Cilium Length and Intraflagellar Transport Regulation by Kinases PKG-1 and GCK-2 in Caenorhabditis elegans Sensory Neurons

ABSTRACT

To understand how ciliopathies such as polycystic kidney disease or Bardet-Biedl syndrome develop, we need to understand the basic molecular mechanisms underlying cilium development. Cilium growth depends on the presence of functional intraflagellar transport (IFT) machinery, and we hypothesized that various kinases and phosphatases might be involved in this regulatory process. A candidate screen revealed two kinases, PKG-1 (a cGMP-dependent protein kinase) and GCK-2 (a mitogen-activated protein kinase kinase kinase kinase 3 [MAP4K3] kinase involved in mTOR signaling), significantly affecting dye filling, chemotaxis, cilium morphology, and IFT component distribution. PKG-1 and GCK-2 show similar expression patterns in Caenorhabditis elegans cilia and colocalize with investigated IFT machinery components. In pkg-1 mutants, a high level of accumulation of kinesin-2 OSM-3 in distal segments was observed in conjunction with an overall reduction of anterograde and retrograde IFT particle A transport, likely as a function of reduced tubulin acetylation. In contrast, in gck-2 mutants, both kinesin-2 motility and IFT particle A motility were significantly elevated in the middle segments, in conjunction with increased tubulin acetylation, possibly the cause of longer cilium growth. Observed effects in mutants can be also seen in manipulating upstream and downstream effectors of the respective cGMP and mTOR pathways. Importantly, transmission electron microscopy (TEM) analysis revealed no structural changes in cilia of pkg-1 and gck-2 mutants.

KEYWORDS:

• CHE-11
• Caenorhabditis elegans
• ciliogenesis
• intraflagellar transport
• KAP-1
• OSM-3
• primary cilia
• XBX-1
• heterotrimeric kinesin-2
• homodimeric kinesin-2

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/MCB.00612-17.

ACKNOWLEDGMENTS

We thank our undergraduate, graduate, and summer interim students Stefan Werner, Hsin-Chu Tsai, Franziska Krautter, Sheng-Han Chen, Hendrik Schürer, and Wang Meng-Chieh for assistance on this project. We thank the C. elegans Core Facility (CECF), funded by National Science Council Taiwan (NSC; now Ministry of Science and Technology), for providing us with worm observation systems and RNAi feeding clones.

This work was funded by NSC grants NSC-102-2311-B-007-006- and NSC-101-2321-B-002-043- as well as by Ministry of Education Taiwan (MOE) grant MOE 99N2455E1 99-100 to O.I.W.

We have no conflict of interest to declare.