Nonmuscle Myosin II-C and its Isoform

本实验室以前的工作揭示了在ATP结合域附近的环I中存在一个外显子，该外显子可以剪接到非肌肉肌球蛋白II-B(NMHC II-B)的重链中。虽然NMHC II-B在体内分布广泛，但插入的异构体只在神经细胞中表达。NMHC II-C还包含一个由24个核苷酸组成的替代外显子，编码8个氨基酸(C1)。这个外显子类似于NMHC II-B中的外显子，被剪接到环I中，但与神经元特异的II-B亚型不同，它存在于各种各样的小鼠组织中。其中包括肝脏、肾脏和睾丸，RT-PCR分析表明，与非插入亚型相比，插入形式的mRNA量大于90%，而脑和肺的编码比例约为50%。成年小鼠骨骼和心肌组织中没有插入的NMHC-IIC亚型，但人骨骼肌组织中有超过50%的NMHC II-C亚型插入。插入的异构体在小鼠发育过程中会发生变化，在胚胎第14期有50%的表达，出生后在肝脏和肾脏中的表达上升到约90%。小鼠大脑中插入的异构体在胚胎14时较少(约20%)，但在出生后一周增加到50%。另一方面，小鼠骨骼肌在胚胎14时表达了超过90%的插入异构体，出生后下降到0%。还检测了一些细胞系，包括小鼠C2C12(分化后，100%未插入异构体)，大鼠PC12(100%插入)和人HepG2细胞(100%插入)。我们克隆了NMHC II-C与GFP蛋白融合的两种异构体，以研究它们在Hela细胞中的亚细胞定位。在Hela细胞分裂过程中，发现它们定位于细胞周质和细胞动态环。Hela细胞胞质分裂过程中的延时视频显微图像清楚地表明，直到两个子代细胞完全生成之前，II-C都存在。当这种插入形式的II-C在人肺癌细胞系(A-549)中被RNAi转染后减少时，细胞出现生长和迁移缺陷，但在COS-7细胞中没有出现NMHC II-B下调后的多核现象。目前，我们正在建立C1Insert基因敲除小鼠，以研究这8个氨基酸在小鼠发育过程中的功能。我们已经产生了必要的嵌合体，目前正在检查生殖系传播。

Previous work from this laboratory has revealed the presence of an exon that can be spliced into the heavy chain of nonmuscle myosin II-B (NMHC II-B) in loop I near the ATP binding domain. Although NMHC II-B has a wide cellular distribution throughout the body, the inserted isoform is only expressed in neuronal cells. NMHC II-C also contains an alternative exon composed of 24 nucleotides encoding 8 amino acids (C1). This exon, similar to that present in NMHC II-B, is spliced into loop I, but unlike the neuronal-specific II-B isoform, is present in a wide variety of mouse tissues. These include liver, kidney and testis, where RT-PCR analysis reveals that the mRNA encoding the inserted form is greater than 90% compared to the noninserted isoform, as well as brain and lung, where it is about 50%. Adult mouse skeletal and cardiac muscle tissues are devoid of the inserted NMHC-IIC isoform, but human skeletal muscle NMHC II-C mRNA is more than 50% inserted isoform. The expression of the inserted isoform changes during mouse development, being 50% at embryonic stage 14 and rising to approximately 90% after birth in the liver and kidney. The mouse cerebrum contains less of the inserted isoform at E14 (approximately 20%), but increases to 50% one week after birth. On the other hand, mouse skeletal muscle expresses over 90% of the inserted isoform at E14, which decreases to 0% after birth. A number of cell lines have also been examined, including mouse C2C12 (after differentiation, 100% noninserted isoform), rat PC12 (100% inserted) and Human HepG2 cells (100% inserted). We have cloned both isoforms of NMHC II-C fused with GFP protein to study their subcellular localization in Hela cells. They are found to localize at the periplasm of the cell and at the cytokinetic ring during cell division of Hela cells. Time-lapse videomicroscpy of Hela cells during cytokinesis clearly indicates that II-C is present until the complete generation of two daughter cells. When this inserted form of II-C was decreased by RNAi transfection in a human lung cancer cell line (A-549), the cells showed growth and migration defects, but no multinucleation as seen following down-regulation of NMHC II-B in COS-7 cells. Presently, we are generating C1 insert knockout mice to investigate the function of this 8 amino acid during mouse development. We have already generated the necessary chimeras and are presently checking for germline transmission.