Fig 1.
The cluster of UNC-9 puncta in PLM neurons represents gap junctions in vivo.
(A) Expression patterns of GFP::UNC-9 fusion proteins represent the localization of gap junctions in PLM neurons. At the top panel, a cartoon picture shows the morphology of PLM neurons. The red rectangles highlight two gap junction zones in PLM neurons. In bottom panels, images of expression patterns of GFP::UNC-9, UNC-9::GFP and GFP::UNC-1 in PLM neurons. Red arrowheads point to GFP puncta at gap junction zones. Yellow stars label ALM cell bodies. CB: PLM cell bodies. (B) Images of immunostaining results show that endogenous UNC-9 forms similar punctate structures at gap junction zone 1. UNC-9 was stained using Rabbit anti-UNC-9 antibody (green color). PLM neurons were labeled using mouse anti-acetylated tubulin antibody (pink color). We confirmed those neurons were PLM neurons based on their morphology and the position of cell bodies. CB: PLM cell bodies. (C) Representative images show that loss-of-function mutations in unc-1 or inx-7 does not affect GFP::UNC-9 puncta. (D) Suppression of chemical synapse formation by mutating rpm-1 and syd-2 does not affect GFP::UNC-9 puncta in PLM neurons. (E) Three types of mutants identified in the genetic screen for gap junctions. Red arrowheads point to GFP::UNC-9 puncta. Yellow stars label ALM cell bodies. In figures C and D, the number displayed at each image shows the number of animals with wild type GFP::UNC-9 puncta/ total animals. Scale bar: 10 μm. Detailed strain information of all figures is listed in the S1 Table.
Fig 2.
UNC-44 and UNC-33 regulate gap junctions.
(A) Confocal images show GFP::UNC-9 puncta in unc-33 and unc-44 mutants, and unc-33(lf) rescue strain. (B) Quantification of the percentage of animals with more than four GFP::UNC-9 puncta at gap junction zone 1 in wild type and mutant strains. Each experiment was performed with N>200 animals at least three times. Pmec-4 promoter was used to express unc-33 cDNA in six mechanosensory neurons including PLM neurons. For transgenic animals the results shown here are generated from at least three independent lines. (C) Quantification of the length of gap junction zone 1 in wild type and mutants with enlarged gap junction zones. N>20 animals. Loss of function of unc-33 or unc-44 disrupts UNC-1 localization (D) and affects the co-localization of UNC-1 with UNC-9(E). (F) Mutating unc-33 and unc-44 increase NSY-5(INX-19) puncta number in adult animals. The number displayed at each images shows the number of animals with showing phenotypes/ total animals. Data are shown as mean ± SD. In comparisons of two groups Student t-test was used. In comparison of three unc-33 allele phenotypes, one-way ANOVA was used. ** p<0.01, ns: no significant difference. Scale bar: 10 μm.
Fig 3.
UNC-44 acts upstream of UNC-33.
Representative images of immunostaining results for FLAG::UNC-33(S)(A) and UNC-44(B) in control and mutant animals. UNC-33(S) localization was determined by a transgene expressing FLAG tagged UNC-33(S) in all neurons (Prgef-1::FALG::unc-33(S)). (C) Overexpression of UNC-33(S) suppresses unc-44(lf) phenotypes. Each experiment was performed with N>200 animals at least three times. For transgenic animals the results shown here are generated from at least three independent lines. Data are shown as mean ± SD. Student’s t-test, ** p<0.01. Scale bar: 10 μm.
Fig 4.
the UNC-44/UNC-33 pathway regulates gap junction dynamics and turnover.
(A) Time kymographs generated from a 30s movie of GFP::UNC-9 dynamics in control, unc-33(lf) and unc-44(lf) animals. In the unc-44 kymograph, the strong GFP::UNC-9 puncta were out of focus to visualize the GFP::UNC-9 movement. (B) Quantification of the percentage of particles moving to (red color) and moving away from gap junctions (blue color) in control (n = 42 animals), unc-33(n = 38) and unc-44 (n = 38) based on the analysis of GFP::UNC-9 movement. Values that differ significantly from wild type (Fisher’s exact test) are denoted on the graphs (* p<0.05). Images (C) and quantification (D) show that photoactivation of PAGFP::UNC-9 at both cell bodies and axons induces 35% decrease of PAGFP::UNC-9 intensity at cell bodies after 3 hours, but does not change PAGFP::UNC-9 intensity at gap junction zone 1. The rainbow bar shows the color code for fluorescence intensity: from blue (low) to white (high). Images (E-G) and quantification (H) show that photoactivation of PAGFP::UNC-9 only at gap junction zone 1 induces 43% decrease of PAGFP::UNC-9 fluorescence intensity at gap junction zone 1 after 3 hours in control animals, and loss of function of unc-44 and unc-33 suppresses the decrease of PAGFP::UNC-9 intensity. In C, E, F and G the top panels show GFP signals from photoactivated PAGFP::UNC-9, and the bottom panels show mCherry signals from expression of Pmec-4::mCherry as internal controls. For quantification in D and H, F3/F0 was calculated based on: (PAGFP intensity/mCherry intensity 3 hours after photoactivation)/ (PAGFP intensity/mCherry intensity right after photoactivation)X100%, N>15 animals for each experiments. Data are shown as mean ± SD. Student’s t-test, ** p<0.01, * p<0.05, ns: no significant difference. Scale bar: 10 μm.
Fig 5.
VAB-8 functions downstream of UNC-44 to regulate gap junction turnover.
(A) Confocal images show the distribution of GFP::UNC-9 puncta in control and vab-8(lf) animals. (B) Quantification of the percentage of animals with more than four GFP::UNC-9 puncta at gap junction zone 1 in mutant and rescue strains. Pmec-4 promoter was used to express vab-8(L) and vab-8(S) cDNA in PLM neurons. (C) Quantification of the length of gap junction zone 1 in wild type and mutants with enlarged gap junction zone. N>20 animals. (D) Quantification shows that after local photoactivation of PAGFP::UNC-9 at gap junction zone 1, the fluorescence intensity decreases about 43% at gap junction zone 1 in control animals, and loss of function of vab-8 suppresses the decrease of PAGFP::UNC-9 intensity. N>15 animals. (E) VAB-8(L) binds to UNC-33(S) in vivo. (F) Overexpression of vab-8(L) suppresses unc-33(lf) and unc-44(lf) phenotypes. Each experiment was performed with N>200 animals at least three times. For transgenic animals the results shown here are generated from at least three independent lines. Data are shown as mean ± SD. Student’s t-test, ** p<0.01, * p<0.05, ns: no significant difference. Scale bar: 10 μm.
Fig 6.
Models for the regulation of gap junctions the UNC-44/UNC-33/VAB-8 pathway.
(A) VAB-8 and UNC-33 function in parallel at the downstream of UNC-44 in gap junction regulation. (B) In gap junction dynamics, UNC-33 works together with an unknown factor at the downstream of UNC-44 to regulate motor protein VAB-8 to control gap junction turnover.