) Centrosomes are organelles of diverse structure that share a common ability to organize microtubule arrays for various cellular functions. They contribute to mitotic spindle organization and orientation in dividing cells. In nondividing cells of many organisms, centrosomes contribute to cell polarity and epithelial morphogenesis, and serve as templates for assembly of motile cilia and sensory (primary) cilia. Both primary and motile cilia are assembled, maintained, and regulated by specific proteins, which have been enumerated in a number of proteomic analyses. One class of ciliary proteins is involved in the process of intraflagellar transport (IFT), where proteins required for cilia assembly are transported by protein carriers (IFT complexes) to the tip of the cilium/flagellum by plus-end-directed motors (kinesin-2), whereas disassociated material is returned to the cilia base by minus-end-directed motors (dynein 1b). Defects in cilia proteins are associated with a number of human diseases termed ciliopathies, and by definition all exhibit defects in cilia structure and/or function. However, ciliopathies are complex disorders characterized by a diversity of cellular abnormalities that make it difficult to precisely define the disease etiology. This complexity is manifest in recent studies suggesting that defects other than cilia may contribute to these disorders. For example, the Doxsey laboratory and others have recently shown that cilia proteins localize to centrosomes and mitotic spindle poles, though mitotic roles of these proteins is poorly understood. During the previous grant period, the Doxsey laboratory began to investigate IFT protein function in mitotic cells. IFT proteins localize to spindle poles as well as kinetochores, midbodies and spindle microtubules. RNAi-mediated depletion of the cilia protein IFT88 in zebrafish embryos and cultured cells disrupts centrosome/spindle pole integrity, astral microtubule organization and spindle organization;depletion of other IFT proteins (IFT20, 52, 57) induces related and additional mitotic defects. A novel mitosis-specific IFT88 complex was identified, which contains proteins linking microtubules to the cell cortex. This event is required for orientation of mitotic spindles and the plane of cell division, and is implicated in cystogenesis and ciliopathy. Based on this work we propose the following specific aims:
Aim 1. Determine the role of IFT88 complexes in mitosis, particularly spindle organization, and test whether IFT88 zebrafish morphants and mouse mutants exhibit mitotic defects prior to cilia expression in early embryos.
Aim 2. Determine functional interactions of IFT88-BPs that contribute to spindle orientation and organization.
Aim 3. Test whether the global ciliogenesis machinery is redirected in dividing cells to perform multiple mitotic functions through the identification of additional mitotic phenotypes and mitosis-specific protein complexes in several different classes of ciliary proteins. This work has the potential to identify new functions for ciliary proteins (mitotic), characterize their molecular underpinnings and provide insights into the etiology of ciliopathies through the study of mitotic abnormalities.
This work has the potential to identify and characterize a contributor to cilia diseases (e.g. Polycystic Kidney Disease, Retinal Degeneration, etc.) that has been largely overlooked, namely functions associated with process of mitosis during cell division. Defects in a diversity of mitotic functions, pathways and structures may explain features of ciliopathies. In the process, novel roles for cilia-associated proteins in mitosis will be identified and hybrids of proteins that have roles in cilia and in mitosis could some day serve as novel drug targets for treating cilia disorders.
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