Dynamic localization of C. elegans TPR-GoLoco proteins mediates mitotic spindle orientation by extrinsic signaling

Abstract

SUMMARYCell divisions are sometimes oriented by extrinsic signals, by mechanisms that are poorly understood. Proteins containing TPR and GoLoco-domains (C. elegans GPR-1/2, Drosophila Pins, vertebrate LGN and AGS3) are candidates for mediating mitotic spindle orientation by extrinsic signals, but the mechanisms by which TPR-GoLoco proteins may localize in response to extrinsic cues are not well defined. The C. elegans TPR-GoLoco protein pair GPR-1/2 is enriched at a site of contact between two cells -the endomesodermal precursor EMS and the germline precursor P 2 -and both cells align their divisions toward this shared cell-cell contact. To determine whether GPR-1/2 is enriched at this site within both cells, we generated mosaic embryos with GPR-1/2 bearing a different fluorescent tag in different cells. We were surprised to find that GPR-1/2 distribution is symmetric in EMS, where GPR-1/2 had been proposed to function as an asymmetric cue for spindle orientation. Instead, GPR-1/2 is asymmetrically distributed only in P 2 . We demonstrate a role for normal GPR-1/2 localization in P 2 division orientation. We show that MES-1/Src signaling plays an instructive role in P 2 for asymmetric GPR-1/2 localization and normal spindle orientation. We ruled out a model in which signaling localizes GPR-1/2 by locally inhibiting LET-99, a GPR-1/2 antagonist. Instead, asymmetric GPR-1/2 distribution is established by destabilization at one cell contact, diffusion, and trapping at another cell contact. Once the mitotic spindle of P 2 is oriented normally, microtubule-dependent removal of GPR-1/2 prevented excess accumulation, in an apparent negative-feedback loop. These results highlight the role of dynamic TPR-GoLoco protein localization as a key mediator of mitotic spindle alignment in response to instructive, external cues.

Wnt and CDK-1 regulate cortical release of WRM-1/β-catenin to control cell division orientation in early Caenorhabditis elegans embryos

“…The finding that WRM-1 T30A S46A synergizes with src-1, but not Wntpathway mutants, suggests that modification by CDK-1 governs the cortical release of WRM-1 in response to Wnt-signaling cues, whereas SRC-1 may independently regulate WRM-1 or some other associated cortical factor(s) to promote EMS spindle rotation. Consistent with this model, the G protein regulators Gα (GPR-1/-2) and LET-99 were shown to asymmetrically localize at the cortex and orient the EMS spindle in response to Src, but not Wnt, signaling (48)(49)(50). LET-99 is also required for the geometry-driven spindle rotation in par-3 mutant embryos (27).…”

Molecular pathways regulating mitotic spindle orientation in animal cells

“…In subsequent divisions, the Fz-Dsh complex (orange) regulates spindle orientation in the EMS cell. Similar to the one-cell stage, PAR complexes show reciprocal polarity in EMS and P2 cells (Arata et al, 2010), whereas GPR-1/2 influences spindle positioning through asymmetric localization in the P2 cell specifically (Werts, Roh-Johnson and Goldstein, 2011). (B) Drosophila neuroblasts polarize along an apical-basal (A-B) axis through the activity of the Par-aPKC complex (red).…”

“…Spindle orientation along the A-P axis and subsequent asymmetric cell division of the EMS cell then generates an E daughter cell, which will give rise to endodermal lineages, and a MS daughter cell, which will form mesodermal lineages. Spindle orientation in the one-cell stage and the P2 blastomere cell requires the activity of GPR-1/2 and LIN-5, which constitute an evolutionarily conserved non-canonical G-protein signaling network (Colombo et al, 2003;Gönczy, 2008;Werts et al, 2011). Mutations in Wnt signaling pathway components result in misalignment of the EMS spindle and mis-specification of germ cell layers (Schlesinger et al, 1999;Walston et al, 2004) (Fig.…”

Identifying Regulators of Morphogenesis Common to Vertebrate Neural Tube Closure andCaenorhabditis elegansGastrulation

“…Contact length ratios: P 1 cells were isolated from wild-type and WM43 gex-3(zu196) embryos at the two-cell stage using the same method of isolation described in Werts et al (2011). The cells were mounted in Shelton's growth medium (Shelton and Bowerman 1996) either as P 1 cells or, after one division, as P 2 -EMS cell pairs.…”