STEM tomography reveals that the canalicular system and -granules remain separate compartments during early secretion stages in blood platelets
STEM tomography reveals that the canalicular system and -granules remain separate compartments during early secretion stages in blood platelets
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DOI：
10.1111/jth.13225
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发表时间：
2016-03-01
影响因子：
10.4
通讯作者：
Storrie, B.
中科院分区：
文献类型：
--
作者：
Pokrovskaya, I. D.;Aronova, M. A.;Storrie, B.
血小板如何组织它们的颗粒货物和使用它们的小管系统仍然存在争议。过去的结构研究由于采样体积小或分辨率降低而受到限制。我们的分析揭示了均匀的颗粒和一个封闭的小管系统，在激活时打开。了解血小板在活化和分泌过程中如何改变其膜有助于止血。血小板检查血管的损伤，作为反应，从它们的颗粒中激活和释放多种蛋白质。α -颗粒可能是生物化学和结构不均匀的；然而，其他研究表明，它们可能更均匀，所观察到的变化反映了颗粒动力学，而不是根本差异。我们的目的是解决-颗粒的结构组织如何支持他们的动力学。方法采用高压冷冻和冷冻替代法制备血小板，以保持天然状态；为了对几乎整个细胞成像，我们在扫描透射电子显微镜（STEM）中记录了层析成像数据。结果和结论在静息血小板中，我们观察到形态学上均匀的颗粒群，在冷冻取代制剂中，总体基质电子密度变化很小（即宏观均匀性）。在静息血小板中，管状颗粒延伸的发生率较低，约为4%，但在血小板分泌的早期阶段，这一发生率增加了10倍。使用STEM，我们观察到最初的去致密颗粒和小管系统仍然是独立的膜结构域。脱密颗粒通过长管状连接与质膜异型融合，或彼此异型融合。小管系统与质膜融合的频率也增加了约三倍。我们的研究结果验证了冷冻替代和STEM断层扫描对表征血小板颗粒分泌的效用，并提出了一种模型，其中血小板颗粒与质膜的融合通过长管连接发生，这可能为-颗粒蛋白的定时释放提供空间限制的通路。
How platelets organize their -granule cargo and use their canalicular system remains controversial. Past structural studies were limited due to small sampling volumes or decreased resolution. Our analyses revealed homogeneous granules and a closed canalicular system that opened on activation. Understanding how platelets alter their membranes during activation and secretion elucidates hemostasis.Summary Background Platelets survey the vasculature for damage and, in response, activate and release a wide range of proteins from their -granules. Alpha-granules may be biochemically and structurally heterogeneous; however, other studies suggest that they may be more homogeneous with the observed variation reflecting granule dynamics rather than fundamental differences.Objectives Our aim was to address how the structural organization of -granules supports their dynamics.Methods To preserve the native state, we prepared platelets by high-pressure freezing and freeze-substitution; and to image nearly entire cells, we recorded tomographic data in the scanning transmission electron microscope (STEM).Results and Conclusions In resting platelets, we observed a morphologically homogeneous -granule population that displayed little variation in overall matrix electron density in freeze-substituted preparations (i.e., macro-homogeneity). In resting platelets, the incidence of tubular granule extensions was low, similar to 4%, but this increased by > 10-fold during early steps in platelet secretion. Using STEM, we observed that the initially decondensing -granules and the canalicular system remained as separate membrane domains. Decondensing -granules were found to fuse heterotypically with the plasma membrane via long, tubular connections or homotypically with each other. The frequency of canalicular system fusion with the plasma membrane also increased by about three-fold. Our results validate the utility of freeze-substitution and STEM tomography for characterizing platelet granule secretion and suggest a model in which fusion of platelet -granules with the plasma membrane occurs via long tubular connections that may provide a spatially limited access route for the timed release of -granule proteins.