A cryo-electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane.
A cryo-electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane.
复制标题
冷冻电子断层扫描工作流程揭示了受损质膜上的细胞介导脱落。
DOI：
10.1126/sciadv.abc6345
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发表时间：
2021-03
期刊：
影响因子：
13.6
通讯作者：
Jensen GJ
中科院分区：
文献类型：
--
作者：
Mageswaran SK;Yang WY;Chakrabarty Y;Oikonomou CM;Jensen GJ
光损伤质膜的Cryo-ET揭示了依赖于F-actin动力学，Myo 1a和Vps 4 B的突起脱落。冷冻电子断层扫描（cryo-ET）为生物过程的分子机制提供了结构背景。虽然简单地实现研究稳定的大分子复合物，使用它来定位短寿命的结构和事件可能是不切实际的。活细胞显微镜、相关光学和电子显微镜以及冷冻ET的组合将缓解这一问题。我们开发了一个工作流程，结合三者来研究HeLa细胞中应对质膜损伤的普遍和动态脱落过程。我们发现丝状伪足样突起在损伤部位富集，并作为脱落的支架，这涉及F-肌动蛋白动力学，肌球蛋白-1a和空泡蛋白分选4 B（运输所需的内体分选复合物的一个组成部分）。总的来说，脱落比目前从平膜囊泡化的模型更复杂。它与肠上皮细胞组成性脱落的相似性证明了其保守机制。我们的工作流程也可以适用于研究其他损伤反应途径和动态细胞事件。
Cryo-ET of photodamaged plasma membrane reveals shedding from protrusions that depends on F-actin dynamics, Myo1a, and Vps4B. Cryo–electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-cell microscopy, correlative light and electron microscopy, and cryo-ET will alleviate this issue. We developed a workflow combining the three to study the ubiquitous and dynamic process of shedding in response to plasma membrane damage in HeLa cells. We found filopodia-like protrusions enriched at damage sites and acting as scaffolds for shedding, which involves F-actin dynamics, myosin-1a, and vacuolar protein sorting 4B (a component of the ‘endosomal sorting complex required for transport’ machinery). Overall, shedding is more complex than current models of vesiculation from flat membranes. Its similarities to constitutive shedding in enterocytes argue for a conserved mechanism. Our workflow can also be adapted to study other damage response pathways and dynamic cellular events.
影响因子：
3.4
作者：
Inoue, Toshihiro;Pattabiraman, Padmanabhan P.;Epstein, David L.;Rao, P. Vasantha
通讯作者：
Rao, P. Vasantha