QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sumiyoshi, E. Articles by Yamamoto, M. Search for Related Content PubMed PubMed Citation Articles by Sumiyoshi, E. Articles by Yamamoto, M.

Protein phosphatase 4 is required for centrosome maturation in mitosis and sperm meiosis in C. elegans

¹ Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan
² PRESTO, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan
³ Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan

View larger version (61K): [in a new window]   Fig. 1. Proteins encoded by two PP4 genes of C. elegans. (A) Alignment of the amino acid sequences of C. elegans PPH-4.1 and PPH-4.2 with other PP4 families (human PPP4, Drosophila PP4 and Arabidopsis PPX1). Black shadowing indicates identical residues. (B) A phylogenic tree of PPP family protein phosphatses.

View larger version (157K): [in a new window]   Fig. 2. Phenotypes of pph-4.1(RNAi) embryos at the one-cell stage. All embryos are shown with the anterior end to the left. (A) A wild-type one-cell embryo showing a male pronucleus (black arrowhead) and a female pronucleus. (B) A wild-type embryo with a bipolar spindle. (C) A pph-4.1(RNAi) embryo with two male pronuclei (black arrowheads). (D) A pph-4.1(RNAi) embryo showing a tetra-polar spindle. (E) A pph-4.1(m-/p+) embryo with a tetra-polar spindle. Three spindle poles are visible in this focal plane. Spindle poles in B, D and E are indicated by white arrowheads. Bar, 10 µm.

View larger version (73K): [in a new window]   Fig. 3. PPH-4.1 is required for proper chromosome segregation during sperm meiosis. Hoechst staining (blue) is overlaid on the Nomarski image. (A) Wild-type spermatids. Each spermatid contains one nucleus. (B) pph-4.1(RNAi) spermatids. White arrowheads indicate anucleated spermatids. Black arrowheads indicate binucleated spermatids. (C-F) Secondary spermatocytes with spermatids (arrowheads) budding from the residual body (arrows). (C,E) Wild-type. (D,F) pph-4.1(RNAi). Chromosomes are not properly segregated in pph-4.1(RNAi) spermatocytes. Bar, 10 µm (A,B); 5 µm (C-F).

View larger version (13K): [in a new window]   Fig. 4. The first mitotic cycle is delayed in both pph-4.1(RNAi) and pph-4.1(M-/p+) embryos. Each symbol represents a single embryo and indicates the time elapsed from pronuclear meeting to the beginning of anaphase B. Open square, wild-type; closed triangle, pph-4.1(RNAi); closed square, pph-4.1(m-/p+).

View larger version (96K): [in a new window]   Fig. 5. PPH-4.1 is required for aster formation and recruitment of -tubulin to centrosomes during mitosis and sperm meiosis. (A,F,K,N) DAPI staining showing chromosomes. (B,G,I,O) Anti--tubulin antibody staining. (C,H,M,P) Anti--tubulin antibody staining. (A-C) Wild-type embryo at pronuclear meeting. (F-H) pph-4.1(RNAi) embryo at pronuclear meeting. (D,E,I,J) Magnified view of the indicated area in B, C, G and H, respectively. (K-M) Wild-type primary spermatocyte showing a meiotic spindle. (N-P) pph-4.1(RNAi) primary spermatocyte showing condensed chromosomes similar to those in (K) and a spindle not properly organized. Note that astral microtubules were poorly organized from the centrosomes (G,I,O) and that -tubulin on the centrosomes was dispersed (H,J,P). Each embryo is aligned with the anterior end to the left. White arrowheads indicate centrosomal localization of -tubulin. Bar, 10 µm (A-C, F-H); 2 µm (D,E,I,J); 5 µm (K-P).

View larger version (99K): [in a new window]   Fig. 6. Loss of PPH-4.1 function affects the localization of PLK-1 during mitosis. (A-C) Wild-type one-cell embryo at prometaphase. (D-F) pph-4.1(RNAi) one-cell embryo after pronuclear meeting. (A,D) DAPI staining showing chromosomes. (B,E) Anti--tubulin antibody staining. (C,F) Anti-PLK-1 antibody staining. White arrowheads indicate centrosomal staining of PLK-1. Black arrowheads indicate perinuclear PLK-1 staining. Black arrows indicate cytoplasmic granules. In pph-4.1(RNAi) embryos, PLK-1 did not localize to centrosomes even though chromosomes are condensed. Embryos are aligned with the anterior end to the left. Bar, 10 µm.

View larger version (78K): [in a new window]   Fig. 7. PPH-4.1 localizes to centrosomes during mitosis. (A) Western blotting against a wild-type worm extract. Lane 1: detection by PPH-4.1 antibodies. Arrows indicate doublet bands corresponding to the predicted molecular mass of PPH-4.1 (37 kDa). Lane 2: detection by the antibodies pretreated with 10xHis-PPH-4.1 protein. (B) Localization of PPH-4.1 during early cleavages. (a,d,g,j,m,p) Anti--tubulin antibody staining. (b,e,h,k,n,q) Anti-PPH-4.1 antibody staining. (c,f,i,l,o,r) Merged images. (a-o) Wild-type embryos. PPH-4.1 was localized to centrosomes from prophase to anaphase and only weakly during telophase (a-1; PPH-4.1 staining on centrosomes is indicated by white arrows). During interphase, centrosomal localization of PPH-4.1 was not observed (m-o; interphase cells are indicated with white arrowheads). (p-r) pph-4.1(RNAi) embryo at prophase. No signal was detected in severely affected pph-4.1(RNAi) embryos. Bar, 10 µm.

View larger version (89K): [in a new window]   Fig. 8. PPH-4.1 may be involved in chiasma formation during meiotic prophase I. (A,B) Oocytes in adult hermaphrodite gonads were stained with DAPI. The distal end is to the left. (A) Wild-type oocytes at the diakinesis stage contain six bivalent chromosomes. (B) In a pph-4.1(RNAi) gonad, oocytes in the distal region contain six bivalents, but oocytes in the proximal region contain twelve univalent chromosomes. (C,D) Magnified view of the indicated areas in (A) and (B), respectively. (A,B) Bar, 10 µm. (C,D) Bar, 2 µm.