Nonmuscle Myosin II-C and its Isoform

本实验室先前的工作揭示了一个外显子的存在，该外显子可以剪接到非肌肉肌球蛋白II-B（NMHC II-B）重链的ATP结合结构域附近的环I中。虽然NMHC II-B在全身具有广泛的细胞分布，但插入的同种型仅在神经元细胞中表达。NMHC II-C还含有由24个核苷酸组成的编码8个氨基酸的选择性外显子（C1）。该外显子与NMHC II-B中存在的外显子相似，剪接成环I，但与神经元特异性II-B亚型不同，存在于多种小鼠组织中。这些包括肝脏，肾脏和睾丸，其中RT-PCR分析显示，与非插入异构体相比，编码插入形式的mRNA大于90%，以及脑和肺，其中约为50%。成年小鼠骨骼肌和心肌组织中没有插入的NMHC-IIC亚型，但人骨骼肌NMHC II-C mRNA的插入亚型超过50%。在小鼠发育过程中，插入的同种型的表达发生变化，在胚胎期14时为50%，出生后在肝脏和肾脏中升高至约90%。小鼠大脑在E14时含有较少的插入同种型（约20%），但在出生后一周增加到50%。另一方面，小鼠骨骼肌在E14时表达超过90%的插入同种型，出生后降至0%。还检查了许多细胞系，包括小鼠C2 C12（分化后，100%未插入同种型）、大鼠PC 12（100%插入）和人HepG 2细胞（100%插入）。我们克隆了两种亚型的NMHC II-C融合GFP蛋白，研究其在Hela细胞中的亚细胞定位。发现它们定位于细胞的周质和细胞分裂过程中的细胞动力学环。Hela细胞在胞质分裂过程中的延时视频显微镜清楚地表明，II-C存在，直到两个子细胞的完整生成。当在人肺癌细胞系（A-549）中通过RNAi转染减少这种插入形式的II-C时，细胞显示出生长和迁移缺陷，但在COS-7细胞中NMHC II-B下调后没有观察到多核化。目前，我们正在产生C1插入基因敲除小鼠，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.