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

Area of Science:

• Cell Biology
• Structural Biology
• Microscopy

Background:

• Biological systems require 3D structural analysis for functional insights.
• Electron microscopy of metal replicas is crucial for high-resolution intracellular visualization.
• Existing methods for studying intracellular structures are often complex and require specialized resources.

Purpose of the Study:

• To present novel, accessible methods for 3D nanometer-resolution analysis of biological structures.
• To investigate the structure of Arabidopsis clathrin-coated vesicles (CCVs), which are poorly preserved by conventional methods.
• To enable detailed structural insights into CCVs within unroofed cells and isolated organelles.

Main Methods:

• Developed scanning transmission electron microscopy tomography for visualizing CCVs in unroofed cells.
• Established a simplified sample preparation protocol avoiding harsh acids, suitable for standard electron microscopy labs.
• Implemented a deep learning method for high-throughput screening of CCV morphologies from 2D images.

Main Results:

• Achieved nanometer resolution visualization of CCV clathrin coat arrangements in unroofed Arabidopsis cells.
• Demonstrated direct comparability between isolated CCV samples and those within cells using standardized preparation.
• Enabled robust screening of CCV "pseudo 3D" morphologies using deep learning analysis.

Conclusions:

• Established accessible 3D electron microscopy techniques for examining biological samples.
• Provided novel structural insights into plant clathrin-coated vesicles.
• Facilitated high-throughput analysis of cellular structures using advanced imaging and deep learning.