Picture from Noblett N et al. (2018) J Cell Biol "DIP-2 suppresses ectopic neurite sprouting and axonal regeneration in mature ...."
(A) Ventral view of anL4 worm showing transcriptional
dip-2p::GFP(zyEx44) expression in VNC neurons (anterior [A]and posterior [A'] segments from same worm).(B-E) Representative images of full-lengthDIP-2::GFP (zyIs47) expression in L4, showingcytoplasmic DIP-2::GFP localization in head (lateral [B] and medial [B'] focal planes from sameworm) as well as neurons in midbody (C and D)and tail (E). Asterisks in B and B' indicate
myo2p::mCherry coinjection marker bleedthrough inpharyngeal muscle. (C' and E') Representativeimages of endogenous GFP::DIP-2 (
zy70) expression showing cytoplasmic localization in mechanosensory neurons. (F) Endogenous GFP::DIP-2
(zy70) in mechanosensory neurons remainscytoplasmic with increasing age. Left: Representative confocal images of ALM in D1, D5, andD7 animals. Right: Quantification of DIP-2 levelsin ALM. Arbitrary units represent GFP intensitylevels (mean +- SEM). Bars: 20 um (A-E); 10 um(C', E', and F).
Picture from Noblett N et al. (2018) J Cell Biol "DIP-2 suppresses ectopic neurite sprouting and axonal regeneration in mature ...."
Figure 4.
dip-2 is expressed in neurons andacts cell autonomously. (A) Ventral view of anL4 worm showing transcriptional
dip-2p::GFP(zyEx44) expression in VNC neurons (anterior [A]and posterior [A'] segments from same worm).(B-E) Representative images of full-lengthDIP-2::GFP (zyIs47) expression in L4, showingcytoplasmic DIP-2::GFP localization in head (lateral [B] and medial [B'] focal planes from sameworm) as well as neurons in midbody (C and D)and tail (E). Asterisks in B and B' indicate
myo2p::mCherry coinjection marker bleedthrough inpharyngeal muscle. (C' and E') Representativeimages of endogenous GFP::DIP-2 (
zy70) expression showing cytoplasmic localization in mechanosensory neurons. (F) Endogenous GFP::DIP-2
(zy70) in mechanosensory neurons remainscytoplasmic with increasing age. Left: Representative confocal images of ALM in D1, D5, andD7 animals. Right: Quantification of DIP-2 levelsin ALM. Arbitrary units represent GFP intensitylevels (mean +- SEM). Bars: 20 um (A-E); 10 um(C', E', and F). (G and H) Neuron-specific expression of DIP-2 rescues neuronal morphologydefects in
dip2(0) (G) and aged WT (H) animals.Error bars (G and H) indicate SEM of proportion(n = 41-386). All statistics, two-tailed t tests.*, P < 0.05; ***, P < 0.001.
Picture from Noblett N et al. (2018) J Cell Biol "DIP-2 suppresses ectopic neurite sprouting and axonal regeneration in mature ...."
Figure S2. Requirements for the DMAP1 binding and AFD domains for DIP-2 neuronal development and protein localization. (A and B) EndogenousGFP::DIP-2 (
zy70) shows cytoplasmic localization in mechanosensory neurons (A) and membrane localization in epidermal cells (B) indistinguishable fromthose expressed in transgenic (zyEx78 and zyIs47) animals. Shown are confocal images of PLM neurons marked by Pmec-4-mKate2 (juSi329) in young adults(A) and L3 stage epidermal seam cells (B, bottom) and wide-field images of embryonic epidermal cells (B, top left) and L2 stage seam cells (B, top right).(C) Schematics of FL, DMAP1-binding, and AFD domain-deleted DIP-2 proteins. (D and E) ALM neuronal morphology and HSN migration defects in
dip-2mutants were rescued with transgenes expressing FL and DMAP1-binding domain-deleted but not AFD-deleted DIP-2 proteins. Error bars indicate SEM of proportion with number of neurons (n = 60-150; D) or animals (n = 27-46; E) indicated above. Significance compared between transgene-negative andtransgene-positive siblings using two-tailed t tests. ***, P < 0.001. (F and G) Representative images of
dip-2 promoter-driven FL DIP-2::GFP and domaindeleted DIP-2::GFP proteins expressed in L4-stage tail neurons and embryonic epidermal cells. FL and DMAP1 and AFD domain deleted DIP-2::GFP proteins show similar localization patterns in late-stage neuronal cells. However, in contrast with FL and DMAP1-deleted DIP-2::GFP, AFD1- and AFD2-deletedDIP-2::GFP proteins displayed a partial or complete loss, respectively, of membrane localization in epidermal cells. Bars: 10 um (A); 20 um (B, F, and G).
Picture from Aviles S et al. (2024) MicroPubl Biol "New alleles of nlp-2 , nlp-22 , and nlp-23 demonstrate ...."
Figure 1. The RPamide neuropeptides are dispensable for stress-induced sleep:(a) Gene structures and alleles for the RPamide neuropeptides. (b) Average minutes of movement quiescence in 10-minute windows during L4 developmentally-timed sleep in wild-type (N=31) and
nlp-22(
stj313) (N=33) animals, wild-type (N=35) and
nlp-23(
stj310) (N=36) animals, and wild-type (N=26),
nlp-22(
stj313) (N=30), and
nlp-22(
stj313);
nlp-23(
stj311) (N=60) animals. (c) Total minutes of movement quiescence during L4 developmentally-timed sleep in wild-type (N=31) and
nlp-22(
stj313) (N=33) animals, wild-type (N=35) and
nlp-23(
stj310) (N=36) animals, wild-type (N=26),
nlp-22(
stj313) (N=30), and
nlp-22(
stj313);
nlp-23(
stj311) (N=60) animals, and wild-type (N=38),
nlp-22(
stj313);
nlp-23(
stj311) (N=34), and
nlp-2(
stj351);
nlp-22(
stj313);
nlp-23(
stj311) (N=82) animals. (d) Total minutes of movement quiescence during UV-induced sleep in wild-type (N=33) and
nlp-22(
stj313) (N=31) animals, wild-type (N=44) and
nlp-23(
stj310) (N=49) animals, wild-type (N=55),
nlp-22(
stj313);
nlp-23(
stj311) (N=41), and
nlp-2(
stj351);
nlp-22(
stj313);
nlp-23(
stj311) (N=50) animals. For both (c) and (d) statistical significance was calculated by Student's t-test (2 genotypes) or one-way ANOVA followed by Tukey's test (3 genotypes)(*p<0.05, **p<0.01, ***p<0.001).
Picture from Vasudevan A et al. (2024) MicroPubl Biol "Physical presence of chemical synapses is necessary for turning behavior of ...."
Figure 1. pre-SVs do not turn into the synaptic branch upon loss of connection to chemical synapses in the C. elegans PLM neuron: For all plots shown in the figure, individual data points are represented as solid circles. The summary statistics are depicted using a boxplot with whiskers. Upper whisker = minimum(maximum value in the distribution, Q3 + 1.5 * IQR) and lower whisker = maximum(minimum value in the distribution, Q1 - 1.5*IQR), where IQR is inter-quartile range.A) Schematic of the C. elegans PLM neuron, representing the laser ablation paradigm for the 'synapse cut'. The red cross marks the site at which laser ablation is performed, such that the connection between the chemical synapses and the synaptic branch is removed. Proportion of 'turning', 'pausing', and 'straight' vesicles is plotted as a percentage of total anterograde pre-SVs that reach the branch point in wild type- jsIs821 animals. N(before cut)=10 animals, n(before cut- 'turning')=103 vesicles, n(before cut-'pausing')= 90 vesicles, n(before cut-'straight')= 20 vesicles. N(after cut) =10 animals, n(after cut-'turning')=6 vesicles, n(after cut-'pausing')= 121 vesicles, n(after cut-'straight')= 15 vesicles. Wilcoxon rank sum test with continuity correction conducted to test for statistical significance, ****p<0.0001. All comparisons are made to the 'before cut' category.B) Schematic of PLM neuron, representing the laser ablation paradigm for the 'control cut'. The red cross marks the site at which laser ablation is performed along the distal process. This site is chosen such that its distance from the branch point is roughly equal to the length of the PLM synaptic branch (~30µm). Proportion of 'turning', 'pausing', and 'straight' vesicles is plotted as a percentage of total anterograde pre-SVs that reach the branch point in wild type- jsIs821 animals. N(before cut)=9 animals, n(before cut- 'turning')=95 vesicles, n(before cut-'pausing')=87 vesicles, n(before cut-'straight')=19 vesicles. N(after cut) =9 animals, n(after cut-'turning')=101 vesicles, n(after cut-'pausing')=95 vesicles, n(after cut-'straight')=15 vesicles. Wilcoxon rank sum test with continuity correction conducted to test for statistical significance. All comparisons are made to the 'before cut' category and are not statistically significant.C) Schematic of the PLM neuron depicting the different categories of pre-SVs transported at the branch point, namely, i) anterograde pre-SVs transported from the cell body to the branch point, ii) retrograde pre-SVs transported from the synaptic branch to the branch point, and iii) retrograde pre-SVs transported from the distal process to the branch point.D) Total flux of pre-SVs reaching the branch point in the anterograde and retrograde directions over a period of 5mins in the 'synapse cut' paradigm. N(before cut)=10 animals, n(before cut- 'anterograde')=213 vesicles, n(before cut- 'retrograde from branch')=26 vesicles, n(after cut- 'anterograde')=142 vesicles, n(after cut-'retrograde from branch')=19 vesicles. Wilcoxon rank sum test with continuity correction conducted to test for statistical significance, all comparisons are non-significant. All comparisons are made to the 'before cut' category.E) Total flux of pre-SVs reaching the branch point in the anterograde and retrograde directions over a period of 5mins in the 'control cut' paradigm. N(before cut)=9 animals, n(before cut- 'anterograde')=201 vesicles, n(before cut- 'retrograde from distal process')=119 vesicles, n(after cut- 'anterograde')=211 vesicles, n(after cut-'retrograde from branch')=72 vesicles. Wilcoxon rank sum test with continuity correction conducted to test for statistical significance, all comparisons are non-significant. All comparisons are made to the 'before cut' category.F) Schematic of the PLM branch point depicting the mobilization of pre-SVs from the paused vesicle cluster at the branch point into different compartments of the neuron.G) Total flux of pre-SVs mobilizing from the paused vesicle cluster at the branch point over a period of 5mins in the 'synapse cut' paradigm. N(before cut)=10 animals, n(before cut-'Into branch')=50 vesicles, n(before cut-'Into distal process')=46 vesicles, n(before cut-'Towards cell body')=24 vesicles. N(after cut) =10 animals, n(after cut-' Into branch')=45 vesicles, n(after cut-'Into distal process')=39 vesicles, n(after cut-'Towards cell body')=21 vesicles. Wilcoxon rank sum test with continuity correction conducted to test for statistical significance, all comparisons are non-significant. All comparisons are made to the 'before cut' category.H) Total flux of pre-SVs mobilising from the paused vesicle cluster at the branch point over a period of 5mins in the 'control cut' paradigm. N(before cut)=9 animals, n(before cut-'Into branch')=45 vesicles, n(before cut-'Into distal process')=46 vesicles, n(before cut-'Towards cell body')=20 vesicles. N(after cut) =9 animals, n(after cut-'Into branch')=42 vesicles, n(after cut-'Into distal process')=41 vesicles, n(after cut-'Towards cell body')=22 vesicles. Wilcoxon rank sum test with continuity correction conducted to test for statistical significance, all comparisons are non-significant. All comparisons are made to the 'before cut' category.