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

非肌肉肌球蛋白 II 在 NB-4 细胞粒细胞分化过程中核分叶中的作用。

• 批准号: 11670992
• 负责人: NISHIKAWA Masakatsu
• 金额: $ 2.24万
• 依托单位: Mie University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670992/
• 关键词: Nuclear lobulation; NB4 cells; granulocytic differentiation; myosin II; all trans retinoic acid; Rho-kinase; myosin phosphatase; phosphorylated myosin light chain; アクチン; HL-60細胞

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.

核分叶形成可增强中性粒细胞的变形性和穿过血管壁进入炎症部位的运动。然而，在粒细胞分化过程中核分裂的机制仍不清楚。非肌肉肌球蛋白II的两种主要亚型A和B（MHC-IIA和MHC-II B）在不同组织中以不同比例存在。肌球蛋白II的活性通过MLC激酶和Rho-激酶在Ser 19处磷酸化肌球蛋白的20 kD轻链（MLC_）来进行功能性调节<20>。我们确定了非肌肉肌球蛋白II是否在全反式维甲酸（ATRA）诱导的人早幼粒细胞白血病细胞系NB 4细胞的粒细胞分化过程中具有核分叶的功能作用。亚型特异性多克隆抗体揭示了这些亚型在间期和分裂NB 4细胞中的细胞分布。NB 4细胞主要表达MHC-IIA和少量的MHC-IIB。在ATRA诱导的粒细胞分化过程中，MHC-IIA的表达水平升高，而MHC-IIB的表达水平不变。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NB 4细胞分化过程中没有变化。免疫荧光共聚焦显微镜显示野生型NB 4细胞中MHC-IIA和MHC-IIB的不同定位。在间期NB 4细胞中，MHC-IIB分布于细胞皮质，弥散分布于胞浆中，而MHC-IIA则定位于核周区。MHC-IIA和磷酸化的MLC_<20>at Ser 19共定位于分裂的NB 4细胞中的断胃沟（收缩环）。在ATRA诱导的NB 4细胞粒细胞中，MHC-IIA和磷酸化的<20>MLC_1共定位于两个核叶之间的桥接区域。Rho激酶和肌球蛋白磷酸酶也共定位于该区域。这些结果表明，MHC-IIA在ATRA诱导的NB 4细胞粒细胞分化过程中的核分叶中具有重要作用。

项目成果

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