spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 4 October 2006
doi: 10.1242/dev.02615

Development 133, 4281-4292 (2006)
Published by The Company of Biologists 2006
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in Development
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lyczak, R.
Right arrow Articles by Bowerman, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lyczak, R.
Right arrow Articles by Bowerman, B.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

The puromycin-sensitive aminopeptidase PAM-1 is required for meiotic exit and anteroposterior polarity in the one-cell Caenorhabditis elegans embryo

Rebecca Lyczak1,2,*, Lynnsey Zweier1, Thomas Group1, Mary Ann Murrow1, Christine Snyder1, Lindsay Kulovitz1, Alexander Beatty1, Kristen Smith1 and Bruce Bowerman2

1 Department of Biology, Ursinus College, Collegeville, PA 19426, USA.
2 Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA.


Figure 1
View larger version (224K):

[in a new window]
  		Fig. 1. pam-1 encodes a puromycin-sensitive aminopeptidase. Alignment of PAM-1 amino acid sequence showing conservation with mouse (GenBank AAH86798) and human (GenBank NP_006301) PSA (see also Brooks et al., 2003Go). Colored blocks mark amino acid identity. Overlined area denotes region of homology with 26S proteasome subunits (Constam et al., 1995Go). The underlined GAMEN motif is characteristic of M1 family metalloproteases and the site of two of the missense mutations A278W in pam-1(or347) and A278T in pam-1(or370) (marked with *). The boxed amino acids in the HEXXH(X)18E motif are necessary for zinc binding. The star denotes the site of deletion in pam-1(or282). The open circle denotes the position of the stop codon present in pam-1(or403), W538X, while the closed circle denotes the position of the stop codon present in pam-1(or547), Q598X.

 

Figure 2
View larger version (87K):

[in a new window]
  		Fig. 2. Meiotic defects in pam-1. (A) Time-lapse images of embryos from H2B::GFP strains. (A, parts a-c) In wild type, the pronuclei were apparent by DIC optics, within a few minutes of meiotic completion. (A, parts d-f) By contrast, the time of pronuclear appearance in pam-1 was significantly delayed. The condensed sperm chromosomes moved toward the oocyte chromosomes before the pronuclear envelopes formed. (B, part a) In wild type, chromosome segregation was clearly visible at the anterior cortex during meiosis II. (B, part b) In some pam-1 embryos, a DNA bridge was observed between the separating chromosomes in meiosis II. (B, part c) In some embryos this bridge persisted and the oocyte pronucleus and second polar body remained joined. Scale bar: 10 µm.

 

Figure 3
View larger version (60K):

[in a new window]
  		Fig. 3. pam-1 embryos lack early signs of AP polarity. Images from a time-lapse DIC series of a single embryo. (A) In wild type, the oocyte and sperm pronuclei appeared at opposite ends of the embryo, with the sperm pronucleus at the posterior pole. The SPCC cued axis polarization and caused changes in the cortex called pseudocleavage. (B) The oocyte pronucleus migrated to meet the sperm pronucleus in the posterior of the embryo. (C,D) The first spindle displaced toward the posterior and the first cleavage was asymmetric, with a larger anterior cell, AB, and a smaller posterior cell, P1. (E) These cells then differed in their cell cycle timing and spindle orientations during the next division. (F) In pam-1 embryos, polarization of the cortex was absent and the SPCC did not contact the posterior cortex as the pronuclei first appeared close to the center of the embryo. (G) The pronuclei met in the center of the cell. (H,I) In about 60% of pam-1 embryos, the first mitotic spindle remained in the center of the embryo, resulting in an equivalent cleavage. (J) When this occurred, the two daughter cells divided synchronously with parallel spindle orientations. (K) The position of pronuclear meeting was plotted for five wild-type and ten pam-1 embryos. On average, the pronuclei in pam-1 embryos met closer to the center of the embryo than in wild type. o, oocyte pronucleus; s, sperm pronucleus. Scale bar: 10 µm.

 

Figure 4
View larger version (64K):

[in a new window]
  		Fig. 4. Polarity markers are disrupted in pam-1. (A-E) Laser scanning confocal images of PAR-2 and PAR-3 in fixed embryos using indirect immunofluorescence. DNA was stained with TOTO. (A,A',D,D') Before and during the first cell division in wild type, PAR-2 localized to the posterior cortex of the embryo and PAR-3 to the anterior cortex. (B,B') In many one-cell pam-1 embryos, PAR-2 did not localize to the cortex, but was found instead on cytoplasmic puncta around the pronuclei. (C,C') When PAR-2 did localize to the cortex, it was often at a lateral position. (E,E') In roughly half of pam-1 embryos, PAR-3 localized around the entire periphery. (F-G') PGL-1 antibody staining of the P granules in fixed embryos using indirect immunofluorescence. (H-I') Fluorescent images of PIE-1::GFP taken in living embryos at the one- and two-cell stage. (F,F') During the one-cell stage in wild type, the germline P granules localized to the posterior pole and were segregated into the posterior daughter cell P1. (H,H') Similarly, the transcription factor PIE-1 localized to the posterior and was found in the cytoplasm and nucleus of the P1 cell. (G,G') In pam-1 embryos, the P granules were frequently mislocalized during the first division and often found in both cells at the two-cell stage. (I,I') PIE-1 failed to become properly partitioned during the first division, and was absent from both daughter cells in embryos that divided symmetrically. Scale bar: 10 µm.

 

Figure 5
View larger version (69K):

[in a new window]
  		Fig. 5. Polarity defects in pam-1 mutants occur in the absence of meiotic exit defects. (A-D) DIC images of cyb-3(RNAi) (A,C) and pam-1; cyb-3(RNAi) (B,D) embryos. cyb-3(RNAi) embryos show normal pseudocleavage (A) and a posteriorly displaced spindle (C), while many pam-1; cyb-3(RNAi) embryos lack pseudocleavage (B) and have a centrally positioned spindle (D). (E-I) Triple-stained confocal images with PGL-1 staining of P granules in red, tubulin staining in green and DAPI DNA staining in blue. While P granules were not localized in meiotic embryos of cyb-3(RNAi) and pam-1; cyb-3(RNAi) (E-F), they were localized to the posterior in all cyb-3(RNAi) embryos during the first mitosis (G). P granules failed to localize posteriorly in most pam-1; cyb-3(RNAi) embryos, being distributed either throughout the cytoplasm (H) or with a slight but incomplete posterior bias (I). Scale bar: 10 µm.

 

Figure 6
View larger version (43K):

[in a new window]
  		Fig. 6. Aberrant centrosome behavior in pam-1. (A) Centrosome positions over time, measured from time-lapse images of embryos expressing ß-TUBULIN::GFP. Each point represents the average of six wild-type or seven pam-1 embryos beginning with the moment of the appearance of the centrosomes. In pam-1 embryos, centrosomes first became visible in the posterior of the embryo, but then quickly moved toward the center of the embryo. (B) A plot of centrosome position relative to the posterior pole from three wild-type and three pam-1 embryos over ten 20 second time points. Each horizontal series represents measurements from one embryo. Measurements were begun when the centrosomes first became visible. Stars indicate the position of the centrosomes when they first separated from one another. The centrosomes in pam-1 moved quickly toward the center of the embryo. (C, parts a-c) Images from a time-lapse ß-TUBULIN::GFP movie of a wild-type embryo show movement of the centrosomes over time. By 200 seconds, the centrosomes clearly flanked the sperm pronucleus near the posterior pole of the embryo. (C, parts d-f) In pam-1 the centrosomes moved quickly into the center of the embryo and remained close together before appearance of the pronuclei. (D, parts a-c) By DIC optics, the centrosomes appeared as small dots flanking the pronuclei until the first spindle assembled (white arrowheads). (D, part d) By contrast, in some pam-1 embryos, the centrosomes began to nucleate microtubules before pronuclear appearance and were visible by DIC optics (white arrowheads). (D, parts e-f) In most cases, the centrosomes reduced in size as the pronuclei formed and then grew larger again as the mitotic spindle assembled. Scale bar: 10 µm.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2006