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 and function of cilia and flagella, the molecular motor dynein and other cytoskeletal assemblies, as well as the development of cryo-electron microscopy and image processing techniques.
Cilia and Flagella:
Cilia and flagella are highly conserved motile organelles containing a microtubule-based scaffold called the axoneme (Figure). Defects in the assembly or function of these organelles are linked to several human diseases, called ciliopathies, such as primary ciliary dyskinesia and polycystic kidney disease. The complexity of cilia and flagella has made it difficult to understand the molecular mechanisms that underlie motility. We are using electron tomography of rapidly frozen cilia and flagella to solve long-standing questions.
Our data and wild-type/mutant comparisons of several major axonemal complexes have revealed the 3D structures of outer and inner dynein arms, doublet microtubules and radial spokes. We discovered several new structures, including links that connect the outer and inner dyneins arms (OID linker), Microtubule Inner Proteins (MIPs), which are periodic structures inside doublet microtubules (Nicastro et al. 2006), the location of the elusive Nexin link (Heuser et al. 2009), a protein tether of a dynein motor domain to the doublet microtubule (Heuser et al. 2011), doublet specific structures (Nicastro 2009, Heuser et al. 2011, Nicastro et al. 2011), and radial spoke heterogeneity (Dymek et al. 2011, Barber et al. 2011).
We will continue to dissect the 3D structure of these remarkable nanomachines, however, many other biological structures will also greatly benefit from studies using cryo-electron tomography. Thus our goal is to apply this powerful technology to diverse biological problems to gain deeper insights into the functional organization of cells. For more details please visit us on our Laboratory Webpage.
Probing Nanoscale Self-Assembly of Nonfluorescent Small Molecules inside Live Mammalian Cells. Gao Y, Berciu C, Kuang Y, Shi J, Nicastro D, Xu B. ACS Nano. 2013 Sep 25. [Epub ahead of print]
Formation of membrane ridges and scallops by the F-BAR protein Nervous Wreck. Becalska AN, Kelley CF, Berciu C, Stanishneva-Konovalova TB, Fu X, Wang S, Sokolova OS, Nicastro D, Rodal AA. Mol Biol Cell. 2013 Aug;24(15):2406-18. doi: 10.1091/mbc.E13-05-0271.
The MIA complex is a conserved and novel dynein regulator essential for normal ciliary motility. Yamamoto R, Song K, Yanagisawa HA, Fox L, Yagi T, Wirschell M, Hirono M, Kamiya R, Nicastro D, Sale WS. J Cell Biol. 2013 Apr 15;201(2):263-78. doi: 10.1083/jcb.201211048.
Conserved structural motifs in the central pair complex of eukaryotic flagella. Carbajal-González BI, Heuser T, Fu X, Lin J, Smith BW, Mitchell DR, Nicastro D. Cytoskeleton (Hoboken). 2013 Feb;70(2):101-20. doi: 10.1002/cm.21094.
One of the nine doublet microtubules of eukaryotic flagella exhibits unique and partially conserved structures. Lin J, Heuser T, Song K, Fu X, Nicastro D. PLoS One. 2012;7(10):e46494. doi: 10.1371/journal.pone.0046494.
The CSC connects three major axonemal complexes involved in dynein regulation. Heuser T, Dymek EE, Lin J, Smith EF, Nicastro D. Mol Biol Cell. 2012 Aug;23(16):3143-55. doi: 10.1091/mbc.E12-05-0357.
Reconfigurable self-assembly through chiral control of interfacial
tension. Gibaud T, Barry E, Zakhary MJ, Henglin M, Ward A, Yang Y, Berciu C,
Oldenbourg R, Hagan MF, Nicastro D, Meyer RB, Dogic Z. Nature. 2012 Jan 4;481(7381):348-51. doi: 10.1038/nature10769.
The structural heterogeneity of radial spokes in cilia and flagella is
conserved. Lin J, Heuser T, Carbajal-González BI, Song K, Nicastro D. Cytoskeleton (Hoboken). 2012 Feb;69(2):88-100. doi:
Three-dimensional structure of the radial spokes reveals heterogeneity
and interactions with dyneins in Chlamydomonas flagella.
Barber CF, Heuser T, Carbajal-González BI, Botchkarev VV Jr, Nicastro D. Mol
Biol Cell. 2012 Jan;23(1):111-20.
Cryo-electron tomography reveals conserved features of doublet
microtubules in flagella. Nicastro D, Fu X, Heuser T, Tso A, Porter ME,
Linck RW. Proc Natl Acad Sci U S A. 2011 Oct 18;108(42):E845-53.
Cilia-like beating of active microtubule bundles. Sanchez T, Welch D, Nicastro D, Dogic Z. Science. 2011 Jul 22;333(6041):456-9.
Building blocks of the nexin-dynein regulatory complex in Chlamydomonas flagella. Lin J, Tritschler D, Song K, Barber CF, Cobb JS, Porter ME, Nicastro D. J Biol Chem. 2011 Aug 19;286(33):29175-91. doi: 10.1074/jbc.M111.241760.
Sas-4 provides a scaffold for cytoplasmic complexes and tethers them in a centrosome. Gopalakrishnan J, Mennella V, Blachon S, Zhai B, Smith AH, Megraw TL, Nicastro D, Gygi SP, Agard DA, Avidor-Reiss T. Nat Commun. 2011 Jun 21;2:359. doi: 10.1038/ncomms1367.
The CSC is required for complete radial spoke assembly and wild-type ciliary motility. Dymek EE, Heuser T, Nicastro D, Smith EF. Mol Biol Cell. 2011 Jul;22(14):2520-31.
Arrangement of photosystem II and ATP synthase in chloroplast membranes of spinach and pea. Daum B, Nicastro D, Austin J 2nd, McIntosh JR, Kühlbrandt W. Plant Cell. 2010 Apr;22(4):1299-312.
The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella. Heuser T, Raytchev M, Krell J, Porter ME, Nicastro D. J Cell Biol. 2009 Dec 14;187(6):921-33.
Cryo-electron microscope tomography to study axonemal organization. Nicastro D. Methods Cell Biol. 2009;91:1-39. Epub 2009 Dec 1.
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.
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.
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.
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.
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.
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.
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.
of cells in 3D: an introduction to electron tomography. McIntosh R., Nicastro D, Mastronarde D (2005) Trends Cell Biol. 15:43-51.
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.
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.
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.
relationships in an ancestral dipteran: a re-examination
of sensillar pathways across the antenna and leg anlagen
(DeGeer, 1776) (Diptera, Chaoboridae). Melzer RR, Sprenger J, Nicastro D, Smola U (1999) Dev Gen Evol. 209: 103-112.
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:
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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