INTERACTION OF NONMUSCLE MYOSIN II WITH PLASMA MEMBRANES

人类基因组序列的可用性促进了对第三种哺乳动物非肌肉肌球蛋白亚型NMHC-IIC的识别，NMHC-IIC在人类和小鼠组织中具有广泛分布。使用NMHC-IIC特异性抗体，我们已经探索了NMHC-IIC在neuro-2a神经母细胞瘤细胞中的表达。未受刺激的neuro-2a细胞表达NMHC-IIA和-IIB，但不表达NMHC-IIC。用诱导神经突形成的dbcAMP分化细胞导致NMHC-IIC的表达。然而，导致神经突生长的其他药物，包括毛喉素和8-溴-cAMP，没有诱导NMHC-IIC，这表明来自dbcAMP分解的丁酸盐可能介导诱导。用丁酸钠处理neuro-2a细胞导致NMHC-IIC的剂量依赖性表达，在0.2mM丁酸盐下具有显著诱导作用，而-IIA和-IIB的表达不受影响。在未刺激的细胞中通过RT-PCR可检测到NMHC-IIC mRNA，但在用2 mM丁酸盐刺激9小时后增加，并在48小时达到最大。NMHC-IIC蛋白表达在18小时时可检测到，并且在3天时达到最大。3天后去除丁酸导致NMHC-IIC蛋白表达减少，表明诱导是可逆的。已知丁酸钠可抑制组蛋白脱乙酰酶，丁酸盐对NMHC-IIC诱导的作用可通过另一种组蛋白脱乙酰酶抑制剂阿司他汀A模拟。在用丁酸盐处理的RAW264.7小鼠巨噬细胞中也发现了剂量依赖性的NMHC-IIC诱导，并且NMHC-IIC的诱导与用10 mM丁酸盐处理的RAW264.7细胞中的细胞凋亡的开始一致。然而，NMHC-IIC的诱导不是对丁酸盐的一般反应，因为丁酸盐处理的HeLa细胞不表达NMHC-IIC，尽管表现出更分化的表型。总之，丁酸钠在经历分化或凋亡的neuro-2a和RAW264.7细胞中诱导NMHC-IIC表达。进一步的研究正在进行中，以阐明诱导的NMHC-IIC的功能。

The availability of the human genome sequence has facilitated the recognition of a third mammalian nonmuscle myosin isoform, NMHC-IIC, which has a widespread distribution in human and mouse tissues. Using an antibody specific for NMHC-IIC, we have explored the expression of NMHC-IIC in neuro-2a neuroblastoma cells. Non-stimulated neuro-2a cells express NMHC-IIA and -IIB, but not NMHC-IIC. Differentiation of the cells with dbcAMP, which induces neurite formation, leads to expression of NMHC-IIC. However, other agents which cause neurite outgrowth, including forskolin and 8-bromo-cAMP, did not induce NMHC-IIC, suggesting that butyrate derived from breakdown of dbcAMP might mediate induction. Treatment of neuro-2a cells with sodium butyrate resulted in the dose-dependent expression of NMHC-IIC, with significant induction at 0.2mM butyrate, while expression of -IIA and -IIB was unaffected. NMHC-IIC mRNA was detectable by RT-PCR in non-stimulated cells, but was increased by 9 hour stimulation with 2mM butyrate and was maximal at 48 hours. NMHC-IIC protein expression was detectable by 18 hours and maximal at 3 days. Removal of butyrate after 3 days resulted in a decrease in NMHC-IIC protein expression, showing induction to be reversible. Sodium butyrate is known to inhibit histone deacetylase and the effects of butyrate on NMHC-IIC induction were mimicked by trichostatin A, another histone deacetylase inhibitor. Dose-dependent induction of NMHC-IIC was also found in RAW264.7 mouse macrophages treated with butyrate and induction of NMHC-IIC coincided with onset of apoptosis in RAW264.7 cells treated with 10mM butyrate. Induction of NMHC-IIC is not, however, a general response to butyrate, since butyrate-treated HeLa cells did not express NMHC-IIC despite demonstrating a more differentiated phenotype. In summary, NMHC-IIC expression is induced by sodium butyrate in neuro-2a and RAW264.7 cells undergoing differentiation or apoptosis. Further studies are in progress to elucidate the function of the induced NMHC-IIC.