- Professor, Cell Biology, Harvard Medical School
- Professor, Pediatrics, Harvard Medical School
- Director, HMS-Portugal Program, Harvard Medical School
DF/HCC PROGRAM AFFILIATION
- Cancer Cell Biology, Member
Our research focuses on the processes that mediate and regulate the movement of membrane proteins throughout cells. In particular we study the molecular mechanisms that underlie the cell’s sorting machineries responsible for receptor-mediated endocytosis and for secretion, and how they are high jacked by toxins, viruses and bacterial pathogens to enter cells. We also study how during cell division, cells control their size and organelle architecture.
These studies led to the first structure determination at atomic resolution of clathrin. By using X-ray crystallography, we determined the structure of its amino-terminal portion, a region critical for interactions controlling coat assembly and cargo sorting. We continued with this structural approach and determined the mode of interaction of ÃŸ-arrestins and adaptors with clathrin. We also used cryo-electronmicroscopy to visualize a complete clathrin coat at 8 Ã… resolution and thereby unveiled the basic structure of the triskelion leg and established the way triskelions pack when they form the clathrin coat, and how auxilin and Hsc70 mediate the ATP-dependent uncoating step. Current high-resolution studies focus on the uncoating step and on the linkage between the clathrin machinery and the non-canonical Wnt-signaling pathway.
Recent developments in fluorescent live-cell imaging techniques geared for single-molecule detection, used in combination with object-identification algorithms, now allow sufficient temporal and spatial resolution to follow the life of a single clathrin coated pit. To use this powerful approach, we have developed and implemented over the past years light-microscopic techniques to analyze the clathrin pathway in living cells. We have shown that it is possible to gather in real time, quantitative, «single-object» data from hundreds of uniquely identified clathrin coated pits and coated vesicles, tracked while they are assembling, recruiting cargo and uncoating, using as probes clathrin, AP-2, auxilin, dynamin and other molecules fused to fluorescent proteins such as EGFP, and fluorescently tagged cargo such as transferrin, LDL, viruses and bacteria.
With these type of dynamic studies we expect to integrate molecular snapshots obtained at high resolution with live-cell processes, in an effort to generate â€˜molecular movies’ to allow us to obtain new frameworks for analyzing some of the molecular contacts and switches that participate in the regulation, availability, and intracellular traffic of the many molecules involved in signal transduction, immune responsiveness, lipid homeostasis, cell-cell recognition and organelle biogenesis. Such biological phenomena have importance for our understanding of such diseases as cancer, viral infection and pathogen invasion, Alzheimer’s, as well as other neurological diseases.