648/j.cb.20200802.11
AB  - 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.
VL  - 8
IS  - 2
ER  -

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