The Ins and Outs of Endocytosis
By Jason Socrates
Bardi
The easiest way to describe endocytosis is to think not
of cells but of sports arenas—crowded with star players,
role players, benchwarmers, waterboys, coaches, referees,
and spectators, and with lots of ticket holders wanting to
come in.
Endocytosis is like an extremely efficient V.I.P. entrance
to the arena: an usher gathers a group of important people
at one of the gates. Then the gate is pulled inward, enveloping
the people and becoming an elevator car that whisks them away
to their skybox. The elevator car then returns the ushers
back to the stadium where they can gather more ticket holders.
To speak less metaphorically, receptor-mediated endocytosis
is an essential cellular process whereby important hormones,
proteins, nutrients, and other macromolecular "cargo" needed
by a cell are collected and transported across plasma membranes—those
lipid bilayers that define the outer edges of eukaryotic cells.
Receptors (ushers) gather proteins and other nutrients (spectators)
and pinch them off in a membrane packet surrounded by a protein
cage. Scientists refer to these packets as "coated vesicles,"
and the machinery that forms them and regulates their formation
is complex, involving numerous structural proteins and accessory
factors. A cell biologist's dream.
"We're interested in all aspects of how that machinery functions,"
says Sandra Schmid, chair of the Department of Cell Biology.
Coated Vesicles
Endocytosis is essential for development, when it plays
a key role in carting cascades of molecules that establish
the gradients necessary for stem and other precursor cells
to develop into specialized cells and tissues. While cells
with mutations that render them unable to undergo endocytosis
can survive in culture, the same mutations are always lethal
to organisms.
Endocytosis also plays a larger role in biology, providing
cells in a mature organism with a way to take in essential
molecules from the bloodstream. Insulin and cholesterol, for
instance, are transported into cells through receptor-mediated
endocytosis.
Endocytosis is also important for medical reasons because
toxins and viruses co-opt the machinery to gain entry into
cells. In contrast, the process may provide a vehicle for
transporting beneficial drugs into cells.
The Receptors and the Cages
The receptors that mediate endocytosis are large proteins
that span the membranes of cells. The outside, "extracellular"
portion has binding sites that allow it to catch molecules
of interest outside the cell. The inside, "intracellular"
portion carries an address label or "sorting signal" that
is its ticket into the endocytic vesicle. These two regions
of the receptor are connected through a "transmembrane"
portion so that binding of cargo to the outside can be sensed
to activated sorting signals on the inside.
"Cargo molecules and their receptors aren't just passengers.
They can also be active drivers in this process controlling
the type of vehicle they take, the speed at which it travels
and the final destination," says Schmid.
The receptors also take cues from the cell. To maximize
the efficiency of receptor-mediated endocytosis, the cell
instructs the receptors to gather and concentrate in a certain
region of the membrane so that they can, in turn, gather and
concentrate the molecules of interest in that small patch
of membrane outside the cell. And while the receptors are
gathering the cargo on the outside of the cell, other molecules
on the inside of the cell are busy making a "vesicle" container
to transport it in.
Vesicles are actually just patches of membrane where receptors
are and where the cargo molecules are being gathered. That
patch of membrane becomes involuted, bulging inward to form
a pit that is surrounded on the inside of the cell by a lattice-like
coat of protein known as clathrin. (Endocytosis is also sometimes
referred to as clathrin-mediated endocytosis in recognition
of this protein's essential role.)
The clathrin surrounds the involuting patch of membrane,
which then pinches off to form the tiny vesicles. One way
to envision the process is to imagine yourself in the fruit
and vegetable aisle of the grocery store. Receptor-mediated
endocytosis is like putting your hand inside a plastic bag,
grabbing a bunch of green beans, and then turning the bag
inside out around them.
Although clathrin is the primary scaffold of the protein
cage, clathrin does not go it alone. Other proteins are also
involved in the formation of the coated vesicle. The assembly
of the clathrin coat is controlled by other regulatory elements
of the cell—such as the regulatory protein dynamin.
"Dynamin is central to the process of clathrin-mediated
endocytosis," says Schmid, and she points to fruit fly (Drosophila)
mutants discovered three decades ago as proof. These particular
mutants have expressed a wounded dynamin protein that is active
at low temperatures but inactive at high temperatures. The
flies are fine at low temperature, but at high temperature
the mutation is lethal and causes cells to lose their ability
to carry out endocytosis. As a result of this loss of function,
the flies never fully develop.
The mutation, as it turns out, is in the dynamin gene—the
same gene that Schmid's laboratory first identified over a
decade ago and that she has been studying ever since.
Conan the Dynamin
Dynamin is responsible for finally pinching off the "neck"
of the budding vesicle, which releases it into the interior
of the cell. Dynamin's control of this essential final step
led Schmid to recently refer to the enzyme as a master regulator
of the late stages of vesicle formation.
Interestingly, dynamin is actually several enzymes in one.
At one end, its amino terminus, dynamin has a GTPase domain—a
portion that when folded correctly can "hydrolyze" or clip
off a phosphate group from a GTP molecule.
Dynamin also carries some of its own activating proteins.
Normally, GTPase enzymes require cofactor proteins (called
"GAP" for GTPase-activating protein) to be active. Dynamin
carries its own GAP.
As far as GTPases go, dynamin is something of a standout.
Small GTPases average around 20,000 Daltons, and large ones
are something like 40,000 Daltons. Dynamin is about 100,000
Daltons—a protein chain of over 800 amino acids.
"It's the Arnold Schwarzenegger of GTPases," says Schmid.
In 1995, Schmid found that dynamin self-assembles at the
neck of budding vesicles, which led her to propose the first
model for how dynamin works. According to this model, the
dynamin self-assembles at the neck of the forming vesicle
and pinches it off, freeing the vesicle to traffic through
the cell. In referring to this mechanism, she likened the
action of dynamin to the assassin's murder weapon the garrote—it
tightens around the neck, and pop.
In subsequent years, Schmid's laboratory went on to probe
this mechanism of action in greater detail, concentrating
on how the dynamin self-assembles and how it tightens around
a budding vesicle. The whole time she was looking for evidence
to support this model.
"In fact," she says, "We found evidence that the model was
wrong."
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