Journal: bioRxiv

Article Title: PP1 phosphatases control PAR-2 localization and polarity establishment in C. elegans embryos

doi: 10.1101/2022.01.19.476913

Figure Lengend Snippet: (A) Representation of the main phases that lead to zygote polarization. Left: early embryo before polarity establishment with anterior PARs uniformly at the cortex and posterior PARs in the cytoplasm. Middle: AIR-1 and microtubules dependent symmetry breaking. Right: polarity maintenance with anterior PARs at the anterior cortex and posterior PARs at the posterior cortex. (B) Embryonic lethality of gfp::par-2 and pkc-3(ne4246); gfp::par-2 embryos after ctrl(RNAi) and gsp-2(RNAi). The reported values correspond to the percentage of un-hatched embryos over the total progeny (larvae and un-hatched embryo). gfp::par- 2; ctrl(RNAi) , n=3132, gfp::par-2; gsp-2(RNAi), n=2405, pkc-3(ne4246); gfp::par-2; ctrl(RNAi), n=1085 and pkc-3(ne4246); gfp::par-2; gsp-2(RNAi ), n=1141. N=4. Mean is shown and error bars indicate SEM. The P-values were determined using 2way ANOVA “Tukey’s multiple comparisons test”. (C) Representative midsection frames of time-lapse imaging of embryos at the polarity maintenance stage: gfp::par- 2; ctrl(RNAi) , n=11, gfp::par-2; gsp-2(RNAi), n=13, pkc-3(ne4246); gfp::par-2; ctrl(RNAi), n=15 and pkc-3(ne4246); gfp::par-2; gsp-2(RNAi ), n=18, N=4. (D) Quantification of the cell cycle time: ctrl(RNAi) , n=12, gsp-2(RNAi) , n=10, pkc-3(ne4246), ctrl(RNAi), n=10, pkc-3(ne4246); gsp-2(RNAi ), n=18. N=3. Mean is shown and error bars indicate SD. The P-values were determined using unpaired Student’s t-test between AB and P1 for each condition and between P1 pkc-3(ne4246); ctrl(RNAi) and P1 pkc-3(ne4246); gsp-2(RNAi) . ns p > 0.05, ***p<0.001, ****p < 0.0001. For all figures, the scale bars are 5 µm, anterior is to the left and posterior to the right.

Article Snippet: The slides were incubated with primary antibodies diluted in PBST with 1% BSA overnight at 4°C (Rabbit anti PLK-1 (1:500) , Rabbit anti-PAR-2 (1:200) , Mouse anti-Tubulin (1:100) (Sigma)).

Techniques: Imaging

Journal: bioRxiv

Article Title: PP1 phosphatases control PAR-2 localization and polarity establishment in C. elegans embryos

doi: 10.1101/2022.01.19.476913

Figure Lengend Snippet: (A) Left: representative midsection frames of time-lapse imaging of gfp::par-2 zygotes at pronuclear meeting, comparing ctrl(RNAi) and gsp-2(RNAi) . N=4. Right: quantification of the gfp::par-2 size domain in live zygotes at pronuclear meeting. Numbers inside the bars indicate sample size. N=4. Mean is shown and error bars indicate SD. The P-values were determined using unpaired Student’s t-test. (B) Embryonic lethality of gfp::par-2 and pkc-3(ne4246); gfp::par-2 embryos after ctrl(RNAi) and gsp-1(RNAi). The reported values correspond to the percentage of un-hatched embryos over the total progeny (larvae and un-hatched embryo). gfp::par- 2; ctrl(RNAi) , n=2040, gfp::par-2; gsp-1(RNAi), n=1586, pkc-3(ne4246); gfp::par-2; ctrl(RNAi), n=751 and pkc-3(ne4246); gfp::par-2; gsp-1(RNAi ), n=382. N=3. Mean is shown and error bars indicate SEM. The P-values were determined using 2way ANOVA “Tukey’s multiple comparisons test”. (C) Representative midsection frames of time-lapse imaging of gfp::par-2 zygotes at pronuclear meeting, comparing ctrl(RNAi) , n= 10, ctrl(RNAi); gsp-1(RNAi) , n= 9, ctrl(RNAi); gsp-2(RNAi), n= 11 and gsp-1/- 2(RNAi) class I, n= 11 and class I,I n=3. N=3. Zygotes co-depleted of both GSP-1 and GSP-2 show PAR-2 uniformly in the cytoplasm in most of the embryos (class I, 78.6%); class II embryos (21.4%) show a weak PAR-2 localization at the cortex compared to ctrl(RNAi) . (D) Quantification of the gfp::par-2 size domain in live zygotes at pronuclear meeting. Numbers inside the bars indicate sample size. N=3. Mean is shown and error bars indicate SD. The P-values were determined using unpaired Student’s t-test. (E) PAR-2 line profile of live zygotes at pronuclear meeting comparing ctrl(RNAi) , n=10, ctrl(RNAi); gsp-1 (RNAi) , n=9 ctrl(RNAi); gsp-2(RNAi) n=11 and gsp-1/-2(RNAi) class I, n=11 and class II, n=3. Mean is shown and error bars indicate SD. N=3. ns p > 0.05, ***p<0.001, ****p < 0.0001.

Article Snippet: The slides were incubated with primary antibodies diluted in PBST with 1% BSA overnight at 4°C (Rabbit anti PLK-1 (1:500) , Rabbit anti-PAR-2 (1:200) , Mouse anti-Tubulin (1:100) (Sigma)).

Techniques: Imaging

Journal: bioRxiv

Article Title: PP1 phosphatases control PAR-2 localization and polarity establishment in C. elegans embryos

doi: 10.1101/2022.01.19.476913

Figure Lengend Snippet: (A) Schematic representation of the N-terminal (1-335aa) of both wild-type PAR-2 (upper panel) and PP1 mutant (RAFA, lower panel). (B) Yeast two-hybrid assay showing the interaction between PAR-2 N (wild type and the RAFA mutant) and GSP-2 and GSP-1. Yeasts were transformed with the indicated plasmids and were grown on selective and non-selective medium. Interaction between bait and prey results in growth on selective medium (50mM 3AT). (C) Alignment of the PP1 motif in PAR-2 for C. elegans , C. nigoni , C. briggsae and C. remanei . Figure re-elaborated from alignment obtained with Blast ( https://blast.ncbi.nlm.nih.gov ).

Article Snippet: The slides were incubated with primary antibodies diluted in PBST with 1% BSA overnight at 4°C (Rabbit anti PLK-1 (1:500) , Rabbit anti-PAR-2 (1:200) , Mouse anti-Tubulin (1:100) (Sigma)).

Techniques: Mutagenesis, Y2H Assay, Transformation Assay

Journal: bioRxiv

Article Title: PP1 phosphatases control PAR-2 localization and polarity establishment in C. elegans embryos

doi: 10.1101/2022.01.19.476913

Figure Lengend Snippet: (A) Representative midsection frames of time-lapse imaging of gfp::par-2° (the symbol ° indicates a mixture between wild-type gfp::par-2 homozygous worms expressing wild-type gfp::par-2 and heterozygous worms expressing wild type gfp::par-2 from one allele and mutant gfp::par-2(RAFA) from the other allele) and gfp::par-2(RAFA) embryos at pronuclear meeting (n=16 and n=17 respectively) N=6. (B) PAR-2 line profile of live zygotes at pronuclear meeting: gfp::par-2° , n= 16, and gfp::par-2(RAFA), n=17, N=6. Mean is shown and error bars indicate SD. (C) wild-type and gfp::par-2(RAFA) dividing two-cell stage embryos. The upper panel show a schematic representation, whereas in the lower panel images from DIC time-lapse movies are shown. In the schematics, the nucleus and the metaphase plate are in red, black lines are microtubules, black circles are centrosomes. (D) Quantification of the cell cycle time in gfp::par-2° and and gfp::par-2(RAFA) embryos. Numbers inside the bars indicate sample size. N=6. Mean is shown and error bars indicate SD. The P-values were determined using Student’s t-test. (E) Representative midsection frames of time-lapse imaging of gfp::par-2° and gfp::par-2(RAFA) one-cell embryos at pronuclear meeting. gfp::par-2° ; ctrl(RNAi) n= 15, pkc-3(RNAi); gfp::par-2° n= 17, gfp::par-2(RAFA); ctrl(RNAi) n= 11 and pkc-3(RNAi); gfp::par-2(RAFA) n=17, N=7. (F) Quantification of PAR-2 intensity obtained as a ratio of PAR-2 posterior cortex intensity over the posterior cytoplasm. Numbers inside the bars indicate sample size. N=7. Mean is shown and error bars indicate SD. The P-values were determined using 2way ANOVA “Tukey’s multiple comparisons test”. ns p > 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: The slides were incubated with primary antibodies diluted in PBST with 1% BSA overnight at 4°C (Rabbit anti PLK-1 (1:500) , Rabbit anti-PAR-2 (1:200) , Mouse anti-Tubulin (1:100) (Sigma)).

Techniques: Imaging, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: PP1 phosphatases control PAR-2 localization and polarity establishment in C. elegans embryos

doi: 10.1101/2022.01.19.476913

Figure Lengend Snippet: (A) Representative midsection frames of time-lapse videos of gfp::par- 2 and gfp::par-2(L165V) embryos at different cell division stages (for embryos before pronuclear migration: n= 9 and n=14 respectively, for the other embryos stage showed n=14 and n=25 respectively). Percentage on the bottom left of each image indicates the percentage of embryos with an anterior PAR-2 domain, except for the top panel (before pronuclear migration) in which indicates the percentage of embryos with PAR-2 around the entire cortex. N=4. (B) Representative midsection frames of time-lapse imaging of gfp::par- 2 and gfp::par-2(L165V) one-cell embryos at different cell division stages, ctrl(RNAi), n=13 and gsp-2(RNAi) , n=16. For the top panel (before pronuclear migration) n= 6 and n=8 for ctrl(RNAi) and gsp-2(RNAi) respectively. N=3. As in panel A, percentage on the bottom left of each embryo indicates the percentage of embryos with an anterior PAR-2 domain, with the exception of the top panel.

Article Snippet: The slides were incubated with primary antibodies diluted in PBST with 1% BSA overnight at 4°C (Rabbit anti PLK-1 (1:500) , Rabbit anti-PAR-2 (1:200) , Mouse anti-Tubulin (1:100) (Sigma)).

Techniques: Migration, Imaging

Journal: bioRxiv

Article Title: PP1 phosphatases control PAR-2 localization and polarity establishment in C. elegans embryos

doi: 10.1101/2022.01.19.476913

Figure Lengend Snippet: Representative images of fixed embryos of the indicated genotypes during pronuclei migration and at pronuclei centration/rotation, stained with anti-tubulin and anti-PAR-2 antibodies. Tubulin is in cyan, PAR-2 in magenta. The number of embryos analyzed is shown on the right in the pie-chart graph. N=5.

Article Snippet: The slides were incubated with primary antibodies diluted in PBST with 1% BSA overnight at 4°C (Rabbit anti PLK-1 (1:500) , Rabbit anti-PAR-2 (1:200) , Mouse anti-Tubulin (1:100) (Sigma)).

Techniques: Migration, Staining

Journal: bioRxiv

Article Title: PP1 phosphatases control PAR-2 localization and polarity establishment in C. elegans embryos

doi: 10.1101/2022.01.19.476913

Figure Lengend Snippet: Schematic representation of a one-cell stage embryo with anterior PAR proteins (in magenta) and posterior PAR proteins (in cyan). At the anterior, phosphorylation of PAR-2 by PKC-3 inhibits PAR-2 cortical localization, whereas at the posterior dephosphorylation of PAR-2 by GSP-2 allows PAR-2 posterior cortical localization. PLK-1 (in pink) downregulates, directly or indirectly, the activity of GSP-2 in the anterior of the embryo.

Article Snippet: The slides were incubated with primary antibodies diluted in PBST with 1% BSA overnight at 4°C (Rabbit anti PLK-1 (1:500) , Rabbit anti-PAR-2 (1:200) , Mouse anti-Tubulin (1:100) (Sigma)).

Techniques: De-Phosphorylation Assay, Activity Assay