F-box protein MEC-15 promotes microtubule stability and neurite growth by antagonizing the activity of HSP90 chaperone network

Abstract

Microtubule (MT) stability plays an important role in regulating neurite growth. We previously found that neomorphic (neo) gain-of-function mutations of mec-7/-tubulin led to increased MT stability and the growth of an ectopic, posteriorly directed neurite in the anterior touch receptor neuron (TRN) ALMs. This clear phenotype provided a sensitized background for identifying novel MT regulators. Through a suppressor screen, we identified a loss-of-function (lf) mutation in mec-15, which suppressed the growth of ALM posterior neurite (ALM-PN) in mec-7(neo) mutants. mec-15 encodes a F-box protein with four WD40 repeats. Rescue experiments showed that mec-15 functions cell autonomously in ALMs and its function requires the F-box. mec-15(lf) single mutants showed the shortening of both PLM anterior and posterior neurites and PLM branching defects, suggesting that MEC-15 is required not only for the growth of ectopic ALM neurites but also for the normal development of TRN morphology. Because MEC-15 physically interacts with SKR-1/Skp1, and TRN-specific RNAi against uba-1 (ubiquitin-activating enzyme) suppressed the generation of ALM-PN in mec-7(neo) mutants, a SCF complex-mediated ubiquitination pathway promotes MT stability and neurite development. To understand the downstream target of MEC-15, we took mec-7(neo); mec-15(lf) as the starter strain and carried out another suppressor screen searching for mutants with recovered ALM-PN. We identified one sti-1(lf), four pph-5(lf), and two dlk-1(lf) alleles in the screen. STI-1/Hop is a cochaperone that physically links Hsp70 and Hsp90 via the tetratricopeptide repeat (TPR)-domain, and the phosphatase PPH-5 is a TPR-containing Hsp90 cofactor. Through a candidate-based approach, we found that similar to mutations in sti-1 and pph-5, the loss of hsp-90, hsp-110/Hsp70, and daf-41/p23 also recovered the growth ALM-PN in mec-15(lf); mec-7(neo) double mutants and rescued the TRN developmental defects in mec-15(lf) single mutants. Electron microscopic studies found that the number of MTs in a cross-section of TRN neurite is dramatically decreased in mec-15(lf) mutants and this defect is rescued in mec-15; pph-5 or mec-15; sti-1 double mutants. Functionally, the reduced touch sensitivity of mec-15 mutants is restored in those double mutants. Thus, our data suggest that MEC-15 promotes MT stability and neuronal growth by inhibiting the Hsp90 chaperone network. One potential target of Hsp90 chaperone is DLK-1, the Dual-Leucine zipper Kinase, which was recently identified as a HSP90 client in mouse neurons. We found that dlk-1(lf) mutations suppressed the defects caused by the loss of mec-15 and overexpression of dlk-1 in the TRNs rescued the lack of Hsp90 chaperones, causing MT instability and neurite growth defects. Therefore, our results revealed an unexpectedly negative regulation of neurodevelopment by the molecular chaperones.