Boyles J, Fox J E, Phillips D R, Stenberg P E
J Cell Biol. 1985 Oct;101(4):1463-72. doi: 10.1083/jcb.101.4.1463.
This study evaluates the structural organization of the cytoskeleton within unactivated, discoid platelets. Previously, such studies have been difficult to interpret because of the ease with which platelets are stimulated, the sensitivity of actin filaments to cell extraction buffers, and the general problem of preserving actin filaments with conventional fixatives, compounded by the density of the cytoplasm in the platelet. In this study we have employed a new fixative containing lysine, which protects actin filaments against damage during fixation and thin-section processing. We used thick (0.25-micron) sections and conventional thin sections of extracted cells (fixed and lysed simultaneously by the addition of 1% Triton X-100 to the initial fixative) as well as thin sections of whole cells to examine three preparations of human platelets: discoid platelets washed by sedimentation; discoid platelets isolated by gel filtration; and circulating platelets collected by dripping blood directly from a vein into fixative. In all of these preparations, long, interwoven actin filaments were observed within the platelet and were particularly concentrated beneath the plasma membrane. These filaments appeared to be linked at irregular intervals to the membrane and to each other via short, approximately 20- to 50-nm-long cross-links of variable width. Although most filaments were outside the circumferential band of microtubules and the cisternae of the open canalicular system, individual filaments dipped down into the cytoplasm and were found between the microtubules and in association with other membranes. The ease with which single actin filaments can be seen in the dense cytoplasm of the human platelet after lysine/aldehyde fixation suggests the great potential of this new fixative for other cells.
本研究评估了未激活的盘状血小板内细胞骨架的结构组织。此前，由于血小板易于被刺激、肌动蛋白丝对细胞提取缓冲液敏感，以及用传统固定剂保存肌动蛋白丝时存在的普遍问题，再加上血小板细胞质密度大，此类研究一直难以解释。在本研究中，我们采用了一种含赖氨酸的新型固定剂，它能在固定和超薄切片处理过程中保护肌动蛋白丝免受损伤。我们使用了提取细胞的厚（0.25微米）切片和传统超薄切片（通过在初始固定剂中添加1% Triton X-100同时进行固定和裂解）以及全细胞的超薄切片，来检查三种人血小板制剂：通过沉降洗涤的盘状血小板；通过凝胶过滤分离的盘状血小板；以及直接从静脉滴血至固定剂中收集的循环血小板。在所有这些制剂中，在血小板内观察到长而交织的肌动蛋白丝，且特别集中在质膜下方。这些丝似乎以不规则的间隔通过短的、宽度可变的约20至50纳米长的交联与膜以及彼此相连。尽管大多数丝位于微管的圆周带和开放小管系统的池之外，但个别丝向下深入细胞质，并在微管之间以及与其他膜相关联处被发现。赖氨酸/醛固定后，在人血小板致密细胞质中能轻易看到单根肌动蛋白丝，这表明这种新型固定剂对其他细胞具有巨大潜力。
