Researchers develop assay that could be applied to drug screening

Using state of the art imaging technology a team from Yale School of Medicine has glimpsed one of the cell's most important 'nano-machines' in action. The work, performed in collaboration with English and French scientists, provides new insight into the machinery cells use to internalize cell surface receptors.

All cells traffic protein cargos across their outer membrane, and one of the most important routes for cargo internalization is clathrin mediated endocytosis (CME). CME is of fundamental importance for many cellular activities including receptor down-regulation, nutrient uptake and maintenance of signal transmission across nerve cell junctions. Mis-regulation of CME has been implicated in some types of cancer and neuro-degenerative disease and the protein machinery of CME has been co-opted by several viruses, including rabies, as a means of entry into healthy cells.

The researchers led by David Zenisek, assistant professor in the Department of Cellular and Molecular Physiology at Yale School of Medicine, used live cell imaging and a novel fluorescence assay to visualize the formation of clathrin-coated vesicles (CCVs) at single clathrin-coated pits (CCPs) with a time resolution of seconds.

"Although the basic model of clathrin-mediated endocytosis was proposed 41years ago, there are many basic questions outstanding," Zenisek said. "For instance, it wasn't known whether single clathrin coated pits give rise to single clathrin coated vesicles. We have now shown directly that coated pits can produce multiple vesicles in succession."

Using a specially adapted microscope, Zenisek and his colleagues Christien Merrifield and David Perrais, simultaneously measured the minute movements made by coated pits as they invaginated and detected membrane scission, the process by which a coated pit is converted into a clathrin coated vesicle. In further experiments, the researchers showed how proteins linked to the actin framework of the cell are brought to sites of coated pit. Actin is a protein polymer used by cells both as a structural element, and to generate force through polymerization.

"The role played by actin in clathrin mediated endocytosis has been controversial for a long time," Zenisek said. "We've now shown in live cells that proteins involved in actin polymerization are recruited to sites of membrane scission, and that disturbing actin polymerization with the toxin latrunculin B, a toxin found in Red Sea sponge, drastically reduces the efficiency of membrane scission and affects many aspects of CCP dynamics."

Source : Yale University