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

Biological systems are the sum of their dynamic three-dimensional(3D)parts.Therefore,it is critical to study biological structures in 3D and at high resolution to gain insights into their physiological functions.Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution.However,many of these methods require specialized equipment and personnel to complete them.Here,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 experiments.First,we establish a method to visualize CCVs in unroofed cells using scanning transmission electron microscopy tomography,providing sufficient resolution to define the clathrin coat arrangements.Critically,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 lab.Secondly,we demonstrate that this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells.Finally,to facilitate the high-throughput and robust screening of metal replicated samples,we provide a deep learning analysis method to screen the“pseudo 3D”morphologies of CCVs imaged with 2D modalities.Collectively,our work establishes accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.

Numerical study of clathrin-mediated endocytosis of nanoparticles by cells under tension

In this study, a three-dimensional mathematical model was used to study the contribution of clathrins during the process of cellular uptake of spherical nanoparticles under different membrane tensions. The clathrin-coated pit (CCP) that forms around the inward budding of the cell membrane was modeled as a vesicle with bending rigidity. An optimization algorithm was proposed for minimizing the total energy of the system, which comprises the deforming nanoparticle, receptor-ligand bonds, cell membrane, and CCP, in which way, the profile of the system is acquired. The results showed that the CCP enable full wrapping of the nanoparticles at various membrane tensions. When the cell membrane tension increases, the total deformation energy also increases, but the ratio of CCP bending to the minimum value of the total energy of the system decreases. The results also showed that the diameter of the endocytic vesicles determined by the competition between the stretching of the cell membrane and confinement of the coated pits are much larger than the nanoparticles, which is quit different as the results in passive endocytosis that is not facilitated by the CCPs. The present results indicate that variations of tension on cell membranes constitutes a biophysical marker for understanding the size distribution of CCPs observed in experiments. The present results also suggest that the early abortion of endocytosis is related to that the receptor-ligand bonds cannot generate adequate force to wrap the nanoparticles into the cell membrane before the clathrins respond to support the endocytic vesicles. Correspondingly, late abortion may relate to the inability of CCPs to confine the nanoparticles until the occurrence of the necking stage of endocytosis.