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Dynamin, Schmid now believes, is a much more sophisticated molecule. "Dynamin is not just the brawn," Schmid says. "It's part of the brains." She thinks that it is integrating the process of endocytosis with other events in the cell. It may be rearranging the actin cytoskeleton of the cell, monitoring what is entering cell-wide, and inducing a stress response.

A Powerful Assay

The biochemical assay that the Schmid laboratory developed involves purifying plasma membranes, stripping them of all their materials, and reconstituting the machinery to generate coated vesicles and recreate each of the steps leading to endocytosis in the test tube. This assay has allowed her to study and understand the action of dynamin and to identify the other cellular machinery that carries out vesicle formation.

"It is different than assays we have used in the past, which use a single receptor and a single ligand," says Schmid. "Now we can look at any receptor we want."

One of the things these studies have revealed is that the regulation of receptor-mediated endocytosis is a highly sophisticated interaction between the cargo molecules, the receptors, dynamin and other enzymes, and the clathrin coat.

In addition to having binding sites outside the cell that recognize and collect the cargo molecules, receptors have binding sites on their portions inside the cell that are used for binding as well—to the clathrin molecules that form the cage around the budding vesicle.

However, clathrin does not recognize the receptors directly. The cell employs "adaptor" proteins that recognize the sorting motifs on the receptors and then "adapt" the cargo molecules to the coat. Like the familiar three-pronged to two-pronged converters people use to plug their toaster ovens into old outlets, adaptor molecules fit the receptors to the clathrin scaffold during vesicle formation.

Moreover, the adaptors exert control over endocytosis because they trigger assembly of the clathrin coat, and they group the receptors together, thus concentrating the cargo. And to make the situation even more elaborate, the adaptors are, in turn, controlled by other parts of the cellular machinery.

About a year ago, the Schmid laboratory discovered a new "kinase" enzyme that is involved in the regulation of cargo selection by controlling the adaptors. The kinase that Schmid found attaches a phosphate group to adaptor molecules and thereby regulates cargo selection by altering the adaptor.

The adaptor molecule is called AP-2, and Schmid's new kinase binds to it and attaches a phosphate group to the portion of AP-2 that is responsible for recognizing the receptor molecule. Schmid found that when the kinase attaches the phosphate to the adaptor molecule the affinity for the cargo molecules increases 25-fold.

"That's a huge difference," she says. "Kinases usually have a 2- to 3-fold effect."

Into the Cell

However, when Schmid and her colleagues went beyond the biochemical assay and designed a series of mutant cells that would allow them to see what happens when they tinker with the kinase, they were surprised.

"We learned things we never could have anticipated from what we had done in the test tube," she says.

What they anticipated was that by overexpressing the kinase, they would be overphosphorylating the AP-2 adaptor molecules. They reasoned that the overphosphorylated adaptors would be randomly distributed and unable to cluster.

Then, since adaptor clustering leads to clathrin clustering, Schmid and her laboratory thought that overexpressing the kinase would shut down vesicle formation and endocytosis.

However it did not.

In fact, to their surprise, they found that the clathrin distribution was not affected by essentially knocking out the function of AP-2. Other adaptors, they concluded may be working independently of AP-2 to accomplish the same goal.

"AP-2 may be just another cargo-specific adaptor," she concludes.

In general, she adds, receptor-mediated endocytosis seems to be more sophisticatedly regulated than was ever previously thought.

"In retrospect this makes sense," says Schmid. "Cells communicate between themselves and with their environment through the plasma membrane. Endocytosis plays a critical role in regulating this communication."


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Yellow LDL particles that carry cholesterol in the bloodstream are captured by receptors on the cell surface. Three-legged, clathrin triskelions (red) assemble into soccer-ball-like lattices to pinch off a piece of the membrane-carrying receptors and cargo into the cell. Electron micrographs (at 100,000X magnification) show flat and deeply curved clathrin coated pits on the inside of the cell. Another protein, dynamin, forms a spiral "collar" around the coated pit and is required, like a purse-string, for sealing the neck and detaching the coated vesicle. Green virus particles can, like Trojan Horses, gain access to the cell through this entryway.