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.
Highlights
Katanin is a heterodimeric hydrolase belonging to the AAA protein family [1]
According to design primers get katanin p60 and katanin p60M1S, and connected to pEGFP-C1 which treated by BgI II and EcoR I
To study the distribution of katanin p60 in cells and the ability to cleave microtubules, the katanin p60 gene was amplified by PCR, and the katanin p60 gene was transferred into the eukaryotic vector
Summary
Katanin is a heterodimeric hydrolase belonging to the AAA protein family [1]. It is ubiquitously expressed in plants and animals, mainly cutting tubulin. Katanin plays an important role in cell division, neural development, cell migration, and the formation of motor organelles [5]. Weather the distribution of katanin effect on its cutting function is not clear. We constructed GFP-p60 and GFP-p60M1S eukaryotic expression plasmids which distribute in nuclear and whole cell in RFL-6 cell to investigate their cutting function on microtubule, which provided a model basis for the subsequent overexpression of p60 gene cleavage research and its mechanism
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