Regulation of cortical contractility and spindle positioning by the protein phosphatase 6 PPH-6 in one-cell stage C. elegans embryos

Abstract

Modulation of the microtubule and the actin cytoskeleton is crucial for proper cell division. Protein phosphorylation is known to be an important regulatory mechanism modulating these cytoskeletal networks. By contrast, there is a relative paucity of information regarding how protein phosphatases contribute to such modulation. Here, we characterize the requirements for protein phosphatase PPH-6 and its associated subunit SAPS-1 in one-cell stage C. elegans embryos. We establish that the complex of PPH-6 and SAPS-1 (PPH-6/SAPS-1) is required for contractility of the actomyosin network and proper spindle positioning. Our analysis demonstrates that PPH-6/SAPS-1 regulates the organization of cortical non-muscle myosin II (NMY-2). Accordingly, we uncover that PPH-6/SAPS-1 contributes to cytokinesis by stimulating actomyosin contractility. Furthermore, we demonstrate that PPH-6/SAPS-1 is required for the proper generation of pulling forces on spindle poles during anaphase. Our results indicate that this requirement is distinct from the role in organizing the cortical actomyosin network. Instead, we uncover that PPH-6/SAPS-1 contributes to the cortical localization of two positive regulators of pulling forces, GPR-1/2 and LIN-5. Our findings provide the first insights into the role of a member of the PP6 family of phosphatases in metazoan development.

Fig. 1.

PPH-6 and SAPS-1 are required for pseudocleavage formation and proper spindle positioning. (A,D,G) Images from DIC time-lapse recordings of wild-type (A), pph-6(RNAi) (D) and saps-1(RNAi) (G) one-cell stage embryos. Black arrowheads indicate the pseudocleavage furrow; open arrowheads, the posterior spindle pole during anaphase/telophase. Elapsed time is indicated in minutes and seconds (minutes:seconds) with t=0 corresponding to nuclear envelope breakdown (NEBD). Unless specified otherwise, scale bars in this and other figures are 10 μm and anterior is to the left. See also corresponding Movies 1, 3 and 5 in the supplementary material. (B,E,H) Representation of the rotation of the nucleo-centrosomal complex (NCC) in wild-type (B), pph-6(RNAi) (E) and saps-1(RNAi) (H) one-cell stage embryos. Each line represents the axis of the NCC at the time of NEBD in one embryo, with 0° indicating complete rotation onto the AP axis. n=25 in each case. (C,F,I) Anaphase posterior spindle pole positions relative to the midline (to mid; +, above; –, below) representative of wild-type (C), pph-6(RNAi) (F) and saps-1(RNAi) (I) one-cell stage embryos. See also Movies 2, 4 and 6 in the supplementary material.

Fig. 2.

PPH-6 and SAPS-1 associate in vivo. (A) Western blot analysis using PPH-6 antibodies on wild-type or pph-6(RNAi) embryonic extracts. The blot was reprobed with α-tubulin antibodies as a loading control (bottom). Note the presence of two species, with the lower one exhibiting the predicted molecular weight of PPH-6 (∼37 kDa). Note also that the ratio between these two species varies among extracts (compare A with inputs in C). Similar variability is observed for SAPS-1 (B,C). The variation might be due to differences in the developmental stages of the embryos in the different preparations. (B) Western blot analysis of wild-type or saps-1(RNAi) embryonic extracts probed with SAPS-1 antibodies. Note presence of two major specific species, exhibiting the predicted molecular weight of the splice variants of SAPS-1 (∼80 kDa and 87 kDa). A non-specific band (NS) served as a loading control. (C) Coimmunoprecipitation from wild-type, pph-6(RNAi) or saps-1(RNAi) embryonic extracts using PPH-6 antibodies. Western blots were probed with antibodies against PPH-6, SAPS-1 or α-tubulin, as indicated. In the second row, the input is exposed 10 times longer than the IP. Input/IP=1:50. In three independent experiments, we observed that PPH-6 antibodies retrieved more PPH-6 from the saps-1(RNAi) extract than from the pph-6(RNAi) extract, despite similar depletion levels of PPH-6. Perhaps PPH-6 not bound to SAPS-1 is more accessible to PPH-6 antibodies. (D) Extract from embryos expressing GFP-SAPS-1 or from wild-type embryos immunoprecipitated with GFP antibodies and analyzed by western blot with GFP or PPH-6 antibodies, as indicated. Note that only the PPH-6 species with the lower molecular weight co-immunoprecipitates with GFP-SAPS-1. Input/IP=1:50.

Fig. 3.

PPH-6 and SAPS-1 distribution in early embryos. One-cell stage (A,B,F,G) or four-cell stage (C-E,H-J) embryos of the indicated genotypes stained with antibodies against PPH-6 (A-E) or SAPS-1 (F-J), shown alone on the left and in red in the merged panels, as well as against α-tubulin (green in the merged panels); DNA is viewed in blue. Arrowheads in A and F indicate enrichment at microtubule asters; arrows in C and H, enrichment at the cell cortex. Note that the perinuclear signal detected by SAPS-1 antibodies does not appear to be specific, as it is not diminished in saps-1(RNAi) embryos (I). Panels E and J are representative of 20 and 17 embryos, respectively, other panels of at least 30 embryos.

Fig. 4.

PPH-6/SAPS-1 is required for clustering of cortical GFP-NMY-2 during pseudocleavage. (A-D) Images from spinning disc confocal recordings of the cortex during pseudocleavage of wild-type and saps-1(RNAi) embryos expressing GFP-MOE or GFP-NMY-2. Images are representative of at least 10 embryos. See also Movies 11-14 in the supplementary material. Note that quantification of the total GFP-MOE signal was unreliable at this stage because segments of the cortex were out of focus in wild-type embryos owing to cortical invaginations (see center of embryo in A). (E,F) Kymographs assembled from images acquired two minutes prior to pronuclear meeting of representative recordings of GFP-NMY-2 in wild-type (E) or saps-1(RNAi) (F) embryos. Flanking kymographs show a region at high magnification over 22.5 seconds. The bright dots at the bottom of F are new NMY-2 foci that also fail to cluster. Scale bars in the magnified images: 1 μm.

Fig. 5.

PPH-6/SAPS-1 plays a role during cytokinesis. (A,B) Kymographs assembled from images acquired during the first two minutes after anaphase onset of representative recordings of GFP-NMY-2 in wild-type (A) or saps-1(RNAi) (B) embryos, representative of 11 embryos each. Arrowheads indicate select foci that intensify in the following frames by the local coalescence of smaller foci. Flanking kymographs show a region at high magnification over 22.5 seconds. Scale bars in the magnified images: 1 μm. (C-F) Images from DIC time-lapse microscopy of embryos of the indicated genotypes. Arrowheads indicate the pseudocleavage furrow; arrows, the extent of ingression of the cleavage furrow at end of the first cycle. Elapsed time relative to NEBD is indicated in minutes and seconds (minutes:seconds). See also corresponding Movies 22-25 in the supplementary material. The maximum extent of furrow formation at the end of the first cell cycle, averaged for the indicated number of embryos and rounded to the nearest integer, is shown below the images. Values are expressed in percentages relative to the width of the embryo at the site of furrow formation. Exact values, ±s.d. are as follows: zen-4(or153), 88.5±5.4%; pph-6(RNAi) zen-4(or153), 6.7±4.5%; saps-1(RNAi); zen-4(or153), 5.0±4.9%.

Fig. 6.

PPH-6/SAPS-1 promotes pulling forces. Average peak velocities ± s.e.m. of anterior (A) and posterior (P) spindle poles after spindle severing in one-cell stage embryos of the indicated genotypes. Spindle severing was performed during metaphase or early anaphase, as indicated. Actual values and number of embryos analyzed are given in Table S1 in the supplementary material. The values for gpb-1(RNAi) are from Afshar et al. (Afshar et al., 2004). The fact that the anterior peak velocity for embryos simultaneously depleted of CSNK-1 and SAPS-1 is slightly higher than that for embryos depleted solely of SAPS-1 might stem from the fact that doubly depleted embryos were analyzed at an earlier time point, as more complete depletion results in sterility.

Fig. 7.

PPH-6/SAPS-1 contributes to cortical enrichment of GPR-1/2. (A,C,E,G,I,K) Embryos of the indicated genotypes were stained with antibodies against GPR-1/2 (shown alone on the left and in red in the merged panels) and against α-tubulin (green in the merged panels); DNA is viewed in blue in the merged panels. (B,D,F,H,J,L) Quantification of cortical GPR-1/2. A line scan was performed across the 0.7×3.4 μm rectangles depicted in red in the corresponding immunofluorescence panels; the total fluorescence intensity along the horizontal axis of this rectangle is shown in the plots, with the y-axis displaying arbitrary gray values. Note that 36% of wild-type embryos (A, n=33), 27% of gpb-1(RNAi) embryos (E, n=20), 20% of gpb-1/saps-1(RNAi) embryos (G, n=17) and 12% of csnk-1(RNAi) embryos (I, n=20) exhibited a slightly weaker distribution than that shown. Conversely, 27% of saps-1(RNAi) embryos (C, n=52) and 10% of csnk-1/saps-1(RNAi) embryos exhibited a slightly higher distribution than that shown, yet weaker than in the wild type. See also Fig. S9 in the supplementary material for quantitative analysis.