The Regulator of Presynaptic Morphology (RPM-1) is known to regulate synaptogenesis and axon termination in different neurons (1,2). To further understand the mechanism of how RPM-1 regulates these processes we immunoprecipitated RPM-1 from worm lysates and identified binding partners by mass spectrometry. Among the candidates, we identified GLO-4, which contains an RCC-1-like GEF domain and is reported to function in gut granule biogenesis (3). We generated GLO-4::GFP and found that GLO-4 is expressed in neurons, and shows partial co-localization with RPM-1.
To assess the neuronal function of
glo-4, we crossed a
glo-4 loss of function (lf) allele,
ok623, with multiple neuronal markers. The pattern of SNB-1::GFP in motor neurons is largely normal in
ok623 animals.
ok623 enhances the SNB-1::GFP defects in
fsn-1(lf), a binding partner of RPM-1 (4).
ok623 animals display strong defects in axon termination in the CAN and the mechanosensory neurons. 75% of ALM axons show extensive overgrowth in
rpm-1(lf) and
glo-4 single mutants. In the PLM neurons,
rpm-1(lf) mutants display axon overgrowth, and also fail to extend synaptic branches to the ventral cord;
glo-4(lf) mutants show similar, but milder phenotypes. Interestingly, double mutants of
glo-4 (lf) and
fsn-1(lf) completely phenocopy
rpm-1(lf) in PLM neurons. These results suggest that
glo-4 and
fsn-1 may function in parallel.
One of the functions of RPM-1 is to inhibit the activation of the Dual Leucine Zipper-bearing Kinase (DLK)-1 (5).
dlk-1(lf) suppressed
rpm-1(lf) strongly in the PLM, and weakly in the ALM neurons. However,
dlk-1(lf) did not suppress
glo-4(lf) in either neuron. The observations that
glo-4(lf) enhances
fsn-1(lf) and is not suppressed by
dlk-1(lf) suggest that it acts in a parallel pathway to the RPM-1/SKR-1/CUL-1/FSN-1 complex that targets proteins for degradation.
Our studies further suggest that RPM-1 is likely using different mechanisms to regulate synaptogenesis and axon termination. The process of synaptogenesis is largely regulated by the ubiquitin ligase activity of RPM-1, while axon termination is principally regulated by RPM-1 via GLO-4