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Abdelhakim AH, Salgado EN, Fu X, Pasham M, Nicastro D, Kirchhausen T and Harrison SC (2014), "Structural correlates of rotavirus cell entry.", PLoS Pathog., Sep, 2014. Vol. 10(9), pp. e1004355.
Abstract: Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex--for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ∼ 700 Å inner capsid particle ("double-layered particle", DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ∼ 10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration.
BibTeX:
@article{Abdelhakim2014,
  author = {Abdelhakim, Aliaa H. and Salgado, Eric N. and Fu, Xiaofeng and Pasham, Mithun and Nicastro, Daniela and Kirchhausen, Tomas and Harrison, Stephen C.},
  title = {Structural correlates of rotavirus cell entry.},
  journal = {PLoS Pathog},
  year = {2014},
  volume = {10},
  number = {9},
  pages = {e1004355},
  url = {http://dx.doi.org/10.1371/journal.ppat.1004355},
  doi = {10.1371/journal.ppat.1004355}
}
Abrusci P, Vergara-Irigaray M, Johnson S, Beeby MD, Hendrixson DR, Roversi P, Friede ME, Deane JE, Jensen GJ, Tang CM and Lea SM (2013), "Architecture of the major component of the type III secretion system export apparatus.", Nat Struct Mol Biol., Jan, 2013. Vol. 20(1), pp. 99-104.
Abstract: Type III secretion systems (T3SSs) are bacterial membrane-embedded nanomachines designed to export specifically targeted proteins from the bacterial cytoplasm. Secretion through T3SS is governed by a subset of inner membrane proteins termed the 'export apparatus'. We show that a key member of the Shigella flexneri export apparatus, MxiA, assembles into a ring essential for secretion in vivo. The ring-forming interfaces are well-conserved in both nonflagellar and flagellar homologs, implying that the ring is an evolutionarily conserved feature in these systems. Electron cryo-tomography revealed a T3SS-associated cytoplasmic torus of size and shape corresponding to those of the MxiA ring aligned to the secretion channel located between the secretion pore and the ATPase complex. This defines the molecular architecture of the dominant component of the export apparatus and allows us to propose a model for the molecular mechanisms controlling secretion.
BibTeX:
@article{Abrusci2013,
  author = {Abrusci, Patrizia and Vergara-Irigaray, Marta and Johnson, Steven and Beeby, Morgan D. and Hendrixson, David R. and Roversi, Pietro and Friede, Miriam E. and Deane, Janet E. and Jensen, Grant J. and Tang, Christoph M. and Lea, Susan M.},
  title = {Architecture of the major component of the type III secretion system export apparatus.},
  journal = {Nat Struct Mol Biol},
  year = {2013},
  volume = {20},
  number = {1},
  pages = {99--104},
  url = {http://dx.doi.org/10.1038/nsmb.2452},
  doi = {10.1038/nsmb.2452}
}
Althoff T, Davies KM, Schulze S, Joos F and Kühlbrandt W (2012), "GRecon: a method for the lipid reconstitution of membrane proteins.", Angew Chem Int Ed Engl., Aug, 2012. Vol. 51(33), pp. 8343-8347.
BibTeX:
@article{Althoff2012,
  author = {Althoff, Thorsten and Davies, Karen M. and Schulze, Sabrina and Joos, Friederike and Kühlbrandt, Werner},
  title = {GRecon: a method for the lipid reconstitution of membrane proteins.},
  journal = {Angew Chem Int Ed Engl},
  year = {2012},
  volume = {51},
  number = {33},
  pages = {8343--8347},
  url = {http://dx.doi.org/10.1002/anie.201202094},
  doi = {10.1002/anie.201202094}
}
Awata J, Song K, Lin J, King SM, Sanderson MJ, Nicastro D and Witman GB (2015), "DRC3 connects the N-DRC to dynein g to regulate flagellar waveform.", Mol Biol Cell., Aug, 2015. Vol. 26(15), pp. 2788-2800.
Abstract: The nexin-dynein regulatory complex (N-DRC), which is a major hub for the control of flagellar motility, contains at least 11 different subunits. A major challenge is to determine the location and function of each of these subunits within the N-DRC. We characterized a Chlamydomonas mutant defective in the N-DRC subunit DRC3. Of the known N-DRC subunits, the drc3 mutant is missing only DRC3. Like other N-DRC mutants, the drc3 mutant has a defect in flagellar motility. However, in contrast to other mutations affecting the N-DRC, drc3 does not suppress flagellar paralysis caused by loss of radial spokes. Cryo-electron tomography revealed that the drc3 mutant lacks a portion of the N-DRC linker domain, including the L1 protrusion, part of the distal lobe, and the connection between these two structures, thus localizing DRC3 to this part of the N-DRC. This and additional considerations enable us to assign DRC3 to the L1 protrusion. Because the L1 protrusion is the only non-dynein structure in contact with the dynein g motor domain in wild-type axonemes and this is the only N-DRC-dynein connection missing in the drc3 mutant, we conclude that DRC3 interacts with dynein g to regulate flagellar waveform.
BibTeX:
@article{Awata2015,
  author = {Awata, Junya and Song, Kangkang and Lin, Jianfeng and King, Stephen M. and Sanderson, Michael J. and Nicastro, Daniela and Witman, George B.},
  title = {DRC3 connects the N-DRC to dynein g to regulate flagellar waveform.},
  journal = {Mol Biol Cell},
  year = {2015},
  volume = {26},
  number = {15},
  pages = {2788--2800},
  url = {http://dx.doi.org/10.1091/mbc.E15-01-0018},
  doi = {10.1091/mbc.E15-01-0018}
}
Barber CF, Heuser T, Carbajal-González BI, Botchkarev Jr VV and Nicastro D (2012), "Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella.", Mol Biol Cell., Jan, 2012. Vol. 23(1), pp. 111-120.
Abstract: Radial spokes (RSs) play an essential role in the regulation of axonemal dynein activity and thus of ciliary and flagellar motility. However, few details are known about the complexes involved. Using cryo-electron tomography and subtomogram averaging, we visualized the three-dimensional structure of the radial spokes in Chlamydomonas flagella in unprecedented detail. Unlike many other species, Chlamydomonas has only two spokes per axonemal repeat, RS1 and RS2. Our data revealed previously uncharacterized features, including two-pronged spoke bases that facilitate docking to the doublet microtubules, and that inner dyneins connect directly to the spokes. Structures of wild type and the headless spoke mutant pf17 were compared to define the morphology and boundaries of the head, including a direct RS1-to-RS2 interaction. Although the overall structures of the spokes are very similar, we also observed some differences, corroborating recent findings about heterogeneity in the docking of RS1 and RS2. In place of a third radial spoke we found an uncharacterized, shorter electron density named "radial spoke 3 stand-in," which structurally bears no resemblance to RS1 and RS2 and is unaltered in the pf17 mutant. These findings demonstrate that radial spokes are heterogeneous in structure and may play functionally distinct roles in axoneme regulation.
BibTeX:
@article{Barber2012,
  author = {Barber, Cynthia F. and Heuser, Thomas and Carbajal-González, Blanca I. and Botchkarev, Jr, Vladimir V and Nicastro, Daniela},
  title = {Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella.},
  journal = {Mol Biol Cell},
  year = {2012},
  volume = {23},
  number = {1},
  pages = {111--120},
  url = {http://dx.doi.org/10.1091/mbc.E11-08-0692},
  doi = {10.1091/mbc.E11-08-0692}
}
Basler M, Pilhofer M, Henderson GP, Jensen GJ and Mekalanos JJ (2012), "Type VI secretion requires a dynamic contractile phage tail-like structure.", Nature., Mar, 2012. Vol. 483(7388), pp. 182-186.
Abstract: Type VI secretion systems are bacterial virulence-associated nanomachines composed of proteins that are evolutionarily related to components of bacteriophage tails. Here we show that protein secretion by the type VI secretion system of Vibrio cholerae requires the action of a dynamic intracellular tubular structure that is structurally and functionally homologous to contractile phage tail sheath. Time-lapse fluorescence light microscopy reveals that sheaths of the type VI secretion system cycle between assembly, quick contraction, disassembly and re-assembly. Whole-cell electron cryotomography further shows that the sheaths appear as long tubular structures in either extended or contracted conformations that are connected to the inner membrane by a distinct basal structure. These data support a model in which the contraction of the type VI secretion system sheath provides the energy needed to translocate proteins out of effector cells and into adjacent target cells.
BibTeX:
@article{Basler2012,
  author = {Basler, M. and Pilhofer, M. and Henderson, G. P. and Jensen, G. J. and Mekalanos, J. J.},
  title = {Type VI secretion requires a dynamic contractile phage tail-like structure.},
  journal = {Nature},
  year = {2012},
  volume = {483},
  number = {7388},
  pages = {182--186},
  url = {http://dx.doi.org/10.1038/nature10846},
  doi = {10.1038/nature10846}
}
Basta T, Wu H-J, Morphew MK, Lee J, Ghosh N, Lai J, Heumann JM, Wang K, Lee YC, Rees DC and Stowell MHB (2014), "Self-assembled lipid and membrane protein polyhedral nanoparticles.", Proc Natl Acad Sci U S A., Jan, 2014. Vol. 111(2), pp. 670-674.
Abstract: We demonstrate that membrane proteins and phospholipids can self-assemble into polyhedral arrangements suitable for structural analysis. Using the Escherichia coli mechanosensitive channel of small conductance (MscS) as a model protein, we prepared membrane protein polyhedral nanoparticles (MPPNs) with uniform radii of ∼ 20 nm. Electron cryotomographic analysis established that these MPPNs contain 24 MscS heptamers related by octahedral symmetry. Subsequent single-particle electron cryomicroscopy yielded a reconstruction at ∼ 1-nm resolution, revealing a conformation closely resembling the nonconducting state. The generality of this approach has been addressed by the successful preparation of MPPNs for two unrelated proteins, the mechanosensitive channel of large conductance and the connexon Cx26, using a recently devised microfluidics-based free interface diffusion system. MPPNs provide not only a starting point for the structural analysis of membrane proteins in a phospholipid environment, but their closed surfaces should facilitate studies in the presence of physiological transmembrane gradients, in addition to potential applications as drug delivery carriers or as templates for inorganic nanoparticle formation.
BibTeX:
@article{Basta2014,
  author = {Basta, Tamara and Wu, Hsin-Jui and Morphew, Mary K. and Lee, Jonas and Ghosh, Nilanjan and Lai, Jeffrey and Heumann, John M. and Wang, Keeshia and Lee, Y. C. and Rees, Douglas C. and Stowell, Michael H B.},
  title = {Self-assembled lipid and membrane protein polyhedral nanoparticles.},
  journal = {Proc Natl Acad Sci U S A},
  year = {2014},
  volume = {111},
  number = {2},
  pages = {670--674},
  url = {http://dx.doi.org/10.1073/pnas.1321936111},
  doi = {10.1073/pnas.1321936111}
}
Beeby M, Ribardo DA, Brennan CA, Ruby EG, Jensen GJ and Hendrixson DR (2016), "Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold.", Proc Natl Acad Sci U S A., Mar, 2016. Vol. 113(13), pp. E1917-E1926.
Abstract: Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, fromCampylobacterandVibriospecies. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes.
BibTeX:
@article{Beeby2016,
  author = {Beeby, Morgan and Ribardo, Deborah A. and Brennan, Caitlin A. and Ruby, Edward G. and Jensen, Grant J. and Hendrixson, David R.},
  title = {Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold.},
  journal = {Proc Natl Acad Sci U S A},
  year = {2016},
  volume = {113},
  number = {13},
  pages = {E1917--E1926},
  url = {http://dx.doi.org/10.1073/pnas.1518952113},
  doi = {10.1073/pnas.1518952113}
}
Briegel A, Ames P, Gumbart JC, Oikonomou CM, Parkinson JS and Jensen GJ (2013), "The mobility of two kinase domains in the Escherichia coli chemoreceptor array varies with signalling state.", Mol Microbiol., Sep, 2013. Vol. 89(5), pp. 831-841.
Abstract: Motile bacteria sense their physical and chemical environment through highly cooperative, ordered arrays of chemoreceptors. These signalling complexes phosphorylate a response regulator which in turn governs flagellar motor reversals, driving cells towards favourable environments. The structural changes that translate chemoeffector binding into the appropriate kinase output are not known. Here, we apply high-resolution electron cryotomography to visualize mutant chemoreceptor signalling arrays in well-defined kinase activity states. The arrays were well ordered in all signalling states, with no discernible differences in receptor conformation at 2-3 nm resolution. Differences were observed, however, in a keel-like density that we identify here as CheA kinase domains P1 and P2, the phosphorylation site domain and the binding domain for response regulator target proteins. Mutant receptor arrays with high kinase activities all exhibited small keels and high proteolysis susceptibility, indicative of mobile P1 and P2 domains. In contrast, arrays in kinase-off signalling states exhibited a range of keel sizes. These findings confirm that chemoreceptor arrays do not undergo large structural changes during signalling, and suggest instead that kinase activity is modulated at least in part by changes in the mobility of key domains.
BibTeX:
@article{Briegel2013,
  author = {Briegel, Ariane and Ames, Peter and Gumbart, James C. and Oikonomou, Catherine M. and Parkinson, John S. and Jensen, Grant J.},
  title = {The mobility of two kinase domains in the Escherichia coli chemoreceptor array varies with signalling state.},
  journal = {Mol Microbiol},
  year = {2013},
  volume = {89},
  number = {5},
  pages = {831--841},
  url = {http://dx.doi.org/10.1111/mmi.12309},
  doi = {10.1111/mmi.12309}
}
Briegel A, Beeby M, Thanbichler M and Jensen GJ (2011), "Activated chemoreceptor arrays remain intact and hexagonally packed.", Mol Microbiol., Nov, 2011. Vol. 82(3), pp. 748-757.
Abstract: Bacterial chemoreceptors cluster into exquisitively sensitive, tunable, highly ordered, polar arrays. While these arrays serve as paradigms of cell signalling in general, it remains unclear what conformational changes transduce signals from the periplasmic tips, where attractants and repellents bind, to the cytoplasmic signalling domains. Conflicting reports support and contest the hypothesis that activation causes large changes in the packing arrangement of the arrays, up to and including their complete disassembly. Using electron cryotomography, here we show that in Caulobacter crescentus, chemoreceptor arrays in cells grown in different media and immediately after exposure to the attractant galactose all exhibit the same 12 nm hexagonal packing arrangement, array size and other structural parameters. ΔcheB and ΔcheR mutants mimicking attractant- or repellent-bound states prior to adaptation also show the same lattice structure. We conclude that signal transduction and amplification must be accomplished through only small, nanoscale conformational changes.
BibTeX:
@article{Briegel2011,
  author = {Briegel, Ariane and Beeby, Morgan and Thanbichler, Martin and Jensen, Grant J.},
  title = {Activated chemoreceptor arrays remain intact and hexagonally packed.},
  journal = {Mol Microbiol},
  year = {2011},
  volume = {82},
  number = {3},
  pages = {748--757},
  url = {http://dx.doi.org/10.1111/j.1365-2958.2011.07854.x},
  doi = {10.1111/j.1365-2958.2011.07854.x}
}
Briegel A, Li X, Bilwes AM, Hughes KT, Jensen GJ and Crane BR (2012), "Bacterial chemoreceptor arrays are hexagonally packed trimers of receptor dimers networked by rings of kinase and coupling proteins.", Proc Natl Acad Sci U S A., Mar, 2012. Vol. 109(10), pp. 3766-3771.
Abstract: Chemoreceptor arrays are supramolecular transmembrane machines of unknown structure that allow bacteria to sense their surroundings and respond by chemotaxis. We have combined X-ray crystallography of purified proteins with electron cryotomography of native arrays inside cells to reveal the arrangement of the component transmembrane receptors, histidine kinases (CheA) and CheW coupling proteins. Trimers of receptor dimers lie at the vertices of a hexagonal lattice in a "two-facing-two" configuration surrounding a ring of alternating CheA regulatory domains (P5) and CheW couplers. Whereas the CheA kinase domains (P4) project downward below the ring, the CheA dimerization domains (P3) link neighboring rings to form an extended, stable array. This highly interconnected protein architecture underlies the remarkable sensitivity and cooperative nature of transmembrane signaling in bacterial chemotaxis.
BibTeX:
@article{Briegel2012,
  author = {Briegel, Ariane and Li, Xiaoxiao and Bilwes, Alexandrine M. and Hughes, Kelly T. and Jensen, Grant J. and Crane, Brian R.},
  title = {Bacterial chemoreceptor arrays are hexagonally packed trimers of receptor dimers networked by rings of kinase and coupling proteins.},
  journal = {Proc Natl Acad Sci U S A},
  year = {2012},
  volume = {109},
  number = {10},
  pages = {3766--3771},
  url = {http://dx.doi.org/10.1073/pnas.1115719109},
  doi = {10.1073/pnas.1115719109}
}
Briegel A, Ortega DR, Huang AN, Oikonomou CM, Gunsalus RP and Jensen GJ (2015), "Structural conservation of chemotaxis machinery across Archaea and Bacteria.", Environ Microbiol Rep., Jun, 2015. Vol. 7(3), pp. 414-419.
Abstract: Chemotaxis allows cells to sense and respond to their environment. In Bacteria, stimuli are detected by arrays of chemoreceptors that relay the signal to a two-component regulatory system. These arrays take the form of highly stereotyped super-lattices comprising hexagonally packed trimers-of-receptor-dimers networked by rings of histidine kinase and coupling proteins. This structure is conserved across chemotactic Bacteria, and between membrane-bound and cytoplasmic arrays, and gives rise to the highly cooperative, dynamic nature of the signalling system. The chemotaxis system, absent in eukaryotes, is also found in Archaea, where its structural details remain uncharacterized. Here we provide evidence that the chemotaxis machinery was not present in the last archaeal common ancestor, but rather was introduced in one of the waves of lateral gene transfer that occurred after the branching of Eukaryota but before the diversification of Euryarchaeota. Unlike in Bacteria, the chemotaxis system then evolved largely vertically in Archaea, with very few subsequent successful lateral gene transfer events. By electron cryotomography, we find that the structure of both membrane-bound and cytoplasmic chemoreceptor arrays is conserved between Bacteria and Archaea, suggesting the fundamental importance of this signalling architecture across diverse prokaryotic lifestyles.
BibTeX:
@article{Briegel2015,
  author = {Briegel, Ariane and Ortega, Davi R. and Huang, Audrey N. and Oikonomou, Catherine M. and Gunsalus, Robert P. and Jensen, Grant J.},
  title = {Structural conservation of chemotaxis machinery across Archaea and Bacteria.},
  journal = {Environ Microbiol Rep},
  year = {2015},
  volume = {7},
  number = {3},
  pages = {414--419},
  url = {http://dx.doi.org/10.1111/1758-2229.12265},
  doi = {10.1111/1758-2229.12265}
}
Briegel A, Wong ML, Hodges HL, Oikonomou CM, Piasta KN, Harris MJ, Fowler DJ, Thompson LK, Falke JJ, Kiessling LL and Jensen GJ (2014), "New insights into bacterial chemoreceptor array structure and assembly from electron cryotomography.", Biochemistry., Mar, 2014. Vol. 53(10), pp. 1575-1585.
Abstract: Bacterial chemoreceptors cluster in highly ordered, cooperative, extended arrays with a conserved architecture, but the principles that govern array assembly remain unclear. Here we show images of cellular arrays as well as selected chemoreceptor complexes reconstituted in vitro that reveal new principles of array structure and assembly. First, in every case, receptors clustered in a trimers-of-dimers configuration, suggesting this is a highly favored fundamental building block. Second, these trimers-of-receptor dimers exhibited great versatility in the kinds of contacts they formed with each other and with other components of the signaling pathway, although only one architectural type occurred in native arrays. Third, the membrane, while it likely accelerates the formation of arrays, was neither necessary nor sufficient for lattice formation. Molecular crowding substituted for the stabilizing effect of the membrane and allowed cytoplasmic receptor fragments to form sandwiched lattices that strongly resemble the cytoplasmic chemoreceptor arrays found in some bacterial species. Finally, the effective determinant of array structure seemed to be CheA and CheW, which formed a "superlattice" of alternating CheA-filled and CheA-empty rings that linked receptor trimers-of-dimer units into their native hexagonal lattice. While concomitant overexpression of receptors, CheA, and CheW yielded arrays with native spacing, the CheA occupancy was lower and less ordered, suggesting that temporal and spatial coordination of gene expression driven by a single transcription factor may be vital for full order, or that array overgrowth may trigger a disassembly process. The results described here provide new insights into the assembly intermediates and assembly mechanism of this massive macromolecular complex.
BibTeX:
@article{Briegel2014,
  author = {Briegel, Ariane and Wong, Margaret L. and Hodges, Heather L. and Oikonomou, Catherine M. and Piasta, Kene N. and Harris, Michael J. and Fowler, Daniel J. and Thompson, Lynmarie K. and Falke, Joseph J. and Kiessling, Laura L. and Jensen, Grant J.},
  title = {New insights into bacterial chemoreceptor array structure and assembly from electron cryotomography.},
  journal = {Biochemistry},
  year = {2014},
  volume = {53},
  number = {10},
  pages = {1575--1585},
  url = {http://dx.doi.org/10.1021/bi5000614},
  doi = {10.1021/bi5000614}
}
Brown JR, Schwartz CL, Heumann JM, Dawson SC and Hoenger A (2016), "A detailed look at the cytoskeletal architecture of the Giardia lamblia ventral disc.", J Struct Biol., Apr, 2016. Vol. 194(1), pp. 38-48.
Abstract: Giardia lamblia is a protistan parasite that infects and colonizes the small intestine of mammals. It is widespread and particularly endemic in the developing world. Here we present a detailed structural study by 3-D negative staining and cryo-electron tomography of a unique Giardia organelle, the ventral disc. The disc is composed of a regular array of microtubules and associated sheets, called microribbons that form a large spiral, held together by a myriad of mostly unknown associated proteins. In a previous study we analyzed by cryo-electron tomography the central microtubule portion (here called disc body) of the ventral disc and found a large portion of microtubule associated inner (MIPs) and outer proteins (MAPs) that render these microtubules hyper-stable. With this follow-up study we expanded our 3-D analysis to different parts of the disc such as the ventral and dorsal areas of the overlap zone, as well as the outer disc margin. There are intrinsic location-specific characteristics in the composition of microtubule-associated proteins between these regions, as well as large differences between the overall architecture of microtubules and microribbons. The lateral packing of microtubule-microribbon complexes varies substantially, and closer packing often comes with contracted lateral tethers that seem to hold the disc together. It appears that the marginal microtubule-microribbon complexes function as outer, laterally contractible lids that may help the cell to clamp onto the intestinal microvilli. Furthermore, we analyzed length, quantity, curvature and distribution between different zones of the disc, which we found to differ from previous publications.
BibTeX:
@article{Brown2016,
  author = {Brown, Joanna R. and Schwartz, Cindi L. and Heumann, John M. and Dawson, Scott C. and Hoenger, Andreas},
  title = {A detailed look at the cytoskeletal architecture of the Giardia lamblia ventral disc.},
  journal = {J Struct Biol},
  year = {2016},
  volume = {194},
  number = {1},
  pages = {38--48},
  url = {http://dx.doi.org/10.1016/j.jsb.2016.01.011},
  doi = {10.1016/j.jsb.2016.01.011}
}
Bui KH, Pigino G and Ishikawa T (2011), "Three-dimensional structural analysis of eukaryotic flagella/cilia by electron cryo-tomography.", J Synchrotron Radiat., Jan, 2011. Vol. 18(1), pp. 2-5.
Abstract: Electron cryo-tomography is a potential approach to analyzing the three-dimensional conformation of frozen hydrated biological macromolecules using electron microscopy. Since projections of each individual object illuminated from different orientations are merged, electron tomography is capable of structural analysis of such heterogeneous environments as in vivo or with polymorphism, although radiation damage and the missing wedge are severe problems. Here, recent results on the structure of eukaryotic flagella, which is an ATP-driven bending organelle, from green algae Chlamydomonas are presented. Tomographic analysis reveals asymmetric molecular arrangements, especially that of the dynein motor proteins, in flagella, giving insight into the mechanism of planar asymmetric bending motion. Methodological challenges to obtaining higher-resolution structures from this technique are also discussed.
BibTeX:
@article{Bui2011,
  author = {Bui, Khanh Huy and Pigino, Gaia and Ishikawa, Takashi},
  title = {Three-dimensional structural analysis of eukaryotic flagella/cilia by electron cryo-tomography.},
  journal = {J Synchrotron Radiat},
  year = {2011},
  volume = {18},
  number = {1},
  pages = {2--5},
  url = {http://dx.doi.org/10.1107/S0909049510036812},
  doi = {10.1107/S0909049510036812}
}
Burgoyne T, Morris EP and Luther PK (2015), "Three-Dimensional Structure of Vertebrate Muscle Z-Band: The Small-Square Lattice Z-Band in Rat Cardiac Muscle.", J Mol Biol., Nov, 2015. Vol. 427(22), pp. 3527-3537.
Abstract: The Z-band in vertebrate striated muscle crosslinks actin filaments of opposite polarity from adjoining sarcomeres and transmits tension along myofibrils during muscular contraction. It is also the location of a number of proteins involved in signalling and myofibrillogenesis; mutations in these proteins lead to myopathies. Understanding the high-resolution structure of the Z-band will help us understand its role in muscle contraction and the role of these proteins in the function of muscle. The appearance of the Z-band in transverse-section electron micrographs typically resembles a small-square lattice or a basketweave appearance. In longitudinal sections, the Z-band width varies more with muscle type than species: slow skeletal and cardiac muscles have wider Z-bands than fast skeletal muscles. As the Z-band is periodic, Fourier methods have previously been used for three-dimensional structural analysis. To cope with variations in the periodic structure of the Z-band, we have used subtomogram averaging of tomograms of rat cardiac muscle in which subtomograms are extracted and compared and similar ones are averaged. We show that the Z-band comprises four to six layers of links, presumably α-actinin, linking antiparallel overlapping ends of the actin filaments from the adjoining sarcomeres. The reconstruction shows that the terminal 5-7nm of the actin filaments within the Z-band is devoid of any α-actinin links and is likely to be the location of capping protein CapZ.
BibTeX:
@article{Burgoyne2015,
  author = {Burgoyne, Thomas and Morris, Edward P. and Luther, Pradeep K.},
  title = {Three-Dimensional Structure of Vertebrate Muscle Z-Band: The Small-Square Lattice Z-Band in Rat Cardiac Muscle.},
  journal = {J Mol Biol},
  year = {2015},
  volume = {427},
  number = {22},
  pages = {3527--3537},
  url = {http://dx.doi.org/10.1016/j.jmb.2015.08.018},
  doi = {10.1016/j.jmb.2015.08.018}
}
Carbajal-González BI, Heuser T, Fu X, Lin J, Smith BW, Mitchell DR and Nicastro D (2013), "Conserved structural motifs in the central pair complex of eukaryotic flagella.", Cytoskeleton (Hoboken)., Feb, 2013. Vol. 70(2), pp. 101-120.
Abstract: Cilia and flagella are conserved hair-like appendages of eukaryotic cells that function as sensing and motility generating organelles. Motility is driven by thousands of axonemal dyneins that require precise regulation. One essential motility regulator is the central pair complex (CPC) and many CPC defects cause paralysis of cilia/flagella. Several human diseases, such as immotile cilia syndrome, show CPC abnormalities, but little is known about the detailed three-dimensional (3D) structure and function of the CPC. The CPC is located in the center of typical [9+2] cilia/flagella and is composed of two singlet microtubules (MTs), each with a set of associated projections that extend toward the surrounding nine doublet MTs. Using cryo-electron tomography coupled with subtomogram averaging, we visualized and compared the 3D structures of the CPC in both the green alga Chlamydomonas and the sea urchin Strongylocentrotus at the highest resolution published to date. Despite the evolutionary distance between these species, their CPCs exhibit remarkable structural conservation. We identified several new projections, including those that form the elusive sheath, and show that the bridge has a more complex architecture than previously thought. Organism-specific differences include the presence of MT inner proteins in Chlamydomonas, but not Strongylocentrotus, and different overall outlines of the highly connected projection network, which forms a round-shaped cylinder in algae, but is more oval in sea urchin. These differences could be adaptations to the mechanical requirements of the rotating CPC in Chlamydomonas, compared to the Strongylocentrotus CPC which has a fixed orientation.
BibTeX:
@article{Carbajal-Gonzalez2013,
  author = {Carbajal-González, Blanca I. and Heuser, Thomas and Fu, Xiaofeng and Lin, Jianfeng and Smith, Brandon W. and Mitchell, David R. and Nicastro, Daniela},
  title = {Conserved structural motifs in the central pair complex of eukaryotic flagella.},
  journal = {Cytoskeleton (Hoboken)},
  year = {2013},
  volume = {70},
  number = {2},
  pages = {101--120},
  url = {http://dx.doi.org/10.1002/cm.21094},
  doi = {10.1002/cm.21094}
}
Chen S, Beeby M, Murphy GE, Leadbetter JR, Hendrixson DR, Briegel A, Li Z, Shi J, Tocheva EI, Müller A, Dobro MJ and Jensen GJ (2011), "Structural diversity of bacterial flagellar motors.", EMBO J., Jul, 2011. Vol. 30(14), pp. 2972-2981.
Abstract: The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species.
BibTeX:
@article{Chen2011,
  author = {Chen, Songye and Beeby, Morgan and Murphy, Gavin E. and Leadbetter, Jared R. and Hendrixson, David R. and Briegel, Ariane and Li, Zhuo and Shi, Jian and Tocheva, Elitza I. and Müller, Axel and Dobro, Megan J. and Jensen, Grant J.},
  title = {Structural diversity of bacterial flagellar motors.},
  journal = {EMBO J},
  year = {2011},
  volume = {30},
  number = {14},
  pages = {2972--2981},
  url = {http://dx.doi.org/10.1038/emboj.2011.186},
  doi = {10.1038/emboj.2011.186}
}
Ciesielski PN, Matthews JF, Tucker MP, Beckham GT, Crowley MF, Himmel ME and Donohoe BS (2013), "3D electron tomography of pretreated biomass informs atomic modeling of cellulose microfibrils.", ACS Nano., Sep, 2013. Vol. 7(9), pp. 8011-8019.
Abstract: Fundamental insights into the macromolecular architecture of plant cell walls will elucidate new structure-property relationships and facilitate optimization of catalytic processes that produce fuels and chemicals from biomass. Here we introduce computational methodology to extract nanoscale geometry of cellulose microfibrils within thermochemically treated biomass directly from electron tomographic data sets. We quantitatively compare the cell wall nanostructure in corn stover following two leading pretreatment strategies: dilute acid with iron sulfate co-catalyst and ammonia fiber expansion (AFEX). Computational analysis of the tomographic data is used to extract mathematical descriptions for longitudinal axes of cellulose microfibrils from which we calculate their nanoscale curvature. These nanostructural measurements are used to inform the construction of atomistic models that exhibit features of cellulose within real, process-relevant biomass. By computational evaluation of these atomic models, we propose relationships between the crystal structure of cellulose Iβ and the nanoscale geometry of cellulose microfibrils.
BibTeX:
@article{Ciesielski2013,
  author = {Ciesielski, Peter N. and Matthews, James F. and Tucker, Melvin P. and Beckham, Gregg T. and Crowley, Michael F. and Himmel, Michael E. and Donohoe, Bryon S.},
  title = {3D electron tomography of pretreated biomass informs atomic modeling of cellulose microfibrils.},
  journal = {ACS Nano},
  year = {2013},
  volume = {7},
  number = {9},
  pages = {8011--8019},
  url = {http://dx.doi.org/10.1021/nn4031542},
  doi = {10.1021/nn4031542}
}
Cope J, Gilbert S, Rayment I, Mastronarde D and Hoenger A (2010), "Cryo-electron tomography of microtubule-kinesin motor complexes.", J Struct Biol., May, 2010. Vol. 170(2), pp. 257-265.
Abstract: Microtubules complexed with molecular motors of the kinesin family or non-motor microtubule associated proteins (MAPs) such as tau or EB1 have been the subject of cryo-electron microcopy based 3-D studies for several years. Most of these studies that targeted complexes with intact microtubules have been carried out by helical 3-D reconstruction, while few were analyzed by single particle approaches or from 2-D crystalline arrays. Helical reconstruction of microtubule-MAP or motor complexes has been extremely successful but by definition, all helical 3-D reconstruction attempts require perfectly helical assemblies, which presents a serious limitation and confines the attempts to 15- or 16-protofilament microtubules, microtubule configurations that are very rare in nature. The rise of cryo-electron tomography within the last few years has now opened a new avenue towards solving 3-D structures of microtubule-MAP complexes that do not form helical assemblies, most importantly for the subject here, all microtubules that exhibit a lattice seam. In addition, not all motor domains or MAPs decorate the microtubule surface regularly enough to match the underlying microtubule lattice, or they adopt conformations that deviate from helical symmetry. Here we demonstrate the power and limitation of cryo-electron tomography using two kinesin motor domains, the monomeric Eg5 motor domain, and the heterodimeric Kar3Vik1 motor. We show here that tomography does not exclude the possibility of post-tomographic averaging when identical sub-volumes can be extracted from tomograms and in both cases we were able to reconstruct 3-D maps of conformations that are not possible to obtain using helical or other averaging-based methods.
BibTeX:
@article{Cope2010,
  author = {Cope, Julia and Gilbert, Susan and Rayment, Ivan and Mastronarde, David and Hoenger, Andreas},
  title = {Cryo-electron tomography of microtubule-kinesin motor complexes.},
  journal = {J Struct Biol},
  year = {2010},
  volume = {170},
  number = {2},
  pages = {257--265},
  url = {http://dx.doi.org/10.1016/j.jsb.2009.12.004},
  doi = {10.1016/j.jsb.2009.12.004}
}
Cope J, Heumann J and Hoenger A (2011), "Cryo-electron tomography for structural characterization of macromolecular complexes.", Curr Protoc Protein Sci., Aug, 2011. Vol. Chapter 17, pp. Unit17.13.
Abstract: Cryo-electron tomography (cryo-ET) is an emerging 3-D reconstruction technology that combines the principles of tomographic 3-D reconstruction with the unmatched structural preservation of biological matter embedded in vitreous ice. Cryo-ET is particularly suited to investigating cell-biological samples and large macromolecular structures that are too polymorphic to be reconstructed by classical averaging-based 3-D reconstruction procedures. This unit aims to make cryo-ET accessible to newcomers and discusses the specialized equipment required, as well as relevant advantages and hurdles associated with sample preparation by vitrification and cryo-ET. Protocols describe specimen preparation, data recording and 3-D data reconstruction for cryo-ET, with a special focus on macromolecular complexes. A step-by-step procedure for specimen vitrification by plunge freezing is provided, followed by the general practicalities of tilt-series acquisition for cryo-ET, including advice on how to select an area appropriate for acquiring a tilt series. A brief introduction to the underlying computational reconstruction principles applied in tomography is described, along with instructions for reconstructing a tomogram from cryo-tilt series data. Finally, a method is detailed for extracting small subvolumes containing identical macromolecular structures from tomograms for alignment and averaging as a means to increase the signal-to-noise ratio and eliminate missing wedge effects inherent in tomographic reconstructions.
BibTeX:
@article{Cope2011,
  author = {Cope, Julia and Heumann, John and Hoenger, Andreas},
  title = {Cryo-electron tomography for structural characterization of macromolecular complexes.},
  journal = {Curr Protoc Protein Sci},
  year = {2011},
  volume = {Chapter 17},
  pages = {Unit17.13},
  url = {http://dx.doi.org/10.1002/0471140864.ps1713s65},
  doi = {10.1002/0471140864.ps1713s65}
}
Davies KM, Anselmi C, Wittig I, Faraldo-Gómez JD and Kühlbrandt W (2012), "Structure of the yeast F1Fo-ATP synthase dimer and its role in shaping the mitochondrial cristae.", Proc Natl Acad Sci U S A., Aug, 2012. Vol. 109(34), pp. 13602-13607.
Abstract: We used electron cryotomography of mitochondrial membranes from wild-type and mutant Saccharomyces cerevisiae to investigate the structure and organization of ATP synthase dimers in situ. Subtomogram averaging of the dimers to 3.7 nm resolution revealed a V-shaped structure of twofold symmetry, with an angle of 86° between monomers. The central and peripheral stalks are well resolved. The monomers interact within the membrane at the base of the peripheral stalks. In wild-type mitochondria ATP synthase dimers are found in rows along the highly curved cristae ridges, and appear to be crucial for membrane morphology. Strains deficient in the dimer-specific subunits e and g or the first transmembrane helix of subunit 4 lack both dimers and lamellar cristae. Instead, cristae are either absent or balloon-shaped, with ATP synthase monomers distributed randomly in the membrane. Computer simulations indicate that isolated dimers induce a plastic deformation in the lipid bilayer, which is partially relieved by their side-by-side association. We propose that the assembly of ATP synthase dimer rows is driven by the reduction in the membrane elastic energy, rather than by direct protein contacts, and that the dimer rows enable the formation of highly curved ridges in mitochondrial cristae.
BibTeX:
@article{Davies2012,
  author = {Davies, Karen M. and Anselmi, Claudio and Wittig, Ilka and Faraldo-Gómez, José D. and Kühlbrandt, Werner},
  title = {Structure of the yeast F1Fo-ATP synthase dimer and its role in shaping the mitochondrial cristae.},
  journal = {Proc Natl Acad Sci U S A},
  year = {2012},
  volume = {109},
  number = {34},
  pages = {13602--13607},
  url = {http://dx.doi.org/10.1073/pnas.1204593109},
  doi = {10.1073/pnas.1204593109}
}
Dent KC, Thompson R, Barker AM, Hiscox JA, Barr JN, Stockley PG and Ranson NA (2013), "The asymmetric structure of an icosahedral virus bound to its receptor suggests a mechanism for genome release.", Structure., Jul, 2013. Vol. 21(7), pp. 1225-1234.
Abstract: Simple, spherical RNA viruses have well-understood, symmetric protein capsids, but little structural information is available for their asymmetric components, such as minor proteins and their genomes, which are vital for infection. Here, we report an asymmetric structure of bacteriophage MS2, attached to its receptor, the F-pilus. Cryo-electron tomography and subtomographic averaging of such complexes result in a structure containing clear density for the packaged genome, implying that the conformation of the genome is the same in each virus particle. The data also suggest that the single-copy viral maturation protein breaks the symmetry of the capsid, occupying a position that would be filled by a coat protein dimer in an icosahedral shell. This capsomere can thus fulfill its known biological roles in receptor and genome binding and suggests an exit route for the genome during infection.
BibTeX:
@article{Dent2013,
  author = {Dent, Kyle C. and Thompson, Rebecca and Barker, Amy M. and Hiscox, Julian A. and Barr, John N. and Stockley, Peter G. and Ranson, Neil A.},
  title = {The asymmetric structure of an icosahedral virus bound to its receptor suggests a mechanism for genome release.},
  journal = {Structure},
  year = {2013},
  volume = {21},
  number = {7},
  pages = {1225--1234},
  url = {http://dx.doi.org/10.1016/j.str.2013.05.012},
  doi = {10.1016/j.str.2013.05.012}
}
Diebolder CA, Beurskens FJ, de Jong RN, Koning RI, Strumane K, Lindorfer MA, Voorhorst M, Ugurlar D, Rosati S, Heck AJR, van de Winkel JGJ, Wilson IA, Koster AJ, Taylor RP, Saphire EO, Burton DR, Schuurman J, Gros P and Parren PWHI (2014), "Complement is activated by IgG hexamers assembled at the cell surface.", Science., Mar, 2014. Vol. 343(6176), pp. 1260-1263.
Abstract: Complement activation by antibodies bound to pathogens, tumors, and self antigens is a critical feature of natural immune defense, a number of disease processes, and immunotherapies. How antibodies activate the complement cascade, however, is poorly understood. We found that specific noncovalent interactions between Fc segments of immunoglobulin G (IgG) antibodies resulted in the formation of ordered antibody hexamers after antigen binding on cells. These hexamers recruited and activated C1, the first component of complement, thereby triggering the complement cascade. The interactions between neighboring Fc segments could be manipulated to block, reconstitute, and enhance complement activation and killing of target cells, using all four human IgG subclasses. We offer a general model for understanding antibody-mediated complement activation and the design of antibody therapeutics with enhanced efficacy.
BibTeX:
@article{Diebolder2014,
  author = {Diebolder, Christoph A. and Beurskens, Frank J. and de Jong, Rob N. and Koning, Roman I. and Strumane, Kristin and Lindorfer, Margaret A. and Voorhorst, Marleen and Ugurlar, Deniz and Rosati, Sara and Heck, Albert J R. and van de Winkel, Jan G J. and Wilson, Ian A. and Koster, Abraham J. and Taylor, Ronald P. and Saphire, Erica Ollmann and Burton, Dennis R. and Schuurman, Janine and Gros, Piet and Parren, Paul W H I.},
  title = {Complement is activated by IgG hexamers assembled at the cell surface.},
  journal = {Science},
  year = {2014},
  volume = {343},
  number = {6176},
  pages = {1260--1263},
  url = {http://dx.doi.org/10.1126/science.1248943},
  doi = {10.1126/science.1248943}
}
Diebolder CA, Halff EF, Koster AJ, Huizinga EG and Koning RI (2015), "Cryoelectron Tomography of the NAIP5/NLRC4 Inflammasome: Implications for NLR Activation.", Structure., Dec, 2015. Vol. 23(12), pp. 2349-2357.
Abstract: Inflammasomes are high molecular weight protein complexes that play a crucial role in innate immunity by activating caspase-1. Inflammasome formation is initiated when molecules originating from invading microorganisms activate nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs) and induce NLR multimerization. Little is known about the conformational changes involved in NLR activation and the structural organization of NLR multimers. Here, we show by cryoelectron tomography that flagellin-induced NAIP5/NLRC4 multimers form right- and left-handed helical polymers with a diameter of 28 nm and a pitch of 6.5 nm. Subtomogram averaging produced an electron density map at 4 nm resolution, which was used for rigid body fitting of NLR subdomains derived from the crystal structure of dormant NLRC4. The resulting structural model of inflammasome-incorporated NLRC4 indicates that a prominent rotation of the LRR domain of NLRC4 is necessary for multimer formation, providing unprecedented insight into the conformational changes that accompany NLR activation.
BibTeX:
@article{Diebolder2015,
  author = {Diebolder, Christoph A. and Halff, Els F. and Koster, Abraham J. and Huizinga, Eric G. and Koning, Roman I.},
  title = {Cryoelectron Tomography of the NAIP5/NLRC4 Inflammasome: Implications for NLR Activation.},
  journal = {Structure},
  year = {2015},
  volume = {23},
  number = {12},
  pages = {2349--2357},
  url = {http://dx.doi.org/10.1016/j.str.2015.10.001},
  doi = {10.1016/j.str.2015.10.001}
}
Dudkina NV, Oostergetel GT, Lewejohann D, Braun H-P and Boekema EJ (2010), "Row-like organization of ATP synthase in intact mitochondria determined by cryo-electron tomography.", Biochim Biophys Acta., Feb, 2010. Vol. 1797(2), pp. 272-277.
Abstract: The fine structure of intact, close-to-spherical mitochondria from the alga Polytomella was visualized by dual-axis cryo-electron tomography. The supramolecular organization of dimeric ATP synthase in the cristae membranes was investigated by averaging subvolumes of tomograms and 3D details at approximately 6 nm resolution were revealed. Oligomeric ATP synthase is composed of rows of dimers at 12 nm intervals; the dimers make a slight angle along the row. In addition, the main features of monomeric ATP synthase, such as the conically shaped F(1) headpiece, central stalk and stator were revealed. This demonstrates the capability of dual-axis electron tomography to unravel details of proteins and their interactions in complete organelles.
BibTeX:
@article{Dudkina2010a,
  author = {Dudkina, Natalya V. and Oostergetel, Gert T. and Lewejohann, Dagmar and Braun, Hans-Peter and Boekema, Egbert J.},
  title = {Row-like organization of ATP synthase in intact mitochondria determined by cryo-electron tomography.},
  journal = {Biochim Biophys Acta},
  year = {2010},
  volume = {1797},
  number = {2},
  pages = {272--277},
  url = {http://dx.doi.org/10.1016/j.bbabio.2009.11.004},
  doi = {10.1016/j.bbabio.2009.11.004}
}
Guichard P, Hamel V, Neves A and Gönczy P (2015), "Isolation, cryotomography, and three-dimensional reconstruction of centrioles.", Methods Cell Biol. Vol. 129, pp. 191-209.
Abstract: Centrioles and basal bodies (referred to hereafter as centrioles for simplicity) are microtubule-based cylindrical organelles that are typically ∼450-nm long and ∼250nm in diameter. The centriole is composed of three distinct regions: the distal part characterized by microtubule doublets, the central core that harbors microtubule triplets, which are also present in the proximal part that also contains the cartwheel, a structure crucial for centriole assembly. The cartwheel was initially revealed by conventional electron microscopy of resin-embedded samples and is thought to impart the near universal ninefold symmetry of centrioles. Deciphering the native architecture of the cartwheel has proven challenging owing to its small dimensions and the difficulties in isolating it. Here, we present a method to purify and analyze the structure of the exceptionally long Trichonympha centriole by cryotomography and subtomogram averaging. Using this method, we revealed the native architecture of the proximal cartwheel-containing region at ∼40Å-resolution. This method can be applied as a general strategy for uncovering the structure of centrioles in other species.
BibTeX:
@article{Guichard2015,
  author = {Guichard, Paul and Hamel, Virginie and Neves, Aitana and Gönczy, Pierre},
  title = {Isolation, cryotomography, and three-dimensional reconstruction of centrioles.},
  journal = {Methods Cell Biol},
  year = {2015},
  volume = {129},
  pages = {191--209},
  url = {http://dx.doi.org/10.1016/bs.mcb.2015.04.003},
  doi = {10.1016/bs.mcb.2015.04.003}
}
Hagen C, Dent KC, Zeev-Ben-Mordehai T, Grange M, Bosse JB, Whittle C, Klupp BG, Siebert CA, Vasishtan D, Bäuerlein FJB, Cheleski J, Werner S, Guttmann P, Rehbein S, Henzler K, Demmerle J, Adler B, Koszinowski U, Schermelleh L, Schneider G, Enquist LW, Plitzko JM, Mettenleiter TC and Grünewald K (2015), "Structural Basis of Vesicle Formation at the Inner Nuclear Membrane.", Cell., Dec, 2015. Vol. 163(7), pp. 1692-1701.
Abstract: Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.
BibTeX:
@article{Hagen2015,
  author = {Hagen, Christoph and Dent, Kyle C. and Zeev-Ben-Mordehai, Tzviya and Grange, Michael and Bosse, Jens B. and Whittle, Cathy and Klupp, Barbara G. and Siebert, C Alistair and Vasishtan, Daven and Bäuerlein, Felix J B. and Cheleski, Juliana and Werner, Stephan and Guttmann, Peter and Rehbein, Stefan and Henzler, Katja and Demmerle, Justin and Adler, Barbara and Koszinowski, Ulrich and Schermelleh, Lothar and Schneider, Gerd and Enquist, Lynn W. and Plitzko, Jürgen M. and Mettenleiter, Thomas C. and Grünewald, Kay},
  title = {Structural Basis of Vesicle Formation at the Inner Nuclear Membrane.},
  journal = {Cell},
  year = {2015},
  volume = {163},
  number = {7},
  pages = {1692--1701},
  url = {http://dx.doi.org/10.1016/j.cell.2015.11.029},
  doi = {10.1016/j.cell.2015.11.029}
}
Hetzel U, Sironen T, Laurinmäki P, Liljeroos L, Patjas A, Henttonen H, Vaheri A, Artelt A, Kipar A, Butcher SJ, Vapalahti O and Hepojoki J (2013), "Isolation, identification, and characterization of novel arenaviruses, the etiological agents of boid inclusion body disease.", J Virol., Oct, 2013. Vol. 87(20), pp. 10918-10935.
Abstract: Boid inclusion body disease (BIBD) is a progressive, usually fatal disease of constrictor snakes, characterized by cytoplasmic inclusion bodies (IB) in a wide range of cell types. To identify the causative agent of the disease, we established cell cultures from BIBD-positive and -negative boa constrictors. The IB phenotype was maintained in cultured cells of affected animals, and supernatants from these cultures caused the phenotype in cultures originating from BIBD-negative snakes. Viruses were purified from the supernatants by ultracentrifugation and subsequently identified as arenaviruses. Purified virus also induced the IB phenotype in naive cells, which fulfilled Koch's postulates in vitro. One isolate, tentatively designated University of Helsinki virus (UHV), was studied in depth. Sequencing confirmed that UHV is a novel arenavirus species that is distinct from other known arenaviruses including those recently identified in snakes with BIBD. The morphology of UHV was established by cryoelectron tomography and subtomographic averaging, revealing the trimeric arenavirus spike structure at 3.2-nm resolution. Immunofluorescence, immunohistochemistry, and immunoblotting with a polyclonal rabbit antiserum against UHV and reverse transcription-PCR (RT-PCR) revealed the presence of genetically diverse arenaviruses in a large cohort of snakes with BIBD, confirming the causative role of arenaviruses. Some snakes were also found to carry arenavirus antibodies. Furthermore, mammalian cells (Vero E6) were productively infected with UHV, demonstrating the potential of arenaviruses to cross species barriers. In conclusion, we propose the newly identified lineage of arenaviruses associated with BIBD as a novel taxonomic entity, boid inclusion body disease-associated arenaviruses (BIBDAV), in the family Arenaviridae.
BibTeX:
@article{Hetzel2013,
  author = {Hetzel, Udo and Sironen, Tarja and Laurinmäki, Pasi and Liljeroos, Lassi and Patjas, Aino and Henttonen, Heikki and Vaheri, Antti and Artelt, Annette and Kipar, Anja and Butcher, Sarah J. and Vapalahti, Olli and Hepojoki, Jussi},
  title = {Isolation, identification, and characterization of novel arenaviruses, the etiological agents of boid inclusion body disease.},
  journal = {J Virol},
  year = {2013},
  volume = {87},
  number = {20},
  pages = {10918--10935},
  url = {http://dx.doi.org/10.1128/JVI.01123-13},
  doi = {10.1128/JVI.01123-13}
}
Heumann JM, Hoenger A and Mastronarde DN (2011), "Clustering and variance maps for cryo-electron tomography using wedge-masked differences.", J Struct Biol., Sep, 2011. Vol. 175(3), pp. 288-299.
Abstract: Cryo-electron tomography provides 3D imaging of frozen hydrated biological samples with nanometer resolution. Reconstructed volumes suffer from low signal-to-noise-ratio (SNR)(1) and artifacts caused by systematically missing tomographic data. Both problems can be overcome by combining multiple subvolumes with varying orientations, assuming they contain identical structures. Clustering (unsupervised classification) is required to ensure or verify population homogeneity, but this process is complicated by the problems of poor SNR and missing data, the factors that led to consideration of multiple subvolumes in the first place. Here, we describe a new approach to clustering and variance mapping in the face of these difficulties. The combined subvolume is taken as an estimate of the true subvolume, and the effect of missing data is computed for individual subvolumes. Clustering and variance mapping then proceed based on differences between expected and observed subvolumes. We show that this new method is faster and more accurate than two current, widely used techniques.
BibTeX:
@article{Heumann2011,
  author = {Heumann, John M. and Hoenger, Andreas and Mastronarde, David N.},
  title = {Clustering and variance maps for cryo-electron tomography using wedge-masked differences.},
  journal = {J Struct Biol},
  year = {2011},
  volume = {175},
  number = {3},
  pages = {288--299},
  url = {http://dx.doi.org/10.1016/j.jsb.2011.05.011},
  doi = {10.1016/j.jsb.2011.05.011}
}
Heuser T, Barber CF, Lin J, Krell J, Rebesco M, Porter ME and Nicastro D (2012), "Cryoelectron tomography reveals doublet-specific structures and unique interactions in the I1 dynein.", Proc Natl Acad Sci U S A., Jul, 2012. Vol. 109(30), pp. E2067-E2076.
Abstract: Cilia and flagella are highly conserved motile and sensory organelles in eukaryotes, and defects in ciliary assembly and motility cause many ciliopathies. The two-headed I1 inner arm dynein is a critical regulator of ciliary and flagellar beating. To understand I1 architecture and function better, we analyzed the 3D structure and composition of the I1 dynein in Chlamydomonas axonemes by cryoelectron tomography and subtomogram averaging. Our data revealed several connections from the I1 dynein to neighboring structures that are likely to be important for assembly and/or regulation, including a tether linking one I1 motor domain to the doublet microtubule and doublet-specific differences potentially contributing to the asymmetrical distribution of dynein activity required for ciliary beating. We also imaged three I1 mutants and analyzed their polypeptide composition using 2D gel-based proteomics. Structural and biochemical comparisons revealed the likely location of the regulatory IC138 phosphoprotein and its associated subcomplex. Overall, our studies demonstrate that I1 dynein is connected to multiple structures within the axoneme, and therefore ideally positioned to integrate signals that regulate ciliary motility.
BibTeX:
@article{Heuser2012a,
  author = {Heuser, Thomas and Barber, Cynthia F. and Lin, Jianfeng and Krell, Jeremy and Rebesco, Matthew and Porter, Mary E. and Nicastro, Daniela},
  title = {Cryoelectron tomography reveals doublet-specific structures and unique interactions in the I1 dynein.},
  journal = {Proc Natl Acad Sci U S A},
  year = {2012},
  volume = {109},
  number = {30},
  pages = {E2067--E2076},
  url = {http://dx.doi.org/10.1073/pnas.1120690109},
  doi = {10.1073/pnas.1120690109}
}
Heuser T, Dymek EE, Lin J, Smith EF and Nicastro D (2012), "The CSC connects three major axonemal complexes involved in dynein regulation.", Mol Biol Cell., Aug, 2012. Vol. 23(16), pp. 3143-3155.
Abstract: Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ-ated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo-electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin-dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility.
BibTeX:
@article{Heuser2012,
  author = {Heuser, Thomas and Dymek, Erin E. and Lin, Jianfeng and Smith, Elizabeth F. and Nicastro, Daniela},
  title = {The CSC connects three major axonemal complexes involved in dynein regulation.},
  journal = {Mol Biol Cell},
  year = {2012},
  volume = {23},
  number = {16},
  pages = {3143--3155},
  url = {http://dx.doi.org/10.1091/mbc.E12-05-0357},
  doi = {10.1091/mbc.E12-05-0357}
}
Heuser T, Raytchev M, Krell J, Porter ME and Nicastro D (2009), "The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella.", J Cell Biol., Dec, 2009. Vol. 187(6), pp. 921-933.
Abstract: Cilia and flagella are highly conserved microtubule (MT)-based organelles with motile and sensory functions, and ciliary defects have been linked to several human diseases. The 9 + 2 structure of motile axonemes contains nine MT doublets interconnected by nexin links, which surround a central pair of singlet MTs. Motility is generated by the orchestrated activity of thousands of dynein motors, which drive interdoublet sliding. A key regulator of motor activity is the dynein regulatory complex (DRC), but detailed structural information is lacking. Using cryoelectron tomography of wild-type and mutant axonemes from Chlamydomonas reinhardtii, we visualized the DRC in situ at molecular resolution. We present the three-dimensional structure of the DRC, including a model for its subunit organization and intermolecular connections that establish the DRC as a major regulatory node. We further demonstrate that the DRC is the nexin link, which is thought to be critical for the generation of axonemal bending.
BibTeX:
@article{Heuser2009,
  author = {Heuser, Thomas and Raytchev, Milen and Krell, Jeremy and Porter, Mary E. and Nicastro, Daniela},
  title = {The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella.},
  journal = {J Cell Biol},
  year = {2009},
  volume = {187},
  number = {6},
  pages = {921--933},
  url = {http://dx.doi.org/10.1083/jcb.200908067},
  doi = {10.1083/jcb.200908067}
}
Hoenger A (2014), "High-resolution cryo-electron microscopy on macromolecular complexes and cell organelles.", Protoplasma., Mar, 2014. Vol. 251(2), pp. 417-427.
Abstract: Cryo-electron microscopy techniques and computational 3-D reconstruction of macromolecular assemblies are tightly linked tools in modern structural biology. This symbiosis has produced vast amounts of detailed information on the structure and function of biological macromolecules. Typically, one of two fundamentally different strategies is used depending on the specimens and their environment. A: 3-D reconstruction based on repetitive and structurally identical unit cells that allow for averaging, and B: tomographic 3-D reconstructions where tilt-series between approximately ± 60 and ± 70° at small angular increments are collected from highly complex and flexible structures that are beyond averaging procedures, at least during the first round of 3-D reconstruction. Strategies of group A are averaging-based procedures and collect large number of 2-D projections at different angles that are computationally aligned, averaged together, and back-projected in 3-D space to reach a most complete 3-D dataset with high resolution, today often down to atomic detail. Evidently, success relies on structurally repetitive particles and an aligning procedure that unambiguously determines the angular relationship of all 2-D projections with respect to each other. The alignment procedure of small particles may rely on their packing into a regular array such as a 2-D crystal, an icosahedral (viral) particle, or a helical assembly. Critically important for cryo-methods, each particle will only be exposed once to the electron beam, making these procedures optimal for highest-resolution studies where beam-induced damage is a significant concern. In contrast, tomographic 3-D reconstruction procedures (group B) do not rely on averaging, but collect an entire dataset from the very same structure of interest. Data acquisition requires collecting a large series of tilted projections at angular increments of 1-2° or less and a tilt range of ± 60° or more. Accordingly, tomographic data collection exposes its specimens to a large electron dose, which is particularly problematic for frozen-hydrated samples. Currently, cryo-electron tomography is a rapidly emerging technology, on one end driven by the newest developments of hardware such as super-stabile microscopy stages as well as the latest generation of direct electron detectors and cameras. On the other end, success also strongly depends on new software developments on all kinds of fronts such as tilt-series alignment and back-projection procedures that are all adapted to the very low-dose and therefore very noisy primary data. Here, we will review the status quo of cryo-electron microscopy and discuss the future of cellular cryo-electron tomography from data collection to data analysis, CTF-correction of tilt-series, post-tomographic sub-volume averaging, and 3-D particle classification. We will also discuss the pros and cons of plunge freezing of cellular specimens to vitrified sectioning procedures and their suitability for post-tomographic volume averaging despite multiple artifacts that may distort specimens to some degree.
BibTeX:
@article{Hoenger2014,
  author = {Hoenger, Andreas},
  title = {High-resolution cryo-electron microscopy on macromolecular complexes and cell organelles.},
  journal = {Protoplasma},
  year = {2014},
  volume = {251},
  number = {2},
  pages = {417--427},
  url = {http://dx.doi.org/10.1007/s00709-013-0600-1},
  doi = {10.1007/s00709-013-0600-1}
}
Hoenger A and McIntosh JR (2009), "Probing the macromolecular organization of cells by electron tomography.", Curr Opin Cell Biol., Feb, 2009. Vol. 21(1), pp. 89-96.
Abstract: A major goal in cell biology is to understand the functional organization of macromolecular complexes in vivo. Electron microscopy is helping cell biologists to achieve this goal, thanks to its ability to resolve structural details in the nanometer range. While issues related to specimen preparation, imaging, and image interpretation make this approach to cell architecture difficult, recent improvements in methods, equipment, and software have facilitated the study of both important macromolecular complexes and comparatively large volumes from cellular specimens. Here, we describe recent progress in electron microscopy of cells and the ways in which the relevant methodologies are helping to elucidate cell architecture.
BibTeX:
@article{Hoenger2009,
  author = {Hoenger, Andreas and McIntosh, J Richard},
  title = {Probing the macromolecular organization of cells by electron tomography.},
  journal = {Curr Opin Cell Biol},
  year = {2009},
  volume = {21},
  number = {1},
  pages = {89--96},
  url = {http://dx.doi.org/10.1016/j.ceb.2008.12.003},
  doi = {10.1016/j.ceb.2008.12.003}
}
Höög JL, Bouchet-Marquis C, McIntosh JR, Hoenger A and Gull K (2012), "Cryo-electron tomography and 3-D analysis of the intact flagellum in Trypanosoma brucei.", J Struct Biol., May, 2012. Vol. 178(2), pp. 189-198.
Abstract: Trypanosoma brucei is a uni-cellular protist that causes African sleeping sickness. These parasites have a flagellum that is attached to the cell body and is indispensible for its motility. The flagellum consists of a canonical 9+2 axoneme and a paraflagellar rod (PFR), an intricate tripartite, fibrous structure that is connected to the axoneme. In this paper we describe results from cryo-electron tomography of unperturbed flagella. This method revealed novel structures that are likely involved in attaching the flagellum to the cell. We also show the first cryo-electron tomographic images of a basal body in situ, revealing electron dense structures inside its triplet microtubules. Sub-tomogram averaging of the PFR revealed that its distal region is organized as an orthorhombic crystal.
BibTeX:
@article{Hoeoeg2012,
  author = {Höög, Johanna L. and Bouchet-Marquis, Cédric and McIntosh, J Richard and Hoenger, Andreas and Gull, Keith},
  title = {Cryo-electron tomography and 3-D analysis of the intact flagellum in Trypanosoma brucei.},
  journal = {J Struct Biol},
  year = {2012},
  volume = {178},
  number = {2},
  pages = {189--198},
  url = {http://dx.doi.org/10.1016/j.jsb.2012.01.009},
  doi = {10.1016/j.jsb.2012.01.009}
}
Kirmse R, Bouchet-Marquis C, Page C and Hoenger A (2010), "Three-dimensional cryo-electron microscopy on intermediate filaments.", Methods Cell Biol. Vol. 96, pp. 565-589.
Abstract: Together with microtubules and actin filaments (F-actin), intermediate filaments (IFs) form the cytoskeleton of metazoan cells. However, unlike the other two entities that are extremely conserved, IFs are much more diverse and are grouped into five different families. In contrast to microtubules and F-actin, IFs do not exhibit a polarity, which may be the reason that no molecular motors travel along them. The molecular structure of IFs is less well resolved than that of the other cytoskeletal systems. This is partially due to their functional variability, tissue-specific expression, and their intrinsic structural properties. IFs are composed mostly of relatively smooth protofibrils formed by antiparallel arranged α-helical coiled-coil bundles flanked by small globular domains at either end. These features make them difficult to study by various electron microscopy methods or atomic force microscopy (AFM). Furthermore, the elongated shape of monomeric or dimeric IF units interferes with the formation of highly ordered three-dimensional (3-D) crystals suitable for atomic resolution crystallography. So far, most of the data we currently have on IF macromolecular structures come from electron microscopy of negatively stained samples, and fragmented α-helical coiled-coil units solved by X-ray diffraction. In addition, AFM allows the observation of the dynamic states of IFs in solution and delivers a new view into the assembly properties of IFs. Here, we discuss the applicability of cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) for the field. Both methods are strongly related and have only recently been applied to IFs. However, cryo-EM revealed distinct new features within IFs that have not been seen before, and cryo-ET adds a 3-D view of IFs revealing the path and number of protofilaments within the various IF assemblies.
BibTeX:
@article{Kirmse2010,
  author = {Kirmse, Robert and Bouchet-Marquis, Cédric and Page, Cynthia and Hoenger, Andreas},
  title = {Three-dimensional cryo-electron microscopy on intermediate filaments.},
  journal = {Methods Cell Biol},
  year = {2010},
  volume = {96},
  pages = {565--589},
  url = {http://dx.doi.org/10.1016/S0091-679X(10)96023-8},
  doi = {10.1016/S0091-679X(10)96023-8}
}
Kováčik L, Kereïche S, Matula P and Raška I (2014), "Sub-volume averaging of repetitive structural features in angularly filtered electron tomographic reconstructions.", Folia Biol (Praha). Vol. 60 Suppl 1, pp. 66-70.
Abstract: Electron tomographic reconstructions suffer from a number of artefacts arising from effects accompanying the processes of acquisition of a set of tilted projections of the specimen in a transmission electron microscope and from its subsequent computational handling. The most pronounced artefacts usually come from imprecise projection alignment, distortion of specimens during tomogram acquisition and from the presence of a region of missing data in the Fourier space, the "missing wedge". The ray artefacts caused by the presence of the missing wedge can be attenuated by the angular image filter, which attenuates the transition between the data and the missing wedge regions. In this work, we present an analysis of the influence of angular filtering on the resolution of averaged repetitive structural motives extracted from three-dimensional reconstructions of tomograms acquired in the single-axis tilting geometry.
BibTeX:
@article{Kovacik2014,
  author = {Kováčik, L. and Kereïche, S. and Matula, P. and Raška, I.},
  title = {Sub-volume averaging of repetitive structural features in angularly filtered electron tomographic reconstructions.},
  journal = {Folia Biol (Praha)},
  year = {2014},
  volume = {60 Suppl 1},
  pages = {66--70}
}
Liljeroos L, Krzyzaniak MA, Helenius A and Butcher SJ (2013), "Architecture of respiratory syncytial virus revealed by electron cryotomography.", Proc Natl Acad Sci U S A., Jul, 2013. Vol. 110(27), pp. 11133-11138.
Abstract: Human respiratory syncytial virus is a human pathogen that causes severe infection of the respiratory tract. Current information about the structure of the virus and its interaction with host cells is limited. We carried out an electron cryotomographic characterization of cell culture-grown human respiratory syncytial virus to determine the architecture of the virion. The particles ranged from 100 nm to 1,000 nm in diameter and were spherical, filamentous, or a combination of the two. The filamentous morphology correlated with the presence of a cylindrical matrix protein layer linked to the inner leaflet of the viral envelope and with local ordering of the glycoprotein spikes. Recombinant viruses with only the fusion protein in their envelope showed that these glycoproteins were predominantly in the postfusion conformation, but some were also in the prefusion form. The ribonucleocapsids were left-handed, randomly oriented, and curved inside the virions. In filamentous particles, they were often adjacent to an intermediate layer of protein assigned to M2-1 (an envelope-associated protein known to mediate association of ribonucleocapsids with the matrix protein). Our results indicate important differences in structure between the Paramyxovirinae and Pneumovirinae subfamilies within the Paramyxoviridae, and provide fresh insights into host cell exit of a serious pathogen.
BibTeX:
@article{Liljeroos2013,
  author = {Liljeroos, Lassi and Krzyzaniak, Magdalena Anna and Helenius, Ari and Butcher, Sarah Jane},
  title = {Architecture of respiratory syncytial virus revealed by electron cryotomography.},
  journal = {Proc Natl Acad Sci U S A},
  year = {2013},
  volume = {110},
  number = {27},
  pages = {11133--11138},
  url = {http://dx.doi.org/10.1073/pnas.1309070110},
  doi = {10.1073/pnas.1309070110}
}
Lin J, Heuser T, Carbajal-González BI, Song K and Nicastro D (2012), "The structural heterogeneity of radial spokes in cilia and flagella is conserved.", Cytoskeleton (Hoboken)., Feb, 2012. Vol. 69(2), pp. 88-100.
Abstract: Radial spokes (RSs) are ubiquitous components of motile cilia and flagella and play an essential role in transmitting signals that regulate the activity of the dynein motors, and thus ciliary and flagellar motility. In some organisms, the 96 nm axonemal repeat unit contains only a pair of spokes, RS1 and RS2, while most organisms have spoke triplets with an additional spoke RS3. The spoke pairs in Chlamydomonas flagella have been well characterized, while spoke triplets have received less attention. Here, we used cryoelectron tomography and subtomogram averaging to visualize the three-dimensional structure of spoke triplets in Strongylocentrotus purpuratus (sea urchin) sperm flagella in unprecedented detail. Only small differences were observed between RS1 and RS2, but the structure of RS3 was surprisingly unique and structurally different from the other two spokes. We observed novel doublet specific features that connect RS2, RS3, and the nexin-dynein regulatory complex, three key ciliary and flagellar structures. The distribution of these doublet specific structures suggests that they could be important for establishing the asymmetry of dynein activity required for the oscillatory movement of cilia and flagella. Surprisingly, a comparison with other organisms demonstrated both that this considerable RS heterogeneity is conserved and that organisms with RS pairs contain the basal part of RS3. This conserved RS heterogeneity may also reflect functional differences between the spokes and their involvement in regulating ciliary and flagellar motility.
BibTeX:
@article{Lin2012,
  author = {Lin, Jianfeng and Heuser, Thomas and Carbajal-González, Blanca I. and Song, Kangkang and Nicastro, Daniela},
  title = {The structural heterogeneity of radial spokes in cilia and flagella is conserved.},
  journal = {Cytoskeleton (Hoboken)},
  year = {2012},
  volume = {69},
  number = {2},
  pages = {88--100},
  url = {http://dx.doi.org/10.1002/cm.21000},
  doi = {10.1002/cm.21000}
}
Lin J, Heuser T, Song K, Fu X and Nicastro D (2012), "One of the nine doublet microtubules of eukaryotic flagella exhibits unique and partially conserved structures.", PLoS One. Vol. 7(10), pp. e46494.
Abstract: The axonemal core of motile cilia and flagella consists of nine doublet microtubules surrounding two central single microtubules. Attached to the doublets are thousands of dynein motors that produce sliding between neighboring doublets, which in turn causes flagellar bending. Although many structural features of the axoneme have been described, structures that are unique to specific doublets remain largely uncharacterized. These doublet-specific structures introduce asymmetry into the axoneme and are likely important for the spatial control of local microtubule sliding. Here, we used cryo-electron tomography and doublet-specific averaging to determine the 3D structures of individual doublets in the flagella of two evolutionarily distant organisms, the protist Chlamydomonas and the sea urchin Strongylocentrotus. We demonstrate that, in both organisms, one of the nine doublets exhibits unique structural features. Some of these features are highly conserved, such as the inter-doublet link i-SUB5-6, which connects this doublet to its neighbor with a periodicity of 96 nm. We also show that the previously described inter-doublet links attached to this doublet, the o-SUB5-6 in Strongylocentrotus and the proximal 1-2 bridge in Chlamydomonas, are likely not homologous features. The presence of inter-doublet links and reduction of dynein arms indicate that inter-doublet sliding of this unique doublet against its neighbor is limited, providing a rigid plane perpendicular to the flagellar bending plane. These doublet-specific features and the non-sliding nature of these connected doublets suggest a structural basis for the asymmetric distribution of dynein activity and inter-doublet sliding, resulting in quasi-planar waveforms typical of 9+2 cilia and flagella.
BibTeX:
@article{Lin2012a,
  author = {Lin, Jianfeng and Heuser, Thomas and Song, Kangkang and Fu, Xiaofeng and Nicastro, Daniela},
  title = {One of the nine doublet microtubules of eukaryotic flagella exhibits unique and partially conserved structures.},
  journal = {PLoS One},
  year = {2012},
  volume = {7},
  number = {10},
  pages = {e46494},
  url = {http://dx.doi.org/10.1371/journal.pone.0046494},
  doi = {10.1371/journal.pone.0046494}
}
Lin J, Okada K, Raytchev M, Smith MC and Nicastro D (2014), "Structural mechanism of the dynein power stroke.", Nat Cell Biol., May, 2014. Vol. 16(5), pp. 479-485.
Abstract: Dyneins are large microtubule motor proteins required for mitosis, intracellular transport and ciliary and flagellar motility. They generate force through a power-stroke mechanism, which is an ATP-consuming cycle of pre- and post-power-stroke conformational changes that cause relative motion between different dynein domains. However, key structural details of dynein's force generation remain elusive. Here, using cryo-electron tomography of intact, active (that is, beating), rapidly frozen sea urchin sperm flagella, we determined the in situ three-dimensional structures of all domains of both pre- and post-power-stroke dynein, including the previously unresolved linker and stalk of pre-power-stroke dynein. Our results reveal that the rotation of the head relative to the linker is the key action in dynein movement, and that there are at least two distinct pre-power-stroke conformations: pre-I (microtubule-detached) and pre-II (microtubule-bound). We provide three-dimensional reconstructions of native dyneins in three conformational states, in situ, allowing us to propose a molecular model of the structural cycle underlying dynein movement.
BibTeX:
@article{Lin2014a,
  author = {Lin, Jianfeng and Okada, Kyoko and Raytchev, Milen and Smith, Maria C. and Nicastro, Daniela},
  title = {Structural mechanism of the dynein power stroke.},
  journal = {Nat Cell Biol},
  year = {2014},
  volume = {16},
  number = {5},
  pages = {479--485},
  url = {http://dx.doi.org/10.1038/ncb2939},
  doi = {10.1038/ncb2939}
}
Linck R, Fu X, Lin J, Ouch C, Schefter A, Steffen W, Warren P and Nicastro D (2014), "Insights into the structure and function of ciliary and flagellar doublet microtubules: tektins, Ca2+-binding proteins, and stable protofilaments.", J Biol Chem., Jun, 2014. Vol. 289(25), pp. 17427-17444.
Abstract: Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, β-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems.
BibTeX:
@article{Linck2014,
  author = {Linck, Richard and Fu, Xiaofeng and Lin, Jianfeng and Ouch, Christna and Schefter, Alexandra and Steffen, Walter and Warren, Peter and Nicastro, Daniela},
  title = {Insights into the structure and function of ciliary and flagellar doublet microtubules: tektins, Ca2+-binding proteins, and stable protofilaments.},
  journal = {J Biol Chem},
  year = {2014},
  volume = {289},
  number = {25},
  pages = {17427--17444},
  url = {http://dx.doi.org/10.1074/jbc.M114.568949},
  doi = {10.1074/jbc.M114.568949}
}
Malet H, Liu K, El Bakkouri M, Chan SWS, Effantin G, Bacia M, Houry WA and Gutsche I (2014), "Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins.", Elife. Vol. 3, pp. e03653.
Abstract: A 3.3 MDa macromolecular cage between two Escherichia coli proteins with seemingly incompatible symmetries-the hexameric AAA+ ATPase RavA and the decameric inducible lysine decarboxylase LdcI-is reconstructed by cryo-electron microscopy to 11 Å resolution. Combined with a 7.5 Å resolution reconstruction of the minimal complex between LdcI and the LdcI-binding domain of RavA, and the previously solved crystal structures of the individual components, this work enables to build a reliable pseudoatomic model of this unusual architecture and to identify conformational rearrangements and specific elements essential for complex formation. The design of the cage created via lateral interactions between five RavA rings is unique for the diverse AAA+ ATPase superfamily.
BibTeX:
@article{Malet2014,
  author = {Malet, Hélène and Liu, Kaiyin and El Bakkouri, Majida and Chan, Sze Wah Samuel and Effantin, Gregory and Bacia, Maria and Houry, Walid A. and Gutsche, Irina},
  title = {Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins.},
  journal = {Elife},
  year = {2014},
  volume = {3},
  pages = {e03653},
  url = {http://dx.doi.org/10.7554/eLife.03653},
  doi = {10.7554/eLife.03653}
}
Martinez R, Schellenberger P, Vasishtan D, Aknin C, Austin S, Dacheux D, Rayne F, Siebert A, Ruzsics Z, Gruenewald K and Wodrich H (2015), "The amphipathic helix of adenovirus capsid protein VI contributes to penton release and postentry sorting.", J Virol., Feb, 2015. Vol. 89(4), pp. 2121-2135.
Abstract: Nuclear delivery of the adenoviral genome requires that the capsid cross the limiting membrane of the endocytic compartment and traverse the cytosol to reach the nucleus. This endosomal escape is initiated upon internalization and involves a highly coordinated process of partial disassembly of the entering capsid to release the membrane lytic internal capsid protein VI. Using wild-type and protein VI-mutated human adenovirus serotype 5 (HAdV-C5), we show that capsid stability and membrane rupture are major determinants of entry-related sorting of incoming adenovirus virions. Furthermore, by using electron cryomicroscopy, as well as penton- and protein VI-specific antibodies, we show that the amphipathic helix of protein VI contributes to capsid stability by preventing premature disassembly and deployment of pentons and protein VI. Thus, the helix has a dual function in maintaining the metastable state of the capsid by preventing premature disassembly and mediating efficient membrane lysis to evade lysosomal targeting. Based on these findings and structural data from cryo-electron microscopy, we suggest a refined disassembly mechanism upon entry.In this study, we show the intricate connection of adenovirus particle stability and the entry-dependent release of the membrane-lytic capsid protein VI required for endosomal escape. We show that the amphipathic helix of the adenovirus internal protein VI is required to stabilize pentons in the particle while coinciding with penton release upon entry and that release of protein VI mediates membrane lysis, thereby preventing lysosomal sorting. We suggest that this dual functionality of protein VI ensures an optimal disassembly process by balancing the metastable state of the mature adenovirus particle.
BibTeX:
@article{Martinez2015,
  author = {Martinez, Ruben and Schellenberger, Pascale and Vasishtan, Daven and Aknin, Cindy and Austin, Sisley and Dacheux, Denis and Rayne, Fabienne and Siebert, Alistair and Ruzsics, Zsolt and Gruenewald, Kay and Wodrich, Harald},
  title = {The amphipathic helix of adenovirus capsid protein VI contributes to penton release and postentry sorting.},
  journal = {J Virol},
  year = {2015},
  volume = {89},
  number = {4},
  pages = {2121--2135},
  url = {http://dx.doi.org/10.1128/JVI.02257-14},
  doi = {10.1128/JVI.02257-14}
}
McIntosh JR, O'Toole E, Zhudenkov K, Morphew M, Schwartz C, Ataullakhanov FI and Grishchuk EL (2013), "Conserved and divergent features of kinetochores and spindle microtubule ends from five species.", J Cell Biol., Feb, 2013. Vol. 200(4), pp. 459-474.
Abstract: Interfaces between spindle microtubules and kinetochores were examined in diverse species by electron tomography and image analysis. Overall structures were conserved in a mammal, an alga, a nematode, and two kinds of yeasts; all lacked dense outer plates, and most kinetochore microtubule ends flared into curved protofilaments that were connected to chromatin by slender fibrils. Analyses of curvature on >8,500 protofilaments showed that all classes of spindle microtubules displayed some flaring protofilaments, including those growing in the anaphase interzone. Curved protofilaments on anaphase kinetochore microtubules were no more flared than their metaphase counterparts, but they were longer. Flaring protofilaments in budding yeasts were linked by fibrils to densities that resembled nucleosomes; these are probably the yeast kinetochores. Analogous densities in fission yeast were larger and less well-defined, but both yeasts showed ring- or partial ring-shaped structures girding their kinetochore microtubules. Flaring protofilaments linked to chromatin are well placed to exert force on chromosomes, assuring stable attachment and reliable anaphase segregation.
BibTeX:
@article{McIntosh2013,
  author = {McIntosh, J Richard and O'Toole, Eileen and Zhudenkov, Kirill and Morphew, Mary and Schwartz, Cindi and Ataullakhanov, Fazly I. and Grishchuk, Ekaterina L.},
  title = {Conserved and divergent features of kinetochores and spindle microtubule ends from five species.},
  journal = {J Cell Biol},
  year = {2013},
  volume = {200},
  number = {4},
  pages = {459--474},
  url = {http://dx.doi.org/10.1083/jcb.201209154},
  doi = {10.1083/jcb.201209154}
}
Nans A, Einheber S, Salzer JL and Stokes DL (2011), "Electron tomography of paranodal septate-like junctions and the associated axonal and glial cytoskeletons in the central nervous system.", J Neurosci Res., Mar, 2011. Vol. 89(3), pp. 310-319.
Abstract: The polarized domains of myelinated axons are specifically organized to maximize the efficiency of saltatory conduction. The paranodal region is directly adjacent to the node of Ranvier and contains specialized septate-like junctions that provide adhesion between axons and glial cells and that constitute a lateral diffusion barrier for nodal components. To complement and extend earlier studies on the peripheral nervous system, electron tomography was used to image paranodal regions from the central nervous system (CNS). Our three-dimensional reconstructions revealed short filamentous linkers running directly from the septate-like junctions to neurofilaments, microfilaments, and organelles within the axon. The intercellular spacing between axons and glia was measured to be 7.4 ± 0.6 nm, over twice the value previously reported in the literature (2.5-3.0 nm). Averaging of individual junctions revealed a bifurcated structure in the intercellular space that is consistent with a dimeric complex of cell adhesion molecules composing the septate-like junction. Taken together, these findings provide new insight into the structural organization of CNS paranodes and suggest that, in addition to providing axo-glial adhesion, cytoskeletal linkage to the septate-like junctions may be required to maintain axonal domains and to regulate organelle transport in myelinated axons.
BibTeX:
@article{Nans2011,
  author = {Nans, Andrea and Einheber, Steven and Salzer, James L. and Stokes, David L.},
  title = {Electron tomography of paranodal septate-like junctions and the associated axonal and glial cytoskeletons in the central nervous system.},
  journal = {J Neurosci Res},
  year = {2011},
  volume = {89},
  number = {3},
  pages = {310--319},
  url = {http://dx.doi.org/10.1002/jnr.22561},
  doi = {10.1002/jnr.22561}
}
Nicastro D, Fu X, Heuser T, Tso A, Porter ME and Linck RW (2011), "Cryo-electron tomography reveals conserved features of doublet microtubules in flagella.", Proc Natl Acad Sci U S A., Oct, 2011. Vol. 108(42), pp. E845-E853.
Abstract: The axoneme forms the essential and conserved core of cilia and flagella. We have used cryo-electron tomography of Chlamydomonas and sea urchin flagella to answer long-standing questions and to provide information about the structure of axonemal doublet microtubules (DMTs). Solving an ongoing controversy, we show that B-tubules of DMTs contain exactly 10 protofilaments (PFs) and that the inner junction (IJ) and outer junction between the A- and B-tubules are fundamentally different. The outer junction, crucial for the initiation of doublet formation, appears to be formed by close interactions between the tubulin subunits of three PFs with unusual tubulin interfaces; other investigators have reported that this junction is weakened by mutations affecting posttranslational modifications of tubulin. The IJ consists of an axially periodic ladder-like structure connecting tubulin PFs of the A- and B-tubules. The recently discovered microtubule inner proteins (MIPs) on the inside of the A- and B-tubules are more complex than previously thought. They are composed of alternating small and large subunits with periodicities of 16 and/or 48 nm. MIP3 forms arches connecting B-tubule PFs, contrary to an earlier report that MIP3 forms the IJ. Finally, the "beak" structures within the B-tubules of Chlamydomonas DMT1, DMT5, and DMT6 are clearly composed of a longitudinal band of proteins repeating with a periodicity of 16 nm. These findings, discussed in relation to genetic and biochemical data, provide a critical foundation for future work on the molecular assembly and stability of the axoneme, as well as its function in motility and sensory transduction.
BibTeX:
@article{Nicastro2011,
  author = {Nicastro, Daniela and Fu, Xiaofeng and Heuser, Thomas and Tso, Alan and Porter, Mary E. and Linck, Richard W.},
  title = {Cryo-electron tomography reveals conserved features of doublet microtubules in flagella.},
  journal = {Proc Natl Acad Sci U S A},
  year = {2011},
  volume = {108},
  number = {42},
  pages = {E845--E853},
  url = {http://dx.doi.org/10.1073/pnas.1106178108},
  doi = {10.1073/pnas.1106178108}
}
Nicastro D, Schwartz C, Pierson J, Gaudette R, Porter ME and McIntosh JR (2006), "The molecular architecture of axonemes revealed by cryoelectron tomography.", Science., Aug, 2006. Vol. 313(5789), pp. 944-948.
Abstract: Eukaryotic flagella and cilia are built on a 9 + 2 array of microtubules plus >250 accessory proteins, forming a biological machine called the axoneme. Here we describe the three-dimensional structure of rapidly frozen axonemes from Chlamydomonas and sea urchin sperm, using cryoelectron tomography and image processing to focus on the motor enzyme dynein. Our images suggest a model for the way dynein generates force to slide microtubules. They also reveal two dynein linkers that may provide "hard-wiring" to coordinate motor enzyme action, both circumferentially and along the axoneme. Periodic densities were also observed inside doublet microtubules; these may contribute to doublet stability.
BibTeX:
@article{Nicastro2006,
  author = {Nicastro, Daniela and Schwartz, Cindi and Pierson, Jason and Gaudette, Richard and Porter, Mary E. and McIntosh, J Richard},
  title = {The molecular architecture of axonemes revealed by cryoelectron tomography.},
  journal = {Science},
  year = {2006},
  volume = {313},
  number = {5789},
  pages = {944--948},
  url = {http://dx.doi.org/10.1126/science.1128618},
  doi = {10.1126/science.1128618}
}
Oda T, Abe T, Yanagisawa H and Kikkawa M (2016), "Docking-complex-independent alignment of Chlamydomonas outer dynein arms with 24-nm periodicity in vitro.", J Cell Sci., Apr, 2016. Vol. 129(8), pp. 1547-1551.
Abstract: The docking complex is a molecular complex necessary for assembly of outer dynein arms (ODAs) on the axonemal doublet microtubules (DMTs) in cilia and flagella. The docking complex is hypothesized to be a 24-nm molecular ruler because ODAs align along the DMTs with 24-nm periodicity. In this study, we rigorously tested this hypothesis using structural and genetic methods. We found that the ODAs can bind to DMTs and porcine microtubules with 24-nm periodicities even in the absence of the docking complexin vitro Using cryo-electron tomography and structural labeling, we observed that the docking complex took an unexpectedly flexible conformation and did not lie along the length of DMTs. In the absence of docking complex, ODAs were released from the DMT at relatively low ionic strength conditions, suggesting that the docking complex strengthens the electrostatic interactions between the ODA and DMT. Based on these results, we conclude that the docking complex serves as a flexible stabilizer of the ODA rather than as a molecular ruler.
BibTeX:
@article{Oda2016,
  author = {Oda, Toshiyuki and Abe, Tatsuki and Yanagisawa, Haruaki and Kikkawa, Masahide},
  title = {Docking-complex-independent alignment of Chlamydomonas outer dynein arms with 24-nm periodicity in vitro.},
  journal = {J Cell Sci},
  year = {2016},
  volume = {129},
  number = {8},
  pages = {1547--1551},
  url = {http://dx.doi.org/10.1242/jcs.184598},
  doi = {10.1242/jcs.184598}
}
Oda T, Abe T, Yanagisawa H and Kikkawa M (2016), "Structure and function of outer dynein arm intermediate and light chain complex.", Mol Biol Cell., Apr, 2016. Vol. 27(7), pp. 1051-1059.
Abstract: The outer dynein arm (ODA) is a molecular complex that drives the beating motion of cilia/flagella.ChlamydomonasODA is composed of three heavy chains (HCs), two ICs, and 11 light chains (LCs). Although the three-dimensional (3D) structure of the whole ODA complex has been investigated, the 3D configurations of the ICs and LCs are largely unknown. Here we identified the 3D positions of the two ICs and three LCs using cryo-electron tomography and structural labeling. We found that these ICs and LCs were all localized at the root of the outer-inner dynein (OID) linker, designated the ODA-Beak complex. Of interest, the coiled-coil domain of IC2 extended from the ODA-Beak to the outer surface of ODA. Furthermore, we investigated the molecular mechanisms of how the OID linker transmits signals to the ODA-Beak, by manipulating the interaction within the OID linker using a chemically induced dimerization system. We showed that the cross-linking of the OID linker strongly suppresses flagellar motility in vivo. These results suggest that the ICs and LCs of the ODA form the ODA-Beak, which may be involved in mechanosignaling from the OID linker to the HCs.
BibTeX:
@article{Oda2016b,
  author = {Oda, Toshiyuki and Abe, Tatsuki and Yanagisawa, Haruaki and Kikkawa, Masahide},
  title = {Structure and function of outer dynein arm intermediate and light chain complex.},
  journal = {Mol Biol Cell},
  year = {2016},
  volume = {27},
  number = {7},
  pages = {1051--1059},
  url = {http://dx.doi.org/10.1091/mbc.E15-10-0723},
  doi = {10.1091/mbc.E15-10-0723}
}
Oda T and Kikkawa M (2013), "Novel structural labeling method using cryo-electron tomography and biotin-streptavidin system.", J Struct Biol., Sep, 2013. Vol. 183(3), pp. 305-311.
Abstract: There are a number of large macromolecular complexes that play important roles in the cell, and identifying the positions of their components is a key step to understanding their structure and function. Several structural labeling methods have been applied to electron microscopy in order to locate a specific component within a macromolecular complex, but each method is associated with problems in specificity, occupancy, signal intensity or precision. Here, we report a novel method for identifying the 3D locations of proteins using biotin-streptavidin labeling and cryo-electron tomography. We labeled a biotinylation-tagged intermediate chain of an axonemal dynein by streptavidin within the Chlamydomonas axoneme and visualized the 3D positions of the labels using subtomogram averaging. Increase of the density attributed to the bound streptavidin was validated by Student's t-test. In conclusion, the combination of the biotin-streptavidin system and cryo-electron tomography is a powerful method to investigate the structure of large macromolecular complexes.
BibTeX:
@article{Oda2013,
  author = {Oda, Toshiyuki and Kikkawa, Masahide},
  title = {Novel structural labeling method using cryo-electron tomography and biotin-streptavidin system.},
  journal = {J Struct Biol},
  year = {2013},
  volume = {183},
  number = {3},
  pages = {305--311},
  url = {http://dx.doi.org/10.1016/j.jsb.2013.07.003},
  doi = {10.1016/j.jsb.2013.07.003}
}
Oda T, Yanagisawa H and Kikkawa M (2015), "Detailed structural and biochemical characterization of the nexin-dynein regulatory complex.", Mol Biol Cell., Jan, 2015. Vol. 26(2), pp. 294-304.
Abstract: The nexin-dynein regulatory complex (N-DRC) forms a cross-bridge between the outer doublet microtubules of the axoneme and regulates dynein motor activity in cilia/flagella. Although the molecular composition and the three-dimensional structure of N-DRC have been studied using mutant strains lacking N-DRC subunits, more accurate approaches are necessary to characterize the structure and function of N-DRC. In this study, we precisely localized DRC1, DRC2, and DRC4 using cryo-electron tomography and structural labeling. All three N-DRC subunits had elongated conformations and spanned the length of N-DRC. Furthermore, we purified N-DRC and characterized its microtubule-binding properties. Purified N-DRC bound to the microtubule and partially inhibited microtubule sliding driven by the outer dynein arms (ODAs). Of interest, microtubule sliding was observed even in the presence of fourfold molar excess of N-DRC relative to ODA. These results provide insights into the role of N-DRC in generating the beating motions of cilia/flagella.
BibTeX:
@article{Oda2015,
  author = {Oda, Toshiyuki and Yanagisawa, Haruaki and Kikkawa, Masahide},
  title = {Detailed structural and biochemical characterization of the nexin-dynein regulatory complex.},
  journal = {Mol Biol Cell},
  year = {2015},
  volume = {26},
  number = {2},
  pages = {294--304},
  url = {http://dx.doi.org/10.1091/mbc.E14-09-1367},
  doi = {10.1091/mbc.E14-09-1367}
}
Oda T, Yanagisawa H, Yagi T and Kikkawa M (2014), "Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity.", J Cell Biol., Mar, 2014. Vol. 204(5), pp. 807-819.
Abstract: Cilia/flagella are conserved organelles that generate fluid flow in eukaryotes. The bending motion of flagella requires concerted activity of dynein motors. Although it has been reported that the central pair apparatus (CP) and radial spokes (RSs) are important for flagellar motility, the molecular mechanism underlying CP- and RS-mediated dynein regulation has not been identified. In this paper, we identified nonspecific intermolecular collision between CP and RS as one of the regulatory mechanisms for flagellar motility. By combining cryoelectron tomography and motility analyses of Chlamydomonas reinhardtii flagella, we show that binding of streptavidin to RS heads paralyzed flagella. Moreover, the motility defect in a CP projection mutant could be rescued by the addition of exogenous protein tags on RS heads. Genetic experiments demonstrated that outer dynein arms are the major downstream effectors of CP- and RS-mediated regulation of flagellar motility. These results suggest that mechanosignaling between CP and RS regulates dynein activity in eukaryotic flagella.
BibTeX:
@article{Oda2014,
  author = {Oda, Toshiyuki and Yanagisawa, Haruaki and Yagi, Toshiki and Kikkawa, Masahide},
  title = {Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity.},
  journal = {J Cell Biol},
  year = {2014},
  volume = {204},
  number = {5},
  pages = {807--819},
  url = {http://dx.doi.org/10.1083/jcb.201312014},
  doi = {10.1083/jcb.201312014}
}
Pilhofer M, Ladinsky MS, McDowall AW, Petroni G and Jensen GJ (2011), "Microtubules in bacteria: Ancient tubulins build a five-protofilament homolog of the eukaryotic cytoskeleton.", PLoS Biol., Dec, 2011. Vol. 9(12), pp. e1001213.
Abstract: Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today. Microtubules have not previously been found in bacteria, and we lack insight into their evolution from the tubulin/FtsZ ancestor. Using electron cryomicroscopy, here we show that the tubulin homologs BtubA and BtubB form microtubules in bacteria and suggest these be referred to as "bacterial microtubules" (bMTs). bMTs share important features with their eukaryotic counterparts, such as straight protofilaments and similar protofilament interactions. bMTs are composed of only five protofilaments, however, instead of the 13 typical in eukaryotes. These and other results suggest that rather than being derived from modern eukaryotic tubulin, BtubA and BtubB arose from early tubulin intermediates that formed small microtubules. Since we show that bacterial microtubules can be produced in abundance in vitro without chaperones, they should be useful tools for tubulin research and drug screening.
BibTeX:
@article{Pilhofer2011,
  author = {Pilhofer, Martin and Ladinsky, Mark S. and McDowall, Alasdair W. and Petroni, Giulio and Jensen, Grant J.},
  title = {Microtubules in bacteria: Ancient tubulins build a five-protofilament homolog of the eukaryotic cytoskeleton.},
  journal = {PLoS Biol},
  year = {2011},
  volume = {9},
  number = {12},
  pages = {e1001213},
  url = {http://dx.doi.org/10.1371/journal.pbio.1001213},
  doi = {10.1371/journal.pbio.1001213}
}
Scharf L, Wang H, Gao H, Chen S, McDowall AW and Bjorkman PJ (2015), "Broadly Neutralizing Antibody 8ANC195 Recognizes Closed and Open States of HIV-1 Env.", Cell., Sep, 2015. Vol. 162(6), pp. 1379-1390.
Abstract: The HIV-1 envelope (Env) spike contains limited epitopes for broadly neutralizing antibodies (bNAbs); thus, most neutralizing antibodies are strain specific. The 8ANC195 epitope, defined by crystal and electron microscopy (EM) structures of bNAb 8ANC195 complexed with monomeric gp120 and trimeric Env, respectively, spans the gp120 and gp41 Env subunits. To investigate 8ANC195's gp41 epitope at higher resolution, we solved a 3.58 Å crystal structure of 8ANC195 complexed with fully glycosylated Env trimer, revealing 8ANC195 insertion into a glycan shield gap to contact gp120 and gp41 glycans and protein residues. To determine whether 8ANC195 recognizes the CD4-bound open Env conformation that leads to co-receptor binding and fusion, one of several known conformations of virion-associated Env, we solved EM structures of an Env/CD4/CD4-induced antibody/8ANC195 complex. 8ANC195 binding partially closed the CD4-bound trimer, confirming structural plasticity of Env by revealing a previously unseen conformation. 8ANC195's ability to bind different Env conformations suggests advantages for potential therapeutic applications.
BibTeX:
@article{Scharf2015,
  author = {Scharf, Louise and Wang, Haoqing and Gao, Han and Chen, Songye and McDowall, Alasdair W. and Bjorkman, Pamela J.},
  title = {Broadly Neutralizing Antibody 8ANC195 Recognizes Closed and Open States of HIV-1 Env.},
  journal = {Cell},
  year = {2015},
  volume = {162},
  number = {6},
  pages = {1379--1390},
  url = {http://dx.doi.org/10.1016/j.cell.2015.08.035},
  doi = {10.1016/j.cell.2015.08.035}
}
Schwartz CL, Heumann JM, Dawson SC and Hoenger A (2012), "A detailed, hierarchical study of Giardia lamblia's ventral disc reveals novel microtubule-associated protein complexes.", PLoS One. Vol. 7(9), pp. e43783.
Abstract: Giardia lamblia is a flagellated, unicellular parasite of mammals infecting over one billion people worldwide. Giardia's two-stage life cycle includes a motile trophozoite stage that colonizes the host small intestine and an infectious cyst form that can persist in the environment. Similar to many eukaryotic cells, Giardia contains several complex microtubule arrays that are involved in motility, chromosome segregation, organelle transport, maintenance of cell shape and transformation between the two life cycle stages. Giardia trophozoites also possess a unique spiral microtubule array, the ventral disc, made of approximately 50 parallel microtubules and associated microribbons, as well as a variety of associated proteins. The ventral disc maintains trophozoite attachment to the host intestinal epithelium. With the help of a combined SEM/microtome based slice and view method called 3View® (Gatan Inc., Pleasanton, CA), we present an entire trophozoite cell reconstruction and describe the arrangement of the major cytoskeletal elements. To aid in future analyses of disc-mediated attachment, we used electron-tomography of freeze-substituted, plastic-embedded trophozoites to explore the detailed architecture of ventral disc microtubules and their associated components. Lastly, we examined the disc microtubule array in three dimensions in unprecedented detail using cryo-electron tomography combined with internal sub-tomogram volume averaging of repetitive domains. We discovered details of protein complexes stabilizing microtubules by attachment to their inner and outer wall. A unique tri-laminar microribbon structure is attached vertically to the disc microtubules and is connected to neighboring microribbons via crossbridges. This work provides novel insight into the structure of the ventral disc microtubules, microribbons and associated proteins. Knowledge of the components comprising these structures and their three-dimensional organization is crucial toward understanding how attachment via the ventral disc occurs in vivo.
BibTeX:
@article{Schwartz2012,
  author = {Schwartz, Cindi L. and Heumann, John M. and Dawson, Scott C. and Hoenger, Andreas},
  title = {A detailed, hierarchical study of Giardia lamblia's ventral disc reveals novel microtubule-associated protein complexes.},
  journal = {PLoS One},
  year = {2012},
  volume = {7},
  number = {9},
  pages = {e43783},
  url = {http://dx.doi.org/10.1371/journal.pone.0043783},
  doi = {10.1371/journal.pone.0043783}
}
Song K, Awata J, Tritschler D, Bower R, Witman GB, Porter ME and Nicastro D (2015), "In situ localization of N and C termini of subunits of the flagellar nexin-dynein regulatory complex (N-DRC) using SNAP tag and cryo-electron tomography.", J Biol Chem., Feb, 2015. Vol. 290(9), pp. 5341-5353.
Abstract: Cryo-electron tomography (cryo-ET) has reached nanoscale resolution for in situ three-dimensional imaging of macromolecular complexes and organelles. Yet its current resolution is not sufficient to precisely localize or identify most proteins in situ; for example, the location and arrangement of components of the nexin-dynein regulatory complex (N-DRC), a key regulator of ciliary/flagellar motility that is conserved from algae to humans, have remained elusive despite many cryo-ET studies of cilia and flagella. Here, we developed an in situ localization method that combines cryo-ET/subtomogram averaging with the clonable SNAP tag, a widely used cell biological probe to visualize fusion proteins by fluorescence microscopy. Using this hybrid approach, we precisely determined the locations of the N and C termini of DRC3 and the C terminus of DRC4 within the three-dimensional structure of the N-DRC in Chlamydomonas flagella. Our data demonstrate that fusion of SNAP with target proteins allowed for protein localization with high efficiency and fidelity using SNAP-linked gold nanoparticles, without disrupting the native assembly, structure, or function of the flagella. After cryo-ET and subtomogram averaging, we localized DRC3 to the L1 projection of the nexin linker, which interacts directly with a dynein motor, whereas DRC4 was observed to stretch along the N-DRC base plate to the nexin linker. Application of the technique developed here to the N-DRC revealed new insights into the organization and regulatory mechanism of this complex, and provides a valuable tool for the structural dissection of macromolecular complexes in situ.
BibTeX:
@article{Song2015,
  author = {Song, Kangkang and Awata, Junya and Tritschler, Douglas and Bower, Raqual and Witman, George B. and Porter, Mary E. and Nicastro, Daniela},
  title = {In situ localization of N and C termini of subunits of the flagellar nexin-dynein regulatory complex (N-DRC) using SNAP tag and cryo-electron tomography.},
  journal = {J Biol Chem},
  year = {2015},
  volume = {290},
  number = {9},
  pages = {5341--5353},
  url = {http://dx.doi.org/10.1074/jbc.M114.626556},
  doi = {10.1074/jbc.M114.626556}
}
Strauss M, Levy HC, Bostina M, Filman DJ and Hogle JM (2013), "RNA transfer from poliovirus 135S particles across membranes is mediated by long umbilical connectors.", J Virol., Apr, 2013. Vol. 87(7), pp. 3903-3914.
Abstract: During infection, the binding of poliovirus to its cell surface receptor at 37°C triggers an expansion of the virus in which internal polypeptides that bind to membranes are externalized. Subsequently, in a poorly understood process, the viral RNA genome is transferred directly across an endosomal membrane, and into the host cell cytoplasm, to initiate infection. Here, cryoelectron tomography demonstrates the results of 37°C warming of a poliovirus-receptor-liposome model complex that was produced using Ni-nitrilotriacetic acid lipids and His-tagged receptor ectodomains. In total, 651 subtomographic volumes were aligned, classified, and averaged to obtain detailed pictures, showing both the conversion of virus into its expanded form and the passage of RNA into intact liposomes. Unexpectedly, the virus and membrane surfaces were located ∼50 Å apart, with the 5-fold axis tilted away from the perpendicular, and the solvent spaces between them were spanned by either one or two long "umbilical" density features that lie at an angle to the virus and membrane. The thinner connector, which sometimes appears alone, is 28 to 30 Å in diameter and has a footprint on the virus surface located close to either a 5-fold or a 3-fold axis. The broader connector has a footprint near the quasi-3-fold hole that opens upon virus expansion and is hypothesized to include RNA, shielded from enzymatic degradation by polypeptides that include the N-terminal extension of VP1 and capsid protein VP4. The implications of these observations for the mechanism of RNase-protected RNA transfer in picornaviruses are discussed.
BibTeX:
@article{Strauss2013,
  author = {Strauss, Mike and Levy, Hazel C. and Bostina, Mihnea and Filman, David J. and Hogle, James M.},
  title = {RNA transfer from poliovirus 135S particles across membranes is mediated by long umbilical connectors.},
  journal = {J Virol},
  year = {2013},
  volume = {87},
  number = {7},
  pages = {3903--3914},
  url = {http://dx.doi.org/10.1128/JVI.03209-12},
  doi = {10.1128/JVI.03209-12}
}
Urbanska P, Song K, Joachimiak E, Krzemien-Ojak L, Koprowski P, Hennessey T, Jerka-Dziadosz M, Fabczak H, Gaertig J, Nicastro D and Wloga D (2015), "The CSC proteins FAP61 and FAP251 build the basal substructures of radial spoke 3 in cilia.", Mol Biol Cell., Apr, 2015. Vol. 26(8), pp. 1463-1475.
Abstract: Dynein motors and regulatory complexes repeat every 96 nm along the length of motile cilia. Each repeat contains three radial spokes, RS1, RS2, and RS3, which transduct signals between the central microtubules and dynein arms. Each radial spoke has a distinct structure, but little is known about the mechanisms of assembly and function of the individual radial spokes. In Chlamydomonas, calmodulin and spoke-associated complex (CSC) is composed of FAP61, FAP91, and FAP251 and has been linked to the base of RS2 and RS3. We show that in Tetrahymena, loss of either FAP61 or FAP251 reduces cell swimming and affects the ciliary waveform and that RS3 is either missing or incomplete, whereas RS1 and RS2 are unaffected. Specifically, FAP251-null cilia lack an arch-like density at the RS3 base, whereas FAP61-null cilia lack an adjacent portion of the RS3 stem region. This suggests that the CSC proteins are crucial for stable and functional assembly of RS3 and that RS3 and the CSC are important for ciliary motility.
BibTeX:
@article{Urbanska2015,
  author = {Urbanska, Paulina and Song, Kangkang and Joachimiak, Ewa and Krzemien-Ojak, Lucja and Koprowski, Piotr and Hennessey, Todd and Jerka-Dziadosz, Maria and Fabczak, Hanna and Gaertig, Jacek and Nicastro, Daniela and Wloga, Dorota},
  title = {The CSC proteins FAP61 and FAP251 build the basal substructures of radial spoke 3 in cilia.},
  journal = {Mol Biol Cell},
  year = {2015},
  volume = {26},
  number = {8},
  pages = {1463--1475},
  url = {http://dx.doi.org/10.1091/mbc.E14-11-1545},
  doi = {10.1091/mbc.E14-11-1545}
}
Vasudevan KK, Song K, Alford LM, Sale WS, Dymek EE, Smith EF, Hennessey T, Joachimiak E, Urbanska P, Wloga D, Dentler W, Nicastro D and Gaertig J (2015), "FAP206 is a microtubule-docking adapter for ciliary radial spoke 2 and dynein c.", Mol Biol Cell., Feb, 2015. Vol. 26(4), pp. 696-710.
Abstract: Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo-electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong. Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo. Thus FAP206 is likely part of the front prong and docks RS2 and dynein c to the microtubule.
BibTeX:
@article{Vasudevan2015,
  author = {Vasudevan, Krishna Kumar and Song, Kangkang and Alford, Lea M. and Sale, Winfield S. and Dymek, Erin E. and Smith, Elizabeth F. and Hennessey, Todd and Joachimiak, Ewa and Urbanska, Paulina and Wloga, Dorota and Dentler, William and Nicastro, Daniela and Gaertig, Jacek},
  title = {FAP206 is a microtubule-docking adapter for ciliary radial spoke 2 and dynein c.},
  journal = {Mol Biol Cell},
  year = {2015},
  volume = {26},
  number = {4},
  pages = {696--710},
  url = {http://dx.doi.org/10.1091/mbc.E14-11-1506},
  doi = {10.1091/mbc.E14-11-1506}
}
Woodward CL, Mendonça LM and Jensen GJ (2015), "Direct visualization of vaults within intact cells by electron cryo-tomography.", Cell Mol Life Sci., Sep, 2015. Vol. 72(17), pp. 3401-3409.
Abstract: The vault complex is the largest cellular ribonucleoprotein complex ever characterized and is present across diverse Eukarya. Despite significant information regarding the structure, composition and evolutionary conservation of the vault, little is know about the complex's actual biological function. To determine if intracellular vaults are morphologically similar to previously studied purified and recombinant vaults, we have used electron cryo-tomography to characterize the vault complexes found in the thin edges of primary human cells growing in tissue culture. Our studies confirm that intracellular vaults are similar in overall size and shape to purified and recombinant vaults previously analyzed. Results from subtomogram averaging indicate that densities within the vault lumen are not ordered, but randomly distributed. We also observe that vaults located in the extreme periphery of the cytoplasm predominately associate with granule-like structures and actin. Our ultrastructure studies augment existing biochemical, structural and genetic information on the vault, and provide important intracellular context for the ongoing efforts to understand the biological function of the native cytoplasmic vault.
BibTeX:
@article{Woodward2015,
  author = {Woodward, Cora L. and Mendonça, Luiza M. and Jensen, Grant J.},
  title = {Direct visualization of vaults within intact cells by electron cryo-tomography.},
  journal = {Cell Mol Life Sci},
  year = {2015},
  volume = {72},
  number = {17},
  pages = {3401--3409},
  url = {http://dx.doi.org/10.1007/s00018-015-1898-y},
  doi = {10.1007/s00018-015-1898-y}
}
Xiong Q, Morphew MK, Schwartz CL, Hoenger AH and Mastronarde DN (2009), "CTF determination and correction for low dose tomographic tilt series.", J Struct Biol., Dec, 2009. Vol. 168(3), pp. 378-387.
Abstract: The resolution of cryo-electron tomography can be limited by the first zero of the microscope's contrast transfer function (CTF). To achieve higher resolution, it is critical to determine the CTF and correct its phase inversions. However, the extremely low signal-to-noise ratio (SNR) and the defocus gradient in the projections of tilted specimens make this process challenging. Two programs, CTFPLOTTER and CTFPHASEFLIP, have been developed to address these issues. CTFPLOTTER obtains a 1D power spectrum by periodogram averaging and rotational averaging and it estimates the noise background with a novel approach, which uses images taken with no specimen. The background-subtracted 1D power spectra from image regions at different defocus values are then shifted to align their first zeros and averaged together. This averaging improves the SNR sufficiently that it becomes possible to determine the defocus for subsets of the tilt series rather than just the entire series. CTFPHASEFLIP corrects images line-by-line by inverting phases appropriately in thin strips of the image at nearly constant defocus. CTF correction by these methods is shown to improve the resolution of aligned, averaged particles extracted from tomograms. However, some restoration of Fourier amplitudes at high frequencies is important for seeing the benefits from CTF correction.
BibTeX:
@article{Xiong2009,
  author = {Xiong, Quanren and Morphew, Mary K. and Schwartz, Cindi L. and Hoenger, Andreas H. and Mastronarde, David N.},
  title = {CTF determination and correction for low dose tomographic tilt series.},
  journal = {J Struct Biol},
  year = {2009},
  volume = {168},
  number = {3},
  pages = {378--387},
  url = {http://dx.doi.org/10.1016/j.jsb.2009.08.016},
  doi = {10.1016/j.jsb.2009.08.016}
}
Zeev-Ben-Mordehai T, Vasishtan D, Hernández Durán A, Vollmer B, White P, Prasad Pandurangan A, Siebert CA, Topf M and Grünewald K (2016), "Two distinct trimeric conformations of natively membrane-anchored full-length herpes simplex virus 1 glycoprotein B.", Proc Natl Acad Sci U S A., Apr, 2016. Vol. 113(15), pp. 4176-4181.
Abstract: Many viruses are enveloped by a lipid bilayer acquired during assembly, which is typically studded with one or two types of glycoproteins. These viral surface proteins act as the primary interface between the virus and the host. Entry of enveloped viruses relies on specialized fusogen proteins to help merge the virus membrane with the host membrane. In the multicomponent herpesvirus fusion machinery, glycoprotein B (gB) acts as this fusogen. Although the structure of the gB ectodomain postfusion conformation has been determined, any other conformations (e.g., prefusion, intermediate conformations) have so far remained elusive, thus restricting efforts to develop antiviral treatments and prophylactic vaccines. Here, we have characterized the full-length herpes simplex virus 1 gB in a native membrane by displaying it on cell-derived vesicles and using electron cryotomography. Alongside the known postfusion conformation, a novel one was identified. Its structure, in the context of the membrane, was determined by subvolume averaging and found to be trimeric like the postfusion conformation, but appeared more condensed. Hierarchical constrained density-fitting of domains unexpectedly revealed the fusion loops in this conformation to be apart and pointing away from the anchoring membrane. This vital observation is a substantial step forward in understanding the complex herpesvirus fusion mechanism, and opens up new opportunities for more targeted intervention of herpesvirus entry.
BibTeX:
@article{Zeev-Ben-Mordehai2016,
  author = {Zeev-Ben-Mordehai, Tzviya and Vasishtan, Daven and Hernández Durán, Anna and Vollmer, Benjamin and White, Paul and Prasad Pandurangan, Arun and Siebert, C Alistair and Topf, Maya and Grünewald, Kay},
  title = {Two distinct trimeric conformations of natively membrane-anchored full-length herpes simplex virus 1 glycoprotein B.},
  journal = {Proc Natl Acad Sci U S A},
  year = {2016},
  volume = {113},
  number = {15},
  pages = {4176--4181},
  url = {http://dx.doi.org/10.1073/pnas.1523234113},
  doi = {10.1073/pnas.1523234113}
}
Zeev-Ben-Mordehai T, Vasishtan D, Siebert CA and Grünewald K (2014), "The full-length cell-cell fusogen EFF-1 is monomeric and upright on the membrane.", Nat Commun. Vol. 5, pp. 3912.
Abstract: Fusogens are membrane proteins that remodel lipid bilayers to facilitate membrane merging. Although several fusogen ectodomain structures have been solved, structural information on full-length, natively membrane-anchored fusogens is scarce. Here we present the electron cryo microscopy three-dimensional reconstruction of the Caenorhabditis elegans epithelial fusion failure 1 (EFF-1) protein natively anchored in cell-derived membrane vesicles. This reveals a membrane protruding, asymmetric, elongated monomer. Flexible fitting of a protomer of the EFF-1 crystal structure, which is homologous to viral class-II fusion proteins, shows that EFF-1 has a hairpin monomeric conformation before fusion. These structural insights, when combined with our observations of membrane-merging intermediates between vesicles, enable us to propose a model for EFF-1 mediated fusion. This process, involving identical proteins on both membranes to be fused, follows a mechanism that shares features of SNARE-mediated fusion while using the structural building blocks of the unilaterally acting class-II viral fusion proteins.
BibTeX:
@article{Zeev-Ben-Mordehai2014a,
  author = {Zeev-Ben-Mordehai, Tzviya and Vasishtan, Daven and Siebert, C Alistair and Grünewald, Kay},
  title = {The full-length cell-cell fusogen EFF-1 is monomeric and upright on the membrane.},
  journal = {Nat Commun},
  year = {2014},
  volume = {5},
  pages = {3912},
  url = {http://dx.doi.org/10.1038/ncomms4912},
  doi = {10.1038/ncomms4912}
}
Zeev-Ben-Mordehai T, Vasishtan D, Siebert CA, Whittle C and Grünewald K (2014), "Extracellular vesicles: a platform for the structure determination of membrane proteins by Cryo-EM.", Structure., Nov, 2014. Vol. 22(11), pp. 1687-1692.
Abstract: Membrane protein-enriched extracellular vesicles (MPEEVs) provide a platform for studying intact membrane proteins natively anchored with the correct topology in genuine biological membranes. This approach circumvents the need to conduct tedious detergent screens for solubilization, purification, and reconstitution required in classical membrane protein studies. We have applied this method to three integral type I membrane proteins, namely the Caenorhabditis elegans cell-cell fusion proteins AFF-1 and EFF-1 and the glycoprotein B (gB) from Herpes simplex virus type 1 (HSV1). Electron cryotomography followed by subvolume averaging allowed the 3D reconstruction of EFF-1 and HSV1 gB in the membrane as well as an analysis of the spatial distribution and interprotein interactions on the membrane. MPEEVs have many applications beyond structural/functional investigations, such as facilitating the raising of antibodies, for protein-protein interaction assays or for diagnostics use, as biomarkers, and possibly therapeutics.
BibTeX:
@article{Zeev-Ben-Mordehai2014,
  author = {Zeev-Ben-Mordehai, Tzviya and Vasishtan, Daven and Siebert, C Alistair and Whittle, Cathy and Grünewald, Kay},
  title = {Extracellular vesicles: a platform for the structure determination of membrane proteins by Cryo-EM.},
  journal = {Structure},
  year = {2014},
  volume = {22},
  number = {11},
  pages = {1687--1692},
  url = {http://dx.doi.org/10.1016/j.str.2014.09.005},
  doi = {10.1016/j.str.2014.09.005}
}