Biology subjects

Smith, J. L. R.

Publications and source records attributed to Smith, J. L. R..

6 recordsLinked to original sources

bioRxiv2026

Femtosecond pulsed laser ablation for in-tissue electron cryo-tomography

In situ electron cryo-tomography (cryo-ET) is a powerful tool for visualising biology inside cells at the molecular scale. The routine application of cryo-ET in tissue samples promises biological insight beyond single cells, but is currently limited by the technical challenge of preparing high quality, electron-transparent specimens from thick (>100 m) biopsies efficiently. Here, we demonstrate that cryogenic femtosecond pulsed laser ablation (cryo-fs-PLA) can accelerate the preparation of vitreous specimens of mammalian brain tissue for cryo-ET. Using cryogenic fluorescence microscopy, regions of interest within high-pressure frozen tissue can be specifically targeted, and cryo-fs-PLA can be employed as a preparative step for serial cryogenic lift-out experiments. We define cryo-fs-PLA parameters and determine ablation rates for two different laser wavelengths, and demonstrate that tilt series collected on lamellae fabricated from sites exposed to cryo-fs-PLA largely maintain their vitrification state. Incorporating cryo-fs-PLA into existing cryo-ET workflows has the potential to unlock the sample preparation of tissues at the throughput necessary for routine in situ cell biology of multicellular samples.

biochemistry↗

bioRxiv2024

Charting the molecular landscape of neuronal organisation within the hippocampus using cryo electron tomography

Cellular cryo-electron tomography (cryoET) enables the capture of detailed structural information within a biologically relevant environment. However, information in more complex samples, such as tissues, is lacking. Importantly, these observations need to be set in context of populations; imaging on the molecular scale to-date is limited to few observations in-situ that struggle to be generalised. This is due to limitations in throughput and versatility employed by current instrumentation. Here, we utilise plasma focused ion beam milling to examine the molecular landscape of mouse hippocampus by cryoET in targeted regions across multiple individuals revealing the complex organisation of macromolecules from the CA1 strata pyramidale (sp) to radiatum (sr). Our data represent a molecular atlas, producing snapshots of hippocampal architecture in adult mouse. The combination of instrumentation and application of technical advancements provides a framework to explore specific structural questions within native tissues in a targeted manner.

biochemistry↗

bioRxiv2022

Ot2Rec: A Semi-Automatic, Extensible, Multi-Software Tomographic Reconstruction Workflow

Electron cryo-tomography (cryo-ET) is an imaging technique for probing 3D structures with at the nanometre scale. This technique has been used extensively in the biomedical field to study the complex structures of proteins and other macromolecules. With the advancement in technology, microscopes are currently capable of producing images amounting to terabytes of data per day, posing great challenges for scientists as the speed of processing of the images cannot keep up with the ever-higher throughput of the microscopes. Therefore, automation is an essential and natural pathway on which image processing - from individual micrographs to full tomograms - is developing. In this paper, we present Ot2Rec, an open-source pipelining tool which aims to enable scientists to build their own processing workflows in a flexible and automatic manner. The basic building blocks of Ot2Rec are plugins which follow a unified API structure, making it simple for scientists to contribute to Ot2Rec by adding features which are not already available. In this paper, we also present three case studies of image processing using Ot2Rec, through which we demonstrate the speedup of using a semi-automatic workflow over a manual one, the possibility of writing and using custom (prototype) plugins, and the flexibility of Ot2Rec which enables the mix-and-match of plugins. We also demonstrate, in the supplementary information, a built-in reporting feature in Ot2Rec which aggregates the metadata from all process being run, and output them in the Jupyter Notebook and/or HTML formats for quick review of image processing quality. Ot2Rec can be found at https://github.com/rosalindfranklininstitute/ot2rec. Impact StatementThe field of cryo electron tomography has grown substantially in recent years, bringing about new advances in hardware and software which enable visualisation of cell and tissue architecture and proteins found in their native context. These same advances have, in some ways, stratified the field into those with access and those without. On the software side, this has emphasised the need for open-source options that do not require high levels of computational literacy to access. Additionally, it has highlighted the need for ways to both mix-and-match software for easy prototyping and comparisons between parameters and methods. Ot2Rec addresses these needs through a simple, unified plugin structure allowing the addition of existing software or the development of new and does so in a way which democratises access.

bioinformatics↗

bioRxiv2022

Cryo-plasma FIB/SEM volume imaging of biological specimens

Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of sub-cellular structures on the mesoscale (10 nm to 10 m). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents hydration shells for in-situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM (cryo-pFIB/SEM). We evaluated the choice of plasma ion source and imaging regimes to produce high quality SEM images of a range of different biological samples. Using an automated workflow we produced three dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20 to 50 nm. Additionally, a tag-free tool is needed to drive the application of in situ structural biology towards tissue. The combination of serial FIB/SEM with plasmabased ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 m) to produce lamella for cryogenic electron tomography.

cell biology↗

bioRxiv2021

Potential coupling between SARS-CoV-2 replicative fitness and interactions of its nucleoprotein with human 14-3-3 proteins

Phosphorylated within its Ser/Arg-rich region, the SARS-CoV-2 nucleoprotein (N) recruits the phosphopeptide-binding human 14-3-3 proteins that play a well-recognized role in replication of many viruses. Here we use genetic code expansion to demonstrate that phosphorylation of SARS-CoV-2 N at either of two pseudo-repeats centered at Ser197 and Thr205 is sufficient for 14-3-3 binding. According to fluorescence anisotropy, the pT205-motif, present in SARS-CoV-2 but not in SARS-CoV, is preferred over the pS197-motif by all seven human 14-3-3 isoforms, which display unforeseen pT205/pS197 binding selectivity hierarchy. Crystal structures demonstrate that pS197 and pT205 are mutually exclusive 14-3-3-binding sites, whereas SAXS and biochemical data indicate 14-3-3 binding occludes the Ser/Arg-rich region, inhibiting its dephosphorylation. This Ser/Arg-rich region of N is highly prone to mutations, as exemplified by the Omicron and Delta variants, with our data suggesting how the strength of its 14-3-3 binding can be linked with the replicative fitness of the virus.

biochemistry↗

bioRxiv2020

The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association can regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, while hijacking cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention. HighlightsSARS-CoV-2 nucleocapsid protein (N) binds to all seven human 14-3-3 isoforms. This association with 14-3-3 strictly depends on phosphorylation of N. The two proteins interact in 2:2 stoichiometry and with the Kd in a M range. Affinity of interaction depends on the specific 14-3-3 isoform. Conserved Ser197-phosphopeptide of N is critical for the interaction.

biochemistry↗