Many cells in the vertebrate body use specialized sensory organelles to sense particular cues in their environment: primary cilia. Proteins that function in cilia are transported into and within cilia by a specialized transport machinery, called intraflagellar transport, IFT. The amphid channel cilia of C. elegans are a good model to study the regulation of cilium length and intraflagellar transport (IFT), since they consist of two segments where different kinesin complexes are used for IFT. Previously, we have shown that coordination of the kinesins is regulated by exposure to dauer pheromone and the heterotrimeric G protein α-subunit GPA-3. Furthermore, animals that carry a dominant active mutant gpa-3 (gpa-3QL) have shorter cilia.
To find out how G protein signalling modulates cilium length, we screened for suppressors of the short cilium phenotype of gpa-3QL animals. This screen identified three components of the DLK/p38 MAP kinase pathway, the MAP3K dlk-1, the p38 MAPK pmk-3 and the ubiquitin-conjugating enzyme variant uev-3. Previous studies have shown that this pathway is important for axon development and regrowth and synapse formation. We show that DLK-1, PMK-3 and UEV-3 localize in cilia or at their base. Surprisingly, mutations in all three genes affect IFT, but these changes do not explain the suppression of the gpa-3QL induced cilium length defect.
p38 MAP kinase has been shown to regulate endocytosis. Interestingly, endocytosis at the base of the cilium is important for proper cilium function. We found that blocking endocytosis by mutating rabx-5 or rme-6, GEFs for RAB-5, or chc-1, the clathrin heavy chain, suppressed the gpa-3QL induced short cilium phenotype. In addition, we found GFP::RAB-5 accumulation at the base of the cilium in pmk-3 and pmk-3; gpa-3QL animals. In contrast, gpa-3QL animals showed relatively low levels of GFP::RAB-5.
Our results identify a new role for the DLK/p38 MAP kinase pathway in the control of cilium length. We propose that environmental signals mediated by GPA-3 and the DLK/MAPK pathway can modulate cilium function by regulating RAB-5 mediated endocytosis.
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