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Fluorescence microscopy shows how living cells form vesicles to carry loads such as growth factors

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Cells have a clever way of transporting goods such as growth factors across the cell membrane and into the cell. This is called clathrin-mediated endocytosis. Clathrin protein molecules aggregate within the cell membrane and distort the membrane to form what looks like a pit from the outside.

Once filled with cargo, the fossa pinches to form a clathrin-coated membrane bound vesicle within the cell, which then proceeds to its proper destination. In cultured cells, hundreds of these clathrin-coated vesicles can form every minute.

However, conflicting models exist for how these vesicles assemble, which has left a critical knowledge gap. One model is the constant curvature model, in which curvature is induced simultaneously with the polymerization of clathrin. Another, the flat-to-curve transition model, states that a flat network of clathrin molecules first assembles within the cell membrane, followed by a conformation change to form the clathrin-coated pit and vesicle.

Evidence for both models can be seen by electron microscopy, but these are static snapshots of fixed cells that do not reveal the true nanoscale dynamics and possible pathways of clathrin-mediated endocytosis.

Today, Alexa Mattheyses and her colleagues from the University of Alabama at Birmingham and Emory University are filling this knowledge gap, using sophisticated fluorescence microscopy images called STAR microscopy. This allowed them to track clathrin-coated vesicle formation in living cells from start to finish for periods of up to 100 seconds.

Their study, reported in the journal Communication on naturesupports what has been termed the flexible pattern of clathrin-coated vesicle formation, which includes both the constant curvature transition and flat-to-curve transition paths.

“We show that clathrin accumulation is preferentially simultaneous with the formation of curvatures in shorter-lived clathrin-coated vesicles, but promotes a flat-to-curve transition in longer-lived clathrin-coated vesicles,” said Mattheyses, professor associate in the UAB Department. Cellular, developmental and integrative biology. “Together, our results provide experimental evidence in favor of the flexible model of endocytosis, in which a single model of curvature formation cannot understand the heterogeneity of the dynamics of cell-mediated endocytosis. “

Simultaneous two-wavelength axial ratiometry, or STAR, microscopy is based on total internal reflection fluorescence, or TIRF, microscopy that allows the visualization of fluorescently labeled proteins at or near the plasma membrane , at a distance of about 100 to 200 nanometers. In STAR microscopy, the protein is labeled with two fluorophores that have different excitation wavelengths to take advantage of the wavelength-dependent depth of penetration of the evanescent field. The result is the ability to simultaneously resolve both protein accumulation, measured by the fluorescence intensity, and the plasma membrane distance, measured by the fluorescence intensity ratio of the two fluorophores. This distance, which represents the z-distribution of the nanometer, is a measure of the curvature in the present study.

Using monkey kidney fibroblast cells, Mattheyses and colleagues labeled the CLCa clathrin light chain with fluorophores that have excitation wavelengths of 488 and 647 nanometers. They then stimulated cells with epidermal growth factor to induce endocytosis. Quantitative analysis using the UAB Cheaha supercomputer produced 1,948 CLCa-STAR accumulations from 13 cells.

They found evidence for three patterns of curvature initiation: nucleation, where curvature formation began less than a second before clathrin appeared; constant curvature, where the formation of the curvature began one to four seconds after the arrival of the clathrin; and the flat-to-curved transition, where curvature began more than four seconds after the accumulation of clathrin.

To further analyze the data, the researchers grouped endocytic events based on clathrin life and vesicle formation patterns. This revealed some interesting features.

“We found that endocytic events of short duration, less than 20 seconds, formed mainly through the constant curvature pattern, while longer events – longer than 20 seconds – favored the transition from flat to curve,” he said. said Mattheyses. “The rate of nucleation events was lower and favored events of short duration. “

To test whether this flexible model of clathrin-mediated endocytosis can be universal across different cell lines, the researchers also imagined human umbilical vein endothelial cells stimulated with vascular endothelial growth factor to stimulate endocytosis. They found a similar range of endocytic events.

“These data show the previously unappreciated heterogeneity in the dynamics of clathrin-mediated endocytosis and the plasticity of clathrin-coated vesicle formation and suggest that different cell types or loads may use this flexibility to influence how the curvature, ”Mattheyses said. Future research using STAR microscopy will define how the recruitment of accessory proteins and the role of biophysical parameters contribute to vesicle formation and how different clathrin dynamics affect cell signaling and homeostasis. “

Co-authors of the study, “Imaging vesicle formation dynamics supports the flexible model of clathrin-mediated endocytosis,” are Tomasz J. Nawara, Yancey D. Williams II, Tejeshwar C. Rao and Elizabeth Sztul, UAB Department of Cell , Developmental and Integrative Biology; and Yuesong Hu and Khalid Salaita, Department of Chemistry, Emory University, Atlanta, Georgia.

Mattheyses and Salaita jointly developed STAR microscopy at Emory in 2015. Mattheyses has worked with TIRF microscopy since his PhD. working at the University of Michigan with Daniel Axelrod, Ph.D., considered by many to be a pioneer of the technique.

Support came from the National Institutes of Health’s GM3099 grant, the National Science Foundation’s CAREER 83200 grant, and the American Heart Association’s 906086 grant.

Cellular, Developmental, and Integrative Biology is a department of UAB’s Marnix E. Heersink School of Medicine.

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