Biology subjects

Oikonomou, C. M.

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

12 recordsLinked to original sources

bioRxiv2022

Dynamic structural adaptations enable the endobiotic predation of bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is an endobiotic microbial predator that offers promise as a living antibiotic for its ability to kill Gram-negative bacteria, including human pathogens. Even after six decades of study, fundamental details of its predation cycle remain mysterious. Here, we used cryo-electron tomography to comprehensively image the lifecycle of B. bacteriovorus at nanometer-scale resolution. In addition to providing the first high-resolution images of predation in a native (hydrated, unstained) state, we also discover several surprising features of the process, including novel macromolecular complexes involved in prey attachment/invasion and a flexible portal structure lining a hole in the prey peptidoglycan that tightly seals the prey outer membrane around the predator during entry. Unexpectedly, we find that B. bacteriovorus does not shed its flagellum during invasion, but rather resorbs it into its periplasm for degradation. Finally, following replication and division in the bdelloplast, we observe a transient and extensive ribosomal lattice on the condensed B. bacteriovorus nucleoid. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/496000v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@35da74org.highwire.dtl.DTLVardef@19923f1org.highwire.dtl.DTLVardef@106a616org.highwire.dtl.DTLVardef@181f2fb_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

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↗

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

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↗

bioRxiv2018

ETDB-Caltech: a blockchain-based distributed public database for electron tomography

Three-dimensional electron microscopy techniques like electron tomography provide valuable insights into cellular structures, and present significant challenges for data storage and dissemination. Here we explored a novel method to publicly release more than 11,000 such datasets, more than 30 TB in total, collected by our group. Our method, based on a peer-to-peer file sharing network built around a blockchain ledger, offers a distributed solution to data storage. In addition, we offer a user-friendly browser-based interface, https://etdb.caltech.edu, for anyone interested to explore and download our data. We discuss the relative advantages and disadvantages of this system and provide tools for other groups to mine our data and/or use the same approach to share their own imaging datasets.

cell biology↗

bioRxiv2018

Simulations suggest a constrictive force is required for Gram-negative bacterial cell division

To divide, Gram-negative bacterial cells must remodel their peptidoglycan cell wall to a smaller and smaller radius at the division site, but how this process occurs remains debated. While the tubulin homolog FtsZ is thought to generate a constrictive force, it has also been proposed that cell wall remodeling alone is sufficient to drive membrane constriction, possibly via a make-before-break mechanism in which new hoops of cell wall are made inside the existing hoops (make) before bonds in the existing wall are cleaved (break). Previously, we constructed software, REMODELER 1, to simulate cell wall remodeling in rod-shaped bacteria during growth. Here, we used this software as the basis for an expanded simulation system, REMODELER 2, which we used to explore different mechanistic models of cell wall division. We found that simply organizing the cell wall synthesis complexes at the midcell was not sufficient to cause wall invagination, even with the implementation of a make-before-break mechanism. Applying a constrictive force at the midcell could drive division if the force was sufficiently large to initially constrict the midcell into a compressed state before new hoops of relaxed cell wall were incorporated between existing hoops. Adding a make-before-break mechanism could drive division with a smaller constrictive force sufficient to bring the midcell peptidoglycan into a relaxed, but not necessarily compressed, state.

biophysics↗

bioRxiv2018

The structural complexity of the Gammaproteobacteria flagellar motor is related to the type of its torque-generating stators

The bacterial flagellar motor is a cell-envelope-embedded macromolecular machine that functions as a propeller to move the cell. Rather than being an invariant machine, the flagellar motor exhibits significant variability between species, allowing bacteria to adapt to, and thrive in, a wide range of environments. For instance, different torque-generating stator modules allow motors to operate in conditions with different pH and sodium concentrations and some motors are adapted to drive motility in high-viscosity environments. How such diversity evolved is unknown. Here we use electron cryo-tomography to determine the in situ macromolecular structures of the flagellar motors of three Gammaproteobacteria species: Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis MR-1, providing the first views of intact motors with dual stator systems. Complementing our imaging with bioinformatics analysis, we find a correlation between the stator system of the motor and its structural complexity. Motors with a single H+-driven stator system have only the core P- and L-rings in their periplasm; those with dual H+-driven stator systems have an extra component elaborating their P-ring; and motors with Na+- (or dual Na+-H+)- driven stator systems have additional rings surrounding both their P- and L-rings. Our results suggest an evolution of structural complexity that may have enabled pathogenic bacteria like L. pneumophila and P. aeruginosa to colonize higher-viscosity environments in animal hosts.

microbiology↗

bioRxiv2018

Stable sub-complexes observed in situ suggest a modular assembly pathway of the bacterial flagellar motor

The self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio-temporal synchronization of gene expression, protein localization and association of a dozen or more unique components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with each subsequent component stabilizing the last. Here, using electron cryo-tomography of intact Legionella pneumophila, Pseudomonas aeruginosa and Shewanella oneidensis cells, we observe stable outer-membrane-embedded sub-complexes of the flagellar motor. These sub-complexes consist of the periplasmic embellished P- and L-rings, in the absence of other flagellar components, and bend the membrane inward dramatically. Additionally, we also observe independent inner-membrane sub-complexes consisting of the C- and MS-rings and export apparatus. These results suggest an alternate model for flagellar motor assembly in which outer- and inner-membrane-associated sub-complexes form independently and subsequently join, enabling later steps of flagellar production to proceed.

microbiology↗

bioRxiv2017

Distinguishing Signal From Autofluorescence In Cryogenic Correlated Light And Electron Microscopy Of Mammalian Cells

Cryogenic correlated light and electron microscopy (cryo-CLEM) is a valuable tool for studying biological processes in situ. In cryo-CLEM, a target protein of interest is tagged with a fluorophore and the location of the corresponding fluorescent signal is used to identify the structure in low-contrast but feature-rich cryo-EM images. To date, cryo-CLEM studies of mammalian cells have relied on very bright organic dyes or fluorescent protein tags concentrated in virus particles. Here we describe a method to expand the application of cryo-CLEM to cells harboring genetically-encoded fluorescent proteins. We discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80K). Compared to fluorescent protein tags, these sources of autofluorescence exhibit a broader spectrum of fluorescence, which we exploited to develop a simple, robust approach to discriminate between the two. We validate this method in INS-1 E cells using a mitochondrial marker, and apply it to study the ultrastructural variability of secretory granules in a near-native state within intact INS-1E pancreatic cells by high-resolution 3D electron cryotomography.

cell biology↗

bioRxiv2017

Uncharacterized bacterial structures revealed by electron cryotomography

SUMMARY STATEMENTHere we present a survey of previously uncharacterized structures we have observed in bacterial cells by electron cryotomography, in the hopes of spurring their identification and study.\n\nABSTRACTElectron cryotomography (ECT) can reveal the native structure and arrangement of macromolecular complexes inside intact cells. This technique has greatly advanced our understanding of the ultrastructure of bacterial cells. Rather than undifferentiated bags of enzymes, we now view bacteria as structurally complex assemblies of macromolecular machines. To date, our group has applied ECT to nearly 90 different bacterial species, collecting more than 15,000 cryotomograms. In addition to known structures, we have observed several, to our knowledge, uncharacterized features in these tomograms. Some are completely novel structures; others expand the features or species range of known structure types. Here we present a survey of these uncharacterized bacterial structures in the hopes of accelerating their identification and study, and furthering our understanding of the structural complexity of bacterial cells.

microbiology↗

bioRxiv2017

Structure of the archaellar motor and associated cytoplasmic cone in Thermococcus kodakaraensis

Archaeal swimming motility is driven by rotary motors called archaella. The structure of these motors, and particularly how they are anchored in the absence of a peptidoglycan cell wall, is unknown. Here, we use electron cryotomography to visualize the archaellar motor in vivo in Thermococcus kodakaraensis. Compared to the homologous bacterial type IV pilus (T4P), we observe structural similarities as well as several unique features. While the position of the cytoplasmic ATPase appears conserved, it is not braced by linkages that extend upward through the cell envelope as in the T4P, but rather by cytoplasmic components that attach it to a large conical frustum up to 500 nm in diameter at its base. In addition to anchoring the lophotrichous bundle of archaella, the conical frustum associates with chemosensory arrays and ribosome-excluding material and may function as a polar organizing center for the coccoid cells.

cell biology↗