Loss of the canonical spindle orientation function in the Pins/ LGN homolog AGS 3
2017; Springer Nature; Volume: 18; Issue: 9 Linguagem: Inglês
10.15252/embr.201643048
ISSN1469-3178
AutoresMehdi Saadaoui, Daijiro Konno, Karine Loulier, Rosette Goïame, Vaibhav Jadhav, Marina Mapelli, Fumio Matsuzaki, Xavier Morin,
Tópico(s)Mechanical Engineering and Vibrations Research
ResumoScientific Report6 July 2017free access Transparent process Loss of the canonical spindle orientation function in the Pins/LGN homolog AGS3 Mehdi Saadaoui Corresponding Author [email protected] Cell Division and Neurogenesis Group, Ecole Normale Supérieure, CNRS, Inserm, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), PSL Research University, Paris, France Search for more papers by this author Daijiro Konno Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Japan Search for more papers by this author Karine Loulier UPMC Université Paris 06, Sorbonne Universités, CNRS, Inserm, Institut de la Vision, Paris, France Search for more papers by this author Rosette Goiame Cell Division and Neurogenesis Group, Ecole Normale Supérieure, CNRS, Inserm, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), PSL Research University, Paris, France Search for more papers by this author Vaibhav Jadhav Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Marina Mapelli orcid.org/0000-0001-8502-0649 Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Fumio Matsuzaki Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Japan Search for more papers by this author Xavier Morin Corresponding Author [email protected] orcid.org/0000-0001-9999-0355 Cell Division and Neurogenesis Group, Ecole Normale Supérieure, CNRS, Inserm, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), PSL Research University, Paris, France Search for more papers by this author Mehdi Saadaoui Corresponding Author [email protected] Cell Division and Neurogenesis Group, Ecole Normale Supérieure, CNRS, Inserm, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), PSL Research University, Paris, France Search for more papers by this author Daijiro Konno Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Japan Search for more papers by this author Karine Loulier UPMC Université Paris 06, Sorbonne Universités, CNRS, Inserm, Institut de la Vision, Paris, France Search for more papers by this author Rosette Goiame Cell Division and Neurogenesis Group, Ecole Normale Supérieure, CNRS, Inserm, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), PSL Research University, Paris, France Search for more papers by this author Vaibhav Jadhav Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Marina Mapelli orcid.org/0000-0001-8502-0649 Department of Experimental Oncology, European Institute of Oncology, Milan, Italy Search for more papers by this author Fumio Matsuzaki Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Japan Search for more papers by this author Xavier Morin Corresponding Author [email protected] orcid.org/0000-0001-9999-0355 Cell Division and Neurogenesis Group, Ecole Normale Supérieure, CNRS, Inserm, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), PSL Research University, Paris, France Search for more papers by this author Author Information Mehdi Saadaoui *,1,†, Daijiro Konno2, Karine Loulier3, Rosette Goiame1, Vaibhav Jadhav4, Marina Mapelli4, Fumio Matsuzaki2 and Xavier Morin *,1 1Cell Division and Neurogenesis Group, Ecole Normale Supérieure, CNRS, Inserm, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), PSL Research University, Paris, France 2Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Japan 3UPMC Université Paris 06, Sorbonne Universités, CNRS, Inserm, Institut de la Vision, Paris, France 4Department of Experimental Oncology, European Institute of Oncology, Milan, Italy †Present address: Morphogenesis in Higher Vertebrates, Developmental and Stem Cell Biology, Institut Pasteur, Paris, France *Corresponding author. Tel: +33 1 45 68 81 54; E-mail: [email protected] *Corresponding author. Tel: +33 1 44 32 37 29; E-mail: [email protected] EMBO Rep (2017)18:1509-1520https://doi.org/10.15252/embr.201643048 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In many cell types, mitotic spindle orientation relies on the canonical “LGN complex” composed of Pins/LGN, Mud/NuMA, and Gαi subunits. Membrane localization of this complex recruits motor force generators that pull on astral microtubules to orient the spindle. Drosophila Pins shares highly conserved functional domains with its two vertebrate homologs LGN and AGS3. Whereas the role of Pins and LGN in oriented divisions is extensively documented, involvement of AGS3 remains controversial. Here, we show that AGS3 is not required for planar divisions of neural progenitors in the mouse neocortex. AGS3 is not recruited to the cell cortex and does not rescue LGN loss of function. Despite conserved interactions with NuMA and Gαi in vitro, comparison of LGN and AGS3 functional domains in vivo reveals unexpected differences in the ability of these interactions to mediate spindle orientation functions. Finally, we find that Drosophila Pins is unable to substitute for LGN loss of function in vertebrates, highlighting that species-specific modulations of the interactions between components of the Pins/LGN complex are crucial in vivo for spindle orientation. Synopsis Using planar divisions in the vertebrate neuroepithelium as a model, the authors explore the proposed conservation of a “spindle orientation” function between LGN and AGS3, two homologs of the Drosophila G-protein regulator Pins. The study uncovers multiple differences that contribute to the functional divergence of AGS3. LGN localizes at the cell cortex in different cell types where spindle orientation relies on its function, while AGS3 remains cytoplasmic. A systematic dissection shows that despite extensive sequence and structure conservation, each of the three TPR, Linker and GPR modules has diverged functionally between LGN and AGS3. Despite their shared role in spindle orientation in fly and vertebrates, Pins and LGN are not functionally interchangeable in vivo. Introduction Oriented cell divisions (OCD) play an essential role in the development, growth, and homeostasis of many tissues 12. They rely on the specific orientation of the mitotic spindle during mitosis, which controls the position of the cleavage plane and hence the position of the two daughter cells within the tissue. In many cell types, OCD relies on an evolutionary conserved protein complex composed of Pins/LGN, Mud/NuMA, and Gαi subunits of heterotrimeric G proteins. In response to intra- or extracellular polarity cues, a local enrichment of this complex at specific cortical regions of the dividing cell is used to recruit motor proteins of the dynein/dynactin complex 345. This creates an imbalance in cortical forces exerted on astral microtubules and drives mitotic spindle movements and orientation 16. A conserved role for Pins and its homolog LGN 7 in mitotic spindle orientation has been largely documented in Drosophila and vertebrates 89101112. In particular, mouse LGN was able to substitute for Pins and rescue both spindle orientation and associated asymmetric cell division defects of embryonic neuroblasts in a Drosophila pins mutant background 13; in addition to this role in spindle orientation, mouse LGN has been involved in the regulation of G protein-activated inwardly rectifying potassium (GIRK) channels 14, and in regulating spine density in cortical neurons 15, a function that requires its ability to interact with MAGUK proteins of the Dlg family. LGN is also essential for the establishment of the planar polarization and the organization of stereocilia bundles in cochlear hair cells 161718, and LGN mutations have been associated with deafness in mice and humans 192021. In addition to LGN, the canonical Drosophila Pins possesses another homologous gene in vertebrates named AGS3 22. AGS3 has been studied in a number of cell types in vitro and in a mouse loss-of-function model, and implicated in a diverse set of cellular, organ, and physiological functions, ranging from autophagy, Golgi apparatus organization, protein trafficking, and drug craving, but a clear picture of its cellular function has yet to emerge 23. In addition, LGN and/or AGS3 show polarized recruitment and may be functionally involved in heterotrimeric G-protein-dependent chemotaxis of mouse neutrophils 24. All three genes belong to the type II class of receptor-independent activator of G-protein signaling (AGS) family 23. They share extensive sequence, structure homology, and biochemical interactions (Fig 1A) 13. Their N-terminal TPR domain (containing eight tetratricopeptide repeats, seven of which contain a leucine–glycine–asparagine motif which gave LGN its name) is involved in multiple protein–protein interactions, and in particular in the interaction with NuMA, which is crucial for the spindle orientation function. The C-terminal GPR (G-protein regulatory) region contains three (in Pins) or four (in LGN and AGS3) GoLoco motifs; GoLoco are 15- to 20-aa Gαi/o-interacting domains with a guanine dissociation inhibitory activity 2526. Within the GPR region, GoLoco motifs are separated by 11- to 25-aa-long sequences that are thus far thought to mainly serve as spacers allowing the simultaneous interaction of the GPR region with multiple Gαi subunits 23. A less conserved linker region separates the TPR and GPR domains. Recently, we have shown that the direct interaction between the phosphorylated linker domain of LGN and the baso-lateral protein Dlg1/SAP97 is crucial for mitotic spindle orientation in chick neuroepithelial progenitors and cultured HeLa cells 27, a function that is conserved in its ortholog Dlg in some fly epithelial tissues 28. Figure 1. The LGN homolog AGS3 is cytoplasmic and does not regulate mitotic spindle orientation in the vertebrate neuroepithelium A. LGN/AGS3 protein structure and functional domains required for interaction with NuMA, Dlg1, and Gαi. The black cross and question mark, respectively, point toward absent or uncharacterized interaction. B. Spindle orientation in anaphase is normal in radial glial cells of AGS3ΔC mice at E14.5 (mean ± SEM, n = 41 and 51 cells from 3 and 4 embryos, respectively; ns = not significant, Mann–Whitney test). C. In mouse radial glial cells at E14.5, both endogenous LGN and a GFP-mLGN fusion protein accumulate at the cell cortex whereas GFP-mAGS3 is cytoplasmic throughout mitosis. D, E. GFP-mLGN is cortical and GFP-mAGS3 is cytoplasmic in dividing chick neural progenitors at E3 (D) and in MDCK cells (E). F. Z-view examples of typical spindle orientation observed upon LGN RNAi and rescue experiments with different LGN and AGS3 constructs in chick neural progenitors at E3. The dashed lines indicate the apical surface and the solid lines the mitotic spindle angle. G, H. Quantification of mitotic spindle angles in LGN RNAi rescue experiment (G) or after misexpression in a wt background (H) in the chick spinal cord (mean ± SEM, n > 50 cells from at least three different embryos per condition; ns = not significant, ****P < 0.0001, Mann–Whitney test). Data information: Scale bars: 5 μm in all panels except panel (E) (10 μm). Download figure Download PowerPoint AGS3 and LGN probably appeared through the duplication of a common Pins-like ancestor and they have clearly evolved new and different functions, through the acquisition of specific interactions 23. This raises the question of whether only one of them, or both, retained the capacity to control spindle orientation. Interestingly, while the linker region of AGS3 does not interact with Dlg family members 1529, one previous study proposed a role for AGS3 during mitotic spindle orientation in the mouse embryonic cortex 30, but the mechanism is unclear. In this study, we explored whether LGN and AGS3 have both retained the “spindle orientation” function of their common ancestor. We first observed that AGS3 is not required for planar spindle orientation in a mouse knockout line, and that AGS3 is not recruited to the cell cortex in mouse neural progenitors. We then used the LGN-dependent planar divisions of neural progenitors in the chick neural tube as an in vivo model to compare the functions of LGN and AGS3, and found that AGS3 cannot substitute for LGN. In-depth analysis through multiple AGS3/LGN chimeras showed that differences in several functional domains contribute to AGS3's loss of the ability to control mitotic spindle orientation. Remarkably, we also found that Drosophila Pins was unable to substitute for LGN in vertebrates, despite the interchangeability of both molecules in the fly. Our study suggests that despite similar binding affinities observed between LGN and AGS3 and their common interaction partners in vitro, other as yet unidentified partners are required to allow these interactions to occur in vivo. Results and Discussion AGS3 is cytoplasmic during progenitor division and is not able to rescue LGN loss of function Since LGN and AGS3 share extensive domain composition and sequence homology (Fig 1A), it has been proposed that both molecules may share functional properties and that both could be involved in spindle orientation. We generated a mouse AGS3 mutant strain lacking the GPR region (AGS3ΔC, Fig EV1) and investigated spindle orientation in mouse cortical progenitors. Remarkably, the orientation of divisions in anaphase was undistinguishable between control and AGS3ΔC mutant cells (Fig 1B), in agreement with our previous observations using siRNA (unpublished and 8) but in contrast to a previous report 30. To better characterize this functional difference, we compared the localization of GFP-LGN and GFP-AGS3 fusion proteins during mitosis in several cell types known to rely on LGN for their spindle orientation 8911. In radial glial cells of the mouse embryonic cortex, in chick embryonic spinal cord neural progenitors, and in MDCK cells, AGS3 failed to be recruited to the cell cortex during mitosis (Fig 1C–E). By contrast, LGN was consistently enriched at the cortex in all three cell types, consistent with its well-documented role in recruiting cortical force generators and its requirement for planar cell division in both mouse and chick neural progenitors. The knockout phenotype and the different localization of the two molecules suggest that AGS3 and LGN do not play a redundant role in spindle orientation in these cells. Click here to expand this figure. Figure EV1. Strategy used to generate the AGS3ΔC mutant mouse strain Download figure Download PowerPoint We previously reported that full-length AGS3 is not produced in the chick, due to a frame shift in the coding sequence 9. We thought of using this characteristic to our advantage by exploring LGN and AGS3 functional divergences in the chick neural tube. Throughout this study, we used mouse AGS3 and mouse LGN cDNAs in functional and localization experiments in vivo. Subcellular distribution was performed in dividing chick neuroepithelial progenitors by in ovo electroporation using low-level expression of GFP-tagged molecules from the weak CMV promoter, in order to avoid saturation of the cell and potential localization artifacts. By contrast, rescue and gain-of-function experiments were performed with “high-level” expression from the strong CAG promoter. In particular, we have previously shown that mouse LGN is able to rescue the LGN RNAi phenotype in the chick neural tube 9 (see also Fig 1F and G). Besides, whereas in vitro studies have described that overexpression of LGN induced defects in spindle orientation relative to the adhesion substrate in HeLa cells 4 and exaggerated spindle rocking in MDCK cells 31, we reported that overexpression of mouse LGN in vivo from the CAG promoter does not prevent planar spindle orientation in the neural tube 9 (see also Fig 1H). In agreement with its cytoplasmic distribution in mitotic neuroepithelial cells, AGS3 was unable to substitute for LGN and failed to rescue spindle orientation defects resulting from LGN knockdown, whereas expression of LGN restored planar spindle orientation (Fig 1F and G for quantification). In addition, we controlled whether AGS3 misexpression may exert a dominant negative effect: like LGN, AGS3 expression in a wild-type (wt) background did not cause any spindle orientation defect (Fig 1H). AGS3 and LGN may compete for the interaction with Gαi subunits at the cell cortex: We therefore analyzed the distribution of GFP-AGS3 in an LGN RNAi background. However, even in the absence of LGN, AGS3 remained cytoplasmic in chick neuroepithelial cells (Fig EV3A). As the two proteins share strong sequence homology, domain composition, and extensive structural similarities, we decided to explore the molecular basis for this functional difference. The linker domain of LGN does not confer the ability to regulate mitotic spindle orientation to AGS3 We have previously established that a direct interaction between LGN and the baso-lateral protein Dlg1 is necessary for the mitotic spindle orientation function of LGN in the chick neural tube 27. This interaction relies on the 130-amino-acid-long linker domain of LGN, and in vitro experiments have defined a short peptide of 18 aa containing a core RRHpS motif inside the linker as a binding interface between LGN and the C-terminal guanylate kinase (GK) domain of Dlg (Fig 2A) 29. Neither the AGS3 linker nor a short AGS3 peptide encompassing the same region is able to interact with Dlg 1529. Phosphorylation of the serine in the RRHpS motif is essential for interaction with Dlg GK domain in vitro 29, and a serine to alanine substitution in this motif strongly reduces the cortical recruitment of full-length LGN and abolishes its spindle orientation capability in vivo 27. The crucial serine residue is conserved between LGN and AGS3. However, the arginine (R) residue localized at position –3, an essential element of the consensus R-X-X-S/T sequence recognized by multiple kinases, is replaced by a glutamine (Q) in AGS3; besides, an alanine substitution at this position in the PhosphoLGN peptide caused a fivefold decrease in the binding affinity between LGN and Dlg, even though the peptide contained a phosphorylated serine in these experiments 29. We therefore reasoned that the ability of AGS3 to be phosphorylated on this particular serine residue is impaired, but that a Q-R substitution in AGS3 may restore phosphorylation of the linker domain, promote interaction with Dlg1, and confer LGN-like spindle orientation properties to AGS3. However, a GFP fusion to AGS3QR still displayed a cytoplasmic localization in neuroepithelial cells (Fig 2B), and AGS3QR was unable to rescue spindle orientation defects in an LGN knockdown background (Fig 2C and D). We then swapped increasing parts of the linker domain between LGN and AGS3 (Figs 2 and EV2). To our surprise, even a complete replacement of the linker did not change the cytoplasmic localization of the GFP-tagged AGS3LGN-linker chimera (Fig 2B). Likewise, AGS3LGN-linker expression did not rescue spindle orientation in an LGN RNAi background (Fig 2C and D). Hence, the inability of AGS3 to interact with Dlg1 is not sufficient to explain its inability to control spindle orientation. Figure 2. The linker domain of LGN does not confer mitotic spindle activity to AGS3 Linker domain sequence alignment of four representative Pins homologs. Green star: phosphorylated serine residue (in Pins and LGN); yellow star: conserved arginine residue in the RRxpS consensus kinase recognition motif. In AGS3, this arginine is replaced with a glutamine residue; black dotted box: peptide sequence used for binding studies in 29; pink dotted box: short linker sequence used in the mAGS3LGNsLinker chimeric construct. Species abbreviations are: Dm Drosophila melanogaster, Gg Gallus gallus, Mm Mus musculus. Replacing increasing portions of AGS3 linker domain with LGN-specific residues is not sufficient to induce cortical localization of GFP-AGS3 fusion constructs in dividing neural progenitors. A scheme above each construct name depicts the structure of the chimeras. Z-view examples of typical spindle orientation observed for each condition. The dashed lines indicate the apical surface and the solid lines the mitotic spindle angle. Quantification of the mitotic spindle angles (mean ± SEM, n > 60 cells; ****P < 0.0001, Mann–Whitney test). Data information: Scale bars: 5 μm in all panels. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. A summary of all the constructs used in this study Download figure Download PowerPoint In line with this conclusion, we also found that overexpression of AGS3QR and AGS3LGN-linker, respectively, caused mild—but significant—and strong spindle misorientation (Fig EV3B). This dominant effect may be explained by a competition between these constructs and endogenous LGN for the interaction with Dlg1 combined to their inability to interact with one or more other LGN partners. Consistent with this hypothesis, expression of AGS3LGN-linker led to the cytoplasmic accumulation of a GFP-Dlg1 construct compared to the control situation, although GFP-Dlg1 was still observed at the cell cortex (Fig EV3C). We therefore set out to identify other important differences between LGN and AGS3. Click here to expand this figure. Figure EV3. Cellular phenotypes associated with the overexpression of AGS3LGNlinker Two mitotic cells with low and high GFP-AGS3 (green) in a cLGN RNAi background in the chick neuroepithelium. mRFP (red) reports electroporation with the LGN miRNA. AGS3 remains cytoplasmic upon cLGN RNAi. Expression of Myc-tagged mAGS3 in the chick neuroepithelium has no effect on planar spindle orientation. By contrast, expression of a Q-R mutant of mAGS3 or a chimeric construct in which the mAGS3 linker sequence is replaced with the mLGN linker, respectively, induce mild and strong dominant spindle misorientation defects (mean ± SEM, n > 46 cells; ns = not significant, ****P < 0.0001, Mann–Whitney test). Overexpression of the AGS3LGNlinker construct in the chick neuroepithelium results in more cytoplasmic GFP-cDlg1 compared to the control situation. Left panel shows representative images; right panel shows the cortical/cytoplasmic ratio of GFP-Dlg1 intensity in cells co-electroporated with either an empty control vector (Ctrl), a vector expressing a 6-Myc-tag (Myc), a vector expressing Myc-tagged AGS3LGN-linker, or a vector expressing Myc-tagged LGNAGS3-GPR (see also Fig 3E for images of GFP-Dlg1 in LGNAGS3-GPR cells). Data show mean ± SEM from n = 20 cells (Ctrl), n = 16 cells (Myc), n = 15 cells (AGS3LGN-linker), and n = 17 cells (LGNAGS3-GPR) with similar level of cortical GFP-Dlg1 intensity. **P < 0.01, ****P < 0.0001, one-way ANOVA test with multiple comparisons. Data information: Scale bars: 5 μm in all panels. Download figure Download PowerPoint Differential cortical recruitment of the GPR domains of LGN and AGS3 in mitotic progenitors in vivo We have previously shown that both linker and GPR domains of LGN are required for its cortical recruitment. In particular, a mutated LGN that is unable to interact with Dlg1 (LGNSA) still displays residual cortical recruitment 2732. By contrast, we did not detect any cortical staining with AGS3 (Figs 1B and 2B). This suggested that the GPR domain of AGS3 may not be able to mediate cortical recruitment and led us to ask whether the GPR domains of LGN and AGS3 are functionally distinct. We generated a chimeric construct in which the GPR domains of LGN were replaced by those of AGS3 (LGNAGS3-GPR). A GFP-tagged version of this chimera displayed a cytoplasmic localization, both in mouse radial glial cells (Fig 3A) and in chick neuroepithelial progenitors (Fig 3B). Remarkably, co-expression of Gαi led to a strong cortical recruitment of LGNAGS3-GPR in mouse radial glial cells (Fig 3A). This suggested that AGS3 GPR domain does not interact with cortical Gαi subunits in vivo. Accordingly, the chimera was unable to rescue the LGN loss-of-function phenotype in the chick neuroepithelium (Fig 3C). In addition, when overexpressed in a wild-type background, LGNAGS3-GPR caused a dominant spindle misorientation phenotype (Fig 3D) similar to the dominant effect of the AGS3LGN-linker construct described above (Fig EV3B). Remarkably, when Myc-tagged LGNAGS3-GPR was expressed in combination with GFP-LGNwt, the latter was poorly recruited to the cell cortex (Fig 3E); this suggests that the dominant phenotype results from a competition with endogenous LGN for interaction with other partners, most likely Dlg1 but also possibly NuMA, reducing LGN cortical recruitment and preventing the formation of a functional Gαi/LGN/NuMA complex at the cortex. Indeed, GFP-Dlg1 was also enriched in the cytoplasm in these cells (Figs 3E and EV3C). The inability of the chimera to localize to the cortex could be due to its failure to switch from an inactive, closed state (where TPR and GPR domains interact together intramolecularly and bind poorly to Gαi and NuMA) to an open active conformation 3133, or to a reduced affinity of the GPR toward Gαi. We addressed the second point by comparing the distribution of GFP-tagged GPR domains alone from LGN and AGS3 during progenitor division. Whereas GPRLGN was enriched at the cell cortex as reported previously 27, GPRAGS3 was cytoplasmic (Fig 3F, upper panels). Figure 3. GPR domains of LGN and AGS3 are functionally distinct in vivo A, B. Replacing the GPR domains of LGN with those of AGS3 (LGNAGS3-GPR chimera) delocalizes LGN from cell cortex to cytoplasm in both mouse (A) and chick (B) neural progenitors. However, overexpressed Gαi1 recruits LGNAGS3-GPR to the cell membrane (A). C. LGNAGS3-GPR is unable to rescue spindle orientation defects in an LGN RNAi background (mean ± SEM, n > 60 cells; ns = not significant, Mann–Whitney test). D. Ectopic expression of LGNAGS3-GPR randomizes spindle orientation in a wt background (mean ± SEM, n = 85 cells; ****P < 0.0001, Mann–Whitney test). E. GFP-LGN is partially delocalized from cell cortex to cytoplasm, and GFP-Dlg1 is stronger in the cytoplasm upon expression of LGNAGS3-GPR. F, G. Localization of GPR domain fusion constructs reveals that binding ability of GoLoco domains to cortical Gαi-GDP requires specific interdomain sequences. AGS3 interdomains induce cytoplasmic localization while LGN interdomains lead to cortical enrichment of LGN/AGS3 GPR domains. The ratio between cortical and cytoplasmic distribution of the different constructs is provided in (G) (see Materials and Methods for detail of quantification; mean ± SEM, n > 8 cells/condition; ns = not significant, **P < 0.01, one-way ANOVA test with multiple comparisons). H. A full-length LGN with AGS3 GoLoco domains (Myc-LGNAGS3-GoLoco) is cortical, (top), whereas full-length LGN with AGS3 interdomains (Myc-LGNAGS3-interdomain) is cytoplasmic (bottom). None of these constructs rescues the LGN RNAi spindle orientation phenotype (mean ± SEM, n > 40 cells; ns = not significant, Mann–Whitney test). I. Overexpressed wt or GDP-bound G203A mutant forms or Gαi1 recruits wt AGS3 to the cell membrane. Data information: Scale bars: 5 μm in all panels. The dashed lines indicate the apical surface and the solid lines the mitotic spindle angle (C, D). Download figure Download PowerPoint Experiments in vitro have reported that individual GoLoco motifs from AGS3 and LGN present similar binding affinity toward Gαi subunits. While the core 19 amino acid GoLoco motif only shows weak affinity, experiments with “extended” GoLoco sequences including six to 35 additional amino acids C-terminal to the core GoLoco motif have shown that these downstream sequences modulate the binding of individual GoLoco motifs 3435, indicating that they may contribute to the global affinity of the protein toward Gαi. However, the in vitro affinity of the whole purified GPR domains toward Gαi appeared very similar between LGN and AGS3, although marginally higher for LGN 36. We therefore wondered whether a conformational difference related to the sequences separating individual GoLoco motifs (thereafter called interdomains) in LGN and AGS3 might be responsible for differential accessibility to cortical Gαi subunits in vivo. To test this idea, we generated complete chimeras within the GPR domains of AGS3 and LGN, in which we swapped the four GoLoco domains between AGS3 and LGN, leaving interdomains unchanged, and vice versa. Indeed, we found that the chimera containing the GoLoco domains of AGS3 and the interdomains of LGN (GPRAGS3interLGN) was recruited to the cortex as efficiently as GPRLGN, while the converse construct (GPRLGNinterAGS3) was essentially cytoplasmic, similar to GPRAGS3 (Fig 3F and G). Similarly, a Myc-tagged version of full-length LGN in which only the GoLoco had been replaced by those from AGS3 (LGNAGS3Goloco) was recruited to the cortex, whereas a construct containing only the interdomains from AGS3 (LGNAGS3interdomain) remained cytoplasmic (Fig 3H). Surprisingly, when expressed in an LGN RNAi background, LGNAGS3Goloco did not rescue the spindle orientation phenotype, indicating that the observed cortical recruitment is not sufficient t
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