scholarly journals In situ structures of periplasmic flagella reveal a distinct cytoplasmic ATPase complex in Borrelia burgdorferi

2018 ◽  
Author(s):  
Zhuan Qin ◽  
Akarsh Manne ◽  
Jiagang Tu ◽  
Zhou Yu ◽  
Kathryn Lees ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Conformational Changes ◽  
Electron Tomography ◽  
Structural Framework ◽  
Structural Rigidity ◽  
Membrane Bound ◽  
Novel Therapeutic Strategies ◽  
Periplasmic Flagella ◽  
Distinct Morphology

ABSTRACTPeriplasmic flagella are essential for the distinct morphology and motility of spirochetes. A flagella-specific Type III secretion system (fT3SS) composed of a membrane-bound export apparatus and a cytosolic ATPase complex is responsible for the assembly of the periplasmic flagella. Here, we combine cryo-electron tomography and mutagenesis approaches to characterize the fT3SS machine in the Lyme disease spirochete Borrelia burgdorferi. We define the fT3SS machine by systematically characterizing mutants lacking key component genes. We discover that a distinct cytosolic ATPase complex is attached to the flagellar C-ring through multiple spoke-like linkers. The ATPase complex not only strengthens structural rigidity of the C-ring, but also undergoes conformational changes in concert with flagellar rotation. Our studies provide structural framework to uncover the unique mechanisms underlying assembly and rotation of the periplasmic flagella and may provide the bases for the development of novel therapeutic strategies against several pathogenic spirochetes.

scholarly journals Characterization of the flagellar collar reveals structural plasticity essential for spirochete motility

2021 ◽  
Author(s):  
Yunjie Chang ◽  
Hui Xu ◽  
Md A. Motaleb ◽  
Jun Liu
Keyword(s):  
Electron Tomography ◽  
Structural Plasticity ◽  
Connective Tissues ◽  
Flagellar Motors ◽  
Specific Complex ◽  
Periplasmic Flagella ◽  
Curved Membrane ◽  
Distinct Morphology

AbstractSpirochetes are a remarkable group of bacteria with distinct morphology and periplasmic flagella that enable motility in viscous environments, such as host connective tissues. The collar, a spirochete-specific complex of the periplasmic flagellum, is required for the unique spirochete motility, yet it has not been clear how the collar assembles and enables spirochetes to transit between complex host environments. Here, we characterize the collar complex in the Lyme disease spirochete Borrelia burgdorferi. We discover as well as delineate the distinct functions of two novel collar proteins, FlcB and FlcC, by combining subtractive bioinformatic, genetic, and cryo-electron tomography approaches. Our high-resolution in-situ structures reveal that the multi-protein collar has a remarkable structural plasticity essential not only for assembly of flagellar motors in the highly curved membrane of spirochetes but also for generation of the high torque necessary for spirochete motility.

scholarly journals In situ structures of membrane-assisted assembly and selective autophagy of enteroviruses

2021 ◽  
Author(s):  
Selma Dahmane ◽  
Adeline Kerviel ◽  
Dustin R. Morado ◽  
Kasturika Shankar ◽  
Björn Ahlman ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Genome Replication ◽  
Lipid Kinase ◽  
Structural Framework ◽  
Cytoplasmic Membranes ◽  
Viral Genome Replication ◽  
Replication Sites ◽  
Membrane Bound ◽  
Secretory Autophagy

SummaryEnteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. New virions are thought to be assembled near the genome replication sites and are released in vesicles through secretory autophagy. Here, we use cryo-electron tomography to show that poliovirus assembles directly on replication membranes. Assembly progression beyond a membrane-bound half-capsid intermediate requires the host lipid kinase VPS34, whereas inhibition of ULK1, the initiator of canonical autophagy, leads to accumulation of virions in vast intracellular arrays followed by an increased release at later time points. We further identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from a second class of autophagic membranes containing protein filaments bundles. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle.

scholarly journals BB0259 Encompasses a Peptidoglycan Lytic Enzyme Function for Proper Assembly of Periplasmic Flagella in Borrelia burgdorferi

2021 ◽  
Vol 12 ◽  
Author(s):  
Hui Xu ◽  
Bo Hu ◽  
David A. Flesher ◽  
Jun Liu ◽  
Md A. Motaleb
Keyword(s):  
Borrelia Burgdorferi ◽  
Electron Tomography ◽  
Lytic Enzyme ◽  
Enzyme Protein ◽  
Lytic Transglycosylase ◽  
Filament Assembly ◽  
Cryo Electron Tomography ◽  
Mutant Strains ◽  
Periplasmic Flagella ◽  
Dual Domain

Assembly of the bacterial flagellar rod, hook, and filament requires penetration through the peptidoglycan (PG) sacculus and outer membrane. In most β- and γ-proteobacteria, the protein FlgJ has two functional domains that enable PG hydrolyzing activity to create pores, facilitating proper assembly of the flagellar rod. However, two distinct proteins performing the same functions as the dual-domain FlgJ are proposed in δ- and ε-proteobacteria as well as spirochetes. The Lyme disease spirochete Borrelia burgdorferi genome possesses a FlgJ and a PG lytic SLT enzyme protein homolog (BB0259). FlgJ in B. burgdorferi is crucial for flagellar hook and filament assembly but not for the proper rod assembly reported in other bacteria. However, BB0259 has never been characterized. Here, we use cryo-electron tomography to visualize periplasmic flagella in different bb0259 mutant strains and provide evidence that the E580 residue of BB0259 is essential for PG-hydrolyzing activity. Without the enzyme activity, the flagellar hook fails to penetrate through the pores in the cell wall to complete assembly of an intact periplasmic flagellum. Given that FlgJ and BB0259 interact with each other, they likely coordinate the penetration through the PG sacculus and assembly of a functional flagellum in B. burgdorferi and other spirochetes. Because of its role, we renamed BB0259 as flagellar-specific lytic transglycosylase or LTaseBb.

scholarly journals Intact Flagellar Motor of Borrelia burgdorferi Revealed by Cryo-Electron Tomography: Evidence for Stator Ring Curvature and Rotor/C-Ring Assembly Flexion

2009 ◽  
Vol 191 (16) ◽  
pp. 5026-5036 ◽  
Author(s):  
Jun Liu ◽  
Tao Lin ◽  
Douglas J. Botkin ◽  
Erin McCrum ◽  
Hanspeter Winkler ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Electron Tomography ◽  
Rotational Symmetry ◽  
Flagellar Motor ◽  
Cell Axis ◽  
Ring Assembly ◽  
Flagellar Motors ◽  
Cryo Electron Tomography ◽  
Flagellar Rotation

ABSTRACT The bacterial flagellar motor is a remarkable nanomachine that provides motility through flagellar rotation. Prior structural studies have revealed the stunning complexity of the purified rotor and C-ring assemblies from flagellar motors. In this study, we used high-throughput cryo-electron tomography and image analysis of intact Borrelia burgdorferi to produce a three-dimensional (3-D) model of the in situ flagellar motor without imposing rotational symmetry. Structural details of B. burgdorferi, including a layer of outer surface proteins, were clearly visible in the resulting 3-D reconstructions. By averaging the 3-D images of ∼1,280 flagellar motors, a ∼3.5-nm-resolution model of the stator and rotor structures was obtained. flgI transposon mutants lacked a torus-shaped structure attached to the flagellar rod, establishing the structural location of the spirochetal P ring. Treatment of intact organisms with the nonionic detergent NP-40 resulted in dissolution of the outermost portion of the motor structure and the C ring, providing insight into the in situ arrangement of the stator and rotor structures. Structural elements associated with the stator followed the curvature of the cytoplasmic membrane. The rotor and the C ring also exhibited angular flexion, resulting in a slight narrowing of both structures in the direction perpendicular to the cell axis. These results indicate an inherent flexibility in the rotor-stator interaction. The FliG switching and energizing component likely provides much of the flexibility needed to maintain the interaction between the curved stator and the relatively symmetrical rotor/C-ring assembly during flagellar rotation.

scholarly journals In situ structure and priming mechanism of the rhoptry secretion system in Plasmodium revealed by cryo-electron tomography

2022 ◽  
Author(s):  
Matthew Martinez ◽  
William David Chen ◽  
Marta Cova ◽  
Petra Andrea Molnár ◽  
Shrawan Kumar Mageswaran ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Electron Tomography ◽  
Secretion System ◽  
Host Cells ◽  
Apicomplexan Parasites ◽  
Significant Difference ◽  
Cryo Electron Tomography ◽  
Parasite Plasmodium Falciparum ◽  
Priming Process

Apicomplexan parasites secrete the contents of rhoptries into host cells to permit their invasion and establishment of an infectious niche. The rhoptry secretory apparatus (RSA), which is critical for rhoptry secretion, was recently discovered in Toxoplasma and Cryptosporidium. It is positioned at the cell apex and associates with an enigmatic apical vesicle (AV), which docks one or two rhoptries at the site of exocytosis. The interplay among the rhoptries, the AV, and the parasite plasma membrane for secretion remains unclear. Moreover, it is unknown if a similar machinery exists in the deadly malaria parasite Plasmodium falciparum. In this study, we use in situ cryo-electron tomography to investigate the rhoptry secretion system in P. falciparum merozoites. We identify the presence of an RSA at the cell apex and a morphologically distinct AV docking the tips of the two rhoptries to the RSA. We also discover two new organizations: one in which the AV is absent with one of the two rhoptry tips docks directly to the RSA, and a second in which the two rhoptries fuse together and the common tip docks directly to the RSA. Interestingly, rhoptries among the three states show no significant difference in luminal volume and density, suggesting that the exocytosis of rhoptry contents has not yet occurred, and that these different organizations likely represent sequential states leading to secretion. Using subtomogram averaging, we reveal different conformations of the RSA structure corresponding to each state, including the opening of a gate-like density in the rhoptry-fused state. These conformational changes of the RSA uncover structural details of a priming process for major rhoptry secretion, which likely occur after initial interaction with a red blood cell. Our results highlight a previously unknown step in the process of rhoptry secretion and indicate a regulatory role for the conserved apical vesicle in host invasion by apicomplexan parasites.

scholarly journals Cryo-electron tomography of periplasmic flagella in Borrelia burgdorferi reveals a distinct cytoplasmic ATPase complex

PLoS Biology ◽  
2018 ◽  
Vol 16 (11) ◽  
pp. e3000050 ◽  
Author(s):  
Zhuan Qin ◽  
Jiagang Tu ◽  
Tao Lin ◽  
Steven J. Norris ◽  
Chunhao Li ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Electron Tomography ◽  
Cryo Electron Tomography ◽  
Periplasmic Flagella

scholarly journals Direct visualization of degradation microcompartments at the ER membrane

2019 ◽  
Vol 117 (2) ◽  
pp. 1069-1080 ◽  
Author(s):  
Sahradha Albert ◽  
Wojciech Wietrzynski ◽  
Chia-Wei Lee ◽  
Miroslava Schaffer ◽  
Florian Beck ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Hot Spots ◽  
Protein Quality ◽  
Electron Tomography ◽  
Interaction Partners ◽  
Cryo Electron Tomography ◽  
Membrane Bound ◽  
Molecular Landscape ◽  
Er Membrane

To promote the biochemical reactions of life, cells can compartmentalize molecular interaction partners together within separated non–membrane-bound regions. It is unknown whether this strategy is used to facilitate protein degradation at specific locations within the cell. Leveraging in situ cryo-electron tomography to image the native molecular landscape of the unicellular alga Chlamydomonas reinhardtii, we discovered that the cytosolic protein degradation machinery is concentrated within ∼200-nm foci that contact specialized patches of endoplasmic reticulum (ER) membrane away from the ER–Golgi interface. These non–membrane-bound microcompartments exclude ribosomes and consist of a core of densely clustered 26S proteasomes surrounded by a loose cloud of Cdc48. Active proteasomes in the microcompartments directly engage with putative substrate at the ER membrane, a function canonically assigned to Cdc48. Live-cell fluorescence microscopy revealed that the proteasome clusters are dynamic, with frequent assembly and fusion events. We propose that the microcompartments perform ER-associated degradation, colocalizing the degradation machinery at specific ER hot spots to enable efficient protein quality control.

scholarly journals HEMNMA-3D: Cryo Electron Tomography Method Based on Normal Mode Analysis to Study Continuous Conformational Variability of Macromolecular Complexes

2021 ◽  
Vol 8 ◽  
Author(s):  
Mohamad Harastani ◽  
Mikhail Eltsov ◽  
Amélie Leforestier ◽  
Slavica Jonic
Keyword(s):  
Data Analysis ◽  
Rigid Body ◽  
Normal Mode ◽  
Conformational Changes ◽  
Electron Tomography ◽  
Normal Mode Analysis ◽  
Mode Analysis ◽  
Macromolecular Complexes ◽  
Conformational Variability

Cryogenic electron tomography (cryo-ET) allows structural determination of biomolecules in their native environment (in situ). Its potential of providing information on the dynamics of macromolecular complexes in cells is still largely unexploited, due to the challenges of the data analysis. The crowded cell environment and continuous conformational changes of complexes make difficult disentangling the data heterogeneity. We present HEMNMA-3D, which is, to the best of our knowledge, the first method for analyzing cryo electron subtomograms in terms of continuous conformational changes of complexes. HEMNMA-3D uses a combination of elastic and rigid-body 3D-to-3D iterative alignments of a flexible 3D reference (atomic structure or electron microscopy density map) to match the conformation, orientation, and position of the complex in each subtomogram. The elastic matching combines molecular mechanics simulation (Normal Mode Analysis of the 3D reference) and experimental, subtomogram data analysis. The rigid-body alignment includes compensation for the missing wedge, due to the limited tilt angle of cryo-ET. The conformational parameters (amplitudes of normal modes) of the complexes in subtomograms obtained through the alignment are processed to visualize the distribution of conformations in a space of lower dimension (typically, 2D or 3D) referred to as space of conformations. This allows a visually interpretable insight into the dynamics of the complexes, by calculating 3D averages of subtomograms with similar conformations from selected (densest) regions and by recording movies of the 3D reference's displacement along selected trajectories through the densest regions. We describe HEMNMA-3D and show its validation using synthetic datasets. We apply HEMNMA-3D to an experimental dataset describing in situ nucleosome conformational variability. HEMNMA-3D software is available freely (open-source) as part of ContinuousFlex plugin of Scipion V3.0 (http://scipion.i2pc.es).

scholarly journals Minicells, Back in Fashion

2016 ◽  
Vol 198 (8) ◽  
pp. 1186-1195 ◽  
Author(s):  
Madeline M. Farley ◽  
Bo Hu ◽  
William Margolin ◽  
Jun Liu
Keyword(s):  
Conformational Changes ◽  
Electron Tomography ◽  
Imaging Techniques ◽  
Three Dimensional ◽  
Model Systems ◽  
Specimen Thickness ◽  
Macromolecular Complexes ◽  
Content Type ◽  
Cellular Environment

Cryo-electron tomography (cryo-ET) has emerged as a leading technique for three-dimensional visualization of large macromolecular complexes and their conformational changes in their native cellular environment. However, the resolution and potential applications of cryo-ET are fundamentally limited by specimen thickness, preventing high-resolutionin situvisualization of macromolecular structures in many bacteria (such asEscherichia coliandSalmonella enterica). Minicells, which were discovered nearly 50 years ago, have recently been exploited as model systems to visualize molecular machinesin situ, due to their smaller size and other unique properties. In this review, we discuss strategies for producing minicells and highlight their use in the study of chemotactic signaling, protein secretion, and DNA translocation. In combination with powerful genetic tools and advanced imaging techniques, minicells provide a springboard for in-depth structural studies of bacterial macromolecular complexesin situand therefore offer a unique approach for gaining novel structural insights into many important processes in microbiology.

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