Three-dimensional visualization of planta clathrin-coated vesicles at ultrastructural resolution

作     者：Alexander Johnson Walter A.Kaufmann Christoph Sommer Tommaso Costanzo Dana A.Dahhan Sebastian Y.Bednarek Jiri Friml Alexander Johnson;Walter A.Kaufmann;Christoph Sommer;Tommaso Costanzo;Dana A.Dahhan;Sebastian Y.Bednarek;Ji(r)í Friml

作者机构：Institute of Science and Technology Austria(ISTA)3400KlosterneuburgAustria UW-MadisonDepartment of Biochemistry433 Babcock Dr.MadisonWI 53706USA 

出 版 物：《Molecular Plant》 (分子植物（英文版）)

年 卷 期：2022年第15卷第10期

页      面：1533-1542页

核心收录：

学科分类：0710[理学-生物学] 071001[理学-植物学] 07[理学] 

基　　金：the Austrian Science Fund I3630B25(to J.F.) 

主　　题：Arabidopsis thaliana clathrin-coated vesicles STEM tomography electron microscopy clathrin-mediated endocytosis 

摘      要：Biological systems are the sum of their dynamic three-dimensional(3D)***,it is critical to study biological structures in 3D and at high resolution to gain insights into their physiological *** microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer ***,many of these methods require specialized equipment and personnel to complete ***,we present novel accessible methods to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolution,focusing on Arabidopsis clathrin-coated vesicles(CCVs).While CCVs are essential trafficking organelles,their detailed structural information is lacking due to their poor preservation when observed via classical electron microscopy protocols ***,we establish a method to visualize CCVs in unroofed cells using scanning transmission electron microscopy tomography,providing sufficient resolution to define the clathrin coat ***,the samples are prepared directly on electron microscopy grids,removing the requirement to use extremely corrosive acids,thereby enabling the use of this method in any electron microscopy ***,we demonstrate that this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in ***,to facilitate the high-throughput and robust screening of metal replicated samples,we provide a deep learning analysis method to screen the“pseudo 3Dmorphologies of CCVs imaged with 2D ***,our work establishes accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.