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author:("pigging, Gaia")
1.  Cryoelectron tomography of radial spokes in cilia and flagella 
The Journal of Cell Biology  2011;195(4):673-687.
Cryo-EM tomography of wild-type and mutant cilia and flagella from Tetrahymena and Chlamydomonas reveals new information on the substructure of radial spokes.
Radial spokes (RSs) are ubiquitous components in the 9 + 2 axoneme thought to be mechanochemical transducers involved in local control of dynein-driven microtubule sliding. They are composed of >23 polypeptides, whose interactions and placement must be deciphered to understand RS function. In this paper, we show the detailed three-dimensional (3D) structure of RS in situ in Chlamydomonas reinhardtii flagella and Tetrahymena thermophila cilia that we obtained using cryoelectron tomography (cryo-ET). We clarify similarities and differences between the three spoke species, RS1, RS2, and RS3, in T. thermophila and in C. reinhardtii and show that part of RS3 is conserved in C. reinhardtii, which only has two species of complete RSs. By analyzing C. reinhardtii mutants, we identified the specific location of subsets of RS proteins (RSPs). Our 3D reconstructions show a twofold symmetry, suggesting that fully assembled RSs are produced by dimerization. Based on our cryo-ET data, we propose models of subdomain organization within the RS as well as interactions between RSPs and with other axonemal components.
PMCID: PMC3257535  PMID: 22065640
2.  Axonemal radial spokes 
Bioarchitecture  2012;2(2):50-58.
The radial spoke (RS) is a complex of at least 23 proteins that works as a mechanochemical transducer between the central‐pair apparatus and the peripheral microtubule doublets in eukaryotic flagella and motile cilia. The RS contributes to the regulation of the activity of dynein motors, and thus to flagellar motility. Despite numerous biochemical, physiological and structural studies, the mechanism of the function of the radial spoke remains unclear. Detailed knowledge of the 3D structure of the RS protein complex is needed in order to understand how RS regulates dynein activity. Here we review the most important findings on the structure of the RS, including results of our recent cryo‐electron tomographic analysis of the RS protein complex.
PMCID: PMC3383722  PMID: 22754630
axoneme; cilia; cryo‐electron tomography; dynein; flagella; motility; radial spokes
3.  Three-dimensional structural analysis of eukaryotic flagella/cilia by electron cryo-tomography 
Journal of Synchrotron Radiation  2010;18(Pt 1):2-5.
Based on the molecular architecture revealed by electron cryo-tomography, the mechanism of the bending motion of eukaryotic flagella/cilia is discussed.
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.
PMCID: PMC3004243  PMID: 21169680
dynein; flagella; axoneme; tomography; cryo-EM
4.  Electron-tomographic analysis of intraflagellar transport particle trains in situ 
The Journal of Cell Biology  2009;187(1):135-148.
Ultrastructural study of Chlamydomonas cilia shows that anterograde IFT particles form trains that are long and narrow, while retrograde IFT form short, compact particle trains.
Intraflagellar transport (IFT) is the bidirectional movement of multipolypeptide particles between the ciliary membrane and the axonemal microtubules, and is required for the assembly, maintenance, and sensory function of cilia and flagella. In this paper, we present the first high-resolution ultrastructural analysis of trains of flagellar IFT particles, using transmission electron microscopy and electron-tomographic analysis of sections from flat-embedded Chlamydomonas reinhardtii cells. Using wild-type and mutant cells with defects in IFT, we identified two different types of IFT trains: long, narrow trains responsible for anterograde transport; and short, compact trains underlying retrograde IFT. Both types of trains have characteristic repeats and patterns that vary as one sections longitudinally through the trains of particles. The individual IFT particles are highly complex, bridged to each other and to the outer doublet microtubules, and are closely apposed to the inner surface of the flagellar membrane.
PMCID: PMC2762096  PMID: 19805633
5.  JUST (Java User Segmentation Tool) for semi-automatic segmentation of tomographic maps 
Journal of structural biology  2007;161(3):287-297.
We are presenting a program for interactive segmentation of tomographic maps, based on objective criteria so as to yield reproducible results. The strategy starts with the automatic segmentation of the entire volume with the watershed algorithm in 3D. The watershed regions are clustered successively by supervised classification, allowing the segmentation of known organelles, such as membranes, vesicles and microtubules. These organelles are processed with topological models and input parameters manually derived from the tomograms. After known organelles are extracted from the volume, all other watershed regions can be organized into homogeneous assemblies on the basis of their densities. To complete the process, all voxels in the volume are assigned either to the background or individual structures, which can then be extracted for visualization with any rendering technique.
The user interface of the program is written in Java, and computational routines are written in C. For some operations, involving the visualization of the tomogram, we refer to existing software, either open or commercial. While the program runs, a history file is created, that allows all parameters and other data to be saved for the purposes of comparison or exchange. Initially, the program was developed for the segmentation of synapses, and organelles belonging to these structures have thus far been the principal targets modeled with JUST. Since each organelle is clustered independently from the rest of the volume, however, the program can accommodate new models of different organelles as well as tomograms of other types of preparations of tissue, such as citoskeletal components in vitreous ice.
PMCID: PMC2692284  PMID: 17707657
Electron tomography; Volume segmentation; Image processing; Feature extraction; Synapsis

Results 1-5 (5)