Centrioles are among the most enigmatic of all cytoskeletal structures. Centriole biogenesis involves a mysterious duplication process whose mechanism is unknown. Centriole function in cell division remains highly controversial, although centrioles are implicated in both mitosis and cytokinesis, and it is thought that centrioles may act as hubs for signaling networks regulating cell cycle progression, checkpoints, and cell proliferation. Abnormalities in centriole copy-number are a standard feature of virtually all solid tumor cells, and appear to be correlated with increased genomic instability. This proposal describes an integrated approach to understanding centriole biology. By combining proteomic analysis, genetic analysis, mathematical modeling, and live-cell imagine, we hope to obtain new insights into centriole duplication and function. The experiments take advantage of the green alga Chlamydomonas reinhardtii, a unicellular organism with genetics similar to yeast, but which has centrioles that are virtually identical to those of animal cells. In addition to its genetic advantages, Chlamydomonas is an excellent system for biochemical isolation of centrioles, and we have exploited these advantages to analyze centriole composition using mass spectrometry, yielding a preliminary list of approximately 200 candidate centriole proteins. By combining reverse-genetic analysis of the centriole proteome, and forward genetic screens, we propose to dissect the molecular pathway of centriole duplication. We have developed a dynamical-systems model for centriole inheritance, and we have used this model to design screens to specifically identify defects in templated and de novo centriole assembly. Preliminary studies on centriole function, using mutants that alter centriole copy number, show increased chromosome loss, cell death, and failures of cleavage. These mutants, as well as mutants altering centriole ultrastructure, will be used to test the role of centrioles in chromosome segregation, mitotic spindle organization, nuclear positioning, and cytokinesis.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Nuclear Dynamics and Transport (NDT)
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Gindhart, Joseph G
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University of California San Francisco
Schools of Medicine
San Francisco
United States
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Ishikawa, Hiroaki; Marshall, Wallace F (2015) Efficient live fluorescence imaging of intraflagellar transport in mammalian primary cilia. Methods Cell Biol 127:189-201
Ishikawa, Hiroaki; Ide, Takahiro; Yagi, Toshiki et al. (2014) TTC26/DYF13 is an intraflagellar transport protein required for transport of motility-related proteins into flagella. Elife 3:e01566
Ishikawa, Hiroaki; Marshall, Wallace F (2013) Isolation of mammalian primary cilia. Methods Enzymol 525:311-25
Apte, Zachary S; Marshall, Wallace F (2013) Statistical method for comparing the level of intracellular organization between cells. Proc Natl Acad Sci U S A 110:E1006-15
Tang, Nan; Marshall, Wallace F (2012) Centrosome positioning in vertebrate development. J Cell Sci 125:4951-61
Ishikawa, Hiroaki; Thompson, James; Yates 3rd, John R et al. (2012) Proteomic analysis of mammalian primary cilia. Curr Biol 22:414-9
Marshall, Wallace F (2012) Centriole asymmetry determines algal cell geometry. Curr Opin Plant Biol 15:632-7
Azimzadeh, Juliette; Wong, Mei Lie; Downhour, Diane Miller et al. (2012) Centrosome loss in the evolution of planarians. Science 335:461-3
Tang, Nan; Marshall, Wallace F; McMahon, Martin et al. (2011) Control of mitotic spindle angle by the RAS-regulated ERK1/2 pathway determines lung tube shape. Science 333:342-345
Rafelski, Susanne M; Keller, Lani C; Alberts, Jonathan B et al. (2011) Apparent diffusive motion of centrin foci in living cells: implications for diffusion-based motion in centriole duplication. Phys Biol 8:026010

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