Majumder Shubhra, Fisk Harold A
Department of Molecular Genetics, The Ohio State University;
Department of Molecular Genetics, The Ohio State University.
J Vis Exp. 2014 Dec 20(94):52030. doi: 10.3791/52030.
Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.
中心体是微小但重要的细胞器，它作为有丝分裂纺锤体的两极来维持基因组完整性，或组装初级纤毛以促进细胞的感觉功能。蛋白质在中心体的水平调控可能与在细胞其他位置不同，并且几种蛋白质在细胞周期不同时间点的中心体水平变化，对于中心粒组装的正确调控似乎至关重要。我们开发了一种定量荧光显微镜检测方法，用于测量来自不同样本（如处于细胞周期不同阶段或用各种试剂处理后）的固定细胞中，中心体处蛋白质水平的相对变化。该检测方法的原理是测量与蛋白质对应的、经过背景校正后的小区域荧光强度，并将该测量值与在所选实验条件下不发生变化的另一种蛋白质的测量值进行归一化。利用该检测方法结合BrdU脉冲和追踪策略来研究未受干扰的细胞周期，我们定量验证了我们最近的观察结果，即VDAC3的中心体库在细胞周期中在中心体处受到调控，可能是通过蛋白酶体介导的、特异性发生在中心体的降解过程来实现的。
