Suliana Manley : Super-resolution imaging and single-molecule tracking, from viruses to chromatin

We apply super-resolution imaging and single-molecule tracking to gain insight into how proteins assemble to form organized structures in cells. We describe several new tools that were developed to study diverse systems, from viruses to chromatin. The HIV structural protein Gag assembles to form spherical particles of radius ~70 nm. During the assembly process, the number of Gag proteins increases over several orders of magnitude, from a few at nucleation to thousands at completion. We demonstrated an approach that permits quantitative morphological and molecular counting analysis of hundreds of HIV-Gag clusters at the cellular plasma membrane, thus elucidating how different fluorescent labels can change the assembly of virions. Higher-order chromatin structure determines the degree of local DNA condensation, which in turn influences gene accessibility and therefore the expression of particular genes. We present two complementary approaches to address this limitation: super-resolution imaging of directly labeled DNA, and singlemolecule high density tracking of proteins participating in DNA packaging. For STORM imaging of DNA, we stained cells with the DNA-specific dye Picogreen, and obtained a ~5-fold improvement in resolution, resolving the sub-diffraction organization of chromatin structures in living cells. For single molecule tracking (sptPALM), we used small chemical tags to target synthetic dyes to specific protein targets, and visualized their dynamics3. The combination of DNA and protein superresolution imaging and single particle tracking will allow us to study chromatin organization in living cells, and rearrangements in response to exogenous signals.

• Category: Nonlinear and Complex Systems
• Duration: 01:31:38
• Date:  September 28, 2012 at 2:45 PM
• Views: 103
• Tags:   seminar, CNCS Seminar