Almost all cell types in the human body demonstrate cellular polarity and extend a microtubule (MT)-based hair-like organelle from the cell surface called the cilium. Polarized cells use the primary cilium as a versatile tool for tissue-specific functions during development, morphogenesis and homeostasis, explaining why cilia/polarity-related disorders (ciliopathies) can affect many organ systems. A number of proteins involved in cystic diseases localize to both the primary cilium and/or its associated basal body (a.k.a. the centrosome) including the intraflagellar transport proteins (IFTs), the Bardet-Biedl syndrome complex that interacts with the small GTPases Rab11 and Rab8, and the vesicle tethering complex, the exocyst. While the consequences of specific ciliopathy mutations are described on an organismal level, the cellular processes are not understood. Since many known ciliopathy mutations lie in proteins that localize to the centrosome, I will examine the concurrent role of th centrosome in polarity establishment and ciliogenesis. The mother centriole appendage protein, centriolin, interacts with the Rab11 effector the exocyst. The exocyst can localize to the Rab11 decorated recycling endosome (RE) and contributes to the formation of the transient apical membrane initiation site (AMIS) in polarized kidney epithelial cells. Furthermore, Rab11, centriolin, and the exocyst are all reported to be involved in primary cilia formation, which is known to occur simultaneously with lumen formation and polarity establishment. Strikingly, I recently found Rab11, the exocyst, and the Rab11 GTPase Activating Protein (GAP) Evi5 to localize to the mother centriole appendages along with REs (Hehnly, Chen, Powers, Liu, &Doxsey, 2012). Based on this, I hypothesize that the mother centriole appendage proteins are required for polarity formation and ciliogenesis during lumenogenesis by regulating Rab11 activity. We will examine 1) whether ciliogenesis and lumenogenesis occur simultaneously and whether lumenogenesis is dependent on ciliogenesis using structured illumination microscopy and live cell imaging. In addition, we will examine whether the AMIS is actually the ciliary vesicl by using EM tomography. 2) We will use super resolution microscopy techniques (e.g. STORM) to examine whether the mother centriole appendage protein cenexin is required to anchor centriolin, Rab11, and the exocyst at the centrosome to regulate lumen formation and ciliogenesis. 3) We will examine whether the mother centrioles ability to regulate Rab11 activity effects MT polarity and dynamics using a novel in vitro binding assay that I have developed in the lab(Hehnly et al., 2012) and super resolution microscopy techniques. In conclusion, our preliminary results provide unexpected insights into the molecular mechanism of endosome organization and function at the centrosome, and future findings will be of importance to a wide audience with interests in centrosomes, membrane trafficking, cilia organization/function, etc. Most importantly, this proposal may provide insights describing how disruption of the proposed molecular interactions may lead to disease phenotypes in ciliopathy patients.

Public Health Relevance

A number of proteins involved in cystic diseases (ciliopathies) localize to two cellular organelles in addition to cilia: the centrosome and the recycling endosome. Both these organelles are required for a cell to make cilia and/or assemble cellular polarity for organ development, but the mechanism is not understood. Our current and future findings will bridge together these two disparate fields, centrosome function and endosome recycling, providing unexpected and novel insights into the molecular mechanism of endosome organization and function through its association with the centrosome.

Agency
National Institute of Health (NIH)
Type
Career Transition Award (K99)
Project #
7K99GM107355-02
Application #
8727084
Study Section
Special Emphasis Panel (ZGM1)
Program Officer
Flicker, Paula F
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Washington
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
Seattle
State
WA
Country
United States
Zip Code
98195
Hehnly, Heidi; Doxsey, Stephen (2014) Rab11 endosomes contribute to mitotic spindle organization and orientation. Dev Cell 28:497-507
Chen, Chun-Ting; Hehnly, Heidi; Yu, Qing et al. (2014) A unique set of centrosome proteins requires pericentrin for spindle-pole localization and spindle orientation. Curr Biol 24:2327-34