Translation of Polarity Cues into Asymmetric Spindle Positioning in Caenorhabditis elegans Embryos Kelly Colombo, Stephan W. Grill, Randall J. Kimple, Francis S. Willard, David P. Siderovski, Pierre Gönczy

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Figure S1
Anterior-posterior polarity cues are not affected in gpr-1/2(RNAi) embryos. Fixed GFP-PAR-6 (A) (S19), GFP-PAR-6 gpr-1/2(RNAi) (B), wild-type (C, E, G, I) or gpr-1/2(RNAi) (D, F, H, J) one-cell stage embryos stained with antibodies against GFP (A, B), PAR-3 (C, D), PAR-2 (E, F), PAR-1 (G, H) or PGL-1 (I, J). In gpr-1/gpr- 2(RNAi) one-cell stage embryos, GFP-PAR-6 and PAR-3 were enriched at the anterior cortex (42/42 and 35/35 embryos, respectively), PAR-2 and PAR-1 at the posterior cortex (93/94 and 51/51 embryos, respectively), and P-granules segregated to the posterior (53/53 embryos). At the four-cell stage, PAR proteins were also correctly localized, while P granules were correctly localized to P₂ in about 50% of embryos, and incorrectly localized to both P₂ and EMS in the remaining embryos at that stage.

Figure S2
Kinetic analysis of GOA-1 binding to GST-GoLoco fusion proteins. Indicated concentrations of GOA-1·GDP were sequentially injected over indicated GST-fusion proteins prebound to an anti-GST sensor chip surface, followed by surface regeneration. Duration of GOA-1 injection (20 l/min, 120 seconds) is denoted by grey shading. Dissociation of bound GOA-1 into running buffer was allowed to continue for 180 seconds. Full curve sets were obtained twice, with identical results. Apparent dissociation constants for GOA-1·GDP were as follows: GST-GPR- 1/GoLoco (KD = 0.31 M, Chi² = 44.3), GST-GPR-1/CT (KD = 0.16 M, Chi² = 44.1), and a GST-fusion with the human GoLoco protein Purkinje-cell protein 2 (GST-Pcp2; K_D = 1.0 M, Chi² = 284).

Figure S3
Distribution of GPR-1/2 in prophase and metaphase one-cell stage embryos. Five wild-type one-cell stage embryos during prophase (A-E) and five wild-type onecell stage embryos during metaphase (F-J) stained with antibodies against GPR and - tubulin (panels in right columns show GPR-1/2 in red and -tubulin in green). All panels at approximately same magnification; bar=10 m. After visual inspection, cortical distribution of GPR-1/2 was deemed to be symmetric in embryos A-C, and slightly asymmetric in embryos D-J. We quantified the ratio of GPR signal at the posterior cortex versus the anterior cortex in these ten embryos, with the following outcome: A=1.02, B= 0.73, C=1.03, D=1.06, E=1.14, average prophase=1.00 (SD=0.16); F=1.63, G= 1.19, H=1.63, I=1.46, J=1.70, average metaphase=1.52 (SD=0.21). Note that intensity of GPR-1/2 at the cell cortex is higher in metaphase embryos compared to prophase embryos. Note also that the focus was changed in embryo B to capture the -tubulin signal.

Figure S4
Working model of spindle positioning in one-cell stage C. elegans embryos. Astral and spindle microtubules are shown with black lines, spindle poles with black disks, active cortical force generators with white rectangles. AP polarity cues (PAR-3 and anterior cortical domain: red; PAR-2 and posterior cortical domain: blue) control the amount of cortical GPR-1/2 (green). GPR-1/2 activates G signaling, which results in cortical force generators acting on astral microtubules. As there is more GPR-1/2 at the posterior cortex, a larger net pulling force (red arrow) is exerted on the posterior spindle pole than on the anterior one. While this working model fits the available data, including the fact that gpr-1/2 and goa-1/gpa-16 are necessary for astral pulling forces, it remains to be determined experimentally whether cortical GPR-1/2 is indeed rate-limiting for force generation.

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• Movie S1    Movie S2
  Time-lapse DIC microscopy of wild-type (Movie S1) and gpr-1/gpr-2(RNAi) (Movie S2) embryo from early pronuclear migration in the one-cell stage until the early four-cell stage. Imaged at 0.2 frames per second, played back at 10 frames per second (i.e. 50x faster than actual events). Time elapsed since the beginning of the sequence is displayed in minutes and seconds.

  We observed other defects in gpr-1/gpr-2(RNAi) embryos using time-lapse DIC microscopy. First, some grp-1/2(RNAi) embryos, especially after prolonged exposure to RNAi, have karyomeres at the two-cell stage (not visible in Movie S2). However, aberrations in chromosome segregation are unlikely to cause the spindle positioning defects because indistinguishable spindle positioning phenotypes are observed in gpr-1/2(RNAi) embryos regardless of them having karyomeres at the twocell stage (Movies S6 and S7). Second, onset of cytokinesis was slightly delayed in one-cell stage embryos. Third, the AB and P1 nuclei remained close to the central cortex for much longer than in wild-type. Fourth, rotation of the centrosomes and associated nucleus did not occur in the P1 blastomere; as a result the spindle set up perpendicular to the AP axis. Fifth, P1 divided ~1.2 min before AB, compared to the usual ~2 min in wild-type. Sixth, cytokinesis in AB failed in 17% gpr-1/gpr-2(RNAi) embryos (n=30).

• Movie S3    Movie S4   Movie S5
  Time-lapse DIC microscopy sequence of spindle severing experiments in wildtype (Movie S3), gpr-1/gpr-2(RNAi) (Movie S4) and goa-1/gpa-16(RNAi) (Movie S5) one-cell stage embryos. Imaged at 2 frames per second, played back at 10 frames per second (i.e. 5x faster than actual events). Small black circles indicate laser shots.

• Movie S6    Movie S7
  Dual DIC (left) and fluorescence (right) microscopy of gpr-1/2(RNAi) embryos carrying a GFP-HIS-11 (Histone2B) transgene (S20). Note that there is a lack of posterior displacement both in the embryo with no apparent chromosome segregation defects (Movie S6, arrows in last frame point to single nucleus in each daughter blastomere), as well as in the one exhibiting abnormal chromosome segregation (Movie S7, arrows in last frame point to two karyomeres in each daughter blastomere). Imaged at 0.1 frames per second, played back at 10 frames per second (i.e. 100x faster than actual events). Time elapsed since the beginning of the sequence is displayed in minutes and seconds.