The N terminus-only (trans) function of the adhesion G protein-coupled receptor latrophilin-1 controls multiple processes in reproduction of Caenorhabditis elegans

Abstract

Adhesion G protein-coupled receptors are unique molecules. They are able to transmit classical signals via G protein activation as well as mediate functions solely through their extracellular N termini, completely independently of the seven transmembrane helices domain and the C terminus. This dual mode of action is highly unusual for G protein-coupled receptors and allows for a plethora of possible cellular consequences. However, the physiological implications and molecular details of this N terminus-mediated signaling are poorly understood. Here, we show that several distinct seven transmembrane helices domain-independent/trans functions of the adhesion G protein-coupled receptor latrophilin homolog latrophilin-1 in the nematode Caenorhabditis elegans together regulate reproduction: sperm guidance, ovulation, and germ cell apoptosis. In these contexts, the receptor elicits its functions in a noncell autonomous manner. The functions might be realized through alternative splicing of the receptor specifically generating N terminus-only variants. Thus, our findings shed light on the versatility of seven transmembrane helices domain-independent/N terminus-only/trans functions of adhesion G protein-coupled receptor and discuss possible molecular details.

Keywords: trans function; adhesion GPCR; apoptosis; germ cells; latrophilin; ovulation; sperm guidance.

Fig. 1.

The C. elegans gonad. a) Shown is one of the two symmetrical U-shaped gonad arms of a C. elegans adult hermaphrodite. In the fourth larval stage (L4), a fixed number of sperm (∼150 per gonad) are produced and stored inside the spermatheca. Subsequently, the gonad switches to continuous oocyte production only. In the distal gonad, germ cell nuclei are not surrounded by a complete membrane but reside in a common cytoplasm. They continually self-renew by mitotic division in the proliferative (mitotic) zone, which is distally enclosed by the DTC. Subsequently, germ cells enter meiotic divisions (crescent-shaped nuclei) and progress further through meiosis, where some germ cells undergo apoptosis. Here, the cells are surrounded by the gonadal sheath cells (not shown). Near the loop, nuclei start to cellularize and to obtain individual plasma membranes. Due to coordinated contraction of the gonadal sheath cells, oocytes are pushed into the spermatheca, fertilized, and eggs are flushed into the uterus. After completing the first set of embryonic divisions, they are laid through the vulva (not shown). Parts of the figure were created in Biorender. Prömel, S. (2024) BioRender.com/u05l303. b) Dissected and DAPI-stained distal gonads of L4 + 1-day-old wild-type and lat-1 hermaphrodites.

Fig. 2.

Nematodes lacking lat-1 display impaired sperm movement. a) Spermathecae of L4 + 1-day-old lat-1 mutant hermaphrodites contain the same amount of sperm (white arrows) as wild-type specimen. Gonads of adult hermaphrodites were dissected and subsequently stained with DAPI. b) Quantification of DAPI stainings as shown in (a) reveals that the number of sperm inside the spermatheca is indifferent from the one in wild-type nematodes. n ≥ 20 in 4 independent experiments. n.s., not significant. c) Residual bodies (black arrowheads) located close to the spermatheca in lat-1 mutant and wild-type gonads. Shown are DIC images of unstained living L4 + 1-day-old hermaphrodites. d) Quantification of anucleate residual bodies based on DIC images as displayed in (c) revealed no significant difference in number between wild-type nematodes and lat-1 mutants. Data from 4 independent experiments, n ≥ 24. n.s., not significant. e) Activation of spermatids by pronase treatment. Dissected spermatids from L4 + 1-day-old lat-1 males showed a similar level of activation as wild-type controls. Data from 3 independent experiments, n ≥ 100. n.s., not significant. f) Sperm amount 48 h after the first ovulation. Sperm was DAPI-stained and sperm loss was calculated (for details, see ‘Materials and methods’). This loss is ameliorated in transgenic lines expressing LNT. It should be noted that due to experimental limitations, the initial sperm count in the spermatheca had to be taken from different animals than the count after 48 h. n ≥ 15. g) Hermaphrodites lacking lat-1 exhibit a continuously smaller brood size over their entire reproductive period with the maximal reduction being visible on the second and third day of egg laying. n ≥ 75 in nine independent replicates. h) The ovulation rate in lat-1 mutants is similar to the one in wild types 24 h after the first ovulation but decreases faster than in wild-type individuals or in lat-1 mutants expressing the LNT. At 96 h after the first ovulation, it is significantly lower. Data of 4 independent experiments, n ≥ 51. n.s., not significant; ***P < 0.001. i) Representative images showing the distribution of male lat-1 and wild-type sperm, respectively, after mating to hermaphrodites of different genotypes. The motility of lat-1 sperm after mating with wild-type hermaphrodites appears to be intact, as indicated by correct localization near the spermatheca (white circle) 1 h postinsemination. Conversely, wild-type sperm inside lat-1 hermaphrodites do not reliably localize next to the spermatheca and remain close to the vulva (white arrowhead). Mating assays were performed with wild-type and lat-1 mutant nematodes in different constellations. Sperm was stained with MitoTracker Red by incubation of living young adult males prior to the mating, and sperm location was monitored 1 h after mating occurred. j) Sperm of wild-type males do not properly migrate to the spermatheca in lat-1 mutant hermaphrodites after mating, while sperm of lat-1 mutant males show no impaired movement toward the spermatheca of wild-type hermaphrodites. The former effect can be rescued by transgenic complementation of the LNT. Quantification was performed from images as shown in (h). Data were acquired in at least three independent experiments with n > 20 biological replicates. n.s., not significant; **P < 0.01; ***P < 0.001.

Fig. 3.

In the absence of LAT-1 function, germ cell apoptosis is increased. a) Germlines of lat-1 mutants exhibit more SYTO 12-stained germ cells compared with wild-type controls. Living adult nematodes 24 h post-L4 were stained with SYTO 12 as a marker for apoptotic cells. Shown are representative images. b) Quantification of SYTO 12 stainings from (a). Reintroduction of the LNT rescues the increased number of apoptotic cells. Given are numbers per gonad. n ≥ 33. **P < 0.01; ***P < 0.001. c) Oocytes (marked with an asterisk) are significantly smaller in lat-1 mutants than in wild-type individuals. Oocyte membranes were visualized in living young adult hermaphrodites by expression of the construct pie-1p::mCherry::PH(PLC1delta1). Shown are representative images. d) Quantification of (c) reveals that oocyte size is decreased in lat-1 mutants. This defect can be ameliorated by expression of the LNT. n ≥ 18. **P < 0.01; ***P < 0.001. e) Oocyte size determination by area measurements confirms data from (d). This way of measuring oocyte dimension was employed to supplement the volume measurements. Data from at least 5 independent experiments of area measurements, n ≥ 39, *P < 0.05. f) The LNT rescues embryonic lethality of lat-1 mutants to a small but significant extent, while amelioration by the full-length lat-1 construct is much stronger and is considered the maximum rescue that can be reached. Assays were performed in 4 independent experiments, n ≥ 30, ***P < 0.001.

Fig. 4.

Expression of lat-1 in the hermaphrodite gonad. a) The receptor is present in all somatic cells of the gonad as well as germ cells. b), d) lat-1 is expressed in the DTC. It is further localized c) in the spermatheca and e) in gonadal sheath cells. For visualizing lat-1 expression, either a CRISPR/Cas9 genome-edited single-copy integrated LNT::GFP (lat-1(1-581)::GFP) was used with signal amplification by an anti-GFP antibody (a–c) (for details, see ‘Materials and methods’) or a CRISPR/Cas9 genome-edited single-copy integrated lat-1p::mCherry (d, e) was employed. The DTC was visualized (d) by expressing lag-2p::myr::GFP and the gonadal sheath cells by using lim-7p::GFP (e). It should be noted that LAT-1 is not visible in germ cells in (d and e) likely due to the weak expression and the fact that no signal amplification by antibodies was employed here.

Fig. 5.

The LNT acts from somatic cells of the C. elegans gonad in a noncell autonomous manner. Tissue-specific expression of the LNT tethered to the membrane (LNT) using promoters with activity restricted to the DTC (lag-2p::LNT), the gonadal sheath cells (lim-7p::LNT), or the spermatheca (plc-1p::LNT) reveals whether a trans mode of the receptor fulfills its functions in different contexts on germ cells and in which location it is required. a) While expression of LNT in the DTC and the gonadal sheath cells generally results in a partial amelioration of the overall brood size defect of lat-1 mutants, sole expression in the spermatheca (plc-1p::LNT) does not. b) The ovulation defect 96 h after the first ovulation of worms lacking lat-1 is rescued by the LNT in both, the DTC and the gonadal sheath cells. c) Sperm migration to the spermatheca is rescued by the sole presence of LAT-1 neither in the DTC nor in the gonadal sheath cells. Data were acquired in at least three independent experiments with n > 20 biological replicates. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Note that some controls in this dataset (wild type, lat-1, lat-1; Ex[LNT]) are the same as in Fig. 2j. d) The increased apoptosis rate in lat-1 mutants is ameliorated by the presence of the receptor in the gonadal sheath cells, but not in the DTC. Numbers are given per gonad. Data from at least 3 independent experiments, n ≥ 28. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Note that the controls in these data (wild type, lat-1, lat-1; Ex[LNT]) are the same as in Fig. 3b. e) The decreased oocyte size observed in lat-1 mutant nematodes can be ameliorated by expressing the LNT in the gonadal sheath cells, but not in the DTC. Data from at least 5 independent experiments of area measurements, n ≥ 39. n.s., not significant; *P < 0.05. Note that the controls in these data (wild type, lat-1, lat-1; Ex[LNT]) are the same as in Fig. 3e.

Fig. 6.

Several transcripts containing only the N terminus of LAT-1 exist. a) Read coverage of the lat-1 locus extracted from available RNA-seq data of day 1 adult wild-type hermaphrodites (Chen et al. 2015) shows an almost uniform distribution of reads over the entire locus. b) Repertoire of lat-1 transcript variants identified from RNA-Seq data (Chen et al. 2015). Relative abundance of each transcript variants was derived from Fragments Per Kilobase of transcript per Million mapped reads values estimated by StringTie (Supplementary Table 2 in Supplementary File 1). Several full-length variants exist which mostly differ in their N-terminal composition. Variants containing only the N terminus seem mostly not to be membrane-anchored. Other variants, which lack the N terminus, constitute about 1%. Only full-length variants with an incidence of more than 0.01% are depicted. The genomic locus of LAT-1 is shown with its longest exons (large boxes) and size-condensed introns (faint lines). All exons found in the analysis are separately plotted above the locus (small blue boxes). The individual exon arrangements of transcripts are shown numbered. Transcripts were defined as a numeric sequence of exons. The longest bona fide ORF is depicted in thick boxes, while the nonprotein coding 5′ and 3′ UTRs are displayed thinner and lighter. 3′ end exons with minor differences in length but identical 5′ splice acceptor sites are considered as one 3′ end exon. Different compositions of the 5′ start exon, 3′ end exon, and/or exons are considered as individual variants. The exact positions of the exons forming the variants are given in Supplementary Table 4 in Supplementary File 1. c) 5′ RACE analyses of wild-type hermaphrodites rendered among others the full-length variant, which has been shown to be the most abundant one in RNA-seq analyses. Further, a variant comprising the N terminus including RBL and HRM domain, but not the GAIN domain was amplified. RBL, rhamnose-binding lectin domain; HRM, hormone-binding domain; GPS, GPCR proteolytic site; GAIN, GPCR autoproteolysis-inducing domain.