Cilia and flagella are evolutionarily conserved organelles with important biological roles in motility, sensation and signaling. They are essential in the life cycles of most eukaryotic organisms, and cilia are found in nearly every human cell. The goal of this research is to determine how regulatory signals change the activity of the molecular motor protein dynein, and how dynein activity is regulated in motile cilia and flagella to generate proper beating. Our laboratory opened a new window into the three-dimensional organization and protein composition of cilia and flagella, and recently discovered previously unseen structures that regulate dynein function and/or are essential for flagellar assembly. Building on a strong base of preliminary data gathered in the preceding project period, this proposal directly addresses key open questions in the field in three specific aims that are directed at understanding the three-dimensional structure, subunit composition, protein interactions and the regulatory functions of the following three major regulatory and signal transduction complexes in cilia and flagella: (1) the nexin-dynein regulatory complex, (2) the I1 inner dynein complex, and (3) radial spoke 3 in organisms with radial spoke triplets. The project uses a multi-disciplinary approach and cutting-edge techniques, combining in situ molecular imaging by cryo-electron tomography and image processing, biochemical and mass spectrometric analyses, integrated structural-genetics approaches and protein labeling techniques to directly visualize gene products in cells. The proposed research will contribute fundamental knowledge and a deeper understanding of the mechanisms underlying motor protein function and control on a molecular level and of the functional organization of cilia and flagella.

Public Health Relevance

The proper function of several vital organs in humans requires the activity of cilia, and defects in ciliary motility and assembly are responsible for a wide variety of life-threatening, congenital disorders, such as polycystic kidney disease, Bardet-Biedl syndrome, primary ciliary dyskinesia, situs inversus, respiratory diseases and heart defects. Our work addresses how the motor protein dynein works and is regulated to give rise to normal movement of cilia. We anticipate that this research will provide new insights into the underlying mechanisms of ciliary-linked disorders in humans, and be applicable also to dynein-driven transport along the microtubule cytoskeleton, which, if defective, can give rise to cancer, brain developmental and neurodegenerative diseases. Such fundamental understanding is a prerequisite to the development of therapeutic protocols capable of attenuating these disease processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM083122-07
Application #
8529556
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Gindhart, Joseph G
Project Start
2007-09-28
Project End
2016-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
7
Fiscal Year
2013
Total Cost
$310,548
Indirect Cost
$117,548
Name
Brandeis University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454
Lin, Jianfeng; Yin, Weining; Smith, Maria C et al. (2014) Cryo-electron tomography reveals ciliary defects underlying human RSPH1 primary ciliary dyskinesia. Nat Commun 5:5727
Linck, Richard; Fu, Xiaofeng; Lin, Jianfeng et al. (2014) Insights into the structure and function of ciliary and flagellar doublet microtubules: tektins, Ca2+-binding proteins, and stable protofilaments. J Biol Chem 289:17427-44
Lin, Jianfeng; Okada, Kyoko; Raytchev, Milen et al. (2014) Structural mechanism of the dynein power stroke. Nat Cell Biol 16:479-85
Yamamoto, Ryosuke; Song, Kangkang; Yanagisawa, Haru-Aki et al. (2013) The MIA complex is a conserved and novel dynein regulator essential for normal ciliary motility. J Cell Biol 201:263-78
Carbajal-Gonzalez, Blanca I; Heuser, Thomas; Fu, Xiaofeng et al. (2013) Conserved structural motifs in the central pair complex of eukaryotic flagella. Cytoskeleton (Hoboken) 70:101-20
Barber, Cynthia F; Heuser, Thomas; Carbajal-González, Blanca I et al. (2012) Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella. Mol Biol Cell 23:111-20
Lin, Jianfeng; Heuser, Thomas; Carbajal-González, Blanca I et al. (2012) The structural heterogeneity of radial spokes in cilia and flagella is conserved. Cytoskeleton (Hoboken) 69:88-100
Sanchez, Timothy; Welch, David; Nicastro, Daniela et al. (2011) Cilia-like beating of active microtubule bundles. Science 333:456-9
Lin, Jianfeng; Tritschler, Douglas; Song, Kangkang et al. (2011) Building blocks of the nexin-dynein regulatory complex in Chlamydomonas flagella. J Biol Chem 286:29175-91
Nicastro, Daniela; Fu, Xiaofeng; Heuser, Thomas et al. (2011) Cryo-electron tomography reveals conserved features of doublet microtubules in flagella. Proc Natl Acad Sci U S A 108:E845-53

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