Diffusion and conformational dynamics of single DNA molecules crowded by cytoskeletal proteins

The high concentrations of proteins crowding cells greatly influence intracellular DNA dynamics. These crowders, ranging from small mobile proteins to large cytoskeletal filaments such as semiflexible actin and rigid microtubules, can hinder diffusion and induce conformational changes in DNA. While previous studies have mainly focused on the effect of small mobile crowders on DNA transport, we focus instead on the role of the cytoskeleton. Specifically, we use fluorescence microscopy and custom single-molecule tracking algorithms to measure center-of-mass transport and time-varying conformational changes of single DNA molecules diffusing in in vitro networks of actin and microtubules. To determine the roles that cytoskeletal filament rigidity and size have on DNA dynamics, we vary the relative concentrations and polymerization states of actin and microtubules crowding DNA and quantify resulting DNA diffusion coefficients, degrees of anomalous diffusion, and changes to conformational size and shape.