Biophysical characterization of nucleo-cytoplasmic transport and short length scale DNA bending using integrated optical probes

Cells are complex systems housing a multitude of coordinated and interconnected functions. Some molecular components and functions are amenable to direct biophysical study by in vitro coupling to external stimuli and/or observation mechanisms, but for many systems of interest this approach is not feasible. Here we detail the creation of integrated optical probes to investigate to two systems that are important in maintaining correct cellular functions: bending of DNA over short regions and transport between the cytoplasm and the nucleus.;DNA, the genetic blueprint of the cell, is not simply a line waiting to be read out, but has a complex topology and set of interactions with various proteins. It is tightly wrapped around nucleosomes, sharply bent by proteins such as the TATA-box Binding Protein, and distal sites are often brought into close proximity as a way to regulate gene expression. The physics of DNA plays an important role in determining how much energy is required to distort the DNA into these correct forms. The wormlike chain polymer physics model has been validated as a way to describe the behavior of long pieces of DNA. The experimentally determined persistence length (the length over which the polymer has a "memory" of it's position) is approximately 150 basepairs (bp), or 50 nanometers (nui) W. In cells, DNA is frequently sharply bent on length scales much shorter than this. For instance 147 bp (roughly one persistence length) is wrapped 1.7 times around the nucleosome core 121. By incorporating a previously calibrated molecular force sensor into a short length of circular DNA we were able to measure the physical strain of highly bent pieces of dsDNA by Forster Resonance Energy Transfer (FRET) and test various models for their applicability to short pieces of DNA. The helical worm-like chain model surprisingly explains the steady-state strain for all but the shortest of bent dsDNA pieces.;The creation of these constructs allowed us the ability to test other properties of highly bent DNA. The strain on the double stranded DNA is shown to not be relieved by either localized melting or transient breathing. We investigated the role of DNA bending strain on the binding affinity of Integration Host Factor (IHF), a bacterial minor groove binding protein induces a 160 degree bend in in the DNA over a 30 basepair stretch of DNA upon binding. The binding affinity of a consensus sequence of bent DNA is an order of magnitude higher than a similar piece of linear DNA. We showed that a relatively modest 3 picoNewtons of bending force is sufficient to enable this tighter binding and that DNA under increased strain does not bind any tighter. Additionally, the binding affinity of mutant sequences, which bind orders of magnitude weaker than the consensus sequence when linear, can be rescued to almost the same tighter binding level by first putting the DNA under strain.;In eukaryotic cells, a nuclear envelope separates the DNA from the rest of the cell body during interphase, when the cell is not dividing Access into and out of the nucleus, however, is required for cellular regulation of gene expression. The Nuclear Porc Complex (NPC) is the only channel connecting the nucleus to the cytoplasm, thus all material moving into or out of the nucleus must pass through an NPC. Small molecules (up to roughly 9 nm in diameter) can freely diffuse through the NPC [3], but larger cargo must bind to a transport receptor in order to translocate. Despite intense study, the mechanism by which the NPC allows for fast, bidirectional, but highly selective transport remains unclear. We created another integrated optical probe, a synthetic protein coupled Quantum Dot cargo, to track at high spatial and temporal precision as it was specifically imported via the importin beta mediated import pathway. We found translocation involves multiple substeps, including a size limit inside the central channel, solubility dependent diffusion inside the central channel, and a Ran-GTP regulated irreversible release step on the nuclear side of the NPC.