Biology subjects

Margolin, B.

Publications and source records attributed to Margolin, B..

3 recordsLinked to original sources

bioRxiv2023

Oblique Line Scan Illumination Enables Expansive, Accurate and Sensitive Single Protein Measurements in Solution and in Living Cells

Single-molecule localization microscopy (SMLM) techniques, such as single-molecule tracking (SMT), enable in situ measurements in cells from which data-rich metrics can be extracted. SMT has been successfully applied to a variety of biological questions and model systems, aiming to unravel the spatiotemporal regulation of molecular mechanisms that govern protein function, downstream pathway effects, and cellular function. While powerful, SMLM often suffers from low throughput and illumination inhomogeneity, along with microscope and user-induced technical biases. Due to technical limitations in scaling SMLM techniques, a tradeoff between spatiotemporal resolution and throughput has been made historically, restricting broad application of these technologies. Here we address these limitations using Oblique Line Scan (OLS), a robust single-objective light-sheet based illumination and detection modality that achieves nanoscale spatial resolution and sub-millisecond temporal resolution across a 250 x 190 m field of view. We demonstrate OLS-enabled SMT on Halo-Tagged proteins in living cells capturing protein motion up to 14 m2 /s. By exploiting the adaptability of the acquisition frame rate and the improved rejection of out of focus light, we extend the utility of OLS beyond cellular compartments with in-solution SMT (isSMT) for single-molecule measurement of ligand-protein interactions and disruption of protein-protein interactions (PPI). We illustrate the versatility of OLS by showcasing two-color SMT, STORM, and single molecule fluorescence recovery after photobleaching (FRAP). OLS expands the range of SMLM applications and paves the way for robust, high-throughput single-molecule investigations of protein dynamics required for drug screening and systems biology studies, both in cells and in solution.

biophysics↗

bioRxiv2023

High-throughput single molecule tracking identifies drug interactions and cellular mechanisms

The regulation of cell physiology depends largely upon interactions of functionally distinct proteins and cellular components. These interactions may be transient or long-lived, but often affect protein motion. Measurement of protein dynamics within a cellular environment, particularly while perturbing protein function with small molecules, may enable dissection of key interactions and facilitate drug discovery; however, current approaches are limited by throughput with respect to data acquisition and analysis. As a result, studies using super-resolution imaging are typically drawing conclusions from tens of cells and a few experimental conditions tested. We addressed these limitations by developing a high-throughput single-molecule tracking (htSMT) platform for pharmacologic dissection of protein dynamics in living cells at an unprecedented scale (capable of imaging > 106 cells/day and screening > 104 compounds). We applied htSMT to measure the cellular dynamics of fluorescently tagged estrogen receptor (ER) and screened a diverse library to identify small molecules that perturbed ER function in real time. With this one experimental modality, we determined the potency, pathway selectivity, target engagement, and mechanism of action for identified hits. Kinetic htSMT experiments were capable of distinguishing between on-target and on-pathway modulators of ER signaling. Integrated pathway analysis recapitulated the network of known ER interaction partners and suggested potentially novel, kinase-mediated regulatory mechanisms. The sensitivity of htSMT revealed a new correlation between ER dynamics and the ability of ER antagonists to suppress cancer cell growth. Therefore, measuring protein motion at scale is a powerful method to investigate dynamic interactions among proteins and may facilitate the identification and characterization of novel therapeutics.

cell biology↗

bioRxiv2019

Locomotor suppression by a monosynaptic amygdala to brainstem circuit

The control of locomotion is fundamental to vertebrate animal survival. Defensive situations require an animal to rapidly decide whether to run away or suppress locomotor activity to avoid detection. While much of the neural circuitry involved in defensive action selection has been elucidated, top-down modulation of brainstem locomotor circuitry remains unclear. Here we provide evidence for the existence and functionality of a monosynaptic connection from the central amygdala (CeA) to the mesencephalic locomotor region (MLR) that inhibits locomotion in unconditioned and conditioned defensive behavior in mice. We show that locomotion stimulated by airpuff coincides with increased activity of MLR glutamatergic neurons. Using retrograde tracing and ex vivo electrophysiology, we find that the CeA makes a monosynaptic connection with the MLR. In the open field, in vivo stimulation of this projection suppressed spontaneous locomotion, whereas inhibition of this projection had no effect. However, inhibiting CeA terminals within the MLR increased both neural activity and locomotor responses to airpuff. Finally, using a conditioned avoidance paradigm known to activate CeA neurons, we find that inhibition of the CeA projection increased successful escape, whereas activating the projection reduced escape. Together these results provide evidence for a new circuit substrate influencing locomotion and defensive behaviors.

neuroscience↗