Role of non-muscle myosin II in the nuclear lobulation during granulocytic differentiation of NB-4 cells.

核分叶化可增强中性粒细胞的可变形性和通过血管壁进入炎症部位的运动。然而，粒细胞分化过程中核分裂的机制尚不清楚。非肌肉肌球蛋白II的两个主要亚型，A和B （MHC-IIA和MHC-IIB）在不同组织中以不同的比例存在。myosin II的活性是通过MLC激酶和rho激酶磷酸化myosin （MLC_<20>）的20kD轻链Ser19位点来进行功能调控的。我们确定了非肌球蛋白II在反式维黄酸（ATRA）诱导的人早幼粒细胞白血病细胞系NB4细胞的粒细胞分化过程中是否在核分叶中发挥功能作用。同种异构体特异性多克隆抗体揭示了这些同种异构体在间期和分裂的NB4细胞中的细胞分布。NB4细胞主要表达MHC-IIA，少量表达MHC-IIB。atra诱导的粒细胞分化过程中，MHC-IIA水平升高，而MHC-IIB水平不变。rho激酶和肌球蛋白磷酸酶（如肌球蛋白结合亚基）的表达在atra诱导的NB4细胞分化过程中没有变化。免疫荧光共聚焦显微镜显示MHC-IIA和MHC-IIB在野生NB4细胞中有明显的定位。在NB4间期细胞中，MHC-IIB存在于细胞皮层并弥散分布在细胞质中，而MHC-IIA定位于核周区。MHC-IIA和Ser19磷酸化的MLC_<20>在分裂的NB4细胞中定位于损伤沟（收缩环）。在atra诱导的NB4细胞粒细胞中，MHC-IIA和磷酸化的MLC_<20>共定位于两个核叶之间的桥接区域。红激酶和肌球蛋白磷酸酶也定位于这一区域。这些结果表明，在atra诱导的NB4细胞粒细胞分化过程中，MHC-IIA在核分叶中起重要作用。

Nuclearlobulation may enhance neutrophil deformability and movement through vessel walls and into sites of inflammation. However the mechanism of nuclear segmentation during granulocytic differentiation remains unknown. Two principal isoforms of non-muscle myosin II, A and B (MHC-IIA and MHC-IIB) are present in different proportions in different tissues. Activity of myosin II is functionally regulated through phosphorylation of 20kD light chain of myosin (MLC_<20>) at Ser19 by MLC kinase and Rho-kinase. We determined whether non-muscle myosin II has a functional role in nuclear lobulation during all trans retinoic acid (ATRA)-induced granulocytic differentiation of human promyelocytic leukemia cell line, NB4 cells. Isoform-specific polyclonal antibodies reveal the cellular distributions of these isoforms in interphase and in dividing NB4 cells. NB4 cells expressed mostly MHC-IIA and a small amount of MHC-IIB.The level of MHC-IIA was increased during ATRA-induced granulocytic differentiation, while that of MHC-IIB was unaltered. Expressions of Rho-kinase and myosin phosphatase (e.g.myosin-binding subunit ) were unchanged during ATRA-induced NB4 cell differentiation. Immunofluorescence confocal microscopy demonstrated distinct localizations for MHC-IIA and MHC-IIB in wild NB4 cells. In interphase NB4 cells, MHC-IIB was present in the cell cortex and diffusely arranged in the cytoplasm, while MHC-IIA was localized in perinuclear region. MHC-IIA and phosphorylated MLC_<20> at Ser19 were colocalized to the deavage furrows (contractile ring) in dividing NB4 cells. In ATRA-induced granulocytes of NB4 cells, MHC-IIA and phosphorylated MLC_<20> were colocalized to the bridging area between two nuclear lobes. Rho-kinase and myosin phosphatase also colocalized to this area. These results suggest that MHC-IIA has a important role in nuclear lobulation during ATRA-induced granulocytic differentiation of NB4 cells.