weisshaa

Quantitative fluorescence microscopy in vivo and in vitro

Graduate Students: Somenath Bakshi, Ken Barns, Heejun Choi, Renee Dalrymple, Trillian Gregg, Wenting Li, and Nambirajan Rangarajan.

Research Interests: Fluorescence microscopy of biological systems. Design and characterization of a fast, SNARE-driven in vitro single-vesicle fusion assay. Tracking of single proteins in live cells with 10 nm spatial resolution. Architecture of vesicle fusion machinery. Spatial biology of the transcription and translation systems in bacterial cells. Direct observation of the attack of antimicrobial agents on bacterial cell membranes.

We participate in the revolution in the fluorescence microscopy of biological systems. It is increasingly possible to observe the motion of proteins and DNA with single-molecule precision in live cells and in contexts approximating natural conditions. The result is an unprecedented, high resolution view of biological structure and activity.  Areas of current focus include:

(1) The structure and function of the proteins responsible for Ca²⁺-triggered synaptic vesicle fusion, the elementary step in neuron-neuron communication. The new PALM methodology enables us to track single copies of Syntaxin in live PC-12 cells with ~20-nm spatial resolution and 20-ms time resolution, for example.

(2) The diffusive motion and spatial distribution of GFP-labeled proteins in live E. coli cells. Species of interest include RNA polymerase, architectural proteins, and 30S ribosomal subunits. FRAP (Fig. 2) and single-molecule tracking (Fig. 3) enable us to directly observe the motion and spatial distributions of key elements of the transcription/translation machinery.

The ribosomes, the nucleoid, and RNAP all exhibit a remarkable level of time-varying spatial organization.  Simple “toy models” developed with the Yethiraj group may help understand the organization based on excluded volume and entropic effects alone (Fig. 4).

(3) The time-resolved attack of antimicrobial agents on bacterial cell membranes.  Examples include LL-37 (Fig. 5), a human antimicrobial peptide, and synthetic random copolymers designed by the Gellman group. Simultaneous two-color imaging of the antimicrobial and cytoplasmic or periplasmic GFP yields unprecedented insight into the mechanism of attack.