RAE-1, a novel PHR binding protein, is required for axon termination and synapse formation in Caenorhabditis elegans - PubMed (original) (raw)

RAE-1, a novel PHR binding protein, is required for axon termination and synapse formation in Caenorhabditis elegans

Brock Grill et al. J Neurosci. 2012.

Abstract

Previous studies in Caenorhabditis elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. To understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam (protein associated with Myc). rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development.

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Figures

Figure 1.

Figure 1.

PHR proteins bind to RAE-1. A, A schematic of the CeRAE-1 protein which is composed of an N-terminal domain and 7 WD repeats. Shown below is the segment of the RAE-1 protein that is deleted by rae-1(tm2784). B, Shown is a 40× DIC image of the vulval region of a wild-type animal and a rae-1(tm2784) mutant. Note the absence of embryos and oocytes, and the protruding vulva in the rae-1 mutant, which can be used to identify these animals. C, coIP of RPM-1::GFP with FLAG::RAE-1. RPM-1::GFP was expressed transgenically alone or in combination with FLAG::RAE-1 in the neurons of C. elegans. D, coIP of myc-Pam and GFP-Rae1. Myc-tagged human Pam and GFP-tagged rat Rae1 or GFP-rat PP2Cα (a protein of similar size to Rae1) were coexpressed in 293 cells. For B and C, a representative of at least three independent experiments is shown. For B, similar results were obtained using a minimum of two transgenic lines.

Figure 2.

Figure 2.

Identification of a Rae1 binding motif in the PHR proteins. A, Schematic representation of human Pam and C. elegans RPM-1. RLD (RCC-1 like domain), PHR (Pam/Highwire/RPM-1 family-specific domains), RBD (Rae1 binding domain), MBD (myc binding domain), NLS (nuclear localization signal), and RING (RING-H2 ubiquitin ligase domain). Shown below is a sequence alignment of the known Rae1 binding protein, Nup98, and the PHR proteins. Residues highlighted in blue are conserved between Nup98 and other previously described Rae1 binding proteins. Residues in Nup98 that are underlined are essential for binding to Rae1. B, coIP of the Pam RBD or the RPM-1 RBD fused to GFP with Flag-tagged rat Rae1. C, Identification of point mutations in the RPM-1 RBD that reduce binding to FLAG-Rae1. D, coIP of RPM-1::GFP or RPM-1::GFP (VIR to AAA) point mutant with FLAG::RAE-1 from transgenic C. elegans protein lysates. Note that the RPM-1::GFP (VIR to AAA) point mutant has greatly reduced binding to FLAG::RAE-1 compared with wild-type RPM-1::GFP. A representative of at least three independent experiments is shown for B–D. For D, at least two independently derived transgenic lines were analyzed for each genotype.

Figure 3.

Figure 3.

_rae-1_−/− mutants have defects in axon termination and synapse formation. A, C, Mechanosensory neurons (ALM and PLM) were visualized using muIs32. A, ALM axon termination defects in _rpm-1_−/− and _rae-1_−/− animals. B, Quantitation of ALM axon termination defects for the indicated genotypes. C, PLM axon termination defects in _rpm-1_−/− and _rae-1_−/− animals. The arrowhead highlights an example of the hook and overextension (hook) defect in rpm-1 mutants and the arrow highlights the less penetrant overextension defect in _rpm-1_−/− mutants. D, Quantitation of PLM axon termination defects for the indicated genotypes. E, Expression of C. elegans rae-1 or a cDNA encoding rat Rae1 in the mechanosensory neurons rescues the axon termination defects (hook) in the PLM neurons of rae-1_−/−;fsn-1_−/− animals. Overexpression of rae-1 was also sufficient to partially rescue defects in _rpm-1_−/− animals. For all extrachromosomal arrays, data from 4 to 10 independently derived transgenic lines was pooled. F, The presynaptic terminals of the DD neurons (dorsal cord) were visualized using SNB-1::GFP. Arrows highlight examples of gaps where presynaptic terminals are absent. G, Quantitation of synapse formation defects in the DD neurons. H, Confocal microscopy was used to visualize the dorsal cord of transgenic animals that express RPM-1::GFP (juIs77) and mCherry::RAE-1 (bggEx76) in the GABAergic motor neurons using the unc-25 promoter. mCherry::RAE-1 forms discrete puncta in the dorsal cord that colocalize with RPM-1::GFP puncta in the perisynatic zone of presynaptic terminals. I, Epifluorescent microscopy was used to visualize RPM-1::GFP driven by its own promoter (juIs58) in the nerve ring, dorsal cord, and SAB neurons of rae-1+/− or _rae-1_−/− animals. No difference in the levels and localization of RPM-1::GFP were observed in rae-1 mutants compared with heterozygous animals. Analysis of axon termination was done at 23°C, and analysis of synapse formation was done at 25°C. Scale bars are 10 μm. For B and D, n is the number of individual mechanosensory neurons that were scored. For G, n is the number of images of the dorsal cord that were quantified. For axon termination defects, error bars represent the SE, and significance was determined using a two-tailed Fisher's exact test. For synapse formation defects, error bars represent SEM, and significance was determined using a t test. **p < 0.005, ***p < 0.001, and ns=not significant.

Figure 4.

Figure 4.

Binding of RAE-1 to RPM-1 is necessary for RPM-1 to be fully functional. Transgenic animals were generated on an _rpm-1_−/− background to quantify rescue of rpm-1 (lf) defects. Rescue was analyzed for transgenic expression of wt RPM-1::GFP (Exrpm-1::GFP), or a point mutant of RPM-1::GFP (Exrpm-1::GFP VIR to AAA). A, ALM axon termination defects were quantified for the indicated genotypes. B, PLM axon termination defects were quantified for the indicated genotypes. Note, that data from 4–6 independently derived transgenic lines was pooled for this analysis, and n is the number of ALM or PLM neurons that were scored. A two-tailed Fisher exact test was used to determine significance. ***p < 0.0005.

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