三基序蛋白34(TRIM34)与微核染色体共定位并阻碍染色体在有丝分裂中期向赤道板的移动

Tripartite motif-containing protein 34(TRIM34) colocalized with micronuclei chromosome and hampers its movement to equatorial plate during the metaphase stage of mitosis

目的观察三基序蛋白34（TRIM34）与微核是否存在共定位关系,及对微核染色体在有丝分裂时移动的影响。方法构建并鉴定TRIM34-p EGFP-N3真核表达载体,转染TRIM34-p EGFP-N3载体至HEK293T细胞,经4’,6-二脒基-2-苯基吲哚（DAPI）染色,用荧光显微镜观察TRIM34与微核间是否存在共定位关系。通过激光共聚焦显微镜技术结合Mito TrackerDeep Red线粒体示踪剂,进一步明确TRIM34、微核与线粒体间的位置关系。观察TRIM34-微核结合体在HEK293T细胞有丝分裂中期与赤道板的位置关系。结果经测序鉴定,成功构建TRIM34-p EGFP-N3真核表达载体。TRIM34-p EGFP-N3载体转染HEK293T细胞后,通过荧光显微镜发现TRIM34与微核可能存在共定位关系。经激光共聚焦显微镜技术进行后续检测发现,存在共定位关系的TRIM34小体与微核间在外形上较一致。TRIM34-微核结合体与线粒体未见明显的共定位关系。在有丝分裂中期,结合TRIM34的微核染色体不能移动至赤道板。结论 TRIM34可以结合微核染色体,阻碍后者在有丝分裂中期向赤道板的移动。

Objective To examine whether tripartite motif-containing protein 34（ TRIM34） is colocalized with micronuclei and investigate the influence on the movement of micronuclei chromosome in mitosis. Methods The eukaryotic expression vector TRIM34-p EGFP-N3 was constructed,identified and then transfected into HEK293 T cel s. With 4＇,6-diamidino-2-phenylindole2HCI（ DAPI） staining,the colocalization between TRIM34 and micronuclei was observed under a fluorescence microscope.Moreover,Mito TrackerDeep Red was used to identify the colocalization between the complex of TRIM34-micronulei and mitochondria under a confocal microscope. Finally,the effect of TRIM34 on the movement of micronuclei chromosome in mitosis was examined. Results DNA sequencing confirmed that the vector TRIM34-p EGFP-N3 was constructed successfully.A fluorescence microscope revealed that TRIM34 could be colocalized with micronuclei in HEK293 T cells transfected with TRIM34-p EGFP-N3. In the same manner, a confocal microscope distinctly showed that TRIM34 was colocalized with micronuclei similarly in appearance. However,there was no distinguished colocalization relationship between the complex of TRIM34-micronulei and mitochondria. Interestingly, the micronuclei chromosome conjugated with TRIM34 was hardly transferred to equatorial plate during the metaphase stage of mitosis. Conclusion TRIM34 is colocalized with micronuclei chromosome and hampers its movement to equatorial plate in mitosis.