Pranchevicius Maria Cristina S, Baqui Munira M A, Ishikawa-Ankerhold Hellen C, Lourenço Elaine V, Leão Ricardo M, Banzi Silmara R, dos Santos Claudia Tavares, Roque-Barreira Maria C, Espreafico Enilza M, Larson Roy E
Department of Cellular and Molecular Biology, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
Cell Motil Cytoskeleton. 2008 Jun;65(6):441-56. doi: 10.1002/cm.20269.
Nuclear actin and nuclear myosins have been implicated in the regulation of gene expression in vertebrate cells. Myosin V is a class of actin-based motor proteins involved in cytoplasmic vesicle transport and anchorage, spindle-pole alignment and mRNA translocation. In this study, myosin-Va, phosphorylated on a conserved serine in the tail domain (phospho-ser(1650) MVa), was localized to subnuclear compartments. A monoclonal antibody, 9E6, raised against a peptide corresponding to phosphoserine(1650) and flanking regions of the murine myosin Va sequence, was immunoreactive to myosin Va heavy chain in cellular and nuclear extracts of HeLa cells, PC12 cells and B16-F10 melanocytes. Immunofluorescence microscopy with this antibody revealed discrete irregular spots within the nucleoplasm that colocalized with SC35, a splicing factor that earmarks nuclear speckles. Phospho-ser(1650) MVa was not detected in other nuclear compartments, such as condensed chromatin, Cajal bodies, gems and perinucleolar caps. Although nucleoli also were not labeled by 9E6 under normal conditions, inhibition of transcription in HeLa cells by actinomycin D caused the redistribution of phospho-ser(1650) MVa to nucleoli, as well as separating a fraction of phospho-ser(1650) MVa from SC35 into near-neighboring particles. These observations indicate a novel role for myosin Va in nuclear compartmentalization and offer a new lead towards the understanding of actomyosin-based gene regulation.
核肌动蛋白和核肌球蛋白与脊椎动物细胞中的基因表达调控有关。肌球蛋白V是一类基于肌动蛋白的运动蛋白，参与细胞质囊泡运输与锚定、纺锤极排列和mRNA转运。在本研究中，在尾部结构域保守丝氨酸位点磷酸化的肌球蛋白-Va（磷酸化丝氨酸(1650) MVa）定位于亚核区室。一种单克隆抗体9E6，是针对与小鼠肌球蛋白Va序列的磷酸丝氨酸(1650)及侧翼区域对应的肽段产生的，在HeLa细胞、PC12细胞和B16-F10黑素细胞的细胞和核提取物中对肌球蛋白Va重链具有免疫反应性。用该抗体进行免疫荧光显微镜观察显示，核质内有离散的不规则斑点，这些斑点与标记核斑点的剪接因子SC35共定位。在其他核区室，如凝聚染色质、卡哈尔体、宝石体和核仁周围帽中未检测到磷酸化丝氨酸(1650) MVa。虽然在正常条件下核仁也未被9E6标记，但放线菌素D对HeLa细胞转录的抑制导致磷酸化丝氨酸(1650) MVa重新分布到核仁，同时使一部分磷酸化丝氨酸(1650) MVa与SC35分离到相邻颗粒中。这些观察结果表明肌球蛋白Va在核区室化中具有新作用，并为理解基于肌动球蛋白的基因调控提供了新线索。
