Fig 1.
ubr-1 mutants exhibit reduced bending during reversal locomotion.
A) Structure of the UBR1 family protein. Position and amino acid substitutions in C. elegans alleles are denoted in the upper panel. Nonsense mutations (red dots), frame shift mutations (blue triangles), and small in-frame deletions (green diamonds) in human UBR1 that lead to JBS are denoted in the lower panel. B) Consecutive images of a wild type and ubr-1 animal during reversals (left to right panels). Wildtype animals generate sinusoidal body bends, whereas ubr-1(hp684, hp821hp833) animals exhibit almost no bending, which was rescued by restoring expression of UBR-1. Dots denote position of tail. Scale bar 200μm. C) Schematic of curvature analysis. A worm skeleton is divided into 29 segments, and relative curvature is calculated for each segment from anterior to posterior, and binned into 4 groups. D, E) In ubr-1 mutants (grey line), bending curvature is reduced across all segments compared to wildtype (black line), and this is rescued by restoring UBR-1 expression (red line). D- ubr-1(hp684); E- ubr-1(hp821hp833). ubr-1 mutants exhibited longer (F) reversal events, and reduced reversal initiation frequency (events/per minute) (G). *P<0.05, ** P<0.01, ***P<0.001 by Two-way RM ANOVA (D, E), and by the Kruskal-Wallis test (F, G). Data are represented as mean ± SEM.
Fig 2.
A critical requirement of UBR-1 in premotor interneurons during reversals.
A) The UBR-1::GFP reporter was expressed in a subset of neurons including the premotor interneurons of the reversal circuit (Premotor INs), the pharyngeal muscles, the body wall muscles, and hypodermal seam cells. Scale bar 10μm. B) Reduced bending in ubr-1 mutants (grey line) was rescued by restoring UBR-1 expression in neurons (red line), but not in muscles (blue line). C) Reduced bending in ubr-1 mutants (grey line) was robustly rescued by restoring UBR-1 expression in glutamate-receptor-expressing, premotor interneurons (INs), not in cholinergic or GABAergic motor neurons (MNs). D) Among the premotor interneurons (INs), a robust rescue required UBR-1 expression in AVE and RIM (red line). *P<0.05, ** P<0.01, ***P<0.001 by Two-way RM ANOVA. Data are represented as mean ± SEM.
Table 1.
The effect of restored expression of UBR-1 and GOT-1 on ubr-1 and ubr-1; got-1’s reversal motor pattern.
Fig 3.
UBR-1 promotes body bending by preventing synchronized A motor neuron activation.
A) Diagram of connectivity of the C. elegans reversal motor circuit, drawn based on White et al. 1986 [45]. Hexagons and the circle denote premotor interneurons, and the A class motor neurons, respectively. Arrows and lines denote chemical and electrical synapses between neurons, respectively. B) Approximated anatomic positions of the three A class motor neurons (VA11, DA7, VA10) and their predicted ventral and dorsal muscle targets, drawn based on Haspel and Donovan, 2011 [79]. C) An example trace of simultaneous calcium imaging of three A-type motor neurons in a moving wildtype animal. Upper panel: activities of VA11, DA7, and VA10 neurons, reflected by the GCaMP6/RFP signal ratio (Upper panel); Lower panel: the instantaneous velocity of the animal, reflected by the displacement of DA7 soma position (Lower panel; positive values indicate moving towards the head; negative values indicate moving towards the tail), during a period of 40s from a 3min recording. Boxed period denotes the reversal period applied for cross-correlation analyses. C’) The cross-correlation of the activity profiles between VA10 and VA11 (left), VA10 and DA7 (center), and DA7 and VA11 (right), respectively. Dotted vertical line denotes the lag time. D, D’) An example trace of simultaneous calcium imaging (D) and cross-correlation analysis (D’) of VA11, DA7 and VA10 in a moving ubr-1 animal. E, E’) An example trace of simultaneous calcium imaging (E) and cross-correlation analysis (E’) of VA11, DA7 and VA10 in a moving transgenic ubr-1 mutant animal with restored UBR-1’s expression in neurons that include AVE/RIM premotor interneurons. F, F’) An example trace of simultaneous calcium imaging (F) and cross-correlation analysis (F’) of VA11, DA7 and VA10 in a moving ubr-1; got-1 mutant animal. G-I) The phase lags between activities of VA10 and VA11 (G), VA10 and DA7 (H), and DA7 and VA11 (I) in animals of respective genotypes. The asynchrony between VA10 and VA11 (G), and between VA10 and DA7 (H) are significantly reduced in ubr-1 mutants compared to wildtype animals, and restored in ubr-1 mutants by both UBR-1 expression in premotor interneurons, and the got-1 mutation. The activation of DA7 and VA11 (I), with higher synchrony than the other two pairs in wildtype animals, was not significantly altered in ubr-1 mutants. *P<0.05, **P<0.01, ***P<0.001 by the Kruskal-Wallis test. Horizontal lines represent mean values.
Fig 4.
Removing GOT-1 transaminase restores bending in ubr-1 mutants.
A) A diagram depicting the structure of GOT-1 and the molecular lesion of the hp731 allele. B) GOT-1 is ubiquitously expressed in all somatic tissues. Scale bar 10μm. C) Mutations in got-1 suppress motor neuron defects in ubr-1. D) Restoring GOT-1 in premotor INs including AVE and RIM (blue line) in ubr-1; got-1 reverted curvature to that of ubr-1. *P<0.05, **P<0.01, ***P<0.001 by the Two-way RM ANOVA test. Data are represented as mean ± SEM.
Fig 5.
GOT-1 synthesizes glutamate from aspartate.
A) GOT transaminases maintain the equilibrium between glutamate and aspartate by reversible transfer of the amino group (NH2) (left panel). Results from our amino acid profiling indicates that in C. elegans, GOT-1 preferentially catalyzes synthesis of glutamate from aspartate (right panel). B) Free amino acid levels measured from whole animal lysates by HPLC. got-1 mutants (Bottom panel) exhibit reduced glutamate and alanine (blue arrows), and increased aspartate, asparagine and cysteine (red arrows) compared to wildtype animals (Top panel). In order to present data of a wide range, the Y-axis utilizes three different scales at different concentration values. Consistent with glutamate being the sole precursor of GABA synthesis, GABA level was also increased in ubr-1 mutants (blue arrow). *P<0.05, **P<0.01 by the Student T test, N: 5 replica. Data are represented as mean ± SEM.
Table 2.
A list of genetic mutants in metabolic and other biological pathways examined for their genetic interactions with ubr-1 mutants.
Fig 6.
Glutamate level is elevated in ubr-1 mutants.
A-C) Free amino acid measured by HPLC, normalized against total protein in the lysate. Levels in mutants were normalized to that of wildtype. A) Glutamate level was increased in ubr-1, but decreased in got-1 mutants; the increase in glutamate was reversed in ubr-1; got-1, but not in ubr-1 got-2 mutants. B) Aspartate exhibited a modest increase in ubr-1, but massive accumulation in got-1 and ubr-1; got-1 mutants. The Y-axis utilizes two different scales (0.5 to 4.0, 4.0 to 40, respectively) to accommodate vastly different values. C) Alanine was significantly decreased in both got-1 and ubr-1; got-1 mutants. D-F) Metabolites measured by LC-MS/MS. D) OAA were significantly decreased in all mutants; α-KG did not exhibit consistent changes. E) The ratio of AMP to ATP was increased in got-1 and ubr-1; got-1 whereas ubr-1 mutants did not show significant changes. F) The ratio of NADP to NADPH was increased in got-1 and ubr-1; got-1 mutants (left panel), while that of glutathione (GSH) to glutathione disulfide (GSSH) was decreased in both mutants (right panel). ubr-1 mutants show no significant change in either ratio. *P<0.05, **P<0.01 by the Student T test. Horizontal lines represent mean values.
Fig 7.
The loss of UBR-1 affects glutamate receptor expression in premotor interneurons AVA and AVE.
A) Representative confocal images of AVA, AVE, and RIM neurons visualized with the Pnmr-1-RFP reporter in wildtype, ubr-1, and ubr-1; got-1 adult animals, as well as in ubr-1 mutants with restored expression of UBR-1 in neurons including AVE and RIM. Note the prominent reduction of fluorescent intensity in AVA in ubr-1 mutants. B-D) Quantification of the fluorescent intensity in AVA, AVE and RIM (B), and relative fluorescent intensity between AVA and RIM (C) and between AVE and RIM (D), in animals with the respective genotypes denoted in A. N: more than 10 animals of each strain. * P<0.05, ** P< 0.01by the two-way ANOVA test. E) Representative confocal images of the GLR-1::GFP signals along the AVA and AVE neurites in the ventral nerve cords of animals with the same genotypes as denoted in panel A. ubr-1 mutants exhibited a marked decrease in fluorescent intensity. F) Quantification of the total GLR-1::GFP intensity along the AVA and AVE ventral cord neurites. N = 10–15, *P<0.05, **P<0.01, ***P<0.001 by the Kruskal-Wallis test. Data are represented as mean ± SEM. Scale bar, 5 μm.
Fig 8.
GOT-1 is not a direct substrate of UBR-1.
A) The total GOT transaminase activity was increased in ubr-1 and ubr-1got-2 mutants, but decreased in got-1 and ubr-1; got-1 mutants. *P<0.05; **P<0.01 by the Kruskal-Wallis test. Horizontal lines represent the mean values. B) The loss of ubr-1 did not lead to changes in GOT-1::GFP protein levels in C. elegans carrying endogenously fused GOT-1::GFP (left panel) or GOT-1::GFP driven by an exogenous pan-neuronal promoter (right panel). C) A working model of UBR-1-mediated negative regulation of glutamate level and bending.