California NanoSystems Institute
CNSI
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March 03, 2016

Masahide Kikkawa, MD, PhD
Professor of Cell Biology & Anatomy,
Graduate School of Medicine,
The Univeristy of Tokyo
“Structural Gentics” of Eukaryotic Cilia/Flagella by Cryo‐Electron Tomography
Abstract: Eukaryotic cilia and flagella are complex cell organelles consisting of more than hundreds of different proteins. They play important roles, such as propeller in trachea and antenna in kidney, in our body. To understand the mechanisms of cilia/flagella, one of the challenges is to determine where each protein is located within the organelle and to identify their functions. For example, it has been a long‐standing question of how dyneins are precisely arranged along the microtubule with 24‐nm or 96‐nm repeats.

To address this challenge, we combine genetics and structural biology. We have developed a novel method for identifying the 3D locations of proteins by combining cryoelectron tomography and genetic manipulation of model organisms to introduce biotin‐tag to specific genes. Using this method, we studied the FAP59/172 complex, which forms a complex, and their absence disrupts 96‐nm repeats in axonemes. Cryo‐electron tomography revealed that the FAP59/172 complex takes a 96‐nm‐long extended conformation along axonemal microtubules. Elongation of the complex resulted in extension of the repeats and duplication of specific axonemal components. We conclude that the FAP59/172 complex is the molecular ruler that defines 96‐nm repeats in cilia/flagella.
March 01, 2016

Julie Biteen, PhD
Assistant Professor of Chemistry,
University of Michigan
Single‐Molecule Imaging and Plasmon‐Enhanced Fluorescence: Understanding Bacterial Function on the Nanoscale
Abstract: By beating the diffraction limit that restricts traditional light microscopy, single‐molecule fluorescence imaging is a precise, noninvasive way to sensitively probe position and dynamics. We are pioneering super‐resolution imaging methods for unraveling important biological processes in live bacteria, and I will discuss how we understood the mechanism of membrane‐bound transcription regulation in a pathogen, and revealed an in􀆟mate and dynamic coupling between DNA mismatch recognition and DNA replication in a highly conserved repair pathway. Still, the resolution of single‐molecule imaging, and thus our ability to understand subcellular dynamics, is limited by the fluorescent probes. Thus, we take advantage of the localized surface plasmon resonances that result from the interaction of light with small metal nanoparticles to improve the brightness and photostability of nearby fluorescent labels. We have measured fundamental properties of plasmon‐enhanced fluorescence with single‐molecule detection, and we have discovered how coupling leads to a predictable shift of the emission position. Finally, we are applying this understanding to biocompatible enhancement of fluorescent protein emission, extending the advantages of metal‐enhanced fluorescence to live‐cell bio‐imaging, and creating a flexible technology for high‐resolution, real‐time imaging.