Abstract
Ciliopathies are life‐threatening human diseases caused by defective cilia. They can often be traced back to mutations of genes encoding transition zone (TZ) proteins demonstrating that the understanding of TZ organisation is of paramount importance. The TZ consists of multimeric protein modules that are subject to a stringent assembly hierarchy. Previous reports place Rpgrip1l at the top of the TZ assembly hierarchy in Caenorhabditis elegans. By performing quantitative immunofluorescence studies in RPGRIP1L−/− mouse embryos and human embryonic cells, we recognise a different situation in vertebrates in which Rpgrip1l deficiency affects TZ assembly in a cell type‐specific manner. In cell types in which the loss of Rpgrip1l alone does not affect all modules, additional truncation or removal of vertebrate‐specific Rpgrip1 results in an impairment of all modules. Consequently, Rpgrip1l and Rpgrip1 synergistically ensure the TZ composition in several vertebrate cell types, revealing a higher complexity of TZ assembly in vertebrates than in invertebrates.
Highlights
Cilia are tiny protrusions of nearly every human cell which function as the cell’s antenna mediating numerous signalling pathways essential for development and homeostasis
To test whether exogenous expression of Rpgrip1l could rescue the amount of Nphp1 at the transition zone (TZ) of Rpgrip1lÀ/À mouse embryonic fibroblasts (MEFs), we transfected these MEFs with a plasmid which encodes human full-length Rpgrip1l. 48 h after transfection, human Rpgrip1l was located at the ciliary TZ of Rpgrip1lÀ/À MEFs and the amount of Nphp1 was restored (Appendix Fig S1A–C)
To test whether the significant reduction of the analysed Nphp proteins at the TZ is based on a decreased total level of these proteins or a reduced expression of the encoding genes in Rpgrip1lÀ/À MEFs, we examined the overall cellular amount of Invs and Cep290 via Western blot studies and the expression of Nphp4 via qRT–PCR
Summary
Cilia are tiny protrusions of nearly every human cell which function as the cell’s antenna mediating numerous signalling pathways essential for development and homeostasis. Ciliopathies are often caused by mutations in genes encoding transition zone (TZ) proteins (Hildebrandt et al, 2011; Czarnecki & Shah, 2012). The TZ represents a compartment at the base of primary cilia at the proximal end of the axoneme controlling ciliary protein entry and exit (Betleja & Cole, 2010; Craige et al, 2010; Omran, 2010; Benzing & Schermer, 2011; Czarnecki & Shah, 2012). The TZ consists of different multiprotein complexes, the Nphp module, the Nphp module, the Mks/ B9 module and the Inversin (Invs alias Nphp2) compartment (Sang et al, 2011; Czarnecki & Shah, 2012). The Nphp module, the Nphp module and the Inversin compartment are summarised as the Nphp modules in this study
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