New Haven, Conn. -- 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
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
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."
Co-workers in the research were Christien Merrifield
of the MRC Laboratory of Molecular Biology in Cambridge,
England, and David Perrais of the Laboratoire de
Physiologie Cellulaire de la Synapse in France.
The research was supported partially by the McKnight
and Kinship Foundations.
Cell: (May 20, 2005)