Fig 1.
Schematic representation of PAR protein-dependent polarity establishment and maintenance during C. elegans early embryogenesis.
The distribution of anterior (PAR-3, PAR-6, PKC-3, in red) and posterior (PAR-1, PAR-2, in green) PAR proteins is depicted during the first two divisions of embryonic blastomeres. In all images, anterior is to the left. M = male pronucleus; F = female pronucleus. See main text for details.
Fig 2.
Depletion of cyb-2.1 and cyb-2.2 suppresses the lethality and polarity defects associated with the loss of par-2.
(A-B) Graphs reporting the viability of embryos of the specified genotypes after RNAi (A) or genetic (B) inactivation of cyb-2.1 and cyb-2.2. The values correspond to the mean percentage of hatching embryos over the total number of embryos ± SEM over three independent assays performed at the specified temperature. (C-D) DIC images from time-lapse movies of embryos grown at 22°C undergoing first (C) or second (D) division. Black arrowheads indicate centrosome positions and white arrowheads point to sites of membrane ingression during cytokinesis. In all panels, anterior is to the left. Scale bar is 10μm. (E-F) Graphs reporting the measurements for spindle position (E) and asynchrony duration (F) in embryos of the specified genotypes grown at the specified temperatures. Spindle position is expressed as a ratio of the distance between the anterior centrosome and the anterior cortex over the distance between the posterior centrosome and the posterior cortex (A/P ratio). Asynchrony duration corresponds to the time difference between the appearance of membrane ingression in AB and in P1. Error bars represent standard deviation over the specified number of events (n). In all panels, asterisks indicate statistical significance with control animals (p≤0.05, Student’s t-test).
Fig 3.
Mutation in the cyclin B-associated kinase CDK-1 suppresses par-2(RNAi) lethality and polarity defects.
(A) Graph reporting the viability of embryos of the specified genotypes after control(RNAi) or par-2(RNAi) treatment. The values correspond to the mean percentage of hatching embryos over the total number of embryos ± SEM over three independent assays performed at 16°C. (B) DIC images from time-lapse movies of par-2(RNAi)-depleted embryos grown at 16°C undergoing first (left) and second (right) division. In all panels, anterior is to the left. Scale bar is 10μm. Black arrowheads indicate centrosome positions and white arrowheads point to sites of membrane ingression during cytokinesis. (C-D) Graphs reporting the measurements for spindle position (C) and asynchrony duration (D) in embryos of the specified genotypes grown at 16°C. Error bars represent standard deviation over the specified number of events (n). In all panels, asterisks indicate statistical significance with control animals (p≤0.05, Student’s t-test).
Fig 4.
CYB-2.1/2 affect anterior PAR protein localization in the 1-cell embryo.
(A, C) Images of embryos of the specified genotypes grown at 25°C (A) or 20°C (C), fixed during mitosis and stained with anti-PAR-6 (left panels) or anti-PAR-3 (right panels) antibodies. In all panels, anterior is to the left. Scale bar is 10μm. (B, D) Graphs reporting the cortical distribution (in % embryo length) of endogenous PAR-6 and PAR-3 along the antero-posterior axis in embryos of the specified genotypes grown at 22°C, 25°C (B) or 20°C (D) and fixed during mitosis. Error bars represent standard deviation over the specified number of events (n). In all panels, asterisks indicate statistical significance with control animals (p≤0.05, Student’s t-test).
Fig 5.
CYB-2.1/2 and CDK-1 regulate PAR-6 levels in the early embryo.
(A) Graph reporting the viability of embryos of the specified genotypes after control(RNAi) or par-2(RNAi) treatment. The values correspond to the mean percentage of hatching embryos over the total number of embryos ± SEM over three independent assays performed at 16°C. (B) Western blot analysis of extracts from embryos of the specified genotypes grown at 20°C and revealed with anti-PAR-6 and anti-alpha-tubulin antibodies. The value under each lane corresponds to the ratio of PAR-6 over alpha-tubulin intensity ± SEM in three independent experiments, with the mean value normalized to WT levels.
Fig 6.
CYB-2.1/2 act with the Cullin CUL-2 but independently of NOS-3 in the embryonic polarity pathway.
(A-B) Graph reporting the viability of embryos of the specified genotypes after control(RNAi) or par-2(RNAi) treatment. The values correspond to the mean percentage of hatching embryos over the total number of embryos ± SEM over three independent assays performed at 15°C (A) or 25°C (B). In all panels, asterisks indicate statistical significance with control animals (p≤0.05, Student’s t-test) (C) Model depicting the proposed dual role of CYB-2.1/2/CDK-1 in the regulation of cell polarity and cell cycle progression during asymmetric division of the C. elegans embryo (see discussion for details).