Cortical localization of the G protein GPA-16 requires RIC-8 function during C. elegans asymmetric cell division

DEV02039 Supplementary Material

Files in this Data Supplement:

• Supplemental Table - Adobe PDF

• Supplemental Figure 1 - Fig. S1. Dendogram showing relative evolutionary distances among select Ga proteins. Highlighed in red are C. elegans GOA-1 and GPA-16. The dendogram was generated using Clustal-W (Aiyar, 2000) and TreeView (Page, 1996) Species abbreviations: Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; Dm, Drosophila melanogaster; Dr, Danio rerio (zebrafish); Ce, Caenorhabditis elegans; Ap, Asterina pectinifera (starfish); Ci, Ciona intestinalis; Ov, Octopus vulgaris; Sp, Strongylocentrotus purpuratus (sea urchin).

  DmCta, Drosophila melanogaster concertina (GenPept accession AAL90272)

  CeGPA-12, Caenorhabditis elegans GPA-12 (GenPept accession AAA81081)

  DrGa12, Danio rerio G-alpha-12 (GenPept accession AAR25616)

  RnGa12, Rattus norvegicus G-alpha-12 (GenPept accession NP_112296)

  MmGa12, Mus musculus G-alpha-12 (GenPept accession NP_034432)

  DrGa13, Danio rerio G-alpha-13 (GenPept accession AAR25617)

  MmGa13, Mus musculus G-alpha-13 (GenPept accession NP_034433)

  RnGa13, Rattus norvegicus G-alpha-13 (GenPept accession AAS64389)

  HsGa13, Homo sapiens G-alpha-13 (GenPept accession AAM12616)

  HsGa12, Homo sapiens G-alpha-12 (GenPept accession EAL23961)

  RnGaz, Rattus norvegicus G-alpha-z (GenPept accession NP_037321)

  MmGaz, Mus musculus G-alpha-z (GenPept accession NP_034441)

  HsGaz, Homo sapiens G-alpha-z (GenPept accession NP_002064)

  DmGas, Drosophila melanogaster G-alpha-s (GenPept accession AAA28579)

  CeGSA-1, Caenorhabditis elegans GSA-1 (GenPept accession AAB96733)

  MmGas, Mus musculus G-alpha-s (GenPept accession AAH80816)

  RnGas, Rattus norvegicus G-alpha-s (GenPept accession AAH61967)

  HsGas, Homo sapiens G-alpha-s (GenPept accession AAP88907)

  DmGaq, Drosophila melanogaster G-alpha-q (GenPept accession AAM49880)

  MmGaq, Mus musculus G-alpha-q (GenPept accession AAH57583)

  RnGaq, Rattus norvegicus G-alpha-q (GenPept accession AAF59930)

  HsGaq, Homo sapiens G-alpha-q (GenPept accession AAM12610)

  CeEGL-30, Caenorhabditis elegans EGL-30 (GenPept accession AAB58071)

  ApGai, Asterina pectinifera G-alpha-i (SwissProt accession P30676)

  SpGai, Strongylocentrotus purpuratus G-alpha-i (GenPept accession AAS38580)

  RnGai3, Rattus norvegicus G-alpha-i3 (GenPept accession NP_037238)

• Supplemental Figure 2 - Fig. S2. Cortical GPR-1/2 distribution in four-cell stage embryos. Four-cell stage embryos of the indicated genotypes stained with antibodies against GPR-1/2 (red) and a-tubulin (green); DNA is shown in blue. Left panels show GPR-1/2 staining alone, right panels the merge of the three signals. Rectangles highlight a region of the cortex at the ABp/EMS boundary to ease comparison of GPR-1/2 cortical staining. Insets represent ~2.53 magnified view of the approximate region indicated by the rectangles. The corresponding quantification of GPR-1/2 cortical distribution is given in Fig. S3. Qualitatively analogous findings were made in two-cell stage embryos (Fig. 4).

• Supplemental Figure 3 - Fig. S3. Quantification of cortical GPR-1/2. Five or more embryos were analyzed as described in the Materials and methods. The results of Student’s t-test to determine the significance of the differences in cortical GPR-1/2 intensities (expressed in arbitrary units, A.U.) were as follows: ric-8 (md1909) versus ric-8 (md1909) goa-1(RNAi), P<0.01; ric-8 (md1909) versus goa-1(RNAi) gpa-16(RNAi), P<0.001; ric-8 (md1909) versus ric-8 (md1909) gpa-16(RNAi), P>0.5; ric-8 (md1909) versus goa-1(RNAi), P>0.5; ric-8 (md1909) versus gpa-16(RNAi), P>0.5; goa-1(RNAi) gpa-16(RNAi)versus ric-8 (md1909) goa-1(RNAi), P>0.05.

• Supplemental Figure 4 - Fig. S4. Western blot analysis of new RIC-8 antibodies. Western blot analysis using RIC-8 antibodies on wild-type or ric-8(RNAi) embryonic extracts. Four bands are detected, one of which appears to be not specific as it is detected in ric-8(RNAi) embryonic extracts. The nature of the ~35 kDa species, as well as that of the minor species migrating just above the main RIC-8 species, remain to be determined. The blot was reprobed with a-tubulin antibodies as a loading control (bottom).

• Movie 1 - Movie 1. Time-lapse DIC microscopy of wild-type one-cell stage embryos showing centration/rotation following meeting of the male and female pronuclei, spindle assembly, oscillations during asymmetric spindle positioning and cell division. Frames were captured every five seconds and the movies are displayed at 10 seconds per frame. Embryos is oriented with anterior to the left and posterior to the right in this and other movies.

• Movie 2 - Movie 2. Time-lapse DIC microscopy of goa-1(sa734) one-cell stage embryos showing centration/rotation following meeting of the male and female pronuclei, spindle assembly, oscillations during asymmetric spindle positioning and cell division. Frames were captured every five seconds and the movies are displayed at 10 seconds per frame. Embryos is oriented with anterior to the left and posterior to the right in this and other movies.

• Movie 3 - Movie 3. Time-lapse DIC microscopy of gpa-16(it143) one-cell stage embryos showing centration/rotation following meeting of the male and female pronuclei, spindle assembly, oscillations during asymmetric spindle positioning and cell division. Frames were captured every five seconds and the movies are displayed at 10 seconds per frame. Embryos is oriented with anterior to the left and posterior to the right in this and other movies.

• Movie 4 - Movie 4. Time-lapse DIC microscopy of gpb-1(RNAi) one-cell stage embryos showing centration/rotation following meeting of the male and female pronuclei, spindle assembly, oscillations during asymmetric spindle positioning and cell division. Frames were captured every five seconds and the movies are displayed at 10 seconds per frame. Embryos is oriented with anterior to the left and posterior to the right in this and other movies. There is abrupt back and forth movements of centrosomes and of associated pronuclei during centration/rotation, and exaggerated spindle oscillations

• Movie 5 - Movie 5. Time-lapse DIC microscopy of goa-1(sa734) gpb-1(RNAi) one-cell stage embryos showing centration/rotation following meeting of the male and female pronuclei, spindle assembly, oscillations during asymmetric spindle positioning and cell division. Frames were captured every five seconds and the movies are displayed at 10 seconds per frame. Embryos is oriented with anterior to the left and posterior to the right in this and other movies. There is abrupt back and forth movements of centrosomes and of associated pronuclei during centration/rotation, and exaggerated spindle oscillations

• Movie 6 - Movie 6. Time-lapse DIC microscopy of gpa-16(it143) gpb-1(RNAi) one-cell stage embryos showing centration/rotation following meeting of the male and female pronuclei, spindle assembly, oscillations during asymmetric spindle positioning and cell division. Frames were captured every five seconds and the movies are displayed at 10 seconds per frame. Embryos is oriented with anterior to the left and posterior to the right in this and other movies. There is abrupt back and forth movements of centrosomes and of associated pronuclei during centration/rotation, and exaggerated spindle oscillations

• Movie 7 - Movie 7. Time-lapse DIC microscopy of goa-1(sa734) gpa-16(RNAi) gpb-1(RNAi) one-cell stage embryos showing centration/rotation following meeting of the male and female pronuclei, spindle assembly, oscillations during asymmetric spindle positioning and cell division. Frames were captured every five seconds and the movies are displayed at 10 seconds per frame. Embryos is oriented with anterior to the left and posterior to the right in this and other movies. There is abrupt back and forth movements of centrosomes and of associated pronuclei during centration/rotation, and exaggerated spindle oscillations

• Movie 8 - Movie 8. Time-lapse DIC microscopy of wild-type one-cell stage embryos from onset of metaphase until early two-cell stage. Frames were captured every 5 seconds and the movies are played at 10 frames per second.

• Movie 9 - Movie 9. Time-lapse DIC microscopy of goa-1(RNAi) one-cell stage embryos from onset of metaphase until early two-cell stage. Frames were captured every 5 seconds and the movies are played at 10 frames per second.

• Movie 10 - Movie 10. Time-lapse DIC microscopy of gpa-16(RNAi) one-cell stage embryos from onset of metaphase until early two-cell stage. Frames were captured every 5 seconds and the movies are played at 10 frames per second.

• Movie 11 - Movie 11. Time-lapse DIC microscopy of ric-8 (md1909) one-cell stage embryos from onset of metaphase until early two-cell stage. Frames were captured every 5 seconds and the movies are played at 10 frames per second.

• Movie 12 - Movie 12. Time-lapse DIC microscopy of ric-8(md1909) gpa-16(RNAi) one-cell stage embryos from onset of metaphase until early two-cell stage. Frames were captured every 5 seconds and the movies are played at 10 frames per second. The distance between spindle poles are similar in ric-8(md1909) and gpa-16(RNAi) ric-8(md1909) embryos, and in goa-1 (RNAi) ric-8(md1909) and goa-1/gpa-16 (RNAi) embryos.

• Movie 13 - Movie 13. Time-lapse DIC microscopy of ric-8(md1909) goa-1(RNAi) one-cell stage embryos from onset of metaphase until early two-cell stage. Frames were captured every 5 seconds and the movies are played at 10 frames per second. The distance between spindle poles are similar in ric-8(md1909) and gpa-16(RNAi) ric-8(md1909) embryos, and in goa-1 (RNAi) ric-8(md1909) and goa-1/gpa-16 (RNAi) embryos.

• Movie 14 - Movie 14. Time-lapse DIC microscopy of goa-1/gpa-16(RNAi) one-cell stage embryos from onset of metaphase until early two-cell stage. Frames were captured every 5 seconds and the movies are played at 10 frames per second.