Caenorhabditis elegans PIG-1/MELK acts in a conserved PAR-4/LKB1 polarity pathway to promote asymmetric neuroblast divisions

Abstract

Asymmetric cell divisions produce daughter cells with distinct sizes and fates, a process important for generating cell diversity during development. Many Caenorhabditis elegans neuroblasts, including the posterior daughter of the Q cell (Q.p), divide to produce a larger neuron or neuronal precursor and a smaller cell that dies. These size and fate asymmetries require the gene pig-1, which encodes a protein orthologous to vertebrate MELK and belongs to the AMPK-related family of kinases. Members of this family can be phosphorylated and activated by the tumor suppressor kinase LKB1, a conserved polarity regulator of epithelial cells and neurons. In this study, we present evidence that the C. elegans orthologs of LKB1 (PAR-4) and its partners STRAD (STRD-1) and MO25 (MOP-25.2) regulate the asymmetry of the Q.p neuroblast division. We show that PAR-4 and STRD-1 act in the Q lineage and function genetically in the same pathway as PIG-1. A conserved threonine residue (T169) in the PIG-1 activation loop is essential for PIG-1 activity, consistent with the model that PAR-4 (or another PAR-4-regulated kinase) phosphorylates and activates PIG-1. We also demonstrate that PIG-1 localizes to centrosomes during cell divisions of the Q lineage, but this localization does not depend on T169 or PAR-4. We propose that a PAR-4-STRD-1 complex stimulates PIG-1 kinase activity to promote asymmetric neuroblast divisions and the generation of daughter cells with distinct fates. Changes in cell fate may underlie many of the abnormal behaviors exhibited by cells after loss of PAR-4 or LKB1.

Figure 1

C. elegans orthologs of MELK, LKB1, STRAD, and MO25 regulate asymmetric cell division of the Q.p neuroblast lineage. (A) A schematic diagram of the Q.p lineage. In wild type, the Q.p neuroblast divides asymmetrically to produce Q.pa, a neuronal precursor, and Q.pp, which is destined for apoptosis. (B) In a cell-fate mutant, the fate of the apoptotic cell Q.pp is transformed to that of its sister Q.pa, and this transformation can result in the production of extra A/PVM and SDQ neurons if Q.pp survives and divides. (C) No extra neurons will be produced in a cell-fate mutant if Q.pp dies. (D) In a cell-death mutant, Q.pp is allowed to survive, but it will not divide. (E) Mutations in C. elegans orthologs of LKB1 (par-4), STRAD (strd-1), and MO25 (mop-25.2) interacted synergistically with ced-3 to produce extra A/PVMs. The number of lineages scored is indicated above the bars for each genotype. The extra A/PVMs were visualized with an integrated transgene zdIs5[Pmec-4::gfp]. The empty feeding vector control is L4440.

Figure 2

PAR-4 and STRD-1 regulate daughter cell size asymmetry in the Q.p division. In the Q.p division, the mitotic precursor Q.pa is three to four times larger than its apoptotic sister Q.pp (Cordes et al. 2006; Singhvi et al. 2011). The transcriptional reporters ayIs9 [Pegl-17::gfp] (A) and rdvIs1 [Pegl-17::myristoylated::mcherry] (B) were used to identify and measure the size of the daughters of the Q.p neuroblast. Ratios of the Q.p daughter cell sizes are depicted as boxplots. The number of divisions scored is indicated above each genotype. Solid circles indicate outliers of the distribution. (A) Mutations in par-4 and strd-1 disrupted the cell-size asymmetry of daughter cells of Q.p neuroblast. pig-1 data (Cordes et al. 2006) are shown here for comparison. (B) A transgene expressing PIG-1(T169D), but not one expressing full-length PIG-1 or PIG-1(T169A), disrupted the cell-size asymmetry of daughter cells of the Q.p neuroblast. All of the analyzed transgenes are in the wild-type background. *P < 0.001 (Student’s t-test).

Figure 3

PAR-4 and STRD-1 act in the Q lineage to promote the Q.p division and function in the same pathway as PIG-1. (A) Mutation of par-4, strd-1, and par-1 enhanced the extra neuron phenotype caused by the weak pig-1 allele gm280, but not by the strong pig-1 allele gm301. Mutation of sad-1 failed to enhance either pig-1(gm301) or pig-1(gm280). Neither pig-1 allele is temperature-sensitive for the extra A/PVM phenotype. (B) A mutation in strd-1, but not in par-4, enhanced the number of extra PLMs in a ced-3-sensitized background. In addition, a mutation in strd-1 enhanced the extra neuron phenotype of a weak but not a strong pig-1 mutant. (C) Expression of the par-4 or strd-1 cDNA from the mab-5 promoter partially rescued the extra PVM but not the AVM defect of ced-3; par-4 and ced-3; strd-1, respectively. The frequency of extra AVMs (open bars) and PVMs (solid bars) is presented separately. We generated two transgenic lines for Pmab-5::par-4::mcherry and two for Pmab-5::strd-1::gfp. Each of the lines for a particular construct was tested and gave similar results. Data for only one line of each type are presented. Number of lineages scored for each genotype is provided. N.S., not significant; **P < 0.005; *P < 0.05 (Fisher’s exact test).

Figure 4

The conserved threonine residue in the activation loop and the kinase-associated 1 domain (KA1) of PIG-1 are essential for its activity. (A) A transgene expressing PIG-1(T169A) failed to rescue the extra neuron phenotype of the strong pig-1 allele gm301 and enhanced the extra neuron phenotype of the weak pig-1 allele gm280. A transgene expressing PIG-1(T169D) partially rescued the extra neuron phenotype of pig-1(gm301). A PIG-1 transgene lacking the KA1 domain [PIG-1(KAΔ)] failed to rescue the extra neuron phenotype of pig-1(gm301). (B) Various PIG-1 transgenes induced extra neurons in a wild-type background. (C) A ced-3 mutation enhanced the extra neuron phenotype of various PIG-1 transgenes. We generated three transgenic lines for PIG-1(T169A), three for PIG-1(T169D), three for PIG-1(KAΔ), and one for PIG-1(KAΔ); K40A. Each of the lines for a particular construct was tested and gave similar results. Data for only one line of each type are presented. Wild-type and mutant PIG-1 proteins were tagged with GFP. All four PIG-1 proteins were expressed at similar levels, so the failure of the PIG-1(T169A) or PIG-1(KAΔ) expression to rescue the extra neuron phenotype of a pig-1 mutant does not result from inappropriate PIG-1 levels. (D) A mutation in par-4 does not suppress the extra PVM phenotype in animals expressing PIG-1(T169D). Number of lineages scored for each genotype are provided. N.S., not significant; **P < 0.005; *P < 0.05 (Fisher’s exact test).

Figure 5

PIG-1 localizes to centrosomes during cell divisions of the Q lineage. (A) Representative fluorescence micrographs showing plasma membrane (PM) and chromosomes (left panels) and PIG-1::GFP (right panels) in QL or QL.p divisions from animals carrying rdvIs1 and gmIs88[Pmab-5::pig-1::gfp]. (B) Representative fluorescence micrographs showing PM and chromosomes (left panels) and PIG-1(T169D)::GFP (right panels) in QL or QL.p divisions from animals carrying rdvIs1 and gmIs87[Pmab-5::pig-1(T169D)::gfp]. Arrows indicate the centrosomes, which are judged by morphological criteria (Ou et al. 2010). For example, during the anaphase of Q.p divisions, chromosomes (labeled by rdvIs1) that are pulled apart appear to wrap around the centrioles (labeled by γ-tubulin::GFP) (Ou et al. 2010). The GFP signals from PIG-1::GFP or PIG-1(T169D)::GFP are consistent with their location near the centrioles, possibly in the pericentriolar material. Scale bar, 3 μm.

Figure 6

A model for how pig-1, par-4, and strd-1 mutants regulate the Q.p lineage. (A) In wild-type animals, the Q.p neuroblast has a posteriorly displaced cleavage plane (vertical dashed line). We propose that anteriorly localized neuronal fate determinants (shaded circles) and posteriorly localized cell-death determinants (“x’s”) specify the fates of the Q.p daughters. The neuroblast divides to produce a large anterior daughter cell that inherits the neuronal fate determinants and becomes a precursor and a small posterior cell that inherits the death-fate determinants and dies. (B) In pig-1 mutants, the neuroblast has a centrally localized cleavage plane and more uniformly distributed determinants. Both daughters of the neuroblast inherit the determinants, leading to the production of two neuronal precursors. (C) In par-4 or strd-1 mutants, the neuroblast has a centrally localized cleavage plane, but the determinants are still asymmetrically localized. This results in the apoptosis of the posterior daughter because it inherits enough cell-death determinants or because it does not inherit enough neuronal-fate determinants.