Hein, K. | Search bioRxiv

bioRxiv2024

EVE is an open modular data analysis software for event-based localization microscopy

Event-based sensors (EBS), or neuromorphic vision sensors, offer a novel approach to imaging by recording light intensity changes asynchronously, unlike conventional cameras that capture light over fixed exposure times. This capability results in high temporal resolution, reduced data redundancy, and a wide dynamic range. This makes EBS ideal for Single-Molecule Localization Microscopy (SMLM) as SMLM relies on the sequential imaging of sparse, blinking fluorescent emitters to achieve super-resolution. Recent studies have shown that EBS can effectively capture these emitters, achieving spatial resolution comparable to traditional cameras. However, existing analyses of event-based SMLM (eveSMLM) data have relied on converting event lists into image frames for conventional analysis, limiting the full potential of the technology. To overcome this limitation, we developed EVE, a specialized software for analyzing eveSMLM data. EVE offers an integrated platform for detection, localization, and post-processing, with various algorithmic options tailored for the unique structure of eveSMLM data. EVE is user-friendly and features an open, modular infrastructure that supports ongoing development and optimization. EVE is the first dedicated tool for event-based SMLM, transforming the analysis process to fully utilize the spatiotemporal data generated by EBS. This allows researchers to explore the full potential of eveSMLM and encourages the development of new analytical methods and experimental improvements.

biophysics↗

bioRxiv2018

Structural and functional studies of the RBPJ-SHARP complex reveal conserved corepressor binding site

The Notch pathway is a conserved signaling mechanism that is essential for cell fate decisions during pre and postnatal development. Dysregulated signaling underlies the pathophysiology of numerous human diseases, most notably T-cell acute lymphoblastic leukemia. Receptor-ligand interactions result in changes in gene expression, which are regulated by the transcription factor CSL. CSL forms a complex with the intracellular domain of the Notch receptor and the transcriptional coactivator Mastermind, which is required to activate transcription of all Notch target genes. CSL can also function as repressor by interacting with corepressor proteins, e.g. SHARP in mammals and Hairless in Drosophila melanogaster; however, its role as a transcriptional repressor is not well understood. Here we determine the high-resolution structure of RBPJ, the mouse CSL ortholog, bound to the corepressor SHARP and DNA, which reveals a new mode of corepressor binding to CSL and an interesting example for how ligand binding sites evolve in proteins. Based on the structure, we designed and tested a number of mutants in biophysical, biochemical, and cellular assays to characterize the role of RBPJ as a repressor of Notch target genes. Our cellular studies clearly demonstrate that RBPJ mutants that are deficient for binding SHARP are incapable of repressing transcription from genes responsive to Notch signaling. Altogether, our structure-function studies of the RBPJ-SHARP corepressor complex bound to DNA provide significant insights into the repressor function of RBPJ and identify a new binding pocket on RBPJ that could be targeted for therapeutic benefit.

biochemistry↗