Abstract
The inositol polyphosphate 5′-phosphatase E (INPP5E) localizes to cilia. We showed that the carrier protein phosphodiesterase 6 delta subunit (PDE6δ) mediates the sorting of farnesylated INPP5E into cilia due to high affinity binding and release by the ADP-ribosylation factor (Arf)-like protein Arl3·GTP. However, the dynamics of INPP5E transport into and inside the ciliary compartment are not fully understood. Here, we investigate the movement of INPP5E using live cell fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) analysis. We show that PDE6δ and the dynein transport system are essential for ciliary sorting and entry of INPP5E. However, its innerciliary transport is regulated solely by the intraflagellar transport (IFT) system, independent from PDE6δ activity and INPP5E farnesylation. By contrast, movement of Arl3 into and within cilia occurs freely by diffusion and IFT-independently. The farnesylation defective INPP5E CaaX box mutant loses the exclusive ciliary localization. The accumulation of this mutant at centrioles after photobleaching suggests an affinity trap mechanism for ciliary entry, that in case of the wild type is overcome by the interaction with PDE6δ. Collectively, we postulate a three-step mechanism regulating ciliary localization of INPP5E, consisting of farnesylation- and PDE6δ-mediated targeting, INPP5E-PDE6δ complex diffusion into the cilium with transfer to the IFT system, and retention inside cilia.
Acknowledgments
AW acknowledges funding by the European Research Council (ERC Grant 268782) and Sonderforschungsbereich-DFG (SFB 642). We cordially thank Prof. Dr. A. Musacchio for the support provided throughout the project. We thank C. Koerner and J. A. Seidel for expert technical assistance, Dr. M. Lokaj for providing templates for plasmid generation and support in generation of stable cell lines, and Dr. E. Zent for CellProfiler analysis development. We are also very thankful to Prof. Dr. M. V. Nachury for providing the IMCD3 Flp-In system.
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Supplemental Material:
The online version of this article offers supplementary material (https://doi.org/10.1515/hsz-2017-0226).
©2018 Walter de Gruyter GmbH, Berlin/Boston
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Artikel in diesem Heft
- Frontmatter
- Reviews
- Host target-based approaches against arboviral diseases
- The role of microRNAs in chronic respiratory disease: recent insights
- Research Articles/Short Communications
- Protein Structure and Function
- Selection of an Anticalin® against the membrane form of Hsp70 via bacterial surface display and its theranostic application in tumour models
- Cell Biology and Signaling
- TGF-β requires the activation of canonical and non-canonical signalling pathways to induce skeletal muscle atrophy
- Impact of protamine I on colon cancer proliferation, invasion, migration, diagnosis and prognosis
- Mechanism and dynamics of INPP5E transport into and inside the ciliary compartment
- Novel Techniques
- A novel design of HA-coated nanoparticles co-encapsulating plasmid METase and 5-Fu shows enhanced application in targeting gastric cancer stem cells