Fratini Marta, Wiegand Tina, Funaya Charlotta, Jiang Zhongxiang, Shah Pranav N M, Spatz Joachim P, Cavalcanti-Adam Elisabetta Ada, Boulant Steeve
Heidelberg University , Department of Infectious Diseases, Virology and German Cancer Research Center , Im Neuenheimer Feld 581 , 69120 Heidelberg , Germany.
Max Planck Institute for Medical Research , Department of Cellular Biophysics , Jahnstrasse 29 , 69120 Heidelberg , Germany.
ACS Infect Dis. 2018 Nov 9;4(11):1585-1600. doi: 10.1021/acsinfecdis.8b00134. Epub 2018 Sep 26.
Clathrin-mediated endocytosis (CME) is an important entry pathway for viruses. Here, we applied click chemistry to covalently immobilize reovirus on surfaces to study CME during early host-pathogen interactions. To uncouple chemical and physical properties of viruses and determine their impact on CME initiation, we used the same strategy to covalently immobilize nanoparticles of different sizes. Using fluorescence live microscopy and electron microscopy, we confirmed that clathrin recruitment depends on particle size and discovered that the maturation into clathrin-coated vesicles (CCVs) is independent from cargo internalization. Surprisingly, we found that the final size of CCVs appears to be imprinted on the clathrin coat at early stages of cargo-cell interactions. Our approach has allowed us to unravel novel aspects of early interactions between viruses and the clathrin machinery that influence late stages of CME and CCVs formation. This method can be easily and broadly applied to the field of nanotechnology, endocytosis, and virology.
网格蛋白介导的内吞作用（CME）是病毒重要的进入途径。在此，我们应用点击化学将呼肠孤病毒共价固定在表面，以研究早期宿主 - 病原体相互作用期间的CME。为了解耦病毒的化学和物理性质并确定它们对CME起始的影响，我们采用相同策略共价固定不同大小的纳米颗粒。通过荧光活细胞显微镜和电子显微镜，我们证实网格蛋白的募集取决于颗粒大小，并发现成熟为网格蛋白包被囊泡（CCV）与货物内化无关。令人惊讶的是，我们发现CCV的最终大小似乎在货物 - 细胞相互作用的早期阶段就印刻在网格蛋白衣被上。我们的方法使我们能够揭示病毒与影响CME后期和CCV形成的网格蛋白机制之间早期相互作用的新方面。该方法可以轻松且广泛地应用于纳米技术、内吞作用和病毒学领域。
