Hartwig J H
Experimental Medicine Division, Brigham and Women's Hospital, Boston, Massachusetts.
J Cell Biol. 1992 Sep;118(6):1421-42. doi: 10.1083/jcb.118.6.1421.
The detergent-insoluble cytoskeleton of the resting human blood platelet contains approximately 2,000 actin filaments approximately 1 micron in length crosslinked at high angles by actin-binding protein and which bind to a spectrin-rich submembrane lamina (Fox, J., J. Boyles, M. Berndt, P. Steffen, and L. Anderson. 1988. J. Cell Biol. 106:1525-1538; Hartwig, J., and M. DeSisto. 1991. J. Cell Biol. 112:407-425). Activation of the platelets by contact with glass results within 30 s in a doubling of the polymerized actin content of the cytoskeleton and the appearance of two distinct new actin structures: bundles of long filaments within filopodia that end at the filopodial tips (filopodial bundles) and a circumferential zone of orthogonally arrayed short filaments within lamellipodia (lamellipodial network). Neither of these structures appears in cells exposed to glass with cytochalasin B present; instead the cytoskeletons have numerous 0.1-0.3-microns-long actin filament fragments attached to the membrane lamina. With the same time course as the glass-induced morphological changes, cytochalasin-sensitive actin nucleating activity, initially low in cytoskeletons of resting platelets, increases 10-fold in cytoskeletons of thrombin-activated platelets. This activity decays with a time course consistent with depolymerization of 0.1-0.3-microns-long actin filaments, and phalloidin inhibits this decay. Cytochalasin-insensitive and calcium-dependent nucleation activity also increases markedly in platelet extracts after thrombin activation of the cells. Prevention of the rise in cytosolic Ca2+ normally associated with platelet activation with the permeant Ca2+ chelator, Quin-2, inhibits formation of lamellipodial networks but not filopodial bundles after glass contact and reduces the cytochalasin B-sensitive nucleation activity by 60% after thrombin treatment. The filopodial bundles, however, are abnormal in that they do not end at the filopodial tips but form loops and return to the cell body. Addition of calcium to chelated cells restores lamellipodial networks, and calcium plus A23187 results in cytoskeletons with highly fragmented actin filaments within seconds. Immunogold labeling with antibodies against gelsolin reveals gelsolin molecules at the ends of filaments attached to the submembrane lamina of resting cytoskeletons and at the ends of some filaments in the lamellipodial networks and filopodial bundles of activated cytoskeletons. Addition of monomeric actin to myosin subfragment 1-labeled activated cytoskeletons leads to new (undecorated) filament growth off the ends of filaments in the filopodial bundles and the lamellipodial network. The simplest explanation for these findings is that gelsolin caps the barbed ends of the filaments in the resting platelet. Uncapping some of these filaments after activation leads to filopodial bundles.(ABSTRACT TRUNCATED AT 400 WORDS)
静息人血血小板的去污剂不溶性细胞骨架含有约2000根肌动蛋白丝，长度约为1微米，由肌动蛋白结合蛋白以大角度交联，并与富含血影蛋白的亚膜层相连（福克斯，J.，J.博伊尔斯，M.伯恩特，P.斯特芬，和L.安德森。1988年。《细胞生物学杂志》106:1525 - 1538；哈特维希，J.，和M.德西斯托。1991年。《细胞生物学杂志》112:407 - 425）。血小板与玻璃接触激活后30秒内，细胞骨架中聚合肌动蛋白含量加倍，并出现两种明显的新肌动蛋白结构：丝状伪足内的长丝束，其末端位于丝状伪足尖端（丝状伪足束），以及片状伪足内正交排列的短丝的周边区域（片状伪足网络）。在存在细胞松弛素B的情况下暴露于玻璃的细胞中，这些结构均未出现；相反，细胞骨架有许多附着在膜层上的0.1 - 0.3微米长的肌动蛋白丝片段。与玻璃诱导的形态变化具有相同的时间进程，细胞松弛素敏感的肌动蛋白成核活性，最初在静息血小板的细胞骨架中较低，在凝血酶激活的血小板的细胞骨架中增加10倍。这种活性的衰减时间进程与0.1 - 0.3微米长的肌动蛋白丝的解聚一致，鬼笔环肽可抑制这种衰减。在细胞经凝血酶激活后，血小板提取物中细胞松弛素不敏感且依赖钙的成核活性也显著增加。用渗透性钙螯合剂喹啉 - 2防止通常与血小板激活相关的胞质Ca2 +升高，可抑制玻璃接触后片状伪足网络的形成，但不影响丝状伪足束的形成，并在凝血酶处理后使细胞松弛素B敏感的成核活性降低60%。然而，丝状伪足束是异常的，因为它们并不在丝状伪足尖端结束，而是形成环并回到细胞体。向螯合细胞中添加钙可恢复片状伪足网络，钙加A23187在数秒内导致细胞骨架中的肌动蛋白丝高度碎片化。用抗凝溶胶蛋白抗体进行免疫金标记显示，凝溶胶蛋白分子存在于附着在静息细胞骨架亚膜层的丝的末端，以及激活细胞骨架的片状伪足网络和丝状伪足束中一些丝的末端。将单体肌动蛋白添加到用肌球蛋白亚片段1标记的激活细胞骨架中，会导致丝状伪足束和片状伪足网络中丝的末端长出新的（未标记的）丝。对这些发现最简单的解释是，凝溶胶蛋白封闭了静息血小板中丝的带刺末端。激活后解开其中一些丝的封闭会导致丝状伪足束的形成。（摘要截短至400字）
