Fluorescence microscopy is an invaluable tool in biomedical research. Unfortunately, the resolution of conventional fluorescence imaging is limited by diffraction, and as a result the details of biological structures are obscured on lengths smaller than a few hundred nanometers. For example, fundamental aspects of dynamic membrane organization below ~250 nm, remain unclear because of this limitation. In particular, the dynamics of protein recruitment during the early stages of clathrin-mediated endocytosis, an essential process for numerous cellular functions including signaling and growth, cannot be resolved spatially. While certain proteins such as dynamin and endophilin have been linked to the endocytic pathway, the sequence of events occurring during the early stages of endocytosis has yet to be fully understood. Recently, the use of stimulated emission depletion (STED) has provided diffraction-unlimited resolution in fluorescence microscopy. However, simultaneously increasing resolution in all three dimensions has proven less than trivial and phototoxicity to living cells is of concern. The long-term objective of this proposal is to realize a novel super-resolution fluorescence microscope and to apply it to the investigation of the dynamic recruitment of proteins involved in clathrin-mediated endocytosis.
The first aim of this project proposes to combine total internal reflection fluorescence (TIRF) microscopy with dual-color STED microscopy to achieve super-resolution imaging in three dimensions at the membrane facing the cover slip. This novel microscope also features less phototoxicity and increases the signal to noise ratio which makes it particularly useful for live cell and dual-color imaging.
The second aim of this project proposes to use this setup to investigate protein recruitment during the early stages of clathrin-mediated endocytosis. Using TIRF-STED, protein distributions in living cells will be visualized in real time to test the hypothesis that recruitment of dynamin to clathrin-coated pits is mediated by prior recruitment of endophilin. Specifically, dual-color super-resolution imaging will be used to determine the sequence of events at clathrin-coated pits and compare the evolution of protein cluster morphologies in control and endophilin knockout cells to determine the role of endophilin in the recruitment of dynamin.

Public Health Relevance

Misregulation in endocytosis, the process by which cells absorb molecules, has been linked to numerous diseases such as Alzheimer's disease, Down syndrome, and muscular dystrophies. Unfortunately, due to the small size of endocytic sites and the limited resolution of conventional fluorescence microscopes it has not been possible to directly visualize their dynamic molecular organization. By combining the two super- resolution techniques total internal reflection fluorescence and stimulated emission depletion, a novel microscope will be developed featuring high-speed acquisition and low phototoxicity for imaging protein recruitment during clathrin-mediated endocytosis. This novel microscope will also have applications to diagnostic imaging.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM096859-02
Application #
8370560
Study Section
Special Emphasis Panel (ZRG1-F14-C (20))
Program Officer
Flicker, Paula F
Project Start
2011-07-01
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
2
Fiscal Year
2012
Total Cost
$52,190
Indirect Cost
Name
Yale University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Wilfling, Florian; Thiam, Abdou Rachid; Olarte, Maria-Jesus et al. (2014) Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting. Elife 3:e01607
Wilfling, Florian; Wang, Huajin; Haas, Joel T et al. (2013) Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets. Dev Cell 24:384-99
Gould, Travis J; Kromann, Emil B; Burke, Daniel et al. (2013) Auto-aligning stimulated emission depletion microscope using adaptive optics. Opt Lett 38:1860-2
Kromann, Emil B; Gould, Travis J; Juette, Manuel F et al. (2012) Quantitative pupil analysis in stimulated emission depletion microscopy using phase retrieval. Opt Lett 37:1805-7
Gould, Travis J; Hess, Samuel T; Bewersdorf, Joerg (2012) Optical nanoscopy: from acquisition to analysis. Annu Rev Biomed Eng 14:231-54
Gould, Travis J; Burke, Daniel; Bewersdorf, Joerg et al. (2012) Adaptive optics enables 3D STED microscopy in aberrating specimens. Opt Express 20:20998-1009