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 8 October 2008
doi: 10.1242/dev.027060

Development 135, 3665-3675 (2008)
Published by The Company of Biologists 2008
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
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 Tenlen, J. R.
Right arrow Articles by Priess, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tenlen, J. R.
Right arrow Articles by Priess, J. R.
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?

MEX-5 asymmetry in one-cell C. elegans embryos requires PAR-4- and PAR-1-dependent phosphorylation

Jennifer R. Tenlen1,2,*, Jeffrey N. Molk2,{dagger}, Nitobe London2,3,{dagger}, Barbara D. Page2,3 and James R. Priess1,2,3,4,{ddagger}

1 Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA.
2 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
3 Howard Hughes Medical Institute, Seattle, WA 98109, USA.
4 Department of Biology, University of Washington, Seattle, WA 98195, USA.


Figure 1
View larger version (51K):
[in this window]
[in a new window]

  		Fig. 1. MEX-5 in oocytes and early embryos. (A) Diagram of MEX-5 (green) and PIE-1 (red) localization from the one-cell to eight-cell stages. At the eight-cell stage, the single germline blastomere expresses PIE-1 and the seven somatic blastomeres express MEX-5; somatic degradation is occurring in the four oldest somatic blastomeres (light green; three are visible in this ventral-up orientation). (B) Diagram of one arm of the gonad showing germ cells progressing through mitosis, meiosis, and forming oocytes. The oldest oocyte (-1 position) enters the spermatheca where it is fertilized by sperm and pushed into the uterus. (C-F'') Each row shows a single one-cell embryo stained for DNA, PAR-3 and MEX-5; arrows indicate nascent maternal (long arrow) and paternal (short arrow) pronuclei. In these wide-field images, fluorescence from cortical PAR-3 is visible in the central focal plane, facilitating comparison with cytoplasmic MEX-5. Anterior is left in all images, and embryos are about 50 µm in length. (G) GFP:MEX-5 in the gonad of a live adult. Embryos a-c are at the one-cell stage; a and b are completing meiosis I and II, c is beginning the first mitotic division (posterior up), and d is at the two-cell stage. (H) Quantitative fluorescence of GFP:MEX-5 at the anterior and posterior poles of five single embryos recorded before and after pronuclear meeting (arrow) as indicated. Error bars indicate s.e.m.

 

Figure 2
View larger version (47K):
[in this window]
[in a new window]

  		Fig. 2. Regions of MEX-5 required for one-cell asymmetry. (A,B) Wild-type or mutant MEX-5 proteins were fused to GFP and scored at the one-cell stage for asymmetry (Asym), P granule localization (Pg), and for degradation after the four-cell stage in somatic blastomeres (Som). ++, wild-type asymmetry; +, reduced asymmetry; (+), weak and variable asymmetry; -, no apparent asymmetry. Representative fluorescence images of some embryos are shown in A. Asterisks by plasmid names indicate fusion proteins with prominent nuclear localization in addition to cytoplasmic localization (arrow in image of pJT35). (B) The C-terminal 22 amino acids of MEX-5 indicating substitutions made within full-length MEX-5. (C) Fluorescence micrographs of transgenic embryos expressing mutant forms of GFP:MEX-5 as listed, at the one-cell, two-cell and eight-cell stages. The eight-cell-stage embryos are labeled and are in the same orientation as in Fig. 1A; asterisks indicate the germline blastomere P3. Arrows point to somatic blastomeres at the eight-cell stage, where MEX-5 normally is degraded. GFP:MEX-5KEN to AAA appeared identical in all respects to GFP:MEX-5 (not shown). The arrowhead points to an example of prominent P granule localization; note abnormally bright P granules in the P3 cell for S458A. (D) Ratio of anteroposterior fluorescence at pronuclear meeting, n>30 embryos each; error bars show s.d., **P<3x10-4 (two-tailed t-test). The type of GFP:MEX-5 fusion protein is indicated in brackets along with the plasmid name. fzr-1(-)=fzr-1(ok380); par-1(-)=par-1(it51). (E) Ratio of anteroposterior fluorescence in single one-cell embryos expressing GFP fusion proteins and recorded at 1-minute intervals before and after pronuclear meeting (arrow).

 

Figure 3
View larger version (109K):
[in this window]
[in a new window]

  		Fig. 3. Kymograph analysis of GFP:MEX-5. (A) Kymograph display of images collected from live recording of a one-cell embryo; line widths are 2 microns. Imaging began before the appearance of the maternal and paternal pronuclei (mpn and ppn), when MEX-5 is symmetrical, and ended at pronuclear meeting (pnm), when MEX-5 is asymmetrical. Arrows indicate examples of GFP:MEX-5-containing particles. (B,C) One-cell embryo as GFP:MEX-5 asymmetry is initiated (decondensation) before (B) and after (C) photobleaching. (D) Kymograph of recovery within the boxed zone indicated in C; the asterisk indicates the time point prior to bleaching and line widths are 8.3 microns. (E,F) Cartoons of idealized kymographs from hypothetical embryos after photobleaching. Scale bars: in A, 4 minutes; in D, 4 seconds.

 

Figure 4
View larger version (38K):
[in this window]
[in a new window]

  		Fig. 4. FRAP and FLIP analysis of GFP:MEX-5 mobility. (A) FRAP results on one-cell embryos with GFP or the fusion protein listed (n=4-10 embryos analyzed per experiment). P-values are from comparisons to pJT02 after pronuclear meeting (pnm); error bars show s.e.m. (B,C) FLIP experiments on embryos at pronuclear meeting. Embryos expressing GFP, NMY-2:GFP, or the GFP:MEX-5 fusion protein listed were photobleached near the posterior pole (asterisk) for the number of cycles indicated. (C) Fluorescence measured at the anterior pole (solid line) and posterior pole (dashed line) for single embryos after each photobleaching cycle for GFP or the GFP:MEX-5 fusion proteins listed. Loss of posterior fluorescence is less for GFP and GFP:MEX-5 [-ZFs; pJT132] than for the other proteins, presumably because a larger fraction of non-bleached, anterior protein moves posteriorly.

 

Figure 5
View larger version (55K):
[in this window]
[in a new window]

  		Fig. 5. S458 is phosphorylated and is required for normal embryonic polarity. (A-C) Western blots of extracts from wild-type adult worms or worms expressing various GFP:MEX-5 fusion proteins as listed. Black triangles indicate 53 kDa (predicted size of endogenous MEX-5); white triangles indicate 82 kDa (predicted size of GFP:MEX-5). (A) Extracts from wild-type worms treated with alkaline phosphatase for 0, 2 or 14 hours and stained with either {alpha}MEX-5 or {alpha}MEX-5(pS458). (B) {alpha}MEX-5(pS458) stains endogenous MEX-5 but not GFP:MEX-5S458A for two independent lines of worms; compare with levels of GFP:MEX-5 stained by {alpha}MEX-5. Note that the levels of all GFP:MEX-5 fusion proteins are much lower than endogenous MEX-5. (C) The GFP:MEX-5(KRRTSL to AAATSA) fusion protein is stained by {alpha}GFP, but not by {alpha}MEX-5(pS458). (D-D'') Single, transgenic mex-5(zu199);mex-6(pk440) embryo expressing GFP:MEX-5S458A and stained for DNA (DAPI, D) and immunostained for GFP (D') and PIE-1 (D''); embryo is at the one-cell stage and in metaphase of first mitosis.

 

Figure 6
View larger version (95K):
[in this window]
[in a new window]

  		Fig. 6. S458 phosphorylation in embryos and oocytes. (A-F') Each row shows a single one-cell stage wild-type or mutant embryo, as listed after staining with {alpha}MEX-5 (left column) and {alpha}MEX-5(pS458) (right column). (G-I') Each row shows one arm of a wild-type or mutant gonad, as listed after staining with {alpha}MEX-5 (left column) and {alpha}MEX-5(pS458) (right column); gonads are oriented as in Fig. 1B. {alpha}MEX-5(pS458) shows non-specific staining of cytoplasmic foci in early embryos (B',D',E',F') and of sperm (sp) in gonads (see H'). par-1(it51), but not par-1(b274), has a mutation in the predicted kinase domain.

 

Figure 7
View larger version (33K):
[in this window]
[in a new window]

  		Fig. 7. Model for MEX-5 asymmetry. Speculative model showing MEX-5 before (left) and after (right) asymmetry. MEX-5 is either bound (green) or unbound (blue) to cytoplasmic actomyosin, or to a component whose mobility is limited by actomyosin. Cortical contraction changes the localization, or dynamics, of actomyosin in the deep cytoplasm, such that MEX-5 concentrates in the anterior. Additional sharpening of the MEX-5 asymmetry might come from factors that modulate MEX-5 binding, such as phosphorylation by posterior-localized PAR-1 (red) or non-localized PAR-4 (not shown).

 

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 2008