Structural analysis of human neutrophil migration. Centriole, microtubule, and microfilament orientation and function during chemotaxis.
Structural analysis of human neutrophil migration. Centriole, microtubule, and microfilament orientation and function during chemotaxis.
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DOI：
10.1083/jcb.75.3.666
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发表时间：
1977-12
期刊：
影响因子：
--
通讯作者：
Gallin JI
中科院分区：
文献类型：
--
作者：
Malech HL;Root RK;Gallin JI
用电子显微镜评价了在化学引诱物梯度（5%大肠杆菌内毒素激活血清）中人中性粒细胞核、中心粒、微管和微丝的取向。将纯化的中性粒细胞（hypaque-Ficoll）置于趋化室的上隔室中。使用小孔（0.45 μm）过滤器允许伪足渗透，但阻碍迁移。在过滤器下方具有活化血清的趋化性的条件下，中性粒细胞群体在过滤器表面取向，核位于远离刺激物的位置，中心粒和核下方的微管的相关径向阵列，以及富含微管的伪足穿透过滤器孔。刺激物（细胞上方的活化血清）的梯度方向的重新定向导致内部结构的重新定向，该内部结构先于伪足形成朝向活化血清并迁移离开过滤器。在缓冲液（随机迁移）或均匀浓度的活化血清（活化随机迁移）中，整个中性粒细胞群体未发生协调定向。激活的随机迁移的条件下，导致运动形态的细胞数量增加，即细胞核，中心粒，微管阵列和伪足的线性排列的细胞不对称性。因此，活化血清增加了表现出运动形态的嗜中性粒细胞的数量，并且活化血清的梯度诱导嗜中性粒细胞的排列，使得这种运动形态在观察到的嗜中性粒细胞群体中是均匀的。在相关的研究中，细胞松弛素B和秋水仙素被用来探讨微丝和微管在中性粒细胞对活化血清的定位和迁移反应中的作用。细胞松弛素B（3.0 μg/ml）可防止迁移并减少所见的微丝，但允许中性粒细胞结构的正常取向。在激活的血清梯度中，秋水仙碱（而不是光秋水仙碱）在低至10（-8）至10（-7）M的浓度下，可降低细胞核和中心粒的方向，并导致中心粒相关微管减少。这些秋水仙素的影响与圆化的细胞和减值的伪足形成。受损的伪足形成的特征在于在没有固体基质的情况下不能形成伪足，在基质内形成狭窄的伪足，以及在活化的血清梯度中伪足取向的缺陷。迁移的功能研究表明，秋水仙素，但不是lumicolchicine，最低限度地减少激活的随机迁移和显着抑制定向迁移，但对随机迁移没有影响。这些研究表明，虽然功能微丝可能是必要的中性粒细胞迁移，完整的微管是必不可少的正常伪足的形成和方向，最大的单向迁移过程中趋化。
Orientation of nucleus, centriole, microtubules, and microfilaments within human neutrophils in a gradient of chemoattractant (5 percent Escherichia coli endotoxin-activated serum) was evaluated by electron microscopy. Purified neutropils (hypaque-Ficoll) were placed in the upper compartment of chemotactic chambers. Use of small pore (0.45 μm) micropore filters permitted pseudopod penetration, but impeded migration. Under conditions of chemotaxis with activated serum beneath the filter, the neutrophil population oriented at the filter surface with nuclei located away from the stimulus, centrioles and associated radial array of microtubules beneath the nuclei, and microfilament-rich pseudopods penetrating the filter pores. Reversal of the direction of the gradient of the stimulus (activated serum above cells) resulted in a reorientation of internal structure which preceded pseudopod formation toward the activated serum and migration off the filter. Coordinated orientation of the entire neutrophil population did not occur in buffer (random migration) or in a uniform concentration of activated serum (activated random migration). Conditions of activated random migration resulted in increased numbers of cells with locomotory morphology, i.e. cellular asymmetry with linear alignment of nucleus, centriole, microtubule array, and pseudopods. Thus, activated serum increased the number of neutrophils exhibiting locomotory morphology, and a gradient of activated serum induced the alignment of neutrophils such that this locomotory morphology was uniform in the observed neutrophil populayion. In related studies, cytochalasin B and colchicines were used to explore the role of microfilaments and microtubules in the neutrophil orientation and migration response to activated serum. Cytochalasin B (3.0 μg/ml) prevented migration and decreased the microfilaments seen, but allowed normal orientation of neutrophil structures. In an activated serum gradient, colchicines, but not lumicolchicine, decreased the orientation of nuclei and centrioles, and caused a decrease in centriole-associated microtubules in concentrations as low as 10(-8) to 10(-7) M. These colchicines effects were associated with the rounding of cells and impairment of pseudopod formation. The impaired pseudopod formation was characterized by an inability to form pseudopods in the absence of a solid substrate, a formation of narrow pseudopods within a substrate, and a defect in pseudopod orientation in an activated serum gradient. Functional studies of migration showed that colchicines, but not lumicolchicine, minimally decreased activated random migration and markedly inhibited directed migration, but had not effect on random migration. These studies show that, although functioning microfilaments are probably necessary for neutrophil migration, intact microtubules are essential for normal pseudopod formation and orientation, and maximal unidirectional migration during chemotaxis.