CALM supports clathrin-coated vesicle completion upon membrane tension increase
CALM supports clathrin-coated vesicle completion upon membrane tension increase
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DOI：
10.1073/pnas.2010438118
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发表时间：
2021-06
期刊：
影响因子：
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通讯作者：
N. Willy;F. Colombo;S. Huber;Anna C. Smith;Erienne G. Norton;C. Kural;Emanuele Cocucci
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文献类型：
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作者：
N. Willy;F. Colombo;S. Huber;Anna C. Smith;Erienne G. Norton;C. Kural;Emanuele Cocucci
网格蛋白包被囊泡（ccv）是负责受体结合配体和细胞外液内化的内吞载体。CCV组装是通过网格蛋白、接头和其他促进曲率形成的辅助分子的连续募集而发生的。为了形成ccv，需要克服在细胞周期、发育和细胞极化过程中变化的局部质膜张力。利用定量荧光显微镜，我们证明了适配器CALM是膜张力增加时CCV形成的主要决定因素。由于CALM是差异表达的，我们的研究结果表明，网格蛋白介导的内吞作用的能力是组织特异性的，这就从机制上解释了为什么CALM的缺失会强烈影响胚胎发育和红细胞分化，而对其他系统的影响很小。网格蛋白包被囊泡（ccv）中最具代表性的成分是网格蛋白三瓣体和接头网格蛋白组装淋巴髓系白血病蛋白（CALM）和异四聚体复合物AP2。在CCV形成过程中，ap180 -氨基末端同源性（ANTH）募集的动态研究一直受到过表达后CALM毒性的阻碍。我们使用敲入基因编辑来表达c端附着的CALM的荧光版本，同时保留其内源性表达水平，并使用尖端的活细胞显微镜方法来研究形成ccv时CALM的募集。我们的研究结果表明，当膜张力增加时，CALM促进了囊泡的完成，这是该适配器存在量的函数。由于接合子的表达，包括CALM，在不同的细胞中是不同的，我们的数据支持一个模型，其中网格蛋白介导的内吞作用的效率是组织特异性的，并解释了为什么CALM在胚胎发生和红细胞发育过程中是必不可少的。
Significance Clathrin-coated vesicles (CCVs) are endocytic carriers responsible for the internalization of receptor-bound ligands and extracellular fluids. CCV assembly occurs by the sequential recruitment of clathrin, adaptors, and other accessory molecules that promote curvature formation. To form, CCVs need to overcome the local plasma membrane tension that varies during cell cycle, development, and cell polarization. Using quantitative fluorescence microscopy, we demonstrate that the adaptor CALM is a major determinant of CCV formation upon membrane tension increase. Since CALM is differentially expressed, our results demonstrate that competence in clathrin-mediated endocytosis is tissue specific, providing mechanistic explanation why CALM depletion strongly affects embryo development and red blood cell differentiation with minor effects in other systems. The most represented components of clathrin-coated vesicles (CCVs) are clathrin triskelia and the adaptors clathrin assembly lymphoid myeloid leukemia protein (CALM) and the heterotetrameric complex AP2. Investigation of the dynamics of AP180-amino-terminal-homology (ANTH) recruitment during CCV formation has been hampered by CALM toxicity upon overexpression. We used knock-in gene editing to express a C-terminal–attached fluorescent version of CALM, while preserving its endogenous expression levels, and cutting-edge live-cell microscopy approaches to study CALM recruitment at forming CCVs. Our results demonstrate that CALM promotes vesicle completion upon membrane tension increase as a function of the amount of this adaptor present. Since the expression of adaptors, including CALM, differs among cells, our data support a model in which the efficiency of clathrin-mediated endocytosis is tissue specific and explain why CALM is essential during embryogenesis and red blood cell development.