Bhan, Prerana et al. (2015) International Worm Meeting ""PKG-1 affects OSM-3 clustering at the distal tip of cilia and negatively affects IFT-A particle transport in the distal segment"."

Shanmugam, Muniesh.M, Huang, Hsin-Yi, Bhan, Prerana, Wagner, Oliver.I, Punjabi, Helly, Hsieh, Ron

[International Worm Meeting, 2015]

Statement of Purpose: Cilia are specialized subcellular organelles which exist in various cell types in most eukaryotic organisms. Non-motile cilia, or primary cilia, are mainly responsible for sensory function and are recognized to inherit essential roles in physiology and development. Ciliary diseases such as polycystic kidney disease (PKD) are one of the most common and life threatening genetic diseases world-wide. However, little is known about the underlying molecular mechanism and pathways that control cilia development and intraflagellar transport (IFT). Based on previous studies revealing critical roles of ciliary kinases such as DYF-5 and DYF-18 in IFT, we expected more kinases and phosphatases to regulate ciliogenesis and IFT. We therefore used data mining tools and data from the literature to identify putative candidates.Methods: For our candidate screen, we adopted a broad range of methods, including dye-filling, chemotaxis assays, IFT component expression pattern, and motility analysis of IFT particles in cilia, to investigate the effect of mutations in genes encoding for ciliary kinases and phosphatases. Here, we report the effect of six kinases and phosphatases on dye-filling and chemotaxis assay, and identified three potential kinases (PKG-1, GCK-2 and DYF-18) that significantly affect ciliogenesis and IFT particle transport.Results: PKG-1 and DYF-18 both affect the distribution of molecular motors kinesin-II and OSM-3. The strongest effect was observed in PKG-1 mutants with obvious clustering of OSM-3 motors at the distal tip of cilia. Also abnormal cilia morphology was observed in PKG-1 mutants while in GCK-2 mutant cilia morphology was similar to wild type animals. We also studied the aberrant clustering of OSM-3 at different time points indicating higher abnormality at the 72 hr stage. On the other hand, GCK-2 affected transport characteristics of IFT particle A (CHE-11) in both the middle and distal cilia segment (similar as observed in DYF-18 mutants), however, PKG-1 affects the transport only in the distal segment. Interestingly, PKG-1 and GCK-2 both affected the retrograde IFT speed (XBX-1 and CHE-11) in the middle and the distal cilia segment. PKG-1 rescue experiments lead to partial recovery of the observed phenotypes concluding that PKG-1 plays a pivotal role in ciliogenesis and intraflagellar transport.

Bhan, Prerana et al. (2017) International Worm Meeting "Cilia length and Intraflagellar transport regulation by kinases PKG-1 and GCK-2 in C. elegans sensory neurons."

Lee, Victor, Shanmugam, Muniesh, Huang, Hsin-Yi, Punjabi, Helly, Bhan, Prerana, Hsieh, Ron

[International Worm Meeting, 2017]

Statement of Purpose: A plethora of human diseases are based on cilia dysfunction including polycystic kidney disease, Bardet-Biedl and Meckel-Gruber syndrome. Fortunately, basic mechanisms underlying cilia development and intraflagellar transport (IFT) became more understandable in recent years. Though at the same time a considerably complex ciliary machinery was unravelled leading to new questions, specifically, how IFT cargo assembles at the cilia base, how it localises to cilia, and how "IFT trains" are regulated. One intriguing recent finding describes the ciliary regulating function of two C.elegans kinases DYF-5 and DYF-18, and we hypothesised that even more kinases and phosphatases may be uncovered. We, therefore employed data mining tools to identify kinases and phosphatases specifically expressing in C.elegans ciliated sensory neurons. Methods: We then used a broad range of methods like dye-filling, Chemotaxis, IFT component expression pattern etc to investigate the effects of selected kinases and phosphatases on ciliogenesis and IFT, and have identified PKG-1 as well as GCK-2 as potential candidates that significantly affect cilia development and cargo transport. Results: In pkg-1 mutants, severe accumulation of homodimeric kinesin-2 OSM-3 at the cilia tip was observed in conjunction with an overall reduction in retrograde speeds of ciliary dynein XBX-1, leading to abnormal cilia morphology, likely as a function of reduced tubulin acetylation. While in gck-2 mutants OSM-3 and IFT-particle A (CHE-11) motility was significantly elevated in conjunction with increased tubulin acetylation, further KAP-1 motility was decreased, confirming a recent model in which the slow KAP-1 motor restricts the motility of the fast OSM-3 motor. Crucially, all observed effects in mutant animals can be rescued by overexpressing the respective protein PKG-1 or GCK-2 (under the cilia specific Posm-5 promoter). Both, PKG-1 and GCK-2 follow similar expression pattern in cilia, localising distally of the middle segment as well as near the tip of the distal segment. Because PKG-1 is related to the cGMP pathways, we knocked down the upstream effectors DAF-11 and ODR-1, respectively, leading to similar observations compared with the pkg-1 mutants. Vice versa, since GCK-2 is related to the mTOR pathway, we used rapamycin to inhibit this pathway leading to similar effects as seen in gck-2 mutants. In summary, we identified and characterised the distinct functions of two novel kinases affecting ciliogenesis and IFT in C.elegans.