Clathrin collaboration

Microscopy shows how cells engulf nutrients and viruses

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BROOKINGS, S.D.—A collaboration between researchers from two South Dakota universities and the National Institutes of Health (NIH) has provided an understanding of a mechanism that enables cells to internalize beneficial nutrients and not-so-beneficial viruses.
South Dakota State University associate professor Adam Hoppe, South Dakota School of Mines & Technology professor Steve Smith and NIH scientists Justin Taraska and Kem Sochacki combined three types of microscopy to trace the method by which a protein called clathrin activates cell membrane bending. The researchers discovered that clathrin, which creates a honeycomb-shaped scaffold on the cell membrane, has an unexpected amount of plasticity when pinching off small portions of the cell membrane.
As the team related in the Jan. 29 issue of Nature Communications, “Clathrin-mediated endocytosis (CME) internalizes plasma membrane by reshaping small regions of the cell surface into spherical vesicles. The key mechanistic question of how coat assembly produces membrane curvature has been studied with molecular and cellular structural biology approaches without direct visualization of the process in living cells; resulting in two competing models for membrane bending. Here we use polarized total internal reflection fluorescence microscopy (pol-TIRF) combined with electron, atomic force and super-resolution optical microscopy to measure membrane curvature during CME.”
They added, “Surprisingly, coat assembly accommodates membrane bending concurrent with or after the assembly of the clathrin lattice. Once curvature began, CME proceeded to scission with robust timing. Four color pol-TIRF showed that CALM accumulated at high levels during membrane bending, implicating its auxiliary role in curvature generation. We conclude that clathrin-coat assembly is versatile and that multiple membrane-bending trajectories likely reflect the energetics of coat assembly relative to competing forces.”
A federally funded intramural research program enabled the contributions of Taraska and Sochacki in Bethesda, Md. Hoppe and Smith work together through the South Dakota BioSystems Networks and Translational Research (BioSNTR) center, funded through the South Dakota Research Innovation Center program and the National Science Foundation’s Established Program to Stimulate Competitive Research. The team hopes that a better understanding of how cells internalize material will assist BioSNTR researchers working with Sioux Falls-based SAB Biotheraputics to develop new alternative treatments for influenza.
According to Hoppe, a cell biologist and BioSNTR director whose research focuses on developing antiviral and anticancer therapeutics, “It was an awesome team science effort and an important model for success. This is a fundamental ‘how does the cell work’ kind of question that has radiating impacts on a multitude of disease processes and important physiological functions in plants, animals and humans. The process, known as endocytosis, is one of the main mechanisms by which cells internalize material from the environment and remodel their surfaces.”
Hoppe and his team filmed live cells internalizing their own membrane using fluorescence on a nanoscale. The team used a laser beam to distinguish between horizontal and vertical orientations. They chemically froze the cells at various stages to garner high-resolution images of membrane bending.
Taraska—a senior investigator in the Laboratory of Molecular and Cellular Imaging at the National Heart, Lung and Blood Institute, part of the NIH—and his team scanned the cells using super resolution light imaging with fluorescence and electron microscopy, correlating the two by putting one image on top of the other. As he explained, “Endocytosis in human cells is primarily driven by clathrin, which forms a honeycomb lattice that grabs a piece of membrane and pulls it inside the cell. Viruses hijack this process to get into cells; it’s also how many drugs are delivered into a cell.”
Smith, a physicist and director of the SD Mines nanoscience and nanoengineering program, used atomic force microscopy to precisely measure the clathrin structure and resulting vesicles, including their height and diameter. Each cell had 50 to 100 vesicles.
According to Smith, “This a big technical accomplishment. Three teams separated by thousands of miles examined the same cell, down to the nanometer level—that’s an incredible accomplishment.”
The three-pronged approach gave the researchers confidence in their findings, and their collaboration will be continuing.

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