Overview

Our research has helped defined the structure, interactions, and assembly-disassembly mechanisms of clathrin and many of its associated proteins, through studies extending over three decades. Our work has been characterized by use of emerging technologies -- from the early days of molecular cloning to contemporary high-resolution structural visualization and live-cell imaging. We have used the tools of x-ray crystallography, cryo electron microscopy, and single-molecule biophysics to create a "molecular movie" of clathrin-mediated endocytosis, and in this way related these molecular events to functional properties of the surfaces of living cells. We also use frontier optical-imaging modalities to examine other cellular membrane remodeling processes exemplified by the formation of intraluminal vesicles and nuclear pores.

Our molecular snapshots led to the first structure determination at atomic resolution of large portions of clathrin and AP-1, key components of the clathrin coat. We used cryo electron microscopy to obtain the first visualization of a complete clathrin coat at 8 Å resolution and thereby unveiled the basic structure of the triskelion leg and established the way triskelions pack when they form the clathrin coat. We then establish how auxilin and Hsc70 mediate the ATP-dependent uncoating step by visualizing a complete clathrin coat bound to auxilin and Hsc70 at 12-15 Å resolution. We continued with this structural approach and determined the mode of interaction of ß-arrestins and adaptors with clathrin and on the linkage between the clathrin machinery and the non-canonical Wnt-signaling pathway.

Direct observation of molecular events in vivo is the ultimate goal of contemporary microscopy. Two recently developed forms of fluorescence microscopy available in our laboratory -- Lattice Light Sheet Microscopy (LLSM) and Lattice Light Sheet Microscopy optimized with Adaptive Optics (AO-LLSM) -- are poised to bridge the gap between molecules and cells, either as independent entities in culture, as components of organoids, or as components of living tissues. The richness and magnitude of the data over periods ranging from seconds to hours, create new challenges for obtaining quantitative representations of the observed dynamics and for deriving accurate and comprehensive models for the underlying developmental mechanisms.

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 other ‘molecular movies' allowing 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 many diseases including cancer, viral infection and pathogen invasion, Alzheimer's, as well as other neurological diseases.

Background

Dr. Kirchhausen is a Professor of Cell Biology and of Pediatrics at Harvard Medical School and the Springer Family Chair of Pediatrics and a Senior investigator at Boston Children’s Hospital. He received his undergraduate degree in Biology from the Universidad Peruana Cayetano Heredia and earned his Ph.D. in Biophysics from the Instituto Venezolano de Investigaciones Cientificas.

The animation by Janet Iwasa and Tom Kirchhausen showing the process of clathrin-mediated endocytosis won the 1st Place Video at the Celldance 2008 contest of the American Society of Cell Biology and was one of the scientific animations highlighted by Erik Olsen in his article "The Animators of Life" published in The New York Times, November 15, 2010.

The YouTube 3D movie by Tsung-Li Liu,, Srigokul (Gokul) Upadhyayula, Eric Betzig, Tom Kirchhausen and colleagues acquired using AO-LLSM and showing the migration of immune cells in the zebrafish inner ear has been downloaded more than 700,000 times.

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