Figure S1 The Enrichment of PAR-1 on the Posterior Cortex Is Disrupted in
pkc-3(RNAi),
par-2(RNAi), and
par-1(
b274) Zygotes, Related to Figure 2 (A) Single-plane confocal images of wild-type and
pkc-3 (RNAi) embryos expressing GFP::PAR-1 or Dendra::MEX-5. Note that in
pkc-3(RNAi) embryos, PAR-1 localizes throughout the cortex and cytoplasm and DendraR::MEX-5 is evenly distributed throughout the cytoplasm. PAR-1 also localizes on centrosomes (bright dots) as reported previously ( Gnczy et al., 2001). (B) Single-plane confocal images of zygotes of the indicated genotypes immunostained with antibodies against PKC-3 (Aono et al., 2004) and PAR-1 (Guo and Kemphues, 1995) or expressing Dendra::MEX-5. PKC-3 is enriched on the anterior cortex in all genotypes. (C) Quantification of the GFP::PAR-1 concentration gradient in wild-type and
par-2 (RNAi) zygotes at pronuclear centration (pronuclei have met at the center of the embryo) and after NEBD. GFP::PAR-1 concentration was determined for multiple embryos (between 7 and 15) at regular positions along the long axis. Obtaining accurate scale measurements is complicated by the fact that GFP::PAR-1 levels approach autofluorescence levels in the anterior cytoplasm. To correct for the background signal from autofluorescence, autofluorescence was measured in embryos not expressing GFP (n= 8) and subtracted from GFP::PAR-1 values. GFP::PAR-1 levels are expressed as the mean concentration relative to the concentration at 2.5% embryo length (normalized to 1 for each embryo). Values between 45% and 65% embryo length were omitted because of signal distortion caused by accumulation of GFP::PAR-1 on centrosomes in this region. Error bars represent SEM. (D) Quantification of endogenous PAR-1 levels in wild-type embryos. Ten mitotic stage embryos were stained with anti-PAR-1 antibody (Guo and Kemphues, 1995) and imaged at the midplane of the cell. Fluorescence levels are expressed as the mean concentration relative to the concentration at 2.5% embryo length (normalized to 1 for each embryo). Values between 35% and 55% embryo length were omitted because of signal distortion caused by the presence of pronuclei and the accumulation of PAR-1 on centrosomes. Unlike the GFP::PAR-1 quantification in panel C, background cytoplasmic staining (Guo and Kemphues, 1995) has not been subtracted from these values. This likely results in the apparently weaker enrichment of endogenous PAR-1 in the posterior cytoplasm relative to GFP::PAR-1. In an additional 10 out of 12 embryos (not included in this quantification), PAR-1 levels were higher in the posterior cytoplasm compared to the anterior cytoplasm. Error bars represent SEM. (E) Western blot analysis of PAR-1 in wild-type,
par-1(
it51) and
par-1(
b274) worms. Extracts from approximately 130 hermaphrodites were separated on SDS-PAGE gels and probed with anti-PAR-1 ( Guo and Kemphues, 1995) and anti-tubulin antibodies (mouse anti-tubulin monoclonal DM1A, Sigma-Aldrich). Position of molecular weight markers are indicated on the left.
par-1(
b274) worms lack full-length PAR-1 and express instead a truncated form (86 kDa) ( Hurd and Kemphues, 2003) at 14% of wild-type levels. (F) Concentration ratio of anterior to posterior DendraR::MEX-5 in wild-type and
par-2(RNAi) embryos at pronuclear centration and following NEBD. Note that DendraR::MEX-5 asymmetry at pronuclear centration is reduced in
par-2(RNAi) embryos compared to wild-type and weakens further following NEBD. This is consistent with the weaker PAR-1 gradient that forms by pronuclear meeting in
par-2(RNAi) embryos and its decay after NEBD (panel C). Error bars represent SEM.