Biology subjects

Hampton, C. M.

Publications and source records attributed to Hampton, C. M..

5 recordsLinked to original sources

bioRxiv2025

Uncovering synaptic and cellular nanoarchitecture of brain tissue via seamless in situ trimming and milling for cryo-electron tomography

Cell-cell communication underlies all emergent properties of the brain, including cognition, learning and memory. The physical basis for these communications is the synapse, a multi-component structure requiring coordinated interactions between diverse cell types. However, many aspects of three-dimensional (3D) synaptic organization remain poorly understood. Here, we developed an approach, seamless in situ trimming and milling (SISTM), to reliably fabricate sufficiently thin lamellae for mapping of the 3D nanoarchitecture of synapses in mouse, monkey and human brain tissue under near-native conditions via cryo-electron tomography (cryo-ET). We validated SISTM in a mouse model of Huntingtons disease, demonstrating distinct 3D alterations to synaptic vesicles and mitochondria. By successfully applying SISTM to macaque brain, we described the 3D architecture of a tripartite synapse within the cortex. Subtomogram averaging (STA) enabled spatial mapping of astrocyte-neuron contacts within the tripartite synapse, revealing neurexin-neuroligin complexes as potential constituents that tether the two cell types. Finally, we showed that the defining features of synaptic nanoarchitecture were conserved across species and evident in human brain tissue obtained postmortem. Combining SISTM with cryo-ET and STA is a starting point for a new understanding of brain organization, disease-induced structural alterations and the development of rational, structure-guided therapeutics.

cell biology↗

bioRxiv2024

Ribosome-Associated Vesicles promote activity-dependent local translation

Local protein synthesis in axons and dendrites underpins synaptic plasticity. However, the composition of the protein synthesis machinery in distal neuronal processes and the mechanisms for its deployment to local translation sites remain unclear. Here, we employed a multi-scale imaging approach combining cryo-electron tomography, volume electron microscopy, and live-cell imaging to identify endoplasmic reticulum-derived Ribosome-Associated Vesicles (RAVs) as a dynamic platform for moving ribosomes to distal processes and promoting activity-dependent local translation. We demonstrate that neuronal stimulation triggers compartment-specific RAV responses: dendrites accumulate stationary RAVs at sites of enhanced translation, while axons accelerate RAV transport. Real-time imaging of translation at single mRNA resolution reveals that RAVs boost local translation output compared to RAV-independent mechanisms. These findings establish RAVs as specialized platforms that integrate activity-dependent signals with local protein synthesis, providing a mechanistic framework for understanding how neurons achieve precise spatiotemporal control of protein synthesis.

neuroscience↗

bioRxiv2023

Cryo-FIB workflow for imaging brain tissue via in situ cryo-electron microscopy

Cryo-electron microscopy (cryo-EM) enables the study of protein complexes, cytoskeletal elements, and organelles in three dimensions without the use of chemical fixation. Most cryo-EM studies focus on vitreously frozen individual cells separated from their native tissue contexts. This reliance on imaging of single cells is primarily due to technical challenges associated with preparing fresh tissue sections at a thinness sufficient for visualization via cryo-EM. Highly heterogenous and specialized tissues, such as brain, are especially affected by this limitation as the cellular, subcellular, and synaptic milieus can significantly vary across neuroanatomical locations. To address this limitation, we established new instrumentation and a workflow that consists of: 1) high-pressure freezing of fresh brain tissue; 2) tissue trimming followed by cryo-focused ion beam milling via the H-bar approach to generate ultrathin lamellae; and 3) cryo-EM imaging. Here, we apply this workflow to visualize the fine ultrastructural details of organelles, as well as cytoskeletal and synaptic elements that comprise the cortical neuropil within fresh, unfixed mouse brain tissue. Moreover, we present initial studies that apply principles of the above workflow to the analysis of postmortem human brain tissue. Overall, our work integrates the strengths of cryo-electron microscopy and tissue-based approaches to produce a generalizable workflow capable of visualizing subcellular structures within complex tissue environments.

neuroscience↗

bioRxiv2020

A Prokaryotic Membrane Sculpting BAR Domain Protein

Bin/Amphiphysin/RVS (BAR) domain proteins belong to a superfamily of coiled-coil proteins influencing membrane curvature in eukaryotes and are associated with vesicle biogenesis, vesicle-mediated protein trafficking, and intracellular signaling. Here we report the first prokaryotic BAR domain protein, BdpA, from Shewanella oneidensis MR-1, known to produce redox-active membrane vesicles and micrometer-scale outer membrane extensions (OMEs). BdpA is required for uniform size distribution of membrane vesicles and scaffolding OMEs into a consistent diameter and curvature. Cryogenic transmission electron microscopy reveals a strain lacking BdpA produces lobed, disordered OMEs rather than membrane tubes produced by the wild type strain. Overexpression of BdpA promotes OME formation during conditions where they are less common. Heterologous expression results in OME production in Marinobacter atlanticus and Escherichia coli. Based on the ability of BdpA to alter membrane curvature in vivo, we propose that BdpA and its homologs comprise a newly identified class of prokaryotic BAR (P-BAR) domains.

microbiology↗

bioRxiv2017

Obstruction of pilus retraction stimulates bacterial surface sensing

Surface association provides numerous fitness advantages to bacteria. Thus, it is critical for bacteria to recognize surface contact and to consequently initiate physiological changes required for a surface-associated lifestyle (1). Ubiquitous microbial appendages called pili are involved in sensing surfaces and mediating downstream surface-associated behaviors (2-6). The mechanism by which pili mediate surface sensing remains unknown, largely due to the difficulty to visualize their dynamic nature and to directly modulate their activity without genetic modification. Here, we show that Caulobacter crescentus pili undergo dynamic cycles of extension and retraction that cease within seconds of surface contact, and this arrest of pilus activity coincides with surface-stimulated holdfast synthesis. By physically blocking pili, we show that imposing resistance to pilus retraction is sufficient to stimulate holdfast synthesis in the absence of surface contact. Thus, resistance to type IV pilus retraction upon surface attachment is used for surface sensing.\n\nOne Sentence SummaryBacteria use the tension imparted on retracting pilus fibers upon their binding to a surface for surface sensing.

microbiology↗