Fluorescence microscopy allows us to create contrast between labeled molecules of interest and background in order to directly monitor labeled molecules. That is, fluorescence microscopy allows us to gather insights on macromolecular actors performing tasks relevant to life. An important factor limiting the spatial resolution accessible by fluorescence microscopy, and ultimately what we can learn from fluorescence microscopy, is set by the wavelength of light emitted by labels on molecules. On account of this limitation, accessing information on spatial scales smaller than light's "diffraction limit", typically hundreds of times bigger than the molecules we study, often requires tuning the photophysical properties of the labels (through methods termed superresolution) and limiting our analysis to static samples in time. As such, monitoring events relevant to life is limited to what we can learn from fixed, static, snapshots. In an ideal world, we would like to monitor life's events as they occur in real, continuous, time at a spatial scale below light’s diffraction limit. Here we propose a means by which to extend the superresolution paradigm to dynamical samples by computation alone. That is, we apply our tools to resolving the tracks of multiple (labeled) molecules operating within a diffraction-limited region. The key idea here is to leverage Bayesian nonparametrics allowing us to systematically learn trajectories of particles as well as the number of particles in a joint optimization simultaneously leveraging information across all frames and pixels within a frame. As time allows, we will expand upon how this tracking method differs from the existing tracking paradigm that has remained diffraction limited.
Animals use behavior to meet their goals of exploration for food and survival. In varying thermal environments, there is an impetus to use behavior to avoid extreme temperatures that could prove fatal. Due to the lack of internal mechanisms of thermoregulation, ectotherms such as the larval zebrafish rely solely on their behavior to navigate their surroundings and achieve homeostasis. This makes them ideal for studying relations between their external stimuli and behavioral response. Additionally, their behavioral repertoire consists primarily of individual bouts interlaced with periods of rest, which makes their movements discrete. Our experiments suggest that zebrafish avoid high temperatures by raising their swim speeds and performing evasive turns when faced with worsening temperatures. In contrast, the fish avoids colder temperatures using targeted movements away from unpleasant temperatures while maintaining a lower swim speed. Interestingly, previous research suggests that fish control their movements across multiple bouts and trajectories differ in their shape at longer timescales. We aim to distinguish between these behaviors at cold and hot temperatures at a behavioral level and the neuronal level.
Transcription factors (TFs) function by binding specific DNA sequences in gene promoters/enhancers to initiate transcription. TF binding sites are frequently found within the nucleosome near the DNA entry-exit region. The TF's DNA binding site position within the nucleosome entry-exit region significantly influences TF occupancy, but how is less quantitatively understood. Recent single-molecule assays show that Cbf1, a budding yeast basic helix-loop-helix leucine zipper (bHLHZ) TF, can access its transiently exposed binding sites within partially unwrapped nucleosomes. To probe the targeting and occupancy of Cbf1dN at different positions within the nucleosomes, we carried out both ensemble and single-molecule fluorescence resonance energy transfer (FRET) measurements. We prepared a set of eight different nucleosomes with different binding site positions in the nucleosomal DNA. First, the ensemble FRET experiments showed a dramatic decrease in TF occupancy to sites extending past 30 base pairs into the nucleosome. This decreased accessibility matches the unwrapping free energy landscape with a boundary at 30bp into the nucleosome where the inner region is over 10-fold less accessible. Second, we found a reduced ensemble FRET changes from binding site position 6-18 to 10-22, which indicates that the binding site orientation can influence how Cbf1dN unwraps DNA away from the histone octamer. The more binding site positions face away from the histone octamer, the less ensemble FRET changes will be. Recent solved structures from Tan lab, using the same nucleosome constructs with different binding site positions, provides the DNA unwrapping conditions consistent with the ensemble FRET data. The structures also revealed that Cbf1dN has favorable interaction with histone H2A tail with binding site position 17-29. SmFRET assays will be used to clarify how this Cbf1dN-H2A interaction will impact Cbf1dN occupancy and kinetics.
Last update: 9/28/2023, Ralf Bundschuh