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As an investigator for the Canadian Institutes of Health Research and an associate professor at the Montreal Neurological Institute, Peter McPherson is often asked the cocktail-party icebreaker, "So, what diseases do you study?" His cheery answer: "I'm not sure yet!"
McPherson and his colleagues are working to understand the role of clathrin-coated vesicles in the complex machinery that controls endocytosis (cellular uptake).
"You've got something outside the cell, whether in the blood or in the gut," he explains, "and it's got to get into the cell. Nutrients, for example, have to get into cells. The cell membrane has to be selectively permeable."
There are different ways to transport nutrients across a membrane, but McPherson is particularly interested in receptor-mediated endocytosis as it relates to cholesterol uptake.
"Cholesterol, which people often think of as something that clogs arteries, is actually a vital nutrient. We need cholesterol in our diet because it's used to build membranes. If your cells don't take up cholesterol, that leads to serious problems. It remains in the blood and clogs up the arteries, of course, but the other problem is that you don't have the cholesterol needed to build these important cell membranes."
Here is a greatly simplified version of this process: clathrin protein molecules inside the cell bind to receptor molecules that span the membrane. The receptor molecules, in turn, bind only to particular cargo on the outside of the cell — lipoprotein particles that contain cholesterol molecules, for example. The clathrin physically bends the membrane inward, creating an invagination. As the membrane bends, the receptor and its bound cholesterol are brought further inside the cell and the invagination deepens to the point of becoming a closed circle, at which point the membrane reseals itself and "cuts loose" the clathrin-coated cholesterol molecule. The result: the membrane has never compromised its integrity (it remains intact throughout the process), yet the cholesterol is now inside the cell, surrounded in a clathrin "cage" (vesicle). To make this drama even more impressive, the clathrin-coated vesicle is only 100 nanometres (a mere one-10-millionth of a metre) in diameter. The clathrin coat then disperses, freeing the cholesterol for cellular consumption.
"This clathrin coat is a really neat little machine," says McPherson, "because of this ability to physically bend membrane. The jury's still out as to how that membrane curvature occurs — whether clathrin somehow pulls it or some other proteins are driving it — but it's true nanoengineering."
Electron microscopes first revealed this process in 1964, but it's remained largely a mystery until recently. When McPherson started studying clathrin-coated vesicles in 1992, while a postdoctoral fellow at Yale University, it was thought that only two molecules (clathrin and the clathrin adaptor molecule, which binds it to the membrane) functioned in the process.
"The idea that there are lots and lots of proteins regulating this process has been slowly evolving," he says. "Now it turns out that there are probably 25, 30, maybe 40 different proteins involved in the curvature, the selection of cargo and the regulation of the whole process."
The key to identifying these proteins is proteomics, a variation of genomics that uses a mass spectrometer (an instrument that measures masses of proteins and other molecules using the basic magnetic force on a moving charged particle) to rapidly identify, localize and analyse a cell's protein make-up. McPherson's team initially focused on the proteins found in clathrin-coated vesicles from rat brains. The brains were put into a homogenizer and "whipped up to rupture the cells." The researchers next put this purée through repeated spins in a centrifuge, exploiting the clathrin-coated vesicle's small size and high density. (Bigger organelles "pellet out" to the bottom of the centrifuge first, so a sequence of spins is an efficient way of isolating the vesicles.) Ten rat brains, which contain approximately 200 grams of protein, yield a few milligrams of clathrin-coated vesicles. The purifying process takes a day to complete.
Next stop: proteomics. "It takes a day to make the samples," says McPherson, and 180 hours of mass spectrometer time to analyze them, then eight months to a year to break down that data. The proteomics is done through the Montreal Proteomics Centre at McGill University, a Genome Quebec/Genome Canada—funded operation that has been a world leader in proteomics studies.
"What we get from the mass-spec is little chunks of peptide, little sequences of amino acid. We take those sequences and we say, ‘OK, what protein did those come from?' We then search the National Centre for Biotechnology Information's GenBank database of genetic sequences, trying to match the sequence from our organelle against a known sequence from a specific gene.
"In many cases, these peptide sequences come from genes that have been put in the database without any direct evidence that they're turned into proteins. They're just sitting there as chunks of DNA, waiting for us to come along and hypothesize that that gene is somehow functioning in the process of endocytosis."
Returning to the cocktail query, this is why McPherson doesn't necessarily know what diseases he's researching. Of course, there are some obvious relationships between endocytosis and disease (for example, hypercholesterolemia, caused by inadequate levels of cholesterol being removed from the blood), but there are likely many big surprises to come — and McPherson revels in the possibilities of the unexpected.
"For so many diseases, the genetic links are still being defined. People still think of Parkinson's disease, for example, as having an environmental cause, but there are now 12 different genes linked to it.
"You could spend a lifetime trying to show exactly how one particular protein functions. For example, we identified a protein three years ago. We got some peptide sequences, and put that into the database. We found the genes that made this protein in GenBank; the genes were listed as having an unknown function. I had a student whose PhD project was to figure out what this particular protein did, and she showed that it absolutely linked back to this process involving clathrin.
Recently, McPherson got an e-mail from a research group at UPenn alerting him to a paper on genetic linkage studies with schizophrenic patients. "They found a region of DNA with certain markers that always got transferred to schizophrenics, and never to non-schizophrenics. There's a gene in there making that protein that has to be linked to schizophrenia, because it's too much of a coincidence in terms of probability. And it turns out to be this same gene that my student identified. Now we have evidence that this membrane uptake process, when mutated, has something to do with schizophrenia.
"You could have sat around all day long thinking, and never come up with this link."