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Cilia project from the surface of most vertebrate cells, and are important for several physiological and developmental processes. Ciliary defects are linked to a wide variety of human diseases, named ciliopathies, underscoring the importance of understanding signaling pathways involved in cilia formation and maintenance. Here we show that Rer1p, a protein quality control receptor residing in the ER-Golgi compartment, is involved in regulating the structure and function of cilia. Both in zebrafish and mammalian cells, loss of Rer1p results inthe shortening of cilium and impairment of its motile and/or sensory function, which is reflected by hearing, vision and left-right asymmetry defects as well as decreased Hedgehog signaling. Importantly, cilia formation is also compromised in cell lines derived from monosomy 1p36 patients, which carry a terminal chromosomal deletion that includes the RER1 gene. A substantial part of the patients clinical picture including hearing problems, congenital heart defects and facial dysmorphism suggests ciliary dysregulation, and can be mimicked in zebrafish by knockdown of Rer1p. We further demonstrate that Rer1p controls ciliogenesis partly through its function in negatively regulating γ-secretase activity and the signaling of at least one of its substrate, Notch, as nanomolar concentrations of a γ-secretase inhibitor partially but significantly rescued cilium length in Rer1p-depleted cells. We therefore identify Rer1p as the first ER-Golgi localized membrane protein involved in ciliogenesis where it exerts this broader physiological function in cells and in vivo models through regulating the assembly and activity of γ-secretase. Acting through the control of ciliogenesis, Rer1p thus may play a pivotal role in contributing to the clinical picture of monosomy 1p36, recognizing it as an underlying ciliopathy.
γ- Secretase is a multimeric protein complex responsible for the proteolytic processing of around 90 different integral membrane proteins. The diverse functions of γ-secretase are therefore attributed to the cleavage of its various substrates and the subsequent signaling properties associated with them. Its involvement in a biologically important process such as ciliogenesis urged us to explore its effectors responsible forthis process. Despite the ubiquitous existence and genuine physiological importance, the protein composition of cilia remains poorly explored. Therefore, we first used a proteomics
approach to map the protein content of isolated cilia; specifically to identify novel cilia localized γ-secretase substrates. Here we demonstrate for the first time that Amyloid Precursor Protein (APP) and APP like protein 2 (APLP2) are localized to the primary cilium. APP plays a central role in the pathogenesis of Alzheimer's disease: APP processing generates β-amyloid (Aβ) peptides, which are deposited in amyloid plaques in brains of Alzheimers disease patients. Despite being intensively investigated, the full scope of the physiological roles of APP still remains enigmatic. Using various cell biological approaches, we demonstrate that APP is proteolytically processed in the cilium and that its sheddase, ADAM10 and γ-secretase complex are located in the cilium as wellas required to maintain proper cilia length. Knockout of APP together with APLP2 leads to short cilia which could be rescued by the stable re-expression of full length/ carboxy terminal fragment of APP (APP-CTF) butnot the APP intracellular domain (AICD). We further show that APP-CTF regulates cilia length through activating the cyclic AMP/Protein Kinase Asignaling pathway, thus establishing for the first time the requirementof APP processing as a novel regulator in cilia length maintenance.
Date:22 Jun 2009 →  4 Jun 2013
Keywords:Cargo receptor Rer1p
Disciplines:Genetics, Systems biology, Molecular and cell biology
Project type:PhD project