The centrosome-cilium complex is a critical organelle of animal cell function. The centrosome is the main microtubule organizing center of animal cells, and the centrioles within the centrosome have a unique structure that allows them to serve as initiators of the assembly of a cilium. Primary cilia are small, antenna-like organelles critical for vertebrate development and physiology. Defects in the centrosome-cilium complex result in human diseases called ciliopathies, characterized by a wide spectrum of phenotypes, highlighting their important role in multiple cell types and organs. This complex forms a distinct compartment of the cell, with a highly polarized microtubule cytoskeleton directing intracellular traffic to and from it, and a specialized segment of the plasma membrane surrounding part of it. In most cells, there is only one centrosome-cilium complex, but this is often altered in cells from a range of diseases, and in cells that have specific developmental programs to amplify centrioles. The function of cilia critically depends on the dynamic changes in protein composition and localization. In particular, Hedgehog (Hh) signaling transduction, essential for embryonic development, adult tissue regeneration and cancer, largely takes place at primary cilia, and involves movements of the signaling proteins into and out of the cilium in response to signal. We have defined six fundamental questions that drive the proposed research. 1) How do the specialized compound microtubules of centriole form, and how do they specifically form only at the centriole? 2) How are centrioles functionalized to carry out the essential roles of cilium formation, centrosome formation, duplication? 3) What are the consequences of failure to maintain centriole number, and what homeostatic mechanisms exist that could facilitate a return to the normal state? 4) What mechanisms promote centriole amplification and loss in specific differentiated contexts? 5) What is the behavior of signaling proteins in the cilium, at the single molecule level, and how does it change in response to activation of the pathway? 6) How are cilia disassembled and is the same mechanism used in different contexts and across evolution? We will use a combination of advanced imaging, cell culture and in vitro differentiation models, combined with sophisticated molecular biology to address these questions. Successful outcomes in these experiments will inform our understanding of centrosome-cilium defects in disease states, including ciliopathies and cancer, and potentially lead to new therapeutic approaches. !
The centrosome-cilium complex is a large organelle, present in most cell types, that functions in reception and transduction of extracellular signals and organization of the internal fiber network of cells; defects in the centrosome and cilium cause a spectrum of human diseases called ciliopathies. The dynamic localization of specific proteins required for signal transduction processes is critical for cilium function as a signaling center. We propose to use state-of-the-art imaging and biochemical methods to understand the structure of the centrosome, how the number of centrosomes is controlled in dividing cells and in specialized cells of the body, and how regulated trafficking and compartmentalization of signaling proteins within cilia is important for the Hedgehog signal transduction pathway.