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

First published online 28 March 2006
doi: 10.1242/jcs.02873

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS 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 Google Scholar Google Scholar Articles by Patalano, S. Articles by Chenevert, J. Search for Related Content PubMed PubMed Citation Articles by Patalano, S. Articles by Chenevert, J. Social Bookmarking                         What's this?

The aPKC–PAR-6–PAR-3 cell polarity complex localizes to the centrosome attracting body, a macroscopic cortical structure responsible for asymmetric divisions in the early ascidian embryo

Solenn Patalano^*, Gérard Prulière, François Prodon, Alexandre Paix, Philippe Dru, Christian Sardet and Janet Chenevert

BioMarCell, Laboratoire de Biologie de Developpement, UMR 7009 CNRS, University Pierre and Marie Curie, Observatoire, Villefranche-sur-mer 06230, France

View larger version (38K): [in a new window]   Fig. 1. Asymmetric divisions directed by the centrosome attracting body (CAB) in the posterior of the ascidian embryo. (A,B) In the 8-cell stage embryo, the two posterior vegetal blastomeres (B4.1 cells) contain both the CAB (cER-mRNA-rich domain in red) and the myoplasm (mitochondria-rich domain in green). (A) B4.1 divides asymmetrically to form a large (B5.1) and a small (B5.2) daughter cell which inherits the CAB. The small daughter cell divides asymmetrically again, generating B6.4 (large cell) and B6.3, the small cell, which contains the CAB and undergoes a final asymmetric division to yield a very small cell (B7.6) which ceases to divide. Blue bars indicate the daughters formed by each CAB-directed asymmetric division. (C) Final position of myoplasm and cER-mRNA domains in the tadpole larva. The myoplasm-containing cells give rise to tail muscle. The B7.6 cells containing the cER-mRNA domain form part of the endodermal strand and are thought to give rise to the germline after metamorphosis (Takamura et al., 2002; Tomioka et al., 2002).

View larger version (135K): [in a new window]   Fig. 2. Visualization of the CAB in posterior vegetal blastomeres of Phallusia mammillata embryos. (A-D) Live Phallusia embryos viewed by DIC optics (A-C) or by fluorescence confocal microscopy (D). (A) Images extracted from a time-lapse sequence (see Movie 1 in supplementary material). t=0: the interphase nucleus (n) appears pinched (arrow) as it migrates towards the CAB; t=5' (5 minutes): the CAB (arrowhead in all panels) appears as a smooth zone, which compacts and thickens during prophase; t=9' (9 minutes): the spindle positions asymmetrically, causing unequal cleavage into large (B6.4) and small (B6.3) cells. (B) The CAB is visible as a smooth cortical zone (arrowhead) late in the cell cycle. (C) A transient surface protrusion forms at the position of the CAB early in the cell cycle. (D) Embryo double labelled for ER (red) and mitochondria (green). The nucleus (n) and centrosome (c) are oriented toward the cER-rich CAB. (E) In situ hybridization showing localization of a postplasmic/PEM mRNA in the CAB (antisense probe for Phallusia Vasa). (F) Embryo fixed with formaldehyde viewed by DIC optics. (G) Scanning electron micrograph shows surface structures protruding at the position of the CAB. The names of the relevant blastomeres are indicated as drawn in Fig. 1. Bars, 10 µm.

View larger version (98K): [in a new window]   Fig. 3. Ascidian aPKCs lack a conserved cyteine-rich domain. (A) Domain structure of aPKCs. PB1, Phox and Bem protein interaction domain which binds PAR-6 protein; Kinase, serine threonine kinase domain; C1, cysteine-rich domain which binds InsPtd(3,4,5)P3. (B) Comparison of aPKC proteins from four ascidian species (Phallusia mammillata, Pm; Ciona intestinalis, Ci; Ciona savigny, Cs; and Halocynthia roretzi, Hr) with aPKCs from human (Hu), frog (Xl), fly (Dm), and worm (Ce). The C1 domain usually present in aPKC but lacking in ascidian aPKCs is underlined. The two boxed areas indicate a normally contiguous region of homology specific to ascidian aPKCs. PmaPKC was obtained by cloning from a cDNA library (this study, GenBank AY987397). CiaPKC was obtained by assembling sequence information from Sasakura et al. (Sasakura et al., 2003) and CLSTR05328r1 from the Ghost genome browser (ghost.zool.kyoto-u.ac.jp). CsaPKC was obtained by analysis of genomic sequence information in http://www2.bioinformatics.tll.org.sg:8082/Ciona_savignyi/ and http://www.broad.mit.edu/annotation/ciona/. HraPKC sequence was obtained from 5' EST data from the Magest genome site accessible via Aniseed (aniseed-ibdm.univ-mrs.fr). The HraPKC protein appears truncated in the kinase domain because the sequence encoding the C-terminal half of the protein is not available.

View larger version (129K): [in a new window]   Fig. 4. aPKC protein but not its mRNA localizes to the CAB. (A) aPKC antibody recognizes a single 65 kDa protein in Phallusia mammillata egg extracts (lane 1). The signal disappears when aPKC antibody is pre-incubated with the specific antigenic peptide (lane 2) but not with an unrelated peptide (lane 3). (B) Immunoblot of protein extracts from different developmental stages (E, egg; 2, 2 cells; 8, 8 cells; 16, 16 cells; 32, 32 cells; G, gastrula; N, neurula; T, tadpole). (C-J) Confocal images of Phallusia embryos immunolabelled for aPKC protein. Arrowhead indicates the pair of CABs. Stages can be compared with drawings in Fig. 1 and posterior vegetal blastomere name is indicated (B4.1, B5.2, B6.3, B7.6). Each image represents a projection of two to four confocal sections containing the brightest aPKC label. (C) 4-cell stage, vegetal view. (D) 8-cell stage, posterior surface view. (E) 8-cell stage, lateral view; the two halves of the embryo are projected into one plane so that both left and right CABs are visible. Inset shows a drawing of the orientation of the embryo as depicted in Fig. 1B. (F) 16-cell stage, vegetal view. (G) 32-cell stage, vegetal view. (H) 64-cell stage, vegetal view. (I) 8-cell stage, high magnification posterior surface view. Movie 2 in supplementary material shows the complete series of confocal z sections corresponding to this image. (J) B5.2 blastomeres from two 16-cell stage embryos showing cell-cycle-dependent compaction of the CAB. (K-N) aPKC mRNA is maternal and ubiquitously distributed. (K) Northern blot. aPKC probe was hybridized to RNA prepared from Phallusia embryos at the indicated stages (E, unfertilized egg; 2: 2 cells; 8: 8 cells; 32: 32 cells; G: gastrula; N, neurula; T, tadpole). (L-N) In situ hybridization using aPKC antisense probe (L,N) or negative control sense probe (M). The CAB is visible as in most fixed embryos (arrowhead in N) but not labelled by the probe for aPKC mRNA. Bars, 15 µm.

View larger version (111K): [in a new window]   Fig. 5. PAR-6 protein localizes to the CAB. (A) Domain structure of PAR-6 proteins. PB1, Phox and Bem protein interaction domain, which binds aPKC protein; CRIB, CDC42 interacting domain; PDZ, protein interaction domain, which binds PAR-3 protein. (B) Comparison of PAR-6 sequences from Phallusia mammillata (Pm), Ciona intestinalis (Ci), Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), Xenopus laevis (Xl) and Mus musculus (Mm). Boxed areas indicate N-terminal (MDKTSSGQRAPSP) and C-terminal (RDVSVKAKRSNEPQD) peptides used to raise antibodies. (C) A single 45 kDa band is detected on a western blot of Phallusia egg extract using sera purified against the synthetic peptide (lane 1) or against the PAR-6 fusion protein (lane 2). (D,E) Both PAR-6 antibodies directed against either the N-terminal or C-terminal peptides label the surface of posterior vegetal blastomeres at the position of the CABs (arrowheads).

View larger version (55K): [in a new window]   Fig. 6. PAR-3 protein localizes to the CAB. (A) Sequence recognized by the PAR-3 antibody. The hatched rectangle represents the region of Drosophila PAR-3 protein (Bazooka) used to raise the PAR-3 antibody (Wodarz et al., 1999). The alignment shows high homology between the first 87 amino acids of this domain and an open reading frame in the Ciona genome. (B) Structure of PAR-3 gene in Ciona genome. The gene covers 20 kb and consists of at least 20 exons, as deduced by comparing conserved coding regions between Ciona intestinalis and Ciona savigny genomes using Vista software. The first three exons (beginning at position 109598) encode the conserved sequence shown in A. The Ciona PAR-3 DNA sequence previously described, which encodes the three PDZ domains (Sasakura et al., 2003) begins 5 kb downstream of the N-terminal homology and reads in the same direction. Numbers below the horizontal line indicate base position on scaffold 159 from version 1.0 of the genome (scaffold 148 in version 1.95). (C) Immunoblot using PAR-3 antibody on protein extract from Phallusia eggs. (D,E) Immunolabelling of Phallusia embryos with PAR-3 antibody. Arrowhead indicates position of the pair of CABs.

View larger version (120K): [in a new window]   Fig. 7. Distribution of mitochondria, microfilaments, microtubules and aPKC protein in CAB-containing blastomeres. (A) B5.2 cells labelled for mitochondria (posterior view). Dotted white line indicates plasma membranes between blastomeres. The dome-shaped CAB is apparent as a mitochondria-free domain (arrow). (B,C) B5.2 cells double labelled for aPKC (green) and mitochondria (red); vegetal views. Arrowhead indicates the cortical face of the CAB; arrow indicates the mitochondria-free space occupied by the cER-mRNA domain and dense granules as in A. Movie 3 in supplementary material displays a series of confocal z sections through a similarly labelled embryo. (D) 8-cell stage embryo double labelled for aPKC (green) and actin microfilaments (red). Posterior vegetal portion of B4.1 cell is shown. (E-G) Isolated cortical fragments from posterior face of 8-cell stage embryo, double-labelled for aPKC (green) and ER (red). (E) One CAB; the aPKC protein (green) is concentrated in particles filling part of the space between ER sheets and tubes (red) which are densely packed in the CAB. (F) Thin confocal sections taken of a pair of CABs, proceeding from the plasma membrane (z1) into the cell (z3, z5), in increments of 0.16 µm, showing that the aPKC-rich layer is closer to the plasma membrane than the ER. (G) Intensity profile of the two signals along the x-z axis; plasma membrane is on the bottom; cytoplasm on top. (H-M) Posterior vegetal blastomeres double-labelled for aPKC (green) and tubulin (red); c, centrosome. (H-J) B5.2 cells in interphase; dotted white lines indicate the position and shape of the nuclei (n) as projected from other focal planes. (K) B4.1 cell in prophase; n, nucleus. Movie 4 in supplementary material shows the complete series of confocal z sections through this cell. (L) B5.2 cells in prometaphase. (M) B5.2 cells in metaphase (above) and anaphase (below). DNA is labelled in blue. Bars, 10 µm.

View larger version (110K): [in a new window]   Fig. 8. aPKC accumulation at the CAB is dependent on actin microfilaments and independent of microtubules. Embryos were incubated with the indicated inhibitor then fixed, immunolabelled and examined by confocal microscopy. (A-D) Treatment at 2-cell stage for 90 minutes, the time equivalent to three cell cycles. Images on the right show aPKC label (surface views); images on the left show Hoechst label in the same embryos (central planes containing chromatin). In embryos treated with cytochalasin or latrunculin, nuclear cycles continue and each blastomere accumulates 8-16 patches of DNA, which are symmetrically arranged and either decondensed as interphase nuclei (A) or condensed in mitotic configurations (B). In embryos treated with nocodazole (C,D) nuclear cycles arrest; DNA is condensed and dispersed randomly along the cleavage furrow. In C (lateral view of one embryo) and D (posterior view of another embryo), arrowheads indicate CAB-like accumulations of aPKC protein. (E-G) Treatment at 16-cell stage for 30 minutes, the time equivalent to one cell cycle. Images show aPKC labelling in posterior blastomeres (B5.2 cells, surface views) from a typical treated embryo. Arrowhead in G indicates a pair of CABs. Graphs show the percentage of embryos in which a concentrated cortical patch of aPKC protein was either present, absent, or weakly apparent in B5.2 cells; at least 20 embryos were scored for each treatment.

View larger version (50K): [in a new window]   Fig. 9. Schematic representation of the structure of the ascidian CAB. The aPKC–PAR-6–PAR-3 domain (PAR domain, purple) is contiguous with the cortical actin microfilament layer (light blue) and sandwiched between the plasma membrane (black) and the cER-mRNA domain (red), all of which is surrounded by the mitochondria-rich myoplasm (green). (A) Dynamic reorganization during asymmetric cleavage. Microtubules (blue) emanating from the proximal centrosome contact the CAB and adjacent cell cortex. During interphase the CAB is widespread and thin as the nucleus migrates towards it. During mitosis the CAB compacts and thickens as the spindle positions asymmetrically. The smaller daughter cell inherits the CAB; centrosome attraction and unequal cleavage are then repeated. (B) Multilayered distribution of the major components of the CAB. Microtubules are not represented since their precise relationship to components of the CAB is not completely understood.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter