Biology subjects

Jensen, G. J.

Publications and source records attributed to Jensen, G. J..

At least 37 records · Page 2Linked to original sources

bioRxiv2021

A novel widespread bacterial structure related to the flagellar type III secretion system

The flagellar type III secretion system (fT3SS) is a suite of membrane-embedded and cytoplasmic proteins responsible for building the bacterial flagellar motility machinery. Homologous proteins form the injectisome machinery bacteria use to deliver effector proteins into eukaryotic cells, and other family members have recently been reported to be involved in the formation of membrane nanotubes. Here we describe a novel, ubiquitous and evolutionarily widespread hat-shaped structure embedded in the inner membrane of bacteria, of yet-unidentified function, that is related to the fT3SS, adding to the already rich repertoire of this family of nanomachines.

microbiology↗

bioRxiv2021

In situ imaging of bacterial membrane projections and associated protein complexes using electron cryo-tomography

The ability to produce membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of [~] 90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified MEs and MVs in 13 species and classified several major ultrastructures: 1) tubes with a uniform diameter (with or without an internal scaffold), 2) tubes with irregular diameter, 3) tubes with a vesicular dilation at their tip, 4) pearling tubes, 5) connected chains of vesicles (with or without neck-like connectors), 6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field.

molecular biology↗

bioRxiv2021

UVC inactivation of pathogenic samples suitable for cryoEM analysis

Cryo-electron microscopy has become an essential tool to understand structure and function of biological samples, from individual proteins to whole cells. Especially for pathogens, such as disease-causing bacteria and viruses, insights gained by cryo-EM can aid in developing cures. However, due to the biosafety restrictions of human pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the delicate ultrastructure of the sample. Alternatively, researchers use in vitro or ex vivo models, which are non-pathogenic but lack the complexity of the pathogen of interest. Here we show that ultraviolet-C (UVC) radiation at cryogenic temperatures can be used to eliminate or dramatically reduce the infectivity of two model organisms, a pathogenic bacterium (Vibrio cholerae) and a virus-like particle (the ICP1 bacteriophage). We show no discernable structural impact of this treatment of either sample using two cryo-EM methods: cryo-electron tomography (cryo-ET) followed by sub-tomogram averaging (STA), and single particle analysis (SPA). Additionally, we applied the UVC irradiation to the protein apoferritin (ApoF), which is a widely used test sample for high resolution SPA studies. The UVC-treated ApoF sample resulted in a 2.1 [A] structure that did not reveal any discernable structural damage. Together, these results show that the UVC irradiation dose that effectively inactivates cryo-EM samples does not negatively impact their structure. This research demonstrates that UVC treatment is an effective and inexpensive addition to the cryo-EM sample preparation toolbox.

molecular biology↗

bioRxiv2021

The stress-sensing domain of activated IRE1α forms helical filaments in narrow ER membrane tubes

The signaling network of the unfolded protein response (UPR) adjusts the protein folding capacity of the endoplasmic reticulum (ER) according to need. The most conserved UPR sensor, IRE1, spans the ER membrane and activates through oligomerization. IRE1 oligomers accumulate in dynamic foci. We determined the in-situ structure of IRE1 foci by cryogenic correlated light and electron microscopy (cryo-CLEM), combined with electron cryo-tomography (cryo-ET) and complementary immuno-electron microscopy. IRE1 oligomers localize to a network of narrow anastomosing ER tubes (diameter ~28 nm) with complex branching. The lumen of the tubes contains protein filaments, likely composed of linear arrays of IRE1 lumenal domain dimers, arranged in two intertwined, left-handed helices. Our findings define a previously unrecognized ER subdomain and suggest positive feedback in IRE1 signaling.

cell biology↗

bioRxiv2021

Measuring gas vesicle dimensions by electron microscopy

Gas vesicles (GVs) are cylindrical or spindle-shaped protein nanostructures filled with air and used for flotation by various cyanobacteria, heterotrophic bacteria, and Archaea. Recently, GVs have gained interest in biotechnology applications due to their ability to serve as imaging agents and actuators for ultrasound, magnetic resonance and several optical techniques. The diameter of GVs is a crucial parameter contributing to their mechanical stability, buoyancy function and evolution in host cells, as well as their properties in imaging applications. Despite its importance, reported diameters for the same types of GV differ depending on the method used for its assessment. Here, we provide an explanation for these discrepancies and utilize electron microscopy (EM) techniques to accurately estimate the diameter of the most commonly studied types of GVs. We show that during air drying on the EM grid, GVs flatten, leading to a ~1.5-fold increase in their apparent diameter. We demonstrate that GVs diameter can be accurately determined by direct measurements from cryo-EM samples or alternatively indirectly derived from widths of flat collapsed and negatively stained GVs. Our findings help explain the inconsistency in previously reported data and provide accurate methods to measure GV dimensions.

biochemistry↗

bioRxiv2020

Challenges in solving structures from radiation-damaged tomograms of protein nanocrystals assessed by simulation

Structure determination methods are needed to resolve the atomic details that underlie protein function. X-ray crystallography has provided most of our knowledge of protein structure but is constrained by the need for large, well-ordered crystals and the loss of phase information. The rapidly developing methods of serial femtosecond crystallography, micro-electron diffraction, and single-particle reconstruction circumvent the first of these limitations by enabling data collection from nanocrystals or purified proteins. However, the first two methods also suffer from the phase problem, while many proteins fall below the molecular weight threshold required by single-particle reconstruction. Cryo-electron tomography of protein nanocrystals has the potential to overcome these obstacles of mainstream structure determination methods. Here we present a data processing scheme that combines routines from X-ray crystallography and new algorithms we developed to solve structures from tomograms of nanocrystals. This pipeline handles image processing challenges specific to tomographic sampling of periodic specimens and is validated using simulated crystals. We also assess the tolerance of this workflow to the effects of radiation damage. Our simulations indicate a trade-off between a wider tilt-range to facilitate merging data from multiple tomograms and a smaller tilt increment to improve phase accuracy. Since phase errors but not merging errors can be overcome with additional datasets, these results recommend distributing the dose over a wide angular range rather than using a finer sampling interval to solve the protein structure.

biophysics↗

bioRxiv2020

Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via -helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by -helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

synthetic biology↗

bioRxiv2020

Rapid Tilt-Series Method for Cryo-Electron Tomography: Characterizing Stage Behavior During FISE Acquisition

We and others recently developed rapid tilt-series acquisition methods for cryo-electron tomography on a Titan Krios G3i equipped with a single axis holder and a K-series direct electron detector and showed that one of these, the fast-incremental single exposure (FISE) method, significantly accelerates tilt-series acquisition when compared to traditional methods while preserving the quality of the images. Here, we characterize the behavior of our single axis holder in detail during a FISE experiment to optimally balance data quality with speed. We explain our methodology in detail so others can characterize their own stages, and conclude with recommendations for projects with different resolution goals.

biophysics↗

bioRxiv2020

In situ structure and organisation of the type IX secretion system

The Bacteroidetes type IX secretion system (T9SS) consists of at least 19 components that translocate proteins with a type A or type B C-terminal domain (CTD) signal across the outer membrane. The overall organisation and architecture of this system including how the secretion pore (Sov) interacts with the other components is unknown. We used cryo-electron tomography to obtain the first images of the T9SS including PorK/N rings inside intact Porphyromonas gingivalis cells. Using proteomics, we identified a novel complex between Sov, PorV and PorA and showed that Sov interacts with the PorK/N rings via PorW and a new component PGN_1783. A separate complex comprising the outer membrane {beta}-barrel protein PorP, PorE, and the type B CTD protein PG1035 was also identified. Similarly, the Flavobacterium johnsoniae PorP-like protein, SprF was found bound to the major gliding motility adhesin, SprB. Based on these data, we propose cell surface anchorage for type B CTD proteins to PorP-like proteins and a unique model where the PorK/N rings function as an outer membrane barrier to maintain the close proximity of the translocon to the shuttle and attachment complexes inside the rings, ensuring the harmonized secretion and cell surface attachment of the T9SS substrates.

microbiology↗

bioRxiv2020

Structure of the bacterial cellulose ribbon and its assembly-guiding cytoskeleton by electron cryotomography

Cellulose is a widespread component of bacterial biofilms, where its properties of exceptional water retention, high tensile strength and stiffness prevents dehydration and mechanical disruption of the biofilm. Bacteria in the Gluconacetobacter genus secrete crystalline cellulose, with a structure very similar to that found in plant cell walls. How this higher-order structure is produced is poorly understood. We used cryo-electron tomography and focused ion beam milling of native bacterial biofilms to image cellulose-synthesizing G. hansenii and G. xylinus bacteria in a frozen-hydrated, near-native state. We confirm previous results suggesting that cellulose crystallization occurs serially following its secretion along one side of the cell, leading to a cellulose ribbon that can reach several microns in length and combine with ribbons from other cells to form a robust biofilm matrix. We were able to take direct measurements in a near-native state of the cellulose sheets. Our results also reveal a novel cytoskeletal structure, that we name the cortical belt, adjacent to the inner membrane and underlying the sites where cellulose is seen emerging from the cell. We find that this structure is not present in other cellulose-synthesizing bacterial species, Agrobacterium tumefaciens and Escherichia coli 1094, which do not produce organized cellulose ribbons. We therefore propose that the cortical belt holds the cellulose synthase complexes in a line, to form higher-order cellulose structures such as sheets and ribbons.

microbiology↗

bioRxiv2020

CryoEM structure of the Vibrio cholerae Type IV competence pilus secretin PilQ

Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, the Type IV competence pilus is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. A lack of structural information has hindered our understanding of this process, however. Here, we solved the first ever high-resolution structure of a Type IV competence pilus secretin. A functional tagged allele of VcPilQ purified from native V. cholerae cells was used to determine the cryoEM structure of the PilQ secretin in amphipol to [~]2.7 [A]. This structure highlights for the first time key differences in the architecture of the Type IV competence pilus secretin from the Type II and Type III Secretin System secretins. Based on our cryoEM structure, we designed a series of mutants to interrogate the mechanism of PilQ. These experiments provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation. We prove that it is possible to reduce pilus biogenesis and natural transformation by sealing the gate, suggesting VcPilQ as a new drug target.

biophysics↗

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↗

bioRxiv2019

Plasma membrane damage removal by F-actin-mediated shedding from repurposed filopodia

Repairing plasma membrane damage is vital to eukaryotic cell survival. Membrane shedding is thought to be key to this repair process, but a detailed view of how the process occurs is still missing. Here we used electron cryotomography to image the ultrastructural details of plasma membrane wound healing. We found that filopodia-like protrusions are built at damage sites, accompanied by retraction of neighboring filopodia, and that these repurposed protrusions act as scaffolds for membrane shedding. This suggests a new role for filopodia as reservoirs of membrane and actin for plasma membrane damage repair. Damage-induced shedding was dependent on F-actin dynamics and Myo1a, as well as Vps4B, an important component of the ESCRT machinery. Thus we find that damage shedding is more complex than current models of simple vesiculation from flat membrane domains. Rather, we observe structural similarities between damage-mediated shedding and constitutive shedding from enterocytes that argue for conservation of a general membrane shedding mechanism.

cell biology↗

bioRxiv2019

Correlated cryogenic fluorescence microscopy and electron cryotomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo

Members of the TRIM protein family have been shown to gather into structures in both the nucleus and cytoplasm. One TRIM protein family member, TRIM5, has been shown to form cytoplasmic bodies involved in restricting retroviruses such as HIV-1. Here we applied cryogenic correlated light and electron microscopy (cryo-CLEM) to intact mammalian cells expressing YFP-rhTRIM5 and found hexagonal nets were present whose arm-lengths were similar to those of the hexagonal nets formed by purified TRIM5 in-vitro. We also observed YFP-rhTRIM5 within a diversity of structures with characteristics expected for organelles involved in different stages of macroautophagy, including disorganized protein aggregations (sequestosomes), sequestosomes flanked by flat double-membraned vesicles (sequestosome:phagophore complexes), sequestosomes within a double-membraned vesicle (autophagosomes), and sequestosomes within multi-vesicular autophagic vacuoles (autolysosomes or amphisomes). Vaults were also seen in these structures, consistent with their role in autophagy. Our data (i) support recent reports that TRIM5 can form both well-organized signaling complexes and non-signaling aggregates, (ii) offer the first images of the macroautophagy pathway in a near-native state, and (iii) reveal that vaults arrive early in macroautophagy.

cell biology↗

bioRxiv2019

Bacterial flagellar motor PL-ring disassembly sub-complexes are widespread and ancient

The bacterial flagellar motor is an amazing nanomachine. Understanding how such complex structures arose is crucial to our understanding of cellular evolution. We and others recently reported that in several Gammaproteobacterial species, a relic sub-complex comprising the decorated P- and L-rings persists in the outer membrane after flagellum disassembly. Imaging nine additional species with cryo-electron tomography, here we show that this sub-complex persists after flagellum disassembly in other phyla as well. Bioinformatic analyses fail to show evidence of any recent horizontal transfers of the P- and L-ring genes, suggesting that this sub-complex and its persistence is an ancient and conserved feature of the flagellar motor. We hypothesize that one function of the P- and L-rings is to seal the outer membrane after motor disassembly.

evolutionary biology↗

bioRxiv2019

Simulations of proposed mechanisms of FtsZ-driven cell constriction

To divide, bacteria must constrict their membranes against significant force from turgor pressure. A tubulin homo-log, FtsZ, is thought to drive constriction, but how FtsZ filaments might generate constrictive force in the absence of motor proteins is not well understood. There are two predominant models in the field. In one, filaments overlap to form complete rings around the circumference of the cell; as filaments slide against each other to maximize lateral contact, the rings constrict. In the other, filaments exert force on the membrane by a GTP-hydrolysis-induced switch in conformation from straight to bent. Here we developed software, ZCONSTRICT, for quantitative 3D simulations of Gram-negative bacterial cell division to test these two models and identify critical conditions required for them to work. We find that the avidity of lateral interactions quickly halts the sliding of filaments, so a mechanism such as depolymerization or treadmilling is required to sustain constriction by filament sliding. For filament bending, we find that a mechanism such as the presence of a rigid linker is required to constrain bending within the division plane and maintain the distance observed in vivo between the filaments and the membrane. We also explored the recent observation of constriction associated with a single FtsZ filament and found that it can be explained by the filament bending model if there is a rigid connection between the filament and the cell wall. Together, our work sheds light on the physical principles underlying bacterial cell division and informs future experiments to elucidate the mechanism of FtsZ.

biophysics↗

bioRxiv2019

Regular Architecture (RegArch): A standard expression language for describing protein architectures

Domain architecture - the arrangement of features in a protein - exhibits syntactic patterns similar to the grammar of a language. This feature enables pattern mining for protein function prediction, comparative genomics, and studies of molecular evolution and complexity. To facilitate such work, here we propose Regular Architecture (RegArch), an expression language to describe syntactic patterns in protein architectures. Like the well-known Regular Expressions for text, RegArchs codify positional and non-positional patterns of elements into nested JSON objects. We describe the standard and provide a reference implementation in JavaScript to parse RegArchs and match annotated proteins.

bioinformatics↗

bioRxiv2019

Repurposing a macromolecular machine: Architecture and evolution of the F7 chemosensory system

How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli. We first identified ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum, characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we then traced the evolution of the system through {gamma}-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost.

microbiology↗