Regulation of cortical contractility and spindle positioning by the protein phosphatase 6 PPH-6 in one-cell stageC. elegansembryos

Abstract

There was an error published in Development 137, 237-247.In Fig. 2D, owing to the PPH-6 film being inadvertently flipped during scanning, the two lanes labelled as input instead showed flowthrough. A revised Fig. 2 with the correct input samples in D is shown below. In addition, a note has been added to the end of the legend to clarify the lack of GFP-SAPS-1 signal in these input lanes.This error does not affect the conclusions of this experiment or of the paper. The authors apologise to readers for this mistake. 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 (overall levels of GFP-SAPS-1 protein in the embryonic lysates are low, hence the lack of detection of GFP-SAPS-1 in the input lanes). 2689

Mechanisms of spindle positioning: cortical force generators in the limelight

“…Thus, in C. elegans, the protein phosphatase 6 family member PPH-6 and its associated subunit SAPS-1 contribute to pulling forces by regulating the levels of GPR-1/2/LIN-5 [54]. The relevant target of PPH-6/ SAPS-1 is also not known and it is furthermore unclear whether the related phosphatase contributes to spindle positioning in other systems.…”

Polarity mediates asymmetric trafficking of the Gβ heterotrimeric G-protein subunit GPB-1 inC. elegansembryos

“…Primary mouse antibodies against -tubulin (1/300; DM1A, Sigma) and GFP (1/50; MAB3580, Millipore) were used together with rabbit antibodies against GPB-1 (1/200; this study) or GPR-1/2 [1/100 (Afshar et al, 2010)]. Secondary antibodies were Alexa 488-conjugated goat anti-mouse (1/500; Molecular Probes) and Cy3-conjugated goat anti-rabbit (1/1000; Dianova).…”

“…This is despite the knowledge of some components that regulate the presence of GPR-1/2 at the cell membrane. Thus, the two G subunits together are crucial for the recruitment of GPR-1/2 to the cell membrane (Colombo et al, 2003), and the PP6 phosphatase PPH-6 as well as its partner SAPS-1 also contribute to this recruitment (Afshar et al, 2010). Moreover, the casein kinase CSNK-1 is a negative regulator of overall GPR-1/2 levels at the cell membrane (Panbianco et al, 2008), whereas the DEP domain protein LET-99 is important for restricting the domain on the cell membrane to which GPR-1/2 is enriched (Panbianco et al, 2008;Tsou et al, 2003).…”

Polarity Control of Spindle Positioning in the C. elegans Embryo

“…PPK-1 or PIP2 may positively regulate the localization of GPR-1/GPR-2-LIN-5, although neither GPR-1/GPR-2 nor LIN-5 has a known PIP2-binding domain (Panbianco et al 2008). The protein phosphatase PPH-6 and its associated subunit Sit4p-associated protein-1 (SAP-1) also promote GPR-1/GPR-2 localization and spindle-pulling forces in anaphase (Afshar et al 2010). Co-depletion of CSNK-1 and PPH-6 resembles the PPH-6/SAPS-1 depleted phenotype of decreased cortical GPR-1/GPR-2 localization and spindle forces.…”

“…This most likely increases cortical rigidity and must therefore decrease the forces generated by anterior pulling force complexes. Indeed, several groups observed substantially increased pulling forces in the anterior after actin depletion by drug treatment (Afshar et al 2010;Redemann et al 2010;Berends et al 2013). Thus, in addition to the regulation of the pulling force complex, a difference in the cortical rigidity caused by actin accumulation provides a possible cause of pulling force asymmetry.…”