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A Colorado State University biochemist is completing the first year of a $985,000, four-year National Science Foundation grant to study how cells take up materials such as proteins from outside their plasma membranes. The research could lead to better understanding of how nutrients, hormones, cholesterol and some viruses enter cells.
Santiago Di Pietro, assistant professor of biochemistry, said cell surface receptors normally capture and internalize molecules from outside the cell. For example, low density lipoproteins (LDL) that transport cholesterol are cleared from the blood by LDL-receptors present on the cell surface, mainly by liver cells. Receptors that carry mutations fail to work properly, which can result in high cholesterol levels in the blood.
How a healthy system works: The cell plasma membrane invaginates a small region forming a vesicle or little cage full of molecules that then pinches off into the cell – a process known as endocytosis. The cell surface displays receptors that select for only certain molecules to be collected in the membrane invagination, so some molecules enter the cell and some do not – a process known as receptor-mediated endocytosis. These receptors have the ability to discriminate between thousands of molecules passing by, so only proper “cargo” molecules enter the cell. Then a series of filaments inside the cell, known as the actin cytoskeleton, join with molecular motors that can actually grab onto the incipient vesicle and move it inside the cell.
Endocytosis is a complex process that requires a specialized cellular machinery composed of hundreds of proteins working in a coordinated fashion. A variety of diseases develop as a consequence of defective components of the general endocytosis machinery or specific receptors.
With his NSF grant, Di Pietro is studying how that little cage – called a clathrin cage – forms, how the cage delivers its goods to the cell with the actin cytoskeleton and the proteins that act as receptors.
“This is a fundamental process for cell life – clathrin-dependent endocytosis is the most important cellular entry pathway,” Di Pietro said. “We’re studying both the machinery that carries out the endocytosis process as well as the proteins that are on the plasma membrane working as receptors.”
Endocytosis is an evolutionarily conserved process such that it is very similar whether it is in human cells or yeast cells. This project uses yeast as a model system to study endocytosis given that it offers a number of experimental advantages.
This NSF grant also has an annual outreach activity involving students at Lesher Middle School, a public school in Fort Collins. In fall 2011, Di Pietro and members of his lab led experimental activities with eighth grade students at Lesher and at CSU, including visualization of endocytosis in live cells by fluorescence microscopy. Students observed and recorded videos of live cells in which a fluorescent molecule labeled the plasma membrane in general or sites of endocytosis in particular. They also observed the nucleus and other cellular compartments.
All these cellular parts were described in a lecture given by Di Pietro and his doctoral students the previous week at Lesher. This activity is intended to get young students excited about science and perhaps spark an interest in a future career in a STEM field, or science, technology, engineering and mathematics disciplines. This activity is planned and carried out in coordination with two teachers at Lesher Middle School, Mrs. Freemyer (science teacher) and Mr. Gallegos (bilingual program director).
Di Pietro has been at Colorado State since 2008. He works with three undergraduate students, three doctoral students and two research scientists in his laboratory. Di Pietro previously has received funding from the American Heart Association to investigate the inner workings of blood clots.