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ABSTRACT: Ciliary compartmentalization plays pivotal roles in ciliogenesis and in various signaling pathways. Here we describe a structure at the ciliary base that appears to have all the features required for compartmentalization and which we thus call the "ciliary partitioning system" (CPS). This complex consists of the terminal plate, which serves as a cytosolic "ciliary pore complex" (CPC), and a membrane region well suited to serve as a diffusion barrier. The CPC is a plate-shaped structure containing nine pores through which the microtubule doublets of the basal body pass. Each pore expands from the doublet B-tubule into an opening well suited for the passage of intraflagellar transport particles. The membrane diffusion barrier encompasses an extended region of detergent-resistant periciliary membrane (ciliary pocket) and a ring complex that connects the CPC to the membrane. Proteomics analysis shows involvement of the ciliary pocket in vesicle trafficking, suggesting that this region plays an active role in membrane transport. The CPC and the ring together form a complete partition defining the ciliary boundary.
Current biology: CB 01/2013; · 10.99 Impact Factor
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ABSTRACT: Iron-reducing bacteria (FeRB) play key roles in anaerobic metal and carbon cycling and carry out biogeochemical transformations that can be harnessed for environmental bioremediation. A subset of FeRB require direct contact with Fe(III)-bearing minerals for dissimilatory growth, yet these bacteria must move between mineral particles. Furthermore, they proliferate in planktonic consortia during biostimulation experiments. Thus, a key question is how such organisms can sustain growth under these conditions. Here we characterized planktonic microbial communities sampled from an aquifer in Rifle, Colorado, USA, close to the peak of iron reduction following in situ acetate amendment. Samples were cryo-plunged on site and subsequently examined using correlated two- and three-dimensional cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission X-ray microscopy (STXM). The outer membranes of most cells were decorated with aggregates up to 150 nm in diameter composed of ∼3 nm wide amorphous, Fe-rich nanoparticles. Fluorescent in situ hybridization of lineage-specific probes applied to rRNA of cells subsequently imaged via cryo-TEM identified Geobacter spp., a well-studied group of FeRB. STXM results at the Fe L(2,3) absorption edges indicate that nanoparticle aggregates contain a variable mixture of Fe(II)-Fe(III), and are generally enriched in Fe(III). Geobacter bemidjiensis cultivated anaerobically in the laboratory on acetate and hydrous ferric oxyhydroxides also accumulated mixed-valence nanoparticle aggregates. In field-collected samples, FeRB with a wide variety of morphologies were associated with nano-aggregates, indicating that cell surface Fe(III) accumulation may be a general mechanism by which FeRB can grow while in planktonic suspension.
The ISME Journal 10/2012; · 7.38 Impact Factor
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ABSTRACT: We present a modern, light portable device specifically designed for environmental samples for cryogenic transmission-electron microscopy (cryo-TEM) by on-site cryo-plunging. The power of cryo-TEM comes from preparation of artifact-free samples. However, in many studies, the samples must be collected at remote field locations, and the time involved in transporting samples back to the laboratory for cryogenic preservation can lead to severe degradation artifacts. Thus, going back to the basics, we developed a simple mechanical device that is light and easy to transport on foot yet effective. With the system design presented here we are able to obtain cryo-samples of microbes and microbial communities not possible to culture, in their near-intact environmental conditions as well as in routine laboratory work, and in real time. This methodology thus enables us to bring the power of cryo-TEM to microbial ecology.
Microscopy Research and Technique 12/2011; 75(6):829-36. · 1.79 Impact Factor
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ABSTRACT: The electrical conductance and the optical density of evaporated carbon films are measured as a function of the thickness of the films. The resulting data show that up to a thickness of approximately 4 nm, carbon films are optically transparent and electrically insulating. The same data also suggest that this insulating character persists near to the surface when the overall thickness is further increased. Since a support film with poor surface conductivity is undesirable for many applications in electron microscopy, we suggest that the usefulness of evaporated carbon films in electron microscopy might be further improved by doping or by other methods that improve the electrical conductivity near the surface.
Journal of Structural Biology 02/2011; 174(2):420-3. · 3.41 Impact Factor
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ABSTRACT: Electron crystallography has become a powerful approach for structural characterization of two-dimensional (2-D) protein crystals. The crystallographic approach provides the simplest route to the type of averaging that is essential for obtaining high resolution structural information from radiation-sensitive samples such as organic molecules. Several atomic or near atomic resolution protein structures have been solved by using cryo-electron crystallography and most of them involved using both image and electron diffraction data. An essential step in either type of work is preparation of specimens suitable for electron microscopy which retain their native state and high degree of order. Methods for preserving samples in a near-native, hydrated state have been developed, with minor variations for different specimens. The major challenge of collecting electron diffraction data particularly at high tilt angle is the blurring of diffraction spots due to imperfect flatness of the crystals. This paper discusses specimen preparation methods for electron crystallographic data collection of 2-D protein crystals with particular emphasis on the factors which affect the flatness of crystals. We also discuss some of the aspects of the data collection protocols which are particular to work with crystals.
Micron 02/2011; 42(2):132-40. · 1.53 Impact Factor
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ABSTRACT: The surface layers (S layers) of those bacteria and archaea that elaborate these crystalline structures have been studied for 40 years. However, most structural analysis has been based on electron microscopy of negatively stained S-layer fragments separated from cells, which can introduce staining artifacts and allow rearrangement of structures prone to self-assemble. We present a quantitative analysis of the structure and organization of the S layer on intact growing cells of the Gram-negative bacterium Caulobacter crescentus using cryo-electron tomography (CET) and statistical image processing. Instead of the expected long-range order, we observed different regions with hexagonally organized subunits exhibiting short-range order and a broad distribution of periodicities. Also, areas of stacked double layers were found, and these increased in extent when the S-layer protein (RsaA) expression level was elevated by addition of multiple rsaA copies. Finally, we combined high-resolution amino acid residue-specific Nanogold labeling and subtomogram averaging of CET volumes to improve our understanding of the correlation between the linear protein sequence and the structure at the 2-nm level of resolution that is presently available. The results support the view that the U-shaped RsaA monomer predicted from negative-stain tomography proceeds from the N terminus at one vertex, corresponding to the axis of 3-fold symmetry, to the C terminus at the opposite vertex, which forms the prominent 6-fold symmetry axis. Such information will help future efforts to analyze subunit interactions and guide selection of internal sites for display of heterologous protein segments.
Journal of bacteriology 11/2010; 192(22):5855-65. · 3.94 Impact Factor
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ABSTRACT: Cell division in Caulobacter crescentus involves constriction and fission of the inner membrane (IM) followed about 20 min later by fission of the outer membrane (OM) and daughter cell separation. In contrast to Escherichia coli, the Caulobacter Tol-Pal complex is essential. Cryo-electron microscopy images of the Caulobacter cell envelope exhibited outer membrane disruption, and cells failed to complete cell division in TolA, TolB, or Pal mutant strains. In wild-type cells, components of the Tol-Pal complex localize to the division plane in early predivisional cells and remain predominantly at the new pole of swarmer and stalked progeny upon completion of division. The Tol-Pal complex is required to maintain the position of the transmembrane TipN polar marker, and indirectly the PleC histidine kinase, at the cell pole, but it is not required for the polar maintenance of other transmembrane and membrane-associated polar proteins tested. Coimmunoprecipitation experiments show that both TolA and Pal interact directly or indirectly with TipN. We propose that disruption of the trans-envelope Tol-Pal complex releases TipN from its subcellular position. The Caulobacter Tol-Pal complex is thus a key component of cell envelope structure and function, mediating OM constriction at the final step of cell division as well as the positioning of a protein localization factor.
Journal of bacteriology 10/2010; 192(19):4847-58. · 3.94 Impact Factor
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ABSTRACT: In the past few years, three-dimensional (3D) subtomogram alignment has become an important tool in cryo-electron tomography (CET). This technique allows one to produce higher resolution images of structures which can not be reconstructed using single-particle methods. Building on previous work, we present a new dissimilarity measure between subtomograms that works well for the noisy images that often occur in CET images. A technique that is more robust to noise provides the ability to analyze macromolecules in thicker samples such as whole cells or lower the defocus in thinner samples to push the first zero of the Contrast Transfer Function (CTF). Our method, Threshold Constrained Cross-Correlation (TCCC), uses statistics of the noise to automatically select only a small percentage of the Fourier coefficients to compute the cross-correlation, which has two main advantages: first, it reduces the influence of the noise by looking at only those peaks dominated by signal; and second, it avoids the missing wedge normalization problem since we consider the same number of coefficients for all possible pairs of subtomograms. We present results with synthetic and real data to compare our approach with other existing methods under different SNR and missing wedge conditions, and show that TCCC improves alignment results for datasets with SNR<0.1. We have made our source code freely available for the community.
Journal of Structural Biology 09/2010; 171(3):332-44. · 3.41 Impact Factor
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ABSTRACT: The diverse functions of microtubules require stiff structures possessing sufficient lateral flexibility to enable bending with high curvature. We used cryo-electron microscopy to investigate the molecular basis for these critical mechanical properties. High-quality structural maps were used to build pseudoatomic models of microtubules containing 11-16 protofilaments, representing a wide range of lateral curvature. Protofilaments in all these microtubules were connected primarily via interprotofilament interactions between the M loops, and the H1'-S2 and H2-S3 loops. We postulate that the tolerance of the loop-loop interactions to lateral deformation provides the capacity for high-curvature bending without breaking. On the other hand, the local molecular architecture that surrounds these connecting loops contributes to the overall rigidity. Interprotofilament interactions in the seam region are similar to those in the normal helical regions, suggesting that the existence of the seam does not significantly affect the mechanical properties of microtubules.
Structure 08/2010; 18(8):1022-31. · 6.35 Impact Factor
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ABSTRACT: Intracellular magnetite crystal formation by magnetotactic bacteria has emerged as a powerful model for investigating the cellular and molecular mechanisms of biomineralization, a process common to all branches of life. Although magnetotactic bacteria are phylogenetically diverse and their crystals morphologically diverse, studies to date have focused on a few, closely related species with similar crystal habits. Here, we investigate the process of magnetite biomineralization in Desulfovibrio magneticus sp. RS-1, the only reported species of cultured magnetotactic bacteria that is outside of the alpha-Proteobacteria and that forms bullet-shaped crystals. Using a variety of high-resolution imaging and analytical tools, we show that RS-1 cells form amorphous, noncrystalline granules containing iron and phosphorus before forming magnetite crystals. Using NanoSIMS (dynamic secondary ion mass spectroscopy), we show that the iron-phosphorus granules and the magnetite crystals are likely formed through separate cellular processes. Analysis of the cellular ultrastructure of RS-1 using cryo-ultramicrotomy, cryo-electron tomography, and tomography of ultrathin sections reveals that the magnetite crystals are not surrounded by membranes but that the iron-phosphorus granules are surrounded by membranous compartments. The varied cellular paths for the formation of these two minerals lead us to suggest that the iron-phosphorus granules constitute a distinct bacterial organelle.
Proceedings of the National Academy of Sciences 07/2010; 107(27):12263-8. · 9.68 Impact Factor
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ABSTRACT: Cell division in Gram-negative organisms requires coordinated invagination of the multilayered cell envelope such that each daughter receives an intact inner membrane, peptidoglycan (PG) layer and outer membrane (OM). Here, we identify DipM, a putative LytM endopeptidase in Caulobacter crescentus, and show that it plays a critical role in maintaining cell envelope architecture during growth and division. DipM localized to the division site in an FtsZ-dependent manner via its PG-binding LysM domains. Although not essential for viability, DeltadipM cells exhibited gross morphological defects, including cell widening and filamentation, indicating a role in cell shape maintenance and division that we show requires its LytM domain. Strikingly, cells lacking DipM also showed OM blebbing at the division site, at cell poles and along the cell body. Cryo electron tomography of sacculi isolated from cells depleted of DipM revealed marked thickening of the PG as compared to wild type, which we hypothesize leads to loss of trans-envelope contacts between components of the Tol-Pal complex. We conclude that DipM is required for normal envelope invagination during division and to maintain a sacculus of constant thickness that allows for maintenance of OM connections throughout the cell envelope.
Molecular Microbiology 05/2010; 77(1):56-73. · 5.01 Impact Factor
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ABSTRACT: Kinesin cytoskeletal motors convert the energy of ATP hydrolysis into stepping movement along microtubules. A partial model of this process has been derived from crystal structures, which show that movement of the motor domain relative to its major microtubule binding element, the switch II helix, is coupled to docking of kinesin's neck linker element along the motor domain. This docking would displace the cargo in the direction of travel and so contribute to a step. However, the crystal structures do not reveal how ATP binding and hydrolysis govern this series of events. We used cryoelectron microscopy to derive 8-9 A-resolution maps of four nucleotide states encompassing the microtubule-attached kinetic cycle of a kinesin motor. The exceptionally high quality of these maps allowed us to build in crystallographically determined conformations of kinesin's key subcomponents, yielding novel arrangements of kinesin's switch II helix and nucleotide-sensing switch loops. The resulting atomic models reveal a seesaw mechanism in which the switch loops, triggered by ATP binding, propel their side of the motor domain down and thereby elicit docking of the neck linker on the opposite side of the seesaw. Microtubules engage the seesaw mechanism by stabilizing the formation of extra turns at the N terminus of the switch II helix, which then serve as an anchor for the switch loops as they modulate the seesaw angle. These observations explain how microtubules activate kinesin's ATP-sensing machinery to promote cargo displacement and inform the mechanism of kinesin's ancestral relative, myosin.
Proceedings of the National Academy of Sciences 02/2010; 107(9):4111-6. · 9.68 Impact Factor
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Grant R Bowman,
Luis R Comolli,
Guido M Gaietta,
Michael Fero,
Sun-Hae Hong,
Ying Jones,
Julie H Lee, Kenneth H Downing,
Mark H Ellisman,
Harley H McAdams,
Lucy Shapiro
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ABSTRACT: The bacterium Caulobacter crescentus has morphologically and functionally distinct cell poles that undergo sequential changes during the cell cycle. We show that the PopZ oligomeric network forms polar ribosome exclusion zones that change function during cell cycle progression. The parS/ParB chromosomal centromere is tethered to PopZ at one pole prior to the initiation of DNA replication. During polar maturation, the PopZ-centromere tether is broken, and the PopZ zone at that pole then switches function to act as a recruitment factor for the ordered addition of multiple proteins that promote the transformation of the flagellated pole into a stalked pole. Stalked pole assembly, in turn, triggers the initiation of chromosome replication, which signals the formation of a new PopZ zone at the opposite cell pole, where it functions to anchor the newly duplicated centromere that has traversed the long axis of the cell. We propose that pole-specific control of PopZ function co-ordinates polar development and cell cycle progression by enabling independent assembly and tethering activities at the two cell poles.
Molecular Microbiology 02/2010; 76(1):173-89. · 5.01 Impact Factor
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ABSTRACT: Cryo-EM is ideally suited for the study of cytoskeleton polymers and their interaction with cellular partners. Our understanding of microtubule (MT) structure and interactions has benefited tremendously from the application of different EM techniques, from the use of electron crystallography to determine the first high-resolution structure of tubulin, to electron tomographic reconstructions of unique MT-based organelles; from molecular details governing the regulated interaction of MTs with kinesin motors, to an atomic description of how antimitotic agents bind to tubulin and affect MT stability. In this chapter, we review these structural findings with an emphasis on how cryo-EM enables our studies.
Methods in enzymology 01/2010; 483:121-42. · 1.90 Impact Factor
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Bong-Gyoon Han,
Ming Dong,
Haichuan Liu,
Lauren Camp,
Jil Geller,
Mary Singer,
Terry C Hazen,
Megan Choi,
H Ewa Witkowska,
David A Ball,
Dieter Typke, Kenneth H Downing,
Maxim Shatsky,
Steven E Brenner,
John-Marc Chandonia,
Mark D Biggin,
Robert M Glaeser
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ABSTRACT: An unbiased survey has been made of the stable, most abundant multi-protein complexes in Desulfovibrio vulgaris Hildenborough (DvH) that are larger than Mr approximately 400 k. The quaternary structures for 8 of the 16 complexes purified during this work were determined by single-particle reconstruction of negatively stained specimens, a success rate approximately 10 times greater than that of previous "proteomic" screens. In addition, the subunit compositions and stoichiometries of the remaining complexes were determined by biochemical methods. Our data show that the structures of only two of these large complexes, out of the 13 in this set that have recognizable functions, can be modeled with confidence based on the structures of known homologs. These results indicate that there is significantly greater variability in the way that homologous prokaryotic macromolecular complexes are assembled than has generally been appreciated. As a consequence, we suggest that relying solely on previously determined quaternary structures for homologous proteins may not be sufficient to properly understand their role in another cell of interest.
Proceedings of the National Academy of Sciences 09/2009; 106(39):16580-5. · 9.68 Impact Factor
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ABSTRACT: Determining the transient chemical properties of the intracellular environment can elucidate the paths through which a biological system adapts to changes in its environment, for example, the mechanisms that enable some obligate anaerobic bacteria to survive a sudden exposure to oxygen. Here we used high-resolution Fourier transform infrared (FTIR) spectromicroscopy to continuously follow cellular chemistry within living obligate anaerobes by monitoring hydrogen bond structures in their cellular water. We observed a sequence of well orchestrated molecular events that correspond to changes in cellular processes in those cells that survive, but only accumulation of radicals in those that do not. We thereby can interpret the adaptive response in terms of transient intracellular chemistry and link it to oxygen stress and survival. This ability to monitor chemical changes at the molecular level can yield important insights into a wide range of adaptive responses.
Proceedings of the National Academy of Sciences 07/2009; 106(31):12599-604. · 9.68 Impact Factor
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ABSTRACT: Since tubulin was discovered in 1967, drug probes have been used to manipulate mechanisms of microtubule polymerization and disassembly. In parallel, advances in optical imagery, electron microscopy, along with both electron and X-ray diffraction have provided ability to "see" the molecular underpinning of these machines. Nanoscale mapping of different tubulin polymers formed in the presence of different drugs and cofactors provide a context for examining the dynamic features relevant to their biological activity. Models built from EM maps have been used to understand the binding of stabilizing drugs such as taxanes and epothilones, to predict more effective molecules, and to explain mutation based resistance. Here, we discuss drug binding in the context of different polymeric forms and propose a trigger mechanism associated with microtubules' dynamic instability.
Topics in current chemistry 01/2009; 286:209-257. · 4.29 Impact Factor
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ABSTRACT: Fully understanding the biology of acid mine drainage (AMD) is central to our ability to control and manipulate its environmental impact. Although genomics and biogeochemical methods are relatively well established in the field, their combination with high-resolution imaging of intact members of microbial biofilm communities has not yet reached its full potential. Here, we used three-dimensional (3D) cryogenic electron tomography to determine the size and ultrastructure of intact ARMAN cells, a novel ultra-small archaeon, and sought evidence for their interactions with other members of its community. Within acid mine drainage biofilms, apparently free-living ARMAN cells from a deeply branched archaeal lineage have volumes of 0.009-0.04 microm(3) (mean approximately 0.03+/-0.01 microm(3)), only approximately 92 ribosomes, yet are frequent hosts for replicating viruses. Organization within the periplasm and partitioning of ribosomes to the inner surface of the cytoplasmic membrane may be factors in size minimization. Most cells contain enigmatic tubular structures of unknown function. The low ribosome copy number per unit volume, indicative of slow growth rates and targeting of cells by diverse viruses may account for the low abundance of ARMAN cells compared with other biofilm community members. Our results provide the first 3D analysis of structural features of these novel and enigmatic cells and their interactions with at least two types of viruses. Our findings also emphasize that new biological phenomena remain to be discovered among lower abundance organisms from novel uncultivated lineages.
The ISME Journal 11/2008; 3(2):159-67. · 7.38 Impact Factor
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ABSTRACT: Bacterial replication origins move towards opposite ends of the cell during DNA segregation. We have identified a proline-rich polar protein, PopZ, required to anchor the separated Caulobacter crescentus chromosome origins at the cell poles, a function that is essential for maintaining chromosome organization and normal cell division. PopZ interacts directly with the ParB protein bound to specific DNA sequences near the replication origin. As the origin/ParB complex is being replicated and moved across the cell, PopZ accumulates at the cell pole and tethers the origin in place upon arrival. The polar accumulation of PopZ occurs by a diffusion/capture mechanism that requires the MreB cytoskeleton. High molecular weight oligomers of PopZ assemble in vitro into a filamentous network with trimer junctions, suggesting that the PopZ network and ParB-bound DNA interact in an adhesive complex, fixing the chromosome origin at the cell pole.
Cell 10/2008; 134(6):945-55. · 32.40 Impact Factor
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ABSTRACT: Kinesin-1 is a molecular motor protein that transports cargo along microtubules. Inside cells, the vast majority of kinesin-1 is regulated to conserve ATP and to ensure its proper intracellular distribution and coordination with other molecular motors. Regulated kinesin-1 folds in half at a hinge in its coiled-coil stalk. Interactions between coiled-coil regions near the enzymatically active heads at the N terminus and the regulatory tails at the C terminus bring these globular elements in proximity and stabilize the folded conformation. However, it has remained a mystery how kinesin-1's microtubule-stimulated ATPase activity is regulated in this folded conformation. Here, we present evidence for a direct interaction between the kinesin-1 head and tail. We photochemically cross-linked heads and tails and produced an 8-A cryoEM reconstruction of the cross-linked head-tail complex on microtubules. These data demonstrate that a conserved essential regulatory element in the kinesin-1 tail interacts directly and specifically with the enzymatically critical Switch I region of the head. This interaction suggests a mechanism for tail-mediated regulation of the ATPase activity of kinesin-1. In our structure, the tail makes simultaneous contacts with the kinesin-1 head and the microtubule, suggesting the tail may both regulate kinesin-1 in solution and hold it in a paused state with high ADP affinity on microtubules. The interaction of the Switch I region of the kinesin-1 head with the tail is strikingly similar to the interactions of small GTPases with their regulators, indicating that other kinesin motors may share similar regulatory mechanisms.
Proceedings of the National Academy of Sciences 08/2008; 105(26):8938-43. · 9.68 Impact Factor
