Defects in cilia have been linked to human pathologies based on aberrant motility (e.g. infertility, respiratory infections) and/or abnormal signaling (e.g. polycystic kidney disease, blindness). Such ciliopathies can result from improper assembly of cilia;therefore, understanding how ciliary precursors are brought to the cilium and into the ciliary compartment may shed new light on their role in disease. How essential structural components are delivered from their site of synthesis to the ciliary axoneme, is a mystery. To address this issue a novel preparation of vesicles has been isolated during flagellar assembly from the cytoplasm of Chlamydomonas, a pivotal organism for the study of assembly and function of cilia. These vesicles carry ciliary membrane proteins (e.g. CrPKD2) as well as components of the ciliary axoneme, including tubulin and radial spoke proteins. Combining high resolution imaging, proteomics and RNAi knockdown, the hypothesis that axonemal proteins are transported to the cilium via peripheral attachment to vesicles targeted to the periciliary membrane is being tested. Once at the base of the cilium, the flagellar transition zone (TZ) is involved in determining which proteins enter/exit the flagellar compartment. Many proteins involved in ciliopathies are found in the TZ. To further characterize the composition of the TZ, TZs of Chlamydomonas have been isolated and analyzed by mass spectrometry. Endosomal Sorting Complexes Required for Transport (ESCRT) proteins, which are involved in membrane dynamics, were identified in this analysis, heralding a previously unknown function of the TZ, which will be studied. These proteins may be involved in removing ubiquitinated proteins from the cilium, the release of ciliary membrane into the surrounding milieu, or in the severing of the organelle from the cell - three poorly understood processes known to occur in cilia. Additional newly identified proteins of the TZ that are conserved in humans will be studied to provide new insights into the role of cilia function and human disease.
A major resurgence in research on cilia and flagella (synonymous terms) occurred following the discovery of Intraflagellar Transport (IFT) and the demonstration that it is essential for the assembly and maintenance of all eukaryotic cilia. Abnormalities in ciliary assembly has, in turn, been shown to underlie many human diseases called ciliopathies, including polycystic kidney disease and blindness. The proposed research will provide new insights into cilia assembly, focusing on how ciliary proteins are transported to the cilia for assembly, as well as identifying new proteins involved in gating proteins into and out of cilia.
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|Wood, Christopher R; Huang, Kaiyao; Diener, Dennis R et al. (2013) The cilium secretes bioactive ectosomes. Curr Biol 23:906-11|
|Gupta, Anjali; Diener, Dennis R; Sivadas, Priyanka et al. (2012) The versatile molecular complex component LC8 promotes several distinct steps of flagellar assembly. J Cell Biol 198:115-26|
|Yuan, Shiaulou; Li, Jade; Diener, Dennis R et al. (2012) Target-of-rapamycin complex 1 (Torc1) signaling modulates cilia size and function through protein synthesis regulation. Proc Natl Acad Sci U S A 109:2021-6|
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