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Cilia are hair like organelles, protruding from virtually any polarized cell within the human body1 where they serve very diverse functions during development, tissue morphogenesis, and homeostasis like signal sensing2, flow generation and protein transport3,4. Due to these divergent functions, diseases caused by malfunction of cilia, the ciliopathies can manifest with various symptoms affecting many organs5. One of the most specialized cilia within the human body is the photoreceptor outer segment. Here, the cilium is not only a sensory organelle for light perception but also serves as a transport highway representing the only connection between the inner and outer segment of the photoreceptors (interconnecting cilium). Because there is no biosynthesis within the outer segment, all material needed, must be transported via the interconnecting cilium. As a consequence of this essential function within photoreceptors, malfunction leads to severe impairment of vision, which is one of the hallmarks of most ciliopathies6.
A prerequisite for normal ciliary function is the interplay between protein machineries within and associated to the cilium. Understanding the assembly and the interplay of these machineries, consisting of multiple protein complexes within the ciliary network is therefore essential to understand ciliary function in health and disease (Figure 1). To contribute to this, I developed and applied different approaches to not only identify the network components and their interconnectivity but also to detect alterations induced by disease causing-mutations7-10.
Lebercilin, for example, is one of the ciliary proteins and is mutated in Leber congenital amaurosis (LCA), a severe form of inherited blindness, affecting children very early in life11. By quantitatively comparing the protein complexes formed by wild type and LCA-associated mutants using affinity purification and quantitative mass spectrometry, I could show that both mutations analysed lead to a complete loss on binding to the intraflagellar transport machinery (IFT) (Figure 2). The IFT is one of the essential ciliary protein transport mechanisms and its malfunction leads to heavy impairment or even loss of photoreceptor outer segment formation. This explains why the loss of interaction between Lebercilin and the IFT leads to impairment of ciliary transport within photoreceptors and ultimately the loss of outer segments and therefore blindness12.
The approaches, used for the dissection of the Lebercilin complexes are currently applied in higher throughput to the whole ciliary protein network (www.syscilia.org) to try to understand principle ciliary functions and especially malfunction on the molecular level of protein interactions that lead to the manifestation of disease. To achieve this, protein complex analysis is combined in a systems biology based project with different other omics technologies and bioinformatics analysis to handle and integrate the large amounts of data generated.
Staff Scientist and Group leader
E-mail:
karsten.boldt[at]uni-tuebingen.de
Phone: +49 (0)7071 29-84950
Fax: +49 (0)7071 29-5777
Website:
www.eye-tuebingen.de/ueffinglab/
Eberhard-Karls University of Tuebingen
Institute for Ophthalmic Research
Nägelestrasse 5
D-72074 Tübingen,
Germany