Biology subjects

Weinberg, R. J.

Publications and source records attributed to Weinberg, R. J..

5 recordsLinked to original sources

bioRxiv2024

Data-driven synapse classification reveals a logic of glutamate receptor composition

The rich diversity of synapses facilitates the capacity of neural circuits to transmit, process and store information. We used multiplex super-resolution proteometric imaging through array tomography to define features of single synapses in mouse neocortex. We find that glutamatergic synapses cluster into subclasses that parallel the distinct biochemical and functional categories of receptor subunits: GluA1/4, GluA2/3 and GluN1/GluN2B. Two of these subclasses align with physiological expectations based on synaptic plasticity: large AMPAR-rich synapses may represent potentiated synapses, whereas small NMDAR-rich synapses suggest "silent" synapses. The NMDA receptor content of large synapses correlates with spine neck diameter, and thus the potential for coupling to the parent dendrite. Overall, ultrastructural features predict receptor content of synapses better than parent neuron identity does, suggesting synapse subclasses act as fundamental elements of neuronal circuits. No barriers prevent future generalization of this approach to other species, or to study of human disorders and therapeutics.

neuroscience↗

bioRxiv2023

Developing a Toolbox of Antibodies Validated for Array Tomography-Based Imaging of Brain Synapses

Antibody-based imaging techniques rely on reagents whose performance may be application-specific. Because commercial antibodies are validated for only a few purposes, users interested in other applications may have to perform extensive in-house antibody testing. Here we present a novel application-specific proxy screening step to efficiently identify candidate antibodies for array tomography (AT), a serial section volume microscopy technique for high-dimensional quantitative analysis of the cellular proteome. To identify antibodies suitable for AT-based analysis of synapses in mammalian brain, we introduce a heterologous cell-based assay that simulates characteristic features of AT, such as chemical fixation and resin embedding that are likely to influence antibody binding. The assay was included into an initial screening strategy to generate monoclonal antibodies that can be used for AT. This approach simplifies the screening of candidate antibodies and has high predictive value for identifying antibodies suitable for AT analyses. In addition, we have created a comprehensive database of AT-validated antibodies with a neuroscience focus and show that these antibodies have a high likelihood of success for postembedding applications in general, including immunogold electron microscopy. The generation of a large and growing toolbox of AT-compatible antibodies will further enhance the value of this imaging technique. Significance StatementArray tomography (AT) is a powerful volume microscopy technique for high-dimensional analysis of complex protein populations in cells and organelles, including synapses. AT involves the use of ultrathin serial sections embedded in resin and subjected to multiple rounds of immunofluorescence antibody (Ab) labeling and imaging. AT relies on antibody-based detection of proteins but because commercial antibodies are typically validated for other applications they often fail for AT. To identify antibodies with high probability of success in AT we developed a novel screening strategy and used this to create a comprehensive database of AT-validated antibodies for neuroscience.

neuroscience↗

bioRxiv2018

Myelinated inhibitory axons in human neocortex

Numerous myelinated axons traverse the human neocortex. In a previous paper (Micheva et al., 2016) we showed that in mouse many of these axons belong to local inhibitory neurons, the parvalbumin-positive basket cells. Here, using samples of neurosurgically-excised cortex, we confirm the presence of myelinated inhibitory axons in all layers of human neocortex. As in mouse, these axons have distinctive features, including high neurofilament content, short nodes of Ranvier, and high content of myelin basic protein in their myelin sheath. We further show that, consistent with the known high-energy demands of parvalbumin interneurons, the inhibitory myelinated axons have more mitochondria, as well more 2,3-cyclic nucleotide 3-phosphodiesterase (a protein enriched in the myelin cytoplasmic channels thought to provide access for trophic support from oligodendrocytes). The distinctive features of myelinated inhibitory axons in human cortical grey matter may have important implications for neurological disorders that involve pathologies of myelinated axons.

neuroscience↗

bioRxiv2018

Automated Antibody Characterization and Screening for Array Tomography via Probabilistic Synapse Detection

Application-specific validation of antibodies is a critical prerequisite for their successful use. Here we introduce an automated framework for characterization and screening of antibodies against synaptic molecules for high-resolution immunofluorescence array tomography (AT). The proposed Synaptic Antibody Screening Tool (SACT), is designed to provide an automatic, robust, flexible, and efficient tool for antibody characterization at scale. By allowing the user to define the molecular composition and size of synapses expected to contain the antigen, the method detects and characterizes puncta and synapses, and outputs automatically computed characteristics such as synapse density and target specificity ratio, which reflect the sensitivity and specificity of immunolabeling with a given antibody. These measurements provide an objective way to characterize and compare the performance of different antibodies against the same target, and can be used to objectively select the antibodies best suited for AT and potentially for other immunolabeling applications.

neuroscience↗

bioRxiv2017

TRIM9-dependent ubiquitination of DCC constrains kinase signaling, exocytosis, and axon branching

Extracellular netrin-1 and its receptor DCC promote axon branching in developing cortical neurons. Netrin-dependent morphogenesis is preceded by multimerization of DCC, activation of FAK and Src family kinases, and increases in exocytic vesicle fusion, yet how these occurrences are linked is unknown. Here we demonstrate that TRIM9-dependent ubiquitination of DCC blocks the interaction with and phosphorylation of FAK. Upon netrin-1 stimulation TRIM9 promotes DCC multimerization, but TRIM9-dependent ubiquitination of DCC is reduced, which promotes an interaction with FAK and subsequent FAK activation. We found that inhibition of FAK activity blocks elevated frequencies of exocytosis in vitro and elevated axon branching in vitro and in vivo. Although FAK inhibition decreased SNARE-mediated exocytosis, assembled SNARE complexes and vesicles adjacent to the plasma membrane were increased, suggesting a novel role for FAK in the progression from assembled SNARE complexes to vesicle fusion in developing murine neurons.\n\nAbbreviations used in this paper

cell biology↗