Our laboratory is studying the three-dimensional structure
of macromolecular machines, organelles and cells using cryo-electron
tomography, with special emphasis on the structure and function
relationships of macromolecular complexes in situ, i.e.
in their native environment. Areas of interest include the
structure of eukaryotic flagella and axonemal dynein, cytoskeleton
and bacteria, as well as the development of cryo-EM and
image processing techniques.
Cryo-Electron Tomography (cryo-ET):
Most natural objects are three-dimensional (3D) in structure.
The higher the complexity of an object for study, the less
revealing are its two-dimensional (2D) projections, due
to superposition of multiple structures in single image
features. The basic idea of tomography is to record a series
of 2D images that are projections of a 3D object at different
angles of tilt (fig. a). A 3D image of this object is then
reconstructed by projecting all the 2D images back into
a common volume with appropriate weighting (fig. b). 
Different
energy sources can be used to produce the projections (x-rays,
ultrasound, electromagnetic waves, etc.) and tomography
has found wide applications in diverse fields, such as diagnostic
medicine and radio astronomy. Electron microscope tomography
(ET) can provide invaluable and novel information about
the 3D ultrastructure of tissues, cells and macromolecules.
The advantage of using rapidly frozen specimen ("cryo")
is the near to native and thus excellent structure preservation
of the biological object. Depending on the thickness of
the sample and the possibilities of post-processing (e.g.
by 3D averaging), a resolution better than 4 nm can be obtained.
We are developing new image processing methods to push the
current limit to higher resolution.
Eukaryotic
Flagella and Axonemal Dynein:
Eukaryotic cilia and flagella are highly conserved motile
systems built on a microtubule-based scaffold called the
axoneme (fig. c). The complexity of this organelle and size
of its major motor protein, dynein, have made it difficult
to understand the molecular mechanisms that underlie flagellar
beating. We are using ET of frozen-hydrated flagella to
solve this problem. 3D reconstructions have enabled us to
clarify aspects of the morphology of the outer dynein arms
and to relate details of dynein's structure to its function
as the motor that moves each doublet microtubule relative
to its neighbor. We have also identified links that connect
dynein arms with one another along the axoneme, as well
as structures that connect inner and outer dynein arms.
Both these novel structural elements are likely to provide
the "hard-wired" controls that help to regulate the speed
and propagation of bends along axonemes. Moreover, we identify
the first periodic structures inside microtubules, objects
that decorate the interior surfaces of both A- and B-subtubules
of the doublets at the sites where stabilizing drugs like
Taxol bind to tubulin, suggesting that these objects may
be examples of the long predicted physiological regulators
that modulate microtubule stability. We also compared mutant
with wild type axonemes, which showed the reproducibility
of our methods and allowed us to characterize the differences
that result from deletion of a single gene for the heavy
chain component of one of the inner dynein arms (fig. d).
Future goals:
Many biological structures will greatly profit from studies
using cryo-ET, and thus our goal is to apply this powerful
technology to diverse biological problems to gain deeper
insights into the functional organization of cells. Most
eukaryotic cells are too thick to be imaged directly by
cryo-ET, thus one focus of our group will be to improve
the technique of frozen-hydrated sectioning to minimize
sectioning artifacts.
Selected Publications:
Kinesin-8 from fission yeast: a heterodimeric, plus-end-directed motor that can couple microtubule depolymerization to cargo movement. Grissom PM, Fiedler T, Grishchuk EL, Nicastro D, West RR, McIntosh JR. Mol Biol Cell. 2009 Feb;20(3):963-72. [abstract]
Drosophila asterless and vertebrate Cep152 Are orthologs essential for centriole duplication. Blachon S, Gopalakrishnan J, Omori Y, Polyanovsky A, Church A, Nicastro D, Malicki J, Avidor-Reiss T. Genetics. 2008 Dec;180(4):2081-94. [abstract]
The structural basis of actin filament branching by the Arp2/3 complex. Rouiller I, Xu XP, Amann KJ, Egile C, Nickell S, Nicastro D, Li R, Pollard TD, Volkmann N, Hanein D. J Cell Biol. 2008 Mar 10;180(5):887-95. [abstract]
Single particle cryoelectron tomography characterization of the structure and structural variability of poliovirus-receptor-membrane complex at 30 A resolution. Bostina M, Bubeck D, Schwartz C, Nicastro D, Filman DJ, Hogle JM. J Struct Biol. 2007 Nov;160(2):200-10. [abstract]
Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching. Schwartz CL, Sarbash VI, Ataullakhanov FI, McIntosh JR, Nicastro D. J Microsc. 2007 Aug;227(Pt 2):98-109. [abstract]
Electron microscopy of microtubule-based cytoskeletal machinery. Hoenger A, Nicastro D. Methods Cell Biol. 2007;79:437-62.
The molecular architecture of axonemes
revealed by cryoelectron tomography. Nicastro D, Schwartz C, Pierson J, Gaudette R, Porter ME,
McIntosh JR. (2006) Science 313:944-8.
[abstract]
3D structure
of eukaryotic flagella in a quiescent state revealed by
cryo-electron tomography. Nicastro D, McIntosh J.R., Baumeister W (2005) Proc Natl Acad Sci USA 102:15889-94.
[abstract]
New views
of cells in 3D: an introduction to electron tomography. McIntosh R., Nicastro D, Mastronarde D (2005) Trends Cell Biol. 15:43-51. [abstract]
Macromolecular architecture in eukaryotic
cells visualized by cryoelectron tomography. Medalia O, Weber I, Frangakis AS, Nicastro D, Gerisch G,
Baumeister W (2002) Science 298:1209-1213.
[abstract]
Identification of
macromolecular complexes in cryoelectron tomograms of phantom
cells. Frangakis AS, Boehm J, Forster F, Nickell S, Nicastro D,
Typke D, Hegerl R, Baumeister W (2002) Proc Natl Acad Sci USA 99:14153-14158. [abstract]
Cryo-electron tomography of Neurospora mitochondria: three-dimensional
organization and ultrastructure of whole ice-embedded organelles. Nicastro D, Frangakis AS, Typke D, Baumeister W (2000)
J Struct Biol. 129: 48-56. [abstract]
Larva-adult
relationships in an ancestral dipteran: a re-examination
of sensillar pathways across the antenna and leg anlagen
of Chaoborus crystallinus (DeGeer, 1776) (Diptera, Chaoboridae). Melzer RR, Sprenger J, Nicastro D, Smola U (1999) Dev Gen Evol. 209: 103-112. [abstract]
Evolution
of small sense organs: sensilla on the larval antennae traced
back to the origin of the Diptera. Nicastro D, Melzer RR, Hruschka H, Smola U (1998) Naturwissenschaften 85:
501-505.
The antennal sensilla
of the carnivorous "phantom" larva of Chaoborus crystallinus
(DeGeer, 1776) (Diptera, Nematocera). Nicastro D, Smola U, Melzer RR (1995) Canadian Journal of
Zoology 73: 15-26.
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