Biology subjects

Briggs, J. A. G.

Publications and source records attributed to Briggs, J. A. G..

27 records · Page 2Linked to original sources

bioRxiv2020

The native structure of the full-length, assembled influenza A virus matrix protein, M1.

Influenza A virus causes millions of severe illnesses during annual epidemics. The most abundant protein in influenza virions is the matrix protein M1 that mediates virus assembly by forming an endoskeleton beneath the virus membrane. The structure of full-length M1, and how it oligomerizes to mediate assembly of virions, is unknown. Here we have determined the complete structure of assembled M1 within intact virus particles, as well as the structure of M1 oligomers reconstituted in vitro. We found that the C-terminal domain of M1 is disordered in solution, but can fold and bind in trans to the N-terminal domain of another M1 monomer, thus polymerising M1 into linear strands which coat the interior surface of the assembling virion membrane. In the M1 polymer, five histidine residues, contributed by three different M1 monomers, form a cluster that can serve as the pH-sensitive disassembly switch after entry into a target cell. These structures therefore provide mechanisms for influenza virus assembly and disassembly.

biochemistry↗

bioRxiv2020

A thermostable, closed, SARS-CoV-2 spike protein trimer.

The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. S exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor binding site, and subsequently from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S which allow production of thermostable, crosslinked, S protein trimers that are trapped in the closed, pre-fusion, state. We have determined the structures of crosslinked and non-crosslinked proteins, identifying two distinct closed conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.

biochemistry↗

bioRxiv2020

Architecture of the AP2:clathrin coat on the membranes of clathrin-coated vesicles

Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cells plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. AP2 is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures, interactions and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P2-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 {beta}2-appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.

molecular biology↗

bioRxiv2019

Complexin suppresses spontaneous exocytosis by capturing the membrane-proximal regions of VAMP2 and SNAP25

The neuronal protein complexin contains multiple domains that exert both clamping and facilitatory functions to tune spontaneous and action potential triggered synaptic release. We address the clamping mechanism and show that the accessory helix of complexin arrests the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that forms the core machinery of intracellular membrane fusion. In a reconstituted fusion assay, site- and stage-specific photo-cross-linking reveals that prior to fusion the complexin accessory helix laterally binds the membrane-proximal C-terminal ends of SNAP25 and VAMP2. Corresponding complexin interface mutants selectively increase spontaneous release of neurotransmitter in living neurons, implying that the accessory helix suppresses final zippering/assembly of the SNARE four-helix bundle by restraining VAMP2 and SNAP25.

neuroscience↗

bioRxiv2019

Structures of virus-like capsids formed by the drosophila neuronal Arc proteins

The neuronal protein Arc is a critical mediator of synaptic plasticity. Arc originated in tetrapods and flies through domestication of retrotransposon Gag genes. Recent studies have suggested that Arc mediates intercellular mRNA transfer and like Gag, can form capsid-like structures. Here we report that Drosophila proteins dArc1 and dArc2 assemble virus-like capsids. We determine the capsid structures to 2.8 [A] and 3.7 [A] resolution, respectively, finding similarity to capsids of retroviruses and retrotransposons. Differences between dArc1 and dArc2 capsids, including the presence of a structured zinc-finger pair in dArc1, are consistent with differential RNA-binding specificity. Our data support a model in which ancestral capsid-forming and RNA-binding properties of Arc remain under positive selection pressure and have been repurposed to function in neuronal signalling.

neuroscience↗

bioRxiv2019

Multiplexed electron microscopy by fluorescent barcoding allows screening for ultrastructural phenotype

Genetic screens performed using high-throughput fluorescent microscopes have generated large datasets that have contributed many insights into cell biology. However, such approaches typically cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) is not subject to this resolution limit, generating detailed images of cellular ultrastructure, but requires time consuming preparation of individual samples, limiting its throughput. Here we overcome this obstacle and describe a robust method for screening by high-throughput electron microscopy. Our approach uses combinations of fluorophores as barcodes to mark the genotype of each cell in mixed populations, and correlative light and electron microscopy to read the fluorescent barcode of each cell before it is imaged by electron microscopy. Coupled with an easy-to-use software workflow for correlation, segmentation and computer image analysis, our method allows to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate the method on several organelles with samples that each contain up to 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher-throughput, and can be utilized in multiple ways to enable electron microscopy to become a powerful screening methodology.

cell biology↗

bioRxiv2018

Fluorescence-Based Detection of Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy

Correlated light and electron microscopy (CLEM) has become a popular technique for combining the protein-specific labeling of fluorescence with electron microscopy, both at room and cryogenic temperatures. Fluorescence applications at cryo-temperature have typically been limited to localization of tagged protein oligomers due to known issues of extended triplet state duration, spectral shifts, and reduced photon capture through cryo-CLEM objectives. Here, we consider fluorophore characteristics and behaviors that could enable more extended applications. We describe how dialkylcarbocanine DiD and its autoquenching by resonant energy transfer can be used to distinguish the fusion state of a lipid bilayer at cryo-temperatures. By adapting an established fusion assay to work under cryo-CLEM conditions, we identified areas of fusion between influenza virus-like particles and fluorescently labeled lipid vesicles on a cryo-EM grid. This result demonstrates that cryo-CLEM can be used to localize functions in addition to tagged proteins, and that fluorescence autoquenching by resonant energy transfer can be incorporated successfully into cryo-CLEM approaches. In the case of membrane fusion applications, this method provides both an orthogonal confirmation of functional state independent of the morphological description from cryo-EM and a way to bridge room-temperature kinetic assays and the cryo-EM images.

biophysics↗

bioRxiv2017

The contributions of the actin machinery to endocytic membrane bending and vesicle formation

Branched and crosslinked actin networks mediate cellular processes that move and shape membranes. To understand how actin contributes during the different stages of endocytic membrane reshaping, we analysed deletion mutants of yeast actin network components using a hybrid imaging approach that combines live imaging with correlative microscopy. We could thereby temporally dissect the effects of different actin network perturbations, revealing distinct stages of actin-based membrane reshaping. Our data show that initiation of membrane bending requires the actin network to be physically linked to the plasma membrane and to be optimally crosslinked. Once initiated, the membrane invagination process is driven by nucleation and polymerization of new actin filaments, independently of the degree of cross-linking and unaffected by a surplus of actin network components. A key transition occurs 2 seconds before scission when the filament nucleation rate drops. From that time point on, invagination growth and vesicle scission are driven by an expansion of the assembled actin network. The expansion is sensitive to the amount of filamentous actin and its crosslinking. Our results suggest that the mechanism by which actin reshapes the membrane adapts to force requirements that vary during the progress of endocytosis.

cell biology↗

bioRxiv2017

Immature HIV-1 Lattice Assembly Dynamics are Regulated by Scaffolding from Nucleic Acid and the Plasma Membrane

The packaging and budding of Gag polyprotein and viral ribonucleic acid (RNA) is a critical step in the human immunodeficiency virus-1 (HIV-1) lifecycle. High-resolution structures of the Gag polyprotein have revealed that the capsid (CA) and spacer peptide 1 (SP1) domains contain important interfaces for Gag self-assembly. However, the molecular details of the multimerization process, especially in the presence of RNA and the cell membrane, have remained unclear. In this work, we investigate the mechanisms that work in concert between the polyproteins, RNA, and membrane to promote immature lattice growth. We develop a coarse-grained (CG) computational model that is derived from sub-nanometer resolution structural data. Our simulations recapitulate contiguous and hexameric lattice assembly driven only by weak anisotropic attractions at the helical CA-SP1 junction. Importantly, analysis from CG and single-particle tracking photoactivated localization (spt-PALM) trajectories indicates that viral RNA and the membrane are critical constituents that actively promote Gag multimerization through scaffolding, while over-expression of short competitor RNA can suppress assembly. We also find that the CA amino-terminal domain imparts intrinsic curvature to the Gag lattice. As a consequence, immature lattice growth appears to be coupled to the dynamics of spontaneous membrane deformation. Our findings elucidate a simple network of interactions that regulate the early stages of HIV-1 assembly and budding.\n\nSIGNIFICANCE STATEMENTIn order for human immunodeficiency virus to proliferate, viral proteins and genomic dimers are assembled at host cell membranes and released as immature virions. Disrupting this key intermediate step in viral replication is a potential target for treatment. However, a detailed molecular view of this process remains lacking. Here, we elucidate a network of constitutive interactions that regulate viral assembly dynamics through a combined computational and experimental approach. Specifically, our analysis reveals the active roles of nucleic acid and the membrane as scaffolds that promote the multimerization of Gag polyprotein which proceeds through multi-step and self-correcting nucleation. Our findings also illustrate the functional importance of the N-terminal, C-terminal, and spacer peptide 1 protein domains.

biophysics↗