Fluorescent microscopy shows how living cells form vesicles to transport cargo such as growth factors – Genetics News

Cells have a clever way to transport goods such as growth factors across the cell membrane and into the cell. This is called clathrin-mediated endocytosis. The clathrin protein molecules aggregate inside the cell membrane and deform the membrane to form a hole-like on the outside.

Once filled with cargo, the pit compresses to form a clathrin-coated vesicle inside the cell, which then travels to its proper destination. In cultured cells, hundreds of such clathrin-coated vesicles can form every minute.

However, conflicting models exist for how these vesicles aggregate, leaving a critical knowledge gap. One of the models is the static bending model, in which the bending occurs simultaneously with the polymerization of clathrin. Another model, the flat-to-curve transition model, says that the flat network of clathrin molecules first assemble within the cell membrane, followed by a change in morphology to form the clathrin-covered pit and vesicle.

Evidence for both models can be seen through electron microscopy – but these are still snapshots of stationary cells that do not reveal true nanodynamic and potential pathways for clathrin-mediated endocytosis.

Today, Alexa Mattheyses and colleagues at the University of Alabama at Birmingham and Emory University are filling this knowledge gap, using complex fluorescent microscopy called STAR microscopy. This allowed them to follow the formation of cadherin-coated vesicles in living cells from start to finish, for periods of up to 100 seconds.

Their study was mentioned in the magazine Connecting with natureIt supports what has been termed the elastic model of caltherine-coated vesicle formation, which includes both a static curvature transition and flat to curvilinear transition pathways.

“We show that clathrin accumulation preferentially synchronizes with curvature formation in short-lived caldhirin-coated vesicles, but promotes a flat-to-curved transition in long-lived clathrin-coated vesicles,” said Mathisis, associate professor at UCLA. UAB department. Cellular, developmental and integrative biology. “Together, our results provide experimental evidence in favor of the flexible model of endocytosis, in which no single model of bend formation can capture the heterogeneity of endocytosis dynamics.”

Two-wave simultaneous axial rate measurement, or STAR, is based on total internal reflection microscopy, or TIRF, which is the microscopy that allows visualization of fluorescently tagged proteins at or near the plasma membrane, at a distance of about 100 to 200 nm. In STAR microscopy, the protein is tagged with a fluorophore of different excitation wavelengths to take advantage of the wavelength-dependent penetration depth of the evanescent field. The result is the ability to simultaneously resolve both protein accumulation, as measured by fluorescence intensity, and plasma membrane distance, as measured by fluorescence intensity ratio. This distance, representing the z-distribution of the nanometer, is a measure of the curvature in the current study.

Using monkey kidney fibroblast cells, Mathisis and colleagues fluorescently labeled the CLCa clathrin light chain that has excitatory wavelengths of 488 and 647 nm. Then they stimulated the cells with epidermal growth factor to induce cellular endocytosis. Quantitative analysis using the UAB Cheaha 1948 supercomputer resulted in CLCa-STAR accumulations from 13 cells.

They found evidence for three patterns of curvature initiation: nucleation, in which curvature formation begins less than a second before clathrin appears; continuous curvature, in which curvature formation begins one to four seconds after the arrival of the clathrin; And the transition from flat to curved, where the bend begins more than four seconds after the clathrin build-up.

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

“We found that short-lived endocytic events, less than 20 seconds, mainly formed via the static bending pattern, while longer events – greater than 20 seconds – favored the transition from flat to curved,” Matises said. “The proportion of nucleation events was smaller and favored short-lived events.”

To test whether this flexible model of clathrin-mediated endocytosis could be universal among different cell lines, the researchers also imaged human umbilical vein endothelial cells stimulated with vascular endothelial growth factor to stimulate endocytosis. They found a similar set of internal events.

“These data demonstrate previously underestimated heterogeneity in the dynamics of clathrin-mediated endocytosis and the plasticity of clathrin-coated vesicle formation, and suggest that different cell types or cargoes may use this flexibility to influence how the curvature is formed,” Matthesis said. Future research using STAR microscopy will determine how additional protein recruitment, 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 the dynamics of vesicle formation supports the flexible model of clathrin-mediated endocytosis,” are Thomass J. and integrative biology. and Yoosung Ho and Khaled Salita, 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 Ph.D. Working at the University of Michigan with Daniel Axelrod, Ph.D., considered by many to be a pioneer in this technology.

Support came from NIH grant GM3099, National Science Foundation grant CAREER 83200, and American Heart Association grant 906086.

Cell, Developmental, and Integrative Biology is a department of the Marnix E. Heersink School of Medicine at UAB.

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