Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

64-POS Board 32 Dynamics of DNA in Living Cells Probed by Fluorescence Correlation Microrheology Cameron Hodges 1 , Rudra Kafle 2 , Jens-christian Meiners 1 . 1 University of Michigan, Ann Arbor, MI, USA, 2 Worcester Polytechnic Institute, Worcester, MA, USA. DNA inside a living cell is highly dynamic. In order to fulfil its biological functions, it has to move to come in contact with distant sites or protein complexes. Yet surprisingly little is known about how DNA actually moves around inside a cell, and what is driving and constraining that motion. We are using fluorescence correlation spectroscopy (FCS) to study the fluctuations of bacterial chromosomal DNA in live and ATP-depleted E. Coli with high temporal resolution. We find that the bacterial chromosome in fully metabolically active cells is softer and more fluid than in the ATP-depleted ones. While this is at odds with the notion of DNA as an entropic spring in the presence of active fluctuations, we can explain our observations with a model in which proteins that weakly crosslink the bacterial DNA are constantly driven off the DNA by processive motor enzymes like RNA polymerase, which keeps the chromosome more liquid than the gel-like state that it assumes in the absence of these processes. For these experiments, we have developed a quantitative live-cell FCS microrheology technique that allows us to measure the viscous and elastic modulus of the DNA over a broad frequency range from hundred microseconds to seconds. We will discuss pitfalls of quantitative live cell FCS, and methods to effectively overcome sources of artifacts, especially photobleaching. We will also present a first application of our technique to HeLa cells.

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