Kin11 Involves In Meiosis Of Germline Micronucleus In Tetrahymena Thermophila

Kinesin is a member of the molecular motor protein superfamily that is widely present in various organisms.It can use the energy generated by the hydrolysis of ATP to move on microtubules.It is mainly involved in the transport of vesicles and organelles,assemble of spindle,mitosis and meiosis.Kinesins are mainly divided into 14 superfamilies,and the functions of different family are different,and the corresponding regulatory mechanisms are not clear.Kinesin-14 family protein Kin141 in Tetrahymena thermophila is a negative directional kinesin,localized on the spindle bipolar,involved in spindle microtubule cross-linking and micronucleus retraction.Kinesin-13 family protein Kin13 Ap is localized on the mitotic micronucleus.Kin11 is the only one Kinesin-6 superfamily member.It is lowly expressed during vegetative reproduction,not expressed during starvation,and highly expressed during early meiotic phase of sexual reproduction.The protein contains 1608 amino acid residues,and 37-440 amino acid residues contain a conserved motor protein domain.In this study,the function of Kin11 in meiosis was mainly analyzed by positioning and knockout phenotypes.The main results obtained are as follows:1.Kin11 is located on the micronucleus and spindle during meiosis.The open reading frame of KIN11?TTHERM₀0637750?is 4824 bp long.There are two nuclear localization sequences?NLS?,NLS1 and NLS2,at the position of 21-29 aa of the N-terminal and 1582-1602 aa of the C-terminal.By constructing a Kin11 mutant cell line Kin11-3HA with a 3HA tag at the C-terminus under the control of its own promoter and performing immunofluorescence localization analysis,there was no localization during vegetative growth.It appeared on the micronucleus and spindle during the Crescent phase of sexual reproduction.The localization of Kin11-3HA is concentrated on the midzone of the spindle at the end of meiosis.2.Overexpression of Kin11 is localized on mitotic micronucleus and spindles.By constructing an HA-Kin11 overexpression mutant cell line regulated by the MTT1 promoter,HA-Kin11 can be overexpressed under Cd2+ induction.Immunofluorescence localization showed that HA-Kin11 localized on micronucleus and mitotic spindles during the vegetative reproduction period.And it localized on micronucleus and spindles during meiosis,and appeared in the newly formed micronucleus at the end of meiosis.3.Mutations in functional domains can affect the localization of Kin11.Construction of Kin11 truncated overexpression mutant cell lines HA-Kin11 Tr N-CU428 and HA-Kin11 Tr C-CU428 regulated by the MTT1 promoter.Immunofluorescent localization analysis revealed that HA-Kin11 Tr N was only localized on the micronucleus and spindle of meiosis after the N-terminal conserved motor domain was truncated,and HA-Kin11 Tr C was localized on the micronucleus and spindle of the growing phase and on the micronucleus at late sexual reproduction phase after the C-terminal coiled-coil domain was truncated.4.Kin11 knockout lead to meiotic chromosome separation and spindle structure abnormalities.Different mating type mutant cell lines KO-Kin11-B2086 and KO-Kin11-CU428 were obtained by homologous recombination.It is confirmed that KIN11 mutant cell lines were completely knockouts by Real-time PCR.In KO-Kin11 mutant cell lines,different degrees of chromosome heterogeneity occurred during meiosis,resulting in the defection of micronucleus after meiosis,gameline micronucleus unable to be selected,and sexual reproduction cannot be completed.Examination of the intracellular spindle morphology revealed that knockout of Kin11 resulted in loose spindle structure in the mutant cell line,and the spindle length of the KO-Kin11 was much shorter than that of the wild-type cell.The absence of Kin11 affects the morphological and structural integrity of the spindle and causes abnormal chromosome segregation.The results show that Kin11 localized on the micronucleus and spindle during meiosis and mitosis,and gather in the central area of the spindle at the end of meiosis.Overexpression of KIN11 can also be localized in newly synthesized micronucleus in later sexual reproduction.The truncation of the C-terminal and N-terminal domains of Kin11 leads to a change in its localization.The loss of Kin11 leads to unequal chromosome separation and abnormal spindle structure during meiosis.All in all,Kin11 is critical in the formation of the meiotic spindle structure and the separation of chromosomes in Tetrahymena thermophila.