Oriented Cell Division in the <i>C</i>. <i>elegans</i> Embryo Is Coordinated by G-Protein Signaling Dependent on the Adhesion GPCR LAT-1

<div><p>Orientation of spindles and cell division planes during development of many species ensures that correct cell-cell contacts are established, which is vital for proper tissue formation. This is a tightly regulated process involving a complex interplay of various signals. The molecular mechanisms underlying several of these pathways are still incompletely understood. Here, we identify the signaling cascade of the <i>C</i>. <i>elegans</i> latrophilin homolog LAT-1, an essential player in the coordination of anterior-posterior spindle orientation during the fourth round of embryonic cell division. We show that the receptor mediates a G protein-signaling pathway revealing that G-protein signaling in oriented cell division is not solely GPCR-independent. Genetic analyses showed that through the interaction with a G_s protein LAT-1 elevates intracellular cyclic AMP (cAMP) levels in the <i>C</i>. <i>elegans</i> embryo. Stimulation of this G-protein cascade in <i>lat-1</i> null mutant nematodes is sufficient to orient spindles and cell division planes in the embryo in the correct direction. Finally, we demonstrate that LAT-1 is activated by an intramolecular agonist to trigger this cascade. Our data support a model in which a novel, GPCR-dependent G protein-signaling cascade mediated by LAT-1 controls alignment of cell division planes in an anterior-posterior direction via a metabotropic G_s-protein/adenylyl cyclase pathway by regulating intracellular cAMP levels.</p></div

An agonistic sequence C-terminal of the GPS cleavage site activates LAT-1 <i>in vitro</i>.

<p>(A) Evolutionary conservation of the putative agonistic region C-terminal of the GPCR proteolytic site (GPS) cleavage site in different latrophilin homologs. The GPS is part of the GPCR-autoproteolysis inducing (GAIN) domain, a characteristic feature of most aGPCRs which is located N-terminal of the first transmembrane domain (TM1). Sequences were retrieved from NCBI/GenBank and aligned utilizing Jalview 2 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005624#pgen.1005624.ref036" target="_blank">36</a>]. (B, F) Peptide-stimulated cAMP response of LAT-1 (B) and rat LPHN1 (F). COS-7 cells transfected with 0.2 μg empty control vector (pcDps) or plasmid encoding the latrophilin homolog, respectively, were stimulated with 1 mM peptide and cAMP levels were measured by cAMP accumulation assay. A mutated peptide served as negative control (CMP12/RMP13). As control for peptide specificity, the human vasopressin type 2 receptor (V₂R) was used, which does not respond to any of the peptides tested. Basal cAMP levels (empty vector, no peptide) are 5.3 ± 1.3 nM. Data are given as means ± SD of five independent experiments, each performed in triplicate. * p < 0.05; ** p < 0.01. (C) LPHN1 is expressed in COS-7 cells. Cells were transfected with 500 ng/well of empty vector (pcDps) or plasmid encoding either rat LPHN1, human P2Y₁₂, or human V₂R. After 48 hours, surface expression levels were determined using surface ELISA and displayed as percentage of positive control P2Y₁₂. Expression of the human vasopressin type 2 receptor V₂R is shown as a comparison. The non-specific OD value (empty vector) was 0.06 ± 0.03 (set 0%) and the OD value of P2Y₁₂ was 1.28 ± 0.12 (set 100%). Data are given as means ± SD of at least three independent experiments. *** p < 0.001. (D) LPHN1 does not yield a basal signal for Gα_i- or Gα_q-protein coupling. Testing G_i and G_q protein-signal specificity of rat LPHN1, IP accumulation assays were performed to detect G_q protein-mediated increase in IP levels. To analyze functional G_i coupling, the chimeric Gα_qi4 protein was applied to reroute a potential G_i-protein pathway to the G_i protein-mediated signaling cascade. For each assay, 1.5 μg/well of plasmid encoding LPHN1 was transfected (for G_i-protein coupling, co-transfection with 100 ng of chimeric Gα_qi4 protein was performed). No activation of any of the signaling pathway was observed. Basal IP levels are: 220 ± 34 CPM/well (empty vector); 234 ± 39 CPM/well (empty vector + Gα_qi4). Data are given as means ± SD of three independent experiments, each performed in triplicate. n.s. not significant. (E) LPHN1 couples to Gα_s. COS-7 cells were transfected with increasing amounts of empty control vector (pcDps) or plasmid encoding either rat LPHN1, human V₂R or human P2Y₁₂, and cAMP levels were measured after 48 hours. cAMP concentrations are shown as fold change over empty control vector, basal cAMP levels: 6.2 ± 2.3 nM (200 ng); 6.1 ± 2.5 nM (300 ng); 5.1 ± 2.6 nM (400 ng); 6.1 ± 4.5 nM (500 ng). The G_s-protein coupling V₂R served as positive and the predominantly G_i-protein coupling P2Y₁₂ as negative control. Data are given as means ± SD of three independent experiments, each performed in triplicate. *** p < 0.001.</p

The agonistic sequence of LAT-1 triggers receptor function <i>in vivo</i>.

<p>(A) Domain architecture of LAT-1 with an exchange of the GPS for the LAT-2 GPS. (B) Peptide-mediated rescue of lethality in <i>lat-1</i> mutant nematodes expressing <i>lat-1</i> with a <i>lat-2</i> GPS. Mothers and subsequently offspring were soaked in 0.1 mM of peptide and individuals reaching adulthood were scored (n ≥ 350). No response to any peptide was observed in <i>lat-1</i> null mutant animals. Negative controls: mutated peptide CMP12 and a peptide derived from rat LPHN1 (RP13). Data are shown as means ± SD, **p<0.01; ***p<0.001. (C) Transgenically expressed <i>lat-1</i>^{<i>T530A/F532A</i>}::<i>gfp</i> exclusively rescues fertility defects of <i>lat-1</i> mutants. Nematodes expressing <i>lat-1</i>^{<i>T530A/F532A</i>}::<i>gfp</i> in a <i>lat-1</i> mutant background display lethality but no fertility defects compared to a <i>lat-1</i>::<i>gfp</i> transgenic control. (D) Expression and protein localization of <i>lat-1</i>::<i>gfp</i> (top) is indistinguishable from <i>lat-1</i>^{<i>T530A/F532A</i>}::<i>gfp</i> (bottom). Arrows indicate expression.</p

Division plane defects in <i>lat-1</i> mutant embryos are rescued by elevation of intracellular cAMP via a G_s protein/adenylyl cyclase pathway.

<p>(A-D) <i>gsa-1</i> RNAi in AB⁴ embryos reveals a similar phenotype to that of <i>lat-1</i>, scale bars = 10 μm. In wild-type embryos treated with the RNAi control vector L4440 ABal divides in an anterior-posterior direction (A) whereas in treated <i>lat-1</i> mutants the division plane is almost perpendicular to MS (B). <i>gsa-1</i> RNAi by feeding over two generations leads to a turning of the F2 ABal division plane in a direction similar to that in <i>lat-1</i> mutants (C). F2 <i>lat-1; gsa-1(RNAi)</i> embryos show a similar ABal division plane to <i>lat-1</i> mutants (D). (E) Cell division plane angles of ABal relative to MS after knockdown of <i>gsa-1</i>. Feeding of <i>gsa-1</i> RNAi on wild-type nematodes resulted in an ABal division plane angle significantly different to the one of control vector-fed wild-type embryos in the F2 generation. Embryos of the F1 generation did not show a significant different angle. Knockdown of <i>gsa-1</i> in <i>lat-1</i> mutants in the F2 generation did not change division plane angles. The vector L4440 served as negative control. Data are shown as means ± SD. * p < 0.05; *** p < 0.001; n ≥ 11. (F) cAMP levels are reduced in <i>lat-1</i> embryos compared to a control wild-type population. cAMP levels were measured after lysis (15 μg protein) by cAMP accumulation assay. Assays were performed in three independent experiments, data are given as means ± SD. ** p < 0.01. (G) Rescue of developmental lethality in <i>lat-1</i> nematodes treated with compounds promoting cAMP accumulation: forskolin (80 μM), IBMX (10 mM) and 8-Br-cAMP (0.25 mM). Mothers and subsequently offspring of wild-type and <i>lat-1</i> individuals were incubated and individuals reaching adulthood were scored (n = 410). As controls, nematodes were incubated in solvent without drug (mock). Data are shown as means ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001. (H) Forskolin increases cAMP levels in <i>C</i>. <i>elegans</i> embryos and adults. <i>lat-1</i> mutants and subsequently embryos were incubated with 80 μM forskolin. Embryos displayed an increase in cAMP levels compared to control animals. Similarly, mutant individuals at the L4 stage also showed an elevation in cAMP concentration. cAMP levels were measured after lysis by cAMP accumulation assay. As controls, nematodes were treated with 0.8% DMSO lacking forskolin (mock). Basal cAMP levels of mock control are: 0.70 ± 0.14 nM (wild-type embryos); 0.37 ± 0.19 nM (<i>lat-1</i> embryos); 1.9 ± 0.7 nM (wild-type adults); 2.3 ± 0.8 nM (<i>lat-1</i> adults). Assays were performed in three independent experiments, data are given as means ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001. (I-L) Relative positions of ABala, ABalp, and MS in an AB⁴ embryo. Schematic representation (top) and DIC microscopy (bottom), scale bars = 10 μm. In wild-type embryos (I) and embryos incubated in 80 μM forskolin for 120 minutes (K) ABal divides in an anterior-posterior direction with only ABalp forming an interface with MS. In <i>lat-1</i> mutants the division plane is skewed resulting in ABala and ABalp contacting MS (J). <i>lat-1</i> embryos treated with 80 μM forskolin for 120 minutes display wild-type division and cell interfaces (L). (M) Arrangement of blastomeres in the 12-cell stage embryo. 3D representation of the data shown in Fig 2I-L using a wild-type, a <i>lat-1</i> embryo and a <i>lat-1</i> embryo treated with 80 μM forskolin (fsk) show that defects in anterior-posterior division plane alignment in <i>lat-1</i> mutant embryos are changed towards wild-type cleavage orientations upon forskolin treatment. Left-hand side shows the positions of the 8-AB descendants and the MS blastomere are shown for the individual embryos. The right-hand side is two views of the mean positions of the blastomeres. The spheres are enlarged to convey an impression of the contacts. Forskolin turns the ABal spindle in the mutant in a more anterior direction. (N) Cell division plane angles of ABal relative to MS of untreated wild-type/<i>lat-1</i> controls and wild-type/<i>lat-1</i> embryos after incubation of mothers and subsequently embryos for 48 hours in 80 μM forskolin. Upon forskolin treatment, mutant embryos display a wild-type division angle. As controls, nematodes were treated with 0.8% DMSO lacking forskolin (mock). Data are shown as average angles ± SD, *** p < 0.001; n ≥ 12. (O) Forskolin reduces the contact length of ABala to MS. Ratio of relative contact lengths of ABala to MS and ABalp to MS cells <i>lat-1</i> embryos untreated (control, n = 10) and after incubation of mothers and subsequently embryos for 48 hours in 80 μM forskolin (n = 11). In wild-type (n = 10) and forskolin-treated wild-type (n = 11) embryos ABala does not contact MS. Data were calculated from pixel analyses utilizing DIC images and are shown as means ± SD, ** p < 0.01.</p

LAT-1 couples to Gα_s, but not to Gα_i or Gα_q.

<p>(A) COS-7 cells were transfected with 500 ng of empty vector (pcDps) or plasmid encoding either human ADP receptor P2Y₁₂, the human vasopressin type 2 receptor V₂R or LAT-1. 48 hours post transfection, surface expression levels were determined with a cell surface ELISA. Expression of the GPCR human vasopressin type 2 receptor V₂R is shown as a comparison. Data are displayed as percentage of P2Y₁₂ (positive control) and given as means ± SD of five independent experiments, each performed in triplicate. The non-specific OD value (empty vector) is 0.02 ± 0.01 (set 0%) and the OD value of P2Y₁₂ is 0.95 ± 0.05 (set 100%). *** p < 0.001. (B) To test for functional coupling of LAT-1 to G_s proteins, COS-7 cells were transfected with increasing amounts of empty control vector (pcDps) or plasmid encoding LAT-1, human vasopressin type 2 receptor V₂R, or human P2Y₁₂, respectively, and cAMP levels were measured 48 hours later by cAMP accumulation assay. cAMP concentrations are shown as fold change over empty control vector, cAMP levels: 6.2 ± 2.3 nM (200 ng); 6.1 ± 2.5 nM (300 ng); 5.1 ± 2.6 nM (400 ng); 6.1 ± 4.5 nM (500 ng). LAT-1 but not P2Y₁₂ causes an increase in cAMP levels. The G_s-protein coupling V₂R served as a positive control and the predominantly G_i-protein coupling P2Y₁₂ as negative control. Data are given as means ± SD of five independent experiments, each performed in triplicate. * p < 0.05; *** p < 0.001. (C) For analyses of G_q-, G_i- and G_s-protein coupling, IP accumulation assays were performed detecting Gα_q-mediated increase in IP levels. To measure functional coupling of LAT-1 to Gα_i, a chimeric Gα_qi4 protein was applied to reroute a G_i-mediated signal to a Gα_q-mediated pathway. Similarly, to validate G_s-protein coupling IP accumulation assays were performed using a Gα_qs4 chimera. For each assay, 1.5 μg of plasmid containing <i>lat-1</i> cDNA were transfected (for G_i- and G_s-coupling, co-transfection with 100 ng of the respective chimeric protein was applied). No signal was detected for G_q or G_i protein-signaling pathways, but for G_s-protein coupling. Basal IP levels are: 220 ± 34 CPM/well (empty vector); 306 ± 20 CPM/well (empty vector + Gα_qi4); 394 ± 53 CPM/well (empty vector + Gα_qs4). Data are given as means ± SD of five independent experiments, each performed in triplicate. n.s. not significant; * p < 0.05.</p

Model for LAT-1 signaling in the early embryo.

<p>LAT-1 resides in an inactive state while the <i>Stachel</i> sequence is not interacting with the 7TM domain. Upon structural changes of the N terminus e.g. binding of an as yet unknown extracellular ligand, the tethered agonist contacts the 7TM domain (1). The G_s protein, likely to be GSA-1, then activates the adenylyl cyclase (2) resulting in an increase of cAMP levels (3). This signal then promotes coordination of anterior-posterior cleavage plane orientation after the fourth round of cell division (4). The cAMP signal within the cell is non-polar.</p