Microtubules are a major component of the cell cytoskeleton, providing cellular structure and facilitating many cellular processes. The tubulin subunits are heterodimers of alpha- and beta-tubulin, and both subunits have multiple isotypes. Differences amongst isotypes occur primarily in the C-terminal tail, which is the intrinsically disordered, negatively charged region at the end of each alpha- and beta-tubulin subunit. Due to their disordered nature, C-terminal tails are not visible in structural studies, but they are known to be primary interaction points for many microtubule-associated proteins and their interactions and conformations are known to be affected by pH.

Microtubules are super structures within the cell that are composed of polymerized alpha and beta tubulin heterodimers and play essential roles in cell division and motility. The alpha and beta tubulin monomers contain short, glutamate-rich, disordered C-terminal tails. Despite their size and flexibility, the C-terminal tails play important roles in microtubule dynamics, regulation, and interactions. The C-terminal tails vary across species and isoforms and generally contain a high net negative charge, indicating an essential role for the polyglutamic acid patches in the C-terminal tails. We hypothesized that these regions are sensitive to the changes in intracellular pH upon cellular stress. Using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics simulations, and binding assays, we determined that the C-terminal tails of alpha and beta tubulins are pH-sensitive in a physiological range. Our results have consequences for cellular regulation of tubulin intra- and intermolecular interactions.

The nonphysiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels affect therapeutic response by performing drug screening in human plasma-like medium. We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds, including rigosertib, an experimental cancer therapeutic that recently failed in phase III clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism end product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. These results demonstrate the broad and dramatic effects nutrient levels can have on drug response and how incorporation of human-specific physiological nutrient medium might help identify compounds whose efficacy could be influenced in humans.

Fluorescence Correlation Spectroscopy (FCS) measures the concentrations and dynamics of fluorescent particles by analyzing the correlation in the concentration fluctuation of particles diffusing in a solution. Due to its diffraction-limited sample volume, the method is only applicable for concentrations that are typically in the nM range. However, biological processes under physiological conditions often involve molecules in the μM concentration range and require sample volumes well below the established limit of about one femtoliter in traditional FCS.

Single molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM dataset is an essential aspect of the image processing and analysis pipeline. Prior work has employed pair auto-correlation functions to directly measure the effective spread of localizations arising from single labeled objects, providing a method for measuring the lateral resolution of an image. Here we incorporate an explicit temporal dependence into this approach, calculating full space-time auto-correlation functions to quantify resolution as a function of the time lapse between localizations. In doing so we find that the past approach reports primarily on localization precision since correlation functions are often dominated by contributions from multiple localizations of the same fluorophore at short time intervals, especially when single molecule blinking kinetics resembles (d)STORM. Examining resolution at longer time-intervals reports on other factors that limit resolution, such as sample drift or imperfect drift correction. This resolution metric reports on how precisely one can measure pairwise distances between labeled objects and is complementary to the commonly used Fourier ring correlation (FRC) metric that also considers spatial sampling. The method is demonstrated on simulated localizations, DNA origami rulers, and antibody labeled cellular structures.

Fluorescence Correlation Spectroscopy (FCS) is a method for investigating particle concentration fluctuations in small volumes that are usually defined by a focused laser beam for excitation and a confocal pinhole for collection resulting in a size of about one femtoliter. Such sampling volumes are suitable for investigating dynamics of molecules in the nM concentration range. However, biological processes on cell membranes that involve molecules in the μM concentration range require sampling volumes well below the conventional FCS limit as well as nanoscale confinement in the longitudinal direction. In this study, we demonstrate that an effective measurement volume as small as one zeptoliter can be achieved via the introduction of a nanowire waveguide resulting in an illumination spot of about 50 nanometers in lateral dimensions and a longitudinal confinement of around 20 nanometers. Using illumination profiles obtained from finite element method simulations of dielectric nanowaveguides, we perform Monte Carlo simulations of fluorescence fluctuation for fluorophores that are confined in a membrane situated directly above the nanowaveguide exit surface with a background of fluorophores freely diffusing in the solution above the membrane. We have developed an analytical model to fit the simulation results and show that the signal is dominated by fluorophores on the membrane due to the extreme confinement of illumination in the longitudinal direction. These nanowaveguide devices are being fabricated and experiments are currently under way to verify the simulation results.