Quantitative Super-Resolution Microscopy is evolving into a powerful technique to study biological processes below the optical diffraction limit. However, a deeper understanding of the biological structures under investigation is often limited by the inability to quantify their molecular composition while resolving their size and shape. I will present our solution to this challenge, an intracellular calibrated Photoactivated Localization Microscopy (PALM) approach, that can measure the size of individual organelles with 20nm resolution as well as count the absolute numbers of molecules they contain.
Using this technique, we simultaneously characterized the size of individual vesicles in the yeast endocytic pathway and the number of accessible PI3P binding sites they contain. The analysis of many steady-state super-resolution snapshots revealed a characteristic vesicle maturation trajectory of composition and size as well as mechanistic information indicating that PI3P production precedes fusion into larger endosomes. Colocalization analysis with known markers of vesicle maturation showed that these regulatory proteins (clathrin, Vps21, Ypt7) associate with different regions of the observed endosome maturation trajectory. I will conclude by presenting ongoing technical developments from my new lab to correct for movement of organelles during PALM imaging in live cells.
In addition I will present the application of single molecule tracking to study the spatial clustering of signaling complexes, which presents an additional regulatory layer that actively tunes pathway gain. We demonstrate that clustering of the signaling complex itself activates the pathway bypassing receptor activation and that the degree of clustering correlates with the adaptive output.
Dr. Puchner obtained his B.S. from Ludwig Maximilians University, Munich Germany and Ph.D. from Ludwig Maximilians University, Munich Germany with Dr. Hermann Gaub. His postdoctoral work was with Dr. Wendell Lim at the University of California, San Francisco.
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MJIS 1001, Purdue University, West Lafayette, IN