Biology subjects

Seeger, R.

Publications and source records attributed to Seeger, R..

3 recordsLinked to original sources

bioRxiv2024

CryoVesNet: A Dedicated Framework for Synaptic Vesicle Segmentation in Cryo Electron Tomograms

Cryo-electron Tomography (Cryo-ET) has the potential to reveal cell structure down to atomic resolution. Nevertheless, cellular cryo-ET data is often highly complex, and visualization, as well as quantification, of subcellular structures require image segmentation. Due to a relatively high level of noise and anisotropic resolution in cryo-ET data, automatic segmentation based on classical computer vision approaches usually does not perform satisfactorily. For this reason, cryo-ET researchers have mostly performed manual segmentation. Communication between neurons relies on neurotransmitter-filled synaptic vesicle (SV) exocytosis. Recruitment of SVs to the plasma membrane is an important means of regulating exocytosis and is influenced by interactions between SVs. Cryo-ET study of the spatial organization of SVs and of their interconnections allows a better understanding of the mechanisms of exocytosis regulation. Extremely accurate SV segmentation is a prerequisite to obtaining a faithful representation of SVs state of connectivity. Hundreds to thousands of SVs are present in a typical synapse, and their time-consuming manual segmentation is a bottleneck in this analysis. Several attempts to automate vesicle segmentation by classical computer vision or machine learning algorithms have not yielded robust results. We addressed this problem by designing a workflow consisting of a U-Net convolutional segmentation network followed by post-processing steps. This combination yields highly accurate results. Furthermore, we provide an interactive tool for accurately segmenting spherical vesicles in a fraction of the time required by available manual segmentation methods. This tool can be used to segment vesicles that were missed by the fully automatic procedure or to quickly segment a handful of vesicles while bypassing the fully automatic procedure. Our pipeline can in principle be used to segment any spherical vesicle in any cell type as well as extracellular vesicles.

neuroscience↗

bioRxiv2022

Targeting the BAG-1 family of co-chaperones in lethal prostate cancer.

Therapies that abrogate persistent androgen receptor (AR) signaling in castration resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain (NTD) of the AR drives transcriptional activity in CRPC but is intrinsically disordered and remains a challenging therapeutic target. Therefore, inhibiting critical co-chaperones, such as BAG-1L, is an attractive alternative strategy. We performed druggability analyses demonstrating the BAG domain to be a challenging drug target. Thio-2, a tool compound, has been reported to bind the BAG domain of BAG-1L and inhibit BAG-1L-mediated AR transactivation. However, despite these data, the mechanism of action of Thio-2 is poorly understood and the BAG domain which is present in all BAG-1 isoforms has not been validated as a therapeutic target. Herein, we demonstrate growth inhibiting activity of Thio-2 in CRPC cell lines and patient derived models with decreased AR genomic binding and AR signaling independent of BAG-1 isoform function. Furthermore, genomic abrogation of BAG-1 isoforms did not recapitulate the described Thio-2 phenotype, and NMR studies suggest that Thio-2 may bind the AR NTD, uncovering a potential alternative mechanism of action, although in the context of low compound solubility. Furthermore, BAG-1 isoform knockout mice are viable and fertile, in contrast to previous studies, and when crossed with prostate cancer mouse models, BAG-1 deletion does not significantly impact prostate cancer development and growth. Overall, these data demonstrate that Thio-2 inhibits AR signaling and growth in CRPC independent of BAG-1 isoforms, and unlike previous studies of the activated AR, therapeutic targeting of the BAG domain requires further validation before being considered a therapeutic strategy for the treatment of CRPC.

molecular biology↗

bioRxiv2022

Morphofunctional changes at the active zone during synaptic vesicle exocytosis

The fusion of synaptic vesicles (SVs) with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. Additionally, the effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Through time-resolved cryo-electron tomography, we ordered events into a sequence. Following stimulation, proximal tethered SVs rapidly form additional tethers with the PM. Simultaneously, fusion initiation occurs by membrane curvature ( buckling) of the SV and PM. It is followed by the formation of a fusion pore, and the collapse of SV membrane. At this time, membrane-proximal, but not membrane-distal, vesicles lose their interconnections, allowing them to move towards the PM. Two mutations of SNAP-25 that arrests or disinhibit spontaneous release, respectively, both caused a loss of interconnectors, while the disinhibiting mutant also caused a loss of membrane proximal multiple-tethered SVs. Overall, tether formation and connector dissolution is triggered by stimulation and respond to the spontaneous fusion rate. These morphological observations likely correspond to the transition of SVs from one functional pool to another.

neuroscience↗