Fig 1.
The two TSP-14 isoforms: TSP-14A and TSP-14B.
(A). Schematic representation of the tsp-14 locus, showing only the alternative and shared coding exons of tsp-14a and tsp-14b. Exons are represented by boxes, and introns by lines. ATG represents the start codon of each isoform. The first coding exon for tsp-14b is located 3511bp upstream of the first coding exon of tsp-14a. The black lines depict the extent of the jj95 deletion, which is close to 2.9kb long. (B). A schematic of TSP-14A and TSP-14B proteins, showing the four transmembrane domains, and the various extracellular and intracellular regions. The only difference between TSP-14A and TSP-14B is at their N-termini, with the red line representing the extra amino acids present in TSP-14B. The CCG and CC motifs in the second extracellular loop are represented by solid circles, with orange solid circle representing cysteine, while the yellow solid circle representing glycine. PM, plasma membrane. Ext, extracellular. (C). Alignment of the amino acid sequences of TSP-14A and TSP-14B. TSP-14B has 24 additional amino acids at its N-terminus (red), which includes the ExxLL motif (blue). The four transmembrane domains are indicated in green and the cysteine residues in the extracellular domain are in red.
Fig 2.
Isoform-specific knockouts of TSP-14 cause distinct phenotypes.
(A). Schematics of the tsp-14 locus, showing isoform-specific knockouts generated in this study. In jj304, the TSP-14A start codon ATG (methionine) is mutated to TTG (isoleucine), knocking out TSP-14A, while introducing a single, although conserved amino acid change in TSP-14B. The tsp-14(jj304) allele is also referred to as tsp-14a(0). In jj317, the TSP-14B start codon ATG and a downstream, in-frame, ATG (see Fig 1C) are mutated to TTG and TTA respectively, knocking out TSP-14B without affecting TSP-14A. The tsp-14(jj317) allele is also referred to as tsp-14b(0). (B). Relative body lengths of synchronized L4 worms of various genotypes. The mean body length of wild-type worms is normalized to 1.0. For each double mutant, data were pooled from two independent isolates. The total number of worms measured for each genotype is at least 60. Tukey’s honestly significant difference (HSD) test following an ANOVA was used to test for differences between different genotypes. ***P < 0.0001. n.s., no significant difference. WT: wild-type. tsp-12(0): tsp-12(jj300). tsp-14(0): tsp-14(jj95). tsp-14a(0): tsp-14(jj304). tsp-14b(0): tsp-14(jj317). (C-N). Differential interference contrast (DIC) micrographs of wild-type (WT, C-E), tsp-12(0); tsp-14(0) (F-H), tsp-12(0); tsp-14a(0) (I-K), tsp-12(0); tsp-14b(0) (L-N) animals at the L4 (C, F, I, L) or young adult stage (D, G, J, M), or their embryos at mid-embryogenesis (E, H, K, N). L4, L4 larva. YA, young adult. Scale bars represent 20 μm. For each genotype, more than 100 animals or embryos were examined. (O). Diagrams depicting the sma-9(0) suppression (Susm) assay. Mutations in the BMP pathway can suppress the loss of M-lineage-derived coelomocytes (CCs) in sma-9(0) mutants. CCs are depicted as green circles. P). Table summarizing the results of the sma-9(0) suppression assay of various tsp-14 alleles. Percentage of suppression was calculated by the number of worms with 1–2 M-derived CCs divided by the total number of worms scored. N represents the total number of worms counted. Data from two independent isolates were combined for each genotype. Groups marked with distinct symbols are significantly different from each other (P<0.001, in all cases when there is a significant difference), while groups with the same symbol are not. Tested using an ANOVA with a Tukey HSD (see Materials and Methods).
Fig 3.
TSP-14A and TSP-14B have distinct and shared functions.
(A) Schematics depicting the single copy transgenes expressing either tsp-14a or tsp-14b under the 3.3kb or 5.2kb tsp-14 promoter at the ttTi4348 locus on chromosome I. (B) Table summarizing the results of the sma-9(0) suppression assay when tsp-14a or tsp-14b was expressed as a single copy transgene. Percentage of suppression was calculated by the number of worms with 1–2 M-derived CCs divided by the total number of worms scored. Groups marked with distinct symbols are significantly different from each other (P<0.001, in all cases when there is a significant difference), while groups with the same symbol are not. Tested using an ANOVA with a Tukey HSD (see Materials and Methods). (C) Relative body lengths of synchronized L4 worms of various genotypes at the Christmas tree stage. The mean body length of wild-type (WT) worms is normalized to 1.0. Numbers within each bar indicate the total number of worms measured. For each double mutant, data were pooled from two independent isolates. The total number of worms measured for each genotype is at least 60. Tukey’s HSD test following an ANOVA was used to test for differences between different genotypes. ***P < 0.0001. WT: wild-type. tsp-12(0): tsp-12(jj300). tsp-14(0): tsp-14(jj95).
Fig 4.
Endogenous TSP-14A ad TSP-14B can be respectively and functionally tagged.
(A). Schematic representations of wild-type and GFP-tagged TSP-14 isoforms. In jj192 (denoted as TSP-14B(NT)) animals, TSP-14B is specifically tagged at its N-terminus with GFP::3xFLAG. In jj183 (denoted as TSP-14A(NT)) animals, TSP-14A specifically tagged at its N-terminus with GFP::3xFLAG. * If spliced properly, the tag should also be inserted into TSP-14B after the first 24aa in jj183 animals. However, no tagged TSP-14B was detectable on western blots (D), suggesting that the expression or splicing of TSP-14B is compromised in jj183 animals. Both TSP-14A and TSP-14B are tagged at their C-termini with GFP::3xFLAG in jj219 (denoted as TSP-14AB(CT) animals. The same GFP::3xFLAG tag as in jj219 is also in jj304 jj319 (denoted as TSP-14B(CT) and jj317 jj377 (denoted as TSP-14A(CT) animals when one of the respective tsp-14 isoforms is knocked out. (B). Relative body lengths of synchronized L4 worms of various genotypes. The mean body length of wild-type worms is normalized to 1.0. For each double mutant, data were pooled from two independent isolates. The total number of worms measured for each genotype is at least 60. Tukey’s HSD test following an ANOVA was used to test for differences between different genotypes. ***P < 0.0001. n.s., no significant difference. As shown, tsp-12(0); tsp-14(jj192) worms are slightly smaller than wild-type (WT) worms, but not as small as tsp-12(0); tsp-14(0) worms. (C). Tables summarizing the results of the sma-9(0) suppression assay of various tsp-14 alleles, in combination with the tsp-12(jj300) null allele. Percentage of suppression was calculated by the number of worms with 1–2 M-derived CCs divided by the total number of worms scored. N represents the total number of worms counted. Data from two independent isolates were combined for each genotype. Groups marked with distinct symbols are significantly different from each other (P<0.001, in all cases when there is a significant difference), while groups with the same symbol are not. Tested using an ANOVA with a Tukey HSD (see Materials and Methods). (D). Western blot showing the various tagged forms of TSP-14A (blue dot) or TSP-14B (white dot) detected by anti-FLAG antibodies. Anti-actin antibodies were used as a loading control. For each lane, 100 synchronized L4 animals were used to prepare the samples. The genotypes of jj322 jj192 and jj368 jj378 are described in Fig 7.
Fig 5.
TSP-14A and TSP-14B exhibit distinct localization patterns.
(A-M) Airyscan confocal images showing the localization of TSP-14A (A-C’ and E), TSP-14B (F-G’ and I), and both TSP-14A and TSP-14B (J-K’ and M) in the head region (GFP: A, F, J, and DIC: A’, F’, J’), hypodermal cells (GFP: C, G, K, and DIC: C’, G’, K’), and the developing vulva (E, I, and M). TSP-14A(NT) is detectable in the tips of anterior sensory cilia (arrow in A), and in sperm cells (arrows in B). * in A and B marks autofluorescence signals. Scale bar in A-M, 10 μm. Panels D, E and L are schematic representations of the different knock-ins. (N-V) Airyscan confocal GFP (N, O, Q, R, T, U) and corresponding DIC (N’, O’, Q’, R’, T’, U’) images of the developing vulva (N, Q, T) and hypodermal cells (O, R, U) from worms expressing different tagged versions of TSP-14. Panels P, S and V are schematic representations of the different knock-ins. Scale bars in O-W, 20 μm.
Fig 6.
TSP-14A is co-localized with TSP-12 and is localized to endosomes.
(A) Schematics of GFP- or TagRFP-tagged TSP-14A. (B-G) Airyscan confocal images of late L4 hypodermal cells expressing both tagged TSP-14A (B, GFP-tagged TSP-14A and E, TagRFP-tagged TSP-14A) and tagged TSP-12 (C, TagRFP-tagged TSP-12 and F, GFP-tagged TSP-12). The corresponding merged images are shown in D and G, respectively. (H) The colocalization between TSP-14A and TSP-12 was quantified using two different methods. PCC, Pearson’s correlation coefficient; MOC, Mander’s overlap coefficient. PCC indicates the correlation of GFP::TSP-14A with TagRFP::TSP-12 or TagRFP::TSP-14A with GFP::TSP-12. MOC indicates the fraction of TagRFP::TSP-12 that overlaps with GFP::TSP-14A (the second bar), or the fraction of GFP::TSP-12 that overlaps with TagRFP::TSP-14A (the forth bar). (I-Q) Airyscan confocal images of L4 stage hypodermal cells expressing N-terminally GFP-tagged TSP-14A (J, M, P) and different endosomal markers, TagRFP::EEA-1 (I, early endosomes), TagRFP::RAB-7 (L, late endosomes and lysosomes), and TagRFP::RAB-11 (O, recycling endosomes). K, N and Q are the corresponding merged images. (R) Bar graphics showing the correlation of TagRFP (EEA-1, RAB-7 or RAB-11) with GFP::TSP-14A (PCC), and the fraction of TSP-14A::GFP that overlaps with TagRFP (MOC). Error bars in H and R represent SEM. Statistical analysis was done using a 2-tailed t test with 95% CIs. Scale bar, 5 μm.
Fig 7.
TSP-14B is sufficient to localize to the basolateral membrane upon forced expression.
Airyscan confocal images of L4 worms expressing either TSP-14A (A) or TSP-14B (B) in the tsp-14(0) background under the snx-1 promoter as single copy transgenes in the ttTi4348 locus on chromosome I. Images shown are of the head region and intestine cells. Arrows point to TSP-14A or TSP-14B signals. Asterisks mark intestinal autofluorescence. TSP-14B::GFP is concentrated on the membrane in the head region and in the basolateral membrane of the intestinal cells, while the TSP-14A::GFP signal is not detectable on cell membranes. Scale bars, 20 μm.
Fig 8.
The 24aa unique to TSP-14B contains a basolateral membrane targeting sequence.
(A, B, E) Schematics of the wild-type (WT) and jj368 mutant tsp-14 loci, either untagged (A), or N-terminally GFP-tagged (B, E), showing the EQCLL sequence in WT TSP-14B (or in jj192, B) mutated to AQCAA in jj368 (A) or jj322 jj192 (E). (C-D, F-G) Airyscan confocal images of L4 larvae showing that wild-type (WT) TSP-14B is localized to the basolateral membrane in the developing vulva (C) and in hypodermal cells (D), while mutated TSP-14B carrying the AQCAA mutation is localized to the apical side of the developing vulva (F) and in intracellular vesicles in hypodermal cells (G). Scale bars, 10 μm. (H) Relative body lengths of synchronized L4 worms at the Christmas tree stage. The mean body length of wild-type worms is normalized to 1.0. For each double mutant, data were pooled from two independent isolates. The total number of worms measured for each genotype is at least 60. Tukey’s HSD following an ANOVA was used to test for differences between different genotypes. ***P < 0.0001. WT: wild-type. tsp-12(0): tsp-12(jj300). tsp-14(0): tsp-14(jj95). As shown, tsp-12(0); tsp-14(jj368) worms are slightly smaller than WT worms, but not as small as tsp-12(0); tsp-14(0) worms. (I) Table summarizing the results of the sma-9(0) suppression assay. Percentage of suppression was calculated by the number of worms with 1–2 M-derived CCs divided by the total number of worms scored. N represents the total number of worms counted. Data from two independent isolates were combined for each genotype. Groups marked with distinct symbols are significantly different from each other (P<0.001, in all cases when there is a significant difference), while groups with the same symbol are not. Tested using an ANOVA with a Tukey HSD (see Materials and Methods). (J) Graph summarizing the brood sizes of different strains. tsp-12(0), tsp-14(0) and tsp-14(jj368) single mutants have the same brood size as wild-type (WT) worms, while tsp-12(0); tsp-14(0) and tsp-12(0); tsp-14(jj368) double mutants have significant reduced brood sizes compared to WT. tsp-12(0): tsp-12(jj300). tsp-14(0): tsp-14(jj95). Tukey’s HSD following an ANOVA was used to test the differences between different genotypes. ***P < 0.0001. (K) Schematic representations of the N-terminally GFP-tagged TSP-14A and N-terminally GFP-tagged TSP-14A with EQCLL mutated to AQCAA (jj368 jj378). (L) Airyscan confocal images of L4 larvae showing the developing vulva and hypodermal cells. The GFP signal in jj368 jj378 worms is very faint and not localized to the basolateral cell membrane. Scale bars, 10 μm.