Capturing membrane trafficking events during 3D angiogenic development in vitro.
Capturing membrane trafficking events during 3D angiogenic development in vitro.
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DOI：
10.1111/micc.12726
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发表时间：
2022-10
期刊：
影响因子：
--
通讯作者：
Kushner EJ
中科院分区：
文献类型：
--
作者：
Francis CR;Kushner EJ
囊泡运输决定了蛋白质定位、功能活性和半衰期，为组织发育提供了至关重要的调控步骤;然而，关于内皮特异性运输特征的信息很少。这部分是由于在内皮组织中可视化运输事件的局限性。我们在这项研究中的目的是探索使用三维（3D）体外发芽模型来成像内皮膜运输事件。在纤维蛋白珠测定中激发内皮细胞生长芽。此后，用荧光蛋白转染喷口，并对各种细胞标记物进行染色。然后使用活细胞和固定细胞显微镜对芽苗的运输事件进行成像。我们的研究结果表明，纤维蛋白珠芽具有很强的apicobasal极性标记的顶端定位的蛋白膜突蛋白和podocalyxin。比较运输介质Rab 27 a和Rab 35之间的3D芽和2D文化表明，泡状载体可以在高分辨率下成像，仅在3D芽中表现出适当的膜极性。最后，我们对血管性血友病因子的胞吐事件进行了成像，并证明了与2D培养相比，3D芽中监测分泌事件的独特成像优势。我们的结果证实，纤维蛋白珠发芽试验非常适合血管生成生长期间运输事件的成像。
Vesicular trafficking dictates protein localization, functional activity, and half‐life, providing a critically important regulatory step in tissue development; however, there is little information detailing endothelial‐specific trafficking signatures. This is due, in part, to limitations in visualizing trafficking events in endothelial tissues. Our aim in this investigation was to explore the use of a 3‐dimensional (3D) in vitro sprouting model to image endothelial membrane trafficking events. Endothelial cells were challenged to grow sprouts in a fibrin bead assay. Thereafter, spouts were transfected with fluorescent proteins and stained for various cell markers. Sprouts were then imaged for trafficking events using live and fixed‐cell microscopy. Our results demonstrate that fibrin bead sprouts have a strong apicobasal polarity marked by apical localization of proteins moesin and podocalyxin. Comparison of trafficking mediators Rab27a and Rab35 between 3D sprouts and 2D culture showed that vesicular carriers can be imaged at high resolution, exhibiting proper membrane polarity solely in 3D sprouts. Lastly, we imaged exocytic events of von Willebrand Factor and demonstrated a distinct imaging advantage for monitoring secretion events in 3D sprouts as compared with 2D culture. Our results establish that the fibrin bead sprouting assay is well‐suited for imaging of trafficking events during angiogenic growth.
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