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First published online 16 January 2008
doi: 10.1242/dev.013425

Development 135, 687-697 (2008)
Published by The Company of Biologists 2008
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Polo kinases regulate C. elegans embryonic polarity via binding to DYRK2-primed MEX-5 and MEX-6

Yuichi Nishi*,{dagger}, Eric Rogers*, Scott M. Robertson and Rueyling Lin{ddagger}

Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.


Figure 1
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  		Fig. 1. Schematic diagram of MEX-5 and PIE-1 proteins in a wild-type C. elegans hermaphrodite gonad, oocytes and early embryos. Germ nuclei (white circles) are in a syncitial cytoplasm of the gonad arm before being enclosed by membranes to form developing oocytes. The oocyte adjacent to the spermatheca undergoes maturation, and is then immediately ovulated and fertilized. Small black ovals: polar bodies. Nuclei are only shown in one-cell embryos. The levels of MEX-5 and PIE-1 proteins are indicated by shades of green and red, respectively. Yellow indicates the presence of both MEX-5 and PIE-1. The blue arrow indicates the proposed time for MBK-2 activation. Embryo images are oriented with the anterior pole to the left, with closed and open arrowheads pointing to the germline blastomere and its somatic sister, respectively. 1C Ana, first mitotic anaphase; 2C, two-cell; 4C, four-cell; 12C, 12-cell; DC, decondensation; MI, meiosis I; MII, meiosis II; ooc, developing oocytes; spm, spermatheca.

 

Figure 2
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  		Fig. 2. PLK-1 cytoplasmic asymmetry in early C. elegans embryos. Merged immunofluorescence micrographs of DAPI staining (red) with anti-PLK-1 (A-F) and anti-PLK-2 (M,N) staining (green). (G-L) GFP fluorescence of GFP::PLK-1PBD. (O,P) GFP fluorescence of GFP::PLK-1FL (O) and GFP::PLK-2FL (P). Closed and open arrowheads indicate the germline blastomere and its somatic sister, respectively. Stages of embryos in A-L are marked to the left; M, 1C; N-P, 2C. Scale bar: 10 µm.

 

Figure 3
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  		Fig. 3. PLK-1 cytoplasmic asymmetry is regulated downstream of MEX-5/6 and upstream of PIE-1 in C. elegans. Micrographs of two- to four-cell stage mutant or RNAi embryos (indicated at top). (A-J) Merged immunofluorescence of anti-PLK-1 (green) and DAPI staining (red). (K-M) GFP fluorescence of GFP::PLK-1PBD. (N-P) Centrosomal and chromosomal anti-PLK-1 staining in one-cell wild-type (N) and par-2(lw32) (P) embryos. A higher magnification photo of wild-type mitotic chromosomes is shown in O. (Q) Centrosomal and chromosomal GFP::PLK-1PBD signal in one-cell mex-5(zu199);mex-6(RNAi) embryos. (R) Quantification of PLK-1 asymmetry. The ratio represents the relative intensity of anterior and posterior portions of one-cell embryos, or somatic versus germline blastomeres in later stage embryos. Numbers in parentheses are standard deviations. n, number of embryos measured. Scale bar: 10 µm in all, except 2.5 µm in O.

 

Figure 4
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  		Fig. 4. T186 is required for interaction with PLK-1PBD and is important for MEX-5 function. (A) Yeast two-hybrid assays. Duplicated strains were incubated on leucine and tryptophan double dropout medium (Leu- Trp-) either without 3AT (left) or with 50 mM 3AT (right). Each strain tested contains a bait plasmid expressing the protein indicated along the top and a prey plasmid expressing the protein indicated to the left. An unrelated protein, ZYG-11, was used as a negative control for both bait and prey. H/A K/M: H542A, K544M. (B) Immunofluorescence micrographs of anti-GFP and anti-PIE-1 stainings in mex-5(zu199), or mex-5(zu199);mex-6(RNAi) embryos expressing GFP::MEX-5 T186A. Stages indicated to the left. Closed and open arrowheads indicate the germline blastomere and its somatic sister, respectively. (C) Rescue of mex-5(zu199) lethality by GFP::MEX-5 versus GFP::MEX-5 T186A, as scored by percentage of viable embryos produced per mex-5 homozygote mother. Scale bar: 10 µm.

 

Figure 5
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  		Fig. 5. MBK-2 and CDK-1 are required for the cytoplasmic asymmetry of PLK-1PBD but not MEX-5 in C. elegans. Immunofluorescence micrographs of anti-GFP (A-E), anti-MEX-5 (F-J) and DAPI staining (K-O) in two-cell wild-type (no RNAi), mbk-2(RNAi), cdk-1(RNAi), mpk-1(RNAi) or gsk-3(RNAi) embryos expressing GFP::PLK-1PBD as indicated. Scale bar: 10 µm.

 

Figure 6
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  		Fig. 6. MBK-2/DYRK2 phosphorylates MEX-5 at T186 and primes for PLK-1 in C. elegans. (A) Kinase assay conducted using indicated substrates and kinases. MEX-5 used here is the fragment containing amino acids 117-270. The positions of MEX-5117-270 (>), OMA-1 (#) and DYRK2 (<) are indicated. Assays with HsDYRK2 and MBK-2 were exposed differentially to get comparable signals for wild-type MEX-5117-270. (B) Tandem kinase assays using MEX-5FL WT or T186A as substrate, first with HsDYRK2 (cold ATP), followed by HsPLK1 (32P-{gamma}ATP). The arrow marks the position of MEX-5FL. Upper panel: 32P incorporation visualized by autoradiography. Lower panel: Coomassie-stained gel. *, a contaminating band co-purified with MBK-2; A186, T186A; D186, T186D; DYRK2, HsDYRK2; KD, kinase dead; WT, wild type.

 

Figure 7
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  		Fig. 7. PLK-1 and PLK-2 are required for MEX-5 and MEX-6 function in C. elegans. (A-L) Immunofluorescence micrographs of anti-MEX-5 and anti-GFP staining and corresponding DAPI staining in one-cell embryos expressing GFP::PIE-1. (A-D) Wild type (no RNAi). (E-H) plk-1(RNAi). (I-L) plk-1(RNAi);plk-2(RNAi). The arrows and arrowheads point to the oocyte- and sperm-derived pronuclei, respectively. Because of defective meiosis, the oocyte pronucleus has a higher DNA content than the sperm pronucleus in plk-1-depleted embryos. (M,O) DIC and (N,P) fluorescence micrographs of GFP::PIE-1 in four-cell wild-type (M,N) or mild plk-1(RNAi); plk-2(RNAi) (O,P) embryos. Closed and open arrowheads indicate the germline blastomere and its somatic sister, respectively. (Q) Quantification of MEX-5 and PIE-1 cytoplasmic asymmetry in one-cell wild-type, plk-1(RNAi), and plk-1(RNAi);plk-2(RNAi) embryos. See Fig. 3R legend for details. Scale bar: 10 µm. *, polar bodies.

 




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