A casein kinase 1 and PAR proteins regulate asymmetry of a PIP(2) synthesis enzyme for asymmetric spindle positioning

Abstract

Spindle positioning is an essential feature of asymmetric cell division. The conserved PAR proteins together with heterotrimeric G proteins control spindle positioning in animal cells, but how these are linked is not known. In C. elegans, PAR protein activity leads to asymmetric spindle placement through cortical asymmetry of Galpha regulators GPR-1/2. Here, we establish that the casein kinase 1 gamma CSNK-1 and a PIP(2) synthesis enzyme (PPK-1) transduce PAR polarity to asymmetric Galpha regulation. PPK-1 is posteriorly enriched in the one-celled embryo through PAR and CSNK-1 activities. Loss of CSNK-1 causes uniformly high PPK-1 levels, high symmetric cortical levels of GPR-1/2 and LIN-5, and increased spindle pulling forces. In contrast, knockdown of ppk-1 leads to low GPR-1/2 levels and decreased spindle forces. Furthermore, loss of CSNK-1 leads to increased levels of PIP(2). We propose that asymmetric generation of PIP(2) by PPK-1 directs the posterior enrichment of GPR-1/2 and LIN-5, leading to posterior spindle displacement.

Figure 1

Excessive Pronuclear and Spindle Movements in csnk-1(RNAi) Embryos Depend on GPR-1/2 and LIN-5 (A–F) Pseudocleavage, centration (just prior to nuclear envelope breakdown), and two-cell stage images taken from time-lapse DIC video recordings in the indicated backgrounds. First row: abnormally strong pseudocleavage is observed in (B) csnk-1(RNAi), (D) csnk-1;gpr-1/2(RNAi), and (F) csnk-1;lin-5(RNAi) compared to (A) wild-type. Second row: pronuclear position at nuclear envelope breakdown (NEBD) in (B) csnk-1(RNAi) embryo is anterior compared to other backgrounds. Third row: (A) a wild-type embryo underwent asymmetric cell division (larger anterior cell), whereas embryos of other backgrounds have two equal sized cells. (G) Traces of anterior and posterior centrosome positions in representative one-cell embryos from pronuclear meeting to cytokinesis onset in the indicated backgrounds. 0% and 100% represent anterior and posterior ends, respectively. Time 0 s indicates NEBD. Note the large and rapid movements of the centrosomes in csnk-1(RNAi) compared to the other backgrounds. (H) Quantification of the phenotypes described in (A)–(F). Asymmetric division is defined as 52%–56% egg length, symmetric division as 48%–52%. In the anterior-most pronuclear position, 0 and 100 represent anterior and posterior ends, respectively. n, number of embryos analyzed. In this and other figures anterior is to the left and posterior to the right. (I and J) A graph (I) and table (J) show mean peak velocities (micrometer/second) of anterior (light gray) and posterior (dark gray) spindle poles measured after spindle severing in one-cell embryos of indicated genotypes. Error bars correspond to SEM. ^∗p < 0.05 compared to corresponding wild-type. Exact p values are given in the table. n, number of embryos analyzed.

Figure 2

CSNK-1 Acts Downstream of PAR-2 and PAR-3 for Control of GPR-1/2 Distribution and Spindle Movements GPR-1/2 (left panels) and tubulin (right panels) staining in embryos of indicated genotypes. (A–D) One-cell embryos at pronuclear meeting. (F–I) One-cell embryos at ana-telophase. (E and J) Quantification of average anterior cortical GPR-1/2 pixel intensities (0%–25% egg length; light gray) and posterior cortical GPR-1/2 pixel intensities (75%–100% egg length; dark gray) in embryos of genotypes indicated at the left. Error bars represent SEM. ^∗p < 0.05; ^#p < 0.056 compared to corresponding wild-type. Overall intensity signifies the average intensity of the whole embryonic cortex. Numbers in brackets show the number of embryos analyzed. In 3/3 csnk-1(RNAi) embryos, pronuclei move anterior to 40% egg length during centration compared to 0/10 for wild-type, 0/4 for par-2 mutants, and 0/5 for par-3 mutants. In 7/7 csnk-1(RNAi);par-2 embryos and 13/17 csnk-1(RNAi);par-3 embryos, pronuclei moved anterior to 40% egg length, similar to csnk-1(RNAi) embryos. In 3/3 csnk-1(RNAi) embryos, the spindle showed jerky unstable movement (rapid spindle movement along both long and short axes) compared to 0/10 for wild-type, 0/4 for par-2, and 0/5 for par-3 embryos. In 7/7 csnk-1(RNAi);par-2 embryos and 17/17 csnk-1(RNAi);par-3 embryos, the spindle showed unstable movements similar to csnk-1(RNAi) embryos.

Figure 3

CSNK-1 Localization (A–D) Wild-type embryos stained for CSNK-1 (left panels) and tubulin (right panels). (A) meiotic embryo, (B) anaphase embryo, (C) four-cell embryo. Staining is specific, as it is absent from (D) csnk-1(RNAi) embryos. (E–G) Projection of two cortical sections of GFP::CSNK-1 at pronuclear centration in indicated genotypes; numbers at the right give the percentage of embryos showing anterior enrichment of GFP::CSNK-1. n, number of embryos analyzed.

Figure 4

Posterior Enrichment of PPK-1 Is Controlled by CSNK-1, PAR-2, and PAR-3 Wild-type (A and B), csnk-1(RNAi) (C), par-2 (D), par-3 (E), and ppk-1(RNAi) (F) embryos stained for PPK-1 (left panels) and tubulin (right panels). (A) One-cell embryo at polarity onset. (B–E) One-cell embryos at anaphase. (F) One-cell embryo at pronuclear centration. PPK-1 is enriched at the posterior in wild-type (A and B) and par-2 (D) embryos. PPK-1 shows symmetric distribution in csnk-1(RNAi) (C) and par-3 (E) embryos. Staining is specific, as it is absent from ppk-1(RNAi) embryos (F). (G) Percent of embryos showing posterior PPK-1 enrichment in indicated backgrounds. Numbers in brackets show the number of embryos analyzed.

Figure 5

Reduced GPR-1/2 and Reduced Spindle Rocking in ppk-1(RNAi) Embryos Wild-type (A) and ppk-1(RNAi) (B) telophase embryos stained for GPR-1/2 (left panels) and tubulin (right panels). GPR-1/2 staining is highly reduced in 90% of ppk-1(RNAi) embryos (n = 10). (C) Posterior centrosome position in the short axis of the egg from metaphase (0 s) to cytokinesis (150 s) in representative wild-type (black line) and ppk-1(RNAi) embryos (gray line). Percentage represents percentage of egg width. ppk-1(RNAi) shows reduced posterior spindle pole rocking compared to wild-type (7.1% average width in ppk-1(RNAi) embryos [n = 6] versus 18.4% in wild-type [n = 5]).

Figure 6

PIP₂ Levels Are Reduced in csnk-1(RNAi) Embryos (A) PI(4)P5-kinase activity in wild-type and ppk-1(RNAi) cytosolic and membrane fractions. ppk-1(RNAi) extracts have approximately 5-fold less activity. (B) Normalized PIP₂ mass in wild-type and csnk-1(RNAi) embryo extracts. PIP₂ mass was measured relative to total phospholipids and set to 1.0 for wild-type. csnk-1(RNAi) embryos show a 1.8-fold increase in PIP₂ levels. ^∗p < 0.01.

Figure 7

Working Model for CSNK-1 and PPK-1 in Spindle Positioning Proposed distribution and activity of proteins along the A/P axis are indicated by their position in the boxes. Lines with bars indicate antagonistic interactions, whereas lines with arrows depict positive interactions. In this model anterior PAR proteins regulate PPK-1 localization through both CSNK-1-dependent and CSNK-1-independent mechanisms. Posterior enrichment of PPK-1 would lead to asymmetric generation of PIP₂, which in turn would lead to posterior enrichment of LIN-5 and GPR-1/2 and asymmetric pulling forces.