Synapse maintenance is impacted by ATAT-2 tubulin acetyltransferase activity and the RPM-1 signaling hub
- The Scripps Research Institute, United States
Figures
Figure 1
RPM-1 functions cell autonomously in mechanosensory neurons to regulate presynaptic bouton maintenance.
(a) Schematic highlighting location of collateral synaptic branch and chemical synapses in PLM mechanosensory neurons. (b) Confocal images at different developmental time points showing presynaptic boutons of PLM neurons are delayed in formation and destabilize in rpm-1 mutants. Note brackets denote PLM presynaptic boutons (PLML and PLMR) and arrows highlight synaptic branch retraction. Note that in confocal images at 16 hr and 60 hr PH, PLML synaptic branch is out of focal plane but bouton is visible. (c) Developmental time course showing synaptic boutons in PLM neurons of rpm-1 mutants are delayed in formation but reach normal levels by 16 hr PH (blue). Subsequently, rpm-1 mutant boutons are progressively lost over time (orange). (d) Quantitation showing bouton maintenance defects in adult rpm-1 mutants, and rescue with transgenic RPM-1 expressed using native rpm-1 promoter or mechanosensory neuron promoter. Significance tested using Fisher’s exact test for c, and Student’s t-test with Bonferroni correction for d. ***p<0.001, **p<0.01 and ns = not significant (p>0.05).
Figure 2
RPM-1 localizes to presynaptic terminals of developing and adult mechanosensory neurons.
(a) Confocal images showing RPM-1::GFP localized at presynaptic boutons of PLM neuron at 16 hr PH. tdTOMATO shows PLM axon and presynaptic terminal morphology. (b) Quantitation of RPM-1::GFP presynaptic localization in PLM neurons at different times in development. (c) Confocal image showing RPM-1 localized to periactive zones adjacent to active zone marker UNC-10::tdTOMATO in adults.
Figure 3 with 1 supplement
RPM-1 regulates synapse maintenance.
(a) Confocal images showing synaptic vesicle marker RAB-3 (green) at presynaptic terminals of PLM mechanosensory neurons during development. RFP shows PLM morphology (magenta). Yellow brackets highlight presynaptic terminals of PLML and PLMR neurons. Note one synaptic branch is out of focal plane. (b) rpm-1 mutants accumulate RAB-3 (green) at presynaptic terminals by 12 hours PH, but presynaptic terminals fail to be maintained leading to synaptic branch retraction by 24 hr PH. (c) Developmental time course of presynaptic boutons and RAB-3::GFP accumulation in wt animals. Full assembly of presynaptic terminals with RAB-3 occurs by 12 hr PH. (d) Developmental time course of presynaptic boutons and RAB-3::GFP in rpm-1 mutants. Presynaptic assembly with RAB-3 is complete by 16 hours PH, but is not maintained and presynaptic terminals are lost at later time points. (e, f) UNC-10::tdTOMATO marks the active zone and assembles at presynaptic terminals in e) wt and (f) rpm-1 mutants at critical synapse assembly time points of 12 and 16 hr PH. (g) Quantitation of boutons containing UNC-10. At 12 hr PH, all boutons contain UNC-10 in wt and rpm-1 mutants. At 16 hr PH, there is a small defect in UNC-10 accumulation at presynaptic terminals of rpm-1 mutants. (h) Quantitation of bouton area. rpm-1 boutons are initially the same size as wt boutons (5 and 7 hours PH). rpm-1 mutants show small decreases in bouton size just prior to synapse loss (12 and 16 hr PH). Significance tested using Fisher’s exact test. ***p<0.001, *p<0.05 and ns = not significant.
Figure 3—figure supplement 1
SYD-2 active zone marker accumulates in rpm-1 mutants at 16 hr PH.
(a) Confocal image showing SYD-2::mScarlet localizes to presynaptic boutons in PLM neurons of wt animals. Top image is merged maximum projection. Bottom images are single confocal slices showing SYD-2::mScarlet (red) and presynaptic bouton (GFP). Note, SYD-2 is visible in the PLMR but not PLML because SYD-2::mScarlet was expressed as an extrachromosomal array which displays mosaicism. (b) In rpm-1 mutants, we observe SYD-2::mScarlet at presynaptic boutons. (c) Quantitation of PLM presynaptic boutons containing SYD-2 for indicated genotypes. Significance tested using Fisher’s exact test. Scale bars are 5 µm.
Figure 4
Drugs that alter microtubule stability affect synapse maintenance defects in rpm-1 mutants.
(a) Decreasing microtubule stability with colchicine enhances synapse maintenance defects in rpm-1 mutants. (b) Increasing microtubule stability with taxol suppresses synapse maintenance defects in rpm-1 mutants. Significance tested using Student’s t-test with Bonferroni correction. ***p<0.001.
Figure 5 with 1 supplement
Several mutants that affect microtubules interact with rpm-1 to affect synapse maintenance.
(a) Confocal images of presynaptic boutons and synaptic branches in adult PLM neurons. In wt animal, presynaptic boutons from PLML and PLMR are visible. Note one synaptic branch is shown and the other is out of the focal plane. atat-2 and rpm-1 mutants lack a synaptic branch and only show PLMR bouton (note loss of PLML, arrow). (b) Quantitation of synapse maintenance defects for indicated genotypes. Note atat-2 shows higher frequency defects than ptrn-1 or mec-17. (c) Quantitation showing synapse maintenance defects are similar in rpm-1; atat-2 and rpm-1; ptrn-1 double mutants compared to rpm-1 single mutants. In contrast, rpm-1; mec-17 and rpm-1; ptl-1 double mutants show enhanced defects compared to rpm-1 single mutants. (d) Quantitation indicates synapse maintenance defects are enhanced in atat-2; ptrn-1 double mutants compared to atat-2 single mutants. (e) Quantitation showing synapse maintenance defects are suppressed in rpm-1; dlk-1 double mutants, but not atat-2; dlk-1 double mutants. Significance tested using Student’s t-test with Bonferroni correction. ***p<0.001 and ns = not significant.
Figure 5—figure supplement 1
Analysis with a second transgenic background indicates atat-2 regulates synapse maintenance.
Quantitation of synapse maintenance defects using zdIs5 (Pmec-4::GFP). This is a different transgenic background than Figure 5, which used muIs32 (Pmec-7::GFP). Note, atat-2 and rpm-1 single mutants show synapse maintenance defects. Frequency of defects is not increased in rpm-1; atat-2 double mutants. Significance assessed by Student’s t-test with Bonferroni correction. ***p<0.001 and ns = not significant.
Figure 6 with 1 supplement
ATAT-2 tubulin acetyltransferase activity functions in mechanosensory neurons to regulate presynaptic maintenance.
(a) Confocal images of presynaptic boutons and synaptic branches in adult PLM neurons. In the wt animal, presynaptic boutons are shown for both PLML and PLMR neurons (brackets). Note one synaptic branch is shown and other is out of focal plane. atat-2 mutant lacks presynaptic boutons from both PLML and PLMR, and synaptic branch absent (arrow). Expression of ATAT-2 in mechanosensory neurons rescues defects. ATAT-2 lacking acetyltransferase activity (ATAT-2 dead) fails to rescue (b) Quantitation of synapse maintenance defects for indicated genotypes. Defects in atat-2 mutants are rescued by using native atat-2 or mechanosensory neuron promoters to transgenically express ATAT-2. No significant rescue occurs with ATAT-2 lacking acetyltransferase activity. (c) Microtubule destabilizing drug colchicine enhances synapse maintenance defects in atat-2 mutants. Significance tested using Student’s t-test with Bonferroni correction. ***p<0.001 and ns = not significant.
Figure 6—figure supplement 1
UNC-10/RIM and RAB-3 accumulate at presynaptic terminals of atat-2 mutants early in development.
(a) UNC-10::tdTOMATO is present at presynaptic boutons of PLM neurons in atat-2 mutants 16 hr PH. (b) Quantitation shows small reduction in frequency of UNC-10 at boutons of atat-2 mutants at 16 hr PH. This is similar to small decrease observed in rpm-1 mutants at the same time point. (c) RAB-3::GFP accumulates at presynaptic boutons of atat-2 mutants by 16 hr PH. Significance tested using Fisher’s exact test. Scale bars are 5 µm. ***p≤0.001.
Figure 7
Habituation to repeated mechanical stimulation is affected by ATAT-2 and RPM-1.
(a) Chemical and electrical synapses in PLM mechanosensory neurons primarily affect habituation to repeated tap stimuli and initial tap sensation, respectively (adapted from Crawley et al., 2017). (b) Multi-worm tracker was used to quantitate tap habituation for indicated adult genotypes. Habituation is defective in atat-2 mutants (red) and rpm-1 mutants (blue) compared to wt animals. rpm-1; atat-2 double mutants (magenta) are not significantly different than rpm-1 single mutants indicating atat-2 and rpm-1 function in a linear pathway to regulate habituation. Habituation level (HL) is shaded in grey. Significance assessed by Student’s t-test with Bonferroni correction. *p<0.05, ***p<0.001 and ns = not significant.
Figure 8
ATAT-2 tubulin acetyltransferase activity functions in a pathway with RPM-1 to regulate synapse maintenance.
Model summarizing genetic and pharmacological results suggesting RPM-1 functions upstream of ATAT-2 acetyltransferase activity to regulate microtubule stability and synapse maintenance. Outcomes indicate the RPM-1/ATAT-2 pathway functions independently of DLK-1 to regulate synapse maintenance.
Additional files
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Supplementary file 1
Transgenes and injection conditions.
- https://doi.org/10.7554/eLife.44040.013
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Transparent reporting form
- https://doi.org/10.7554/eLife.44040.014
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Synapse maintenance is impacted by ATAT-2 tubulin acetyltransferase activity and the RPM-1 signaling hub
eLife 8:e44040.
https://doi.org/10.7554/eLife.44040
