Analyzing Intracellular Localization Of Structural Domain In Kinesin Motor Protein Kif18A

Objective:To explore the intracellular localization of structural domains in kinesin motor protein Kifl 8A in eukaryotic cells.Methods:1. Construct the GFP-expressing plasmids of Kif18A's wild type and each domain including motor, stalk, tail and motor with part of stalk domain(1) Using BIOZOL total RNA extraction kit to extract total RNA of MCF7,and reverse-transcript RNA to cDNA.Design up-stream and down-stream primers of wild Kif18A.The cDNA of Kif18A was generated by PCR.The product was resolved on 1.5% agarose gel and purified by Biospin PCR product purification kit.Sal I and EcoR I cut the product,and then linked it with expressing vector pEGFP.Transfected it into competent cells Top10,picked the positive colony to LB (Lurie-Bertani),in which the final concentration of Amp is 50μg/mL.Purify plasmid by Biospin DNA extraction kit.Tested it through enzyme cutting,electrophoresis on agarose gel and sequencing.(2) Designed primers of every domain,generated the nucleic acid sequences of every domain through PCR with the constructed plasmid of wild Kif18A as template.As the method in (1),get the expressing plasmids of every domain.2. Cell culture and transfectionPlated 3×104 f MCF7 cells into each well of 24-well plate and culturde in RPMI-1640 including 10% etal serum overnight in incubator.The second day,transfected cells with Lipofectamine2000 when the fusion degree receive 70%.0.5μg of plasmid was transfected into the cells with 0.5μL Lipofectamine2000 and 100 PMI-1640.4-6hours later,changed to RPMI-1640 with 10% fetal serum,cultured overnight.3.Western blot analysis for the expressing productCells were collected and lysed in lysis buffer for 40 minutes on ice.40μg of cell extract was resolved on 7.5% polyacrylamide gels for 1.5 hours using minigel apparatus, transferred to PVDF membrane for 2 hours and sealed off in 5% nonfat milk power for 1 hour.Affinity-purified rabbit antibodies against GFP incubate PVDF membrane for 2 hours to detect fusion proteins. Then exposed to HRP-conjugated IgG followed by development using ECL reagent.4.Transfection and immunofluorescence staining detection of cells grown on coverslips3×104 cells were seeded into each well of 24-well plates and culture for transfection on the next day. Cells were fixed with cold methanol for 5 minutes and then blocked with PBS for 20 minutes contained 10% bovine serum and 0.1% Triton X-100. The staining was performed using mouse anti-a-tubulin (1:10000 dilution) for 2 hours followed by Texas Red conjugated goat anti-mouse antibodies (1:10000 dilution) for 1 hours at room temperature respectively. Finally, DNA was stained with DAPI (1μg/ml) for 5 minutes. After washing with PBS, coverslips were amounted on clean slides with FluoroGuard Antifade Reagent and sealed with Cytoseal 60.5.The intracellular localization of GFP-fused proteins was determined under fluorescent microscopyResults:In interphase cells, GFP-motor domain distributes along microtubules. GFP-tail domain localizes in the nucleus as well as along microtubules and GFP-stalk domain scatters throughout the cytoplasm. In telophase cells, GFP-motor domain still distributes along microtubules. In contrast, GFP-motor with part of stalk domain concentrates on the midbody.Conclusion:The motor and tail domain of Kif18A can both associate with microtubules and the tail domain contained nuclear localization signal (NLS). Only with part of stalk region, motor domain of Kif18 A is able to localize on the midbody. This study identified the localization of kinesin molecule Kif18A in eukaryotic cells interphase and mitotic metaphase,discussed the localization pattern of every domain.This can lead us to explore the specific functions of Kifl 8A in cell mitosis.