Different Functionals Domain of SpastinM87V Affect Cellular Microtubule Cutting
Yang Yuhao,
Zhang Guowei,
Ji Zhisheng,
Cai Zhenbin,
Liu Qiuling,
Li Shaojin,
Lin Hongsheng
Issue:
Volume 7, Issue 1, June 2019
Pages:
1-9
Received:
14 January 2019
Accepted:
25 February 2019
Published:
20 March 2019
Abstract: 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).
Abstract: 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 sh...
Show More
Different Location of Katanin P60 Effect on Cellular Microtubule Cutting
Li Shaojin,
Luo Jianxian,
Ji Zhisheng,
Yang Yuhao,
Zhang Guowei,
Tan Minghui,
Yang Hua
Issue:
Volume 7, Issue 1, June 2019
Pages:
10-13
Received:
12 August 2019
Accepted:
28 August 2019
Published:
19 September 2019
Abstract: Katanin is a heterodimeric hydrolase belonging to the AAA protein family and its function is cutting microtubules. It plays an important role in cell division, neural development, cell migration, and the formation of motor organelles. Katanin has two subunits including katanin p80 and p60. This research is aimed to study the effect of different location of katanin p60 on cellular microtubule cutting. First, katanin p60 gene was amplified by PCR, and the eukaryotic recombinant plasmid GFP-katanin p60 and GFP- katanin p60M1S were constructed. The recombinant plasmid was identified by enzyme digestion and sequencing. The expression of katanin in RFL-6 cells was detected by western blot analysis. And, the distribution of katanin p60 in RFL-6 cells and its effect on microtubule were further examined. The results showed that the GFP-katanin p60 and GFP-katanin p60M1S recombinant plasmids was successfully constructed. By western blot analysis, GFP-katanin p60 and GFP-katanin p60M1S recombinant plasmids could be successfully expressed in RFL-6 cells. The microtubule cutting ability of GFP-katanin p60M1S which distribute in whole-cell was stronger than GFP-katanin p60 which distribute in nuclear. Therefore, the cutting function on microtubule of katanin p60 depends on its distribution, and the whole cell distribution is beneficial to katanin p60 in cutting microtubule.
Abstract: Katanin is a heterodimeric hydrolase belonging to the AAA protein family and its function is cutting microtubules. It plays an important role in cell division, neural development, cell migration, and the formation of motor organelles. Katanin has two subunits including katanin p80 and p60. This research is aimed to study the effect of different loc...
Show More
