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Asymmetric enrichment of PIE-1 in the Caenorhabditis elegans zygote mediated by binary counterdiffusion.

Daniels BR, Perkins EM, Dobrowsky TM, Sun SX, Wirtz D - J. Cell Biol. (2009)

Bottom Line: Despite its critical involvement in cell fate determination, the enrichment of germline determinants remains poorly understood.Here, combining live-cell fluorescence methods and kinetic modeling, we demonstrate that the enrichment process does not involve protein immobilization, intracellular compartmentalization, or localized protein degradation.Instead, our results support a heterogeneous reaction/diffusion model for PIE-1 enrichment in which the diffusion coefficient of PIE-1 is reversibly reduced in the posterior, resulting in a stable protein gradient across the zygote at steady state.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA.

ABSTRACT
To generate cellular diversity in developing organisms while simultaneously maintaining the developmental potential of the germline, germ cells must be able to preferentially endow germline daughter cells with a cytoplasmic portion containing specialized cell fate determinants not inherited by somatic cells. In Caenorhabditis elegans, germline inheritance of the protein PIE-1 is accomplished by first asymmetrically localizing the protein to the germplasm before cleavage and subsequently degrading residual levels of the protein in the somatic cytoplasm after cleavage. Despite its critical involvement in cell fate determination, the enrichment of germline determinants remains poorly understood. Here, combining live-cell fluorescence methods and kinetic modeling, we demonstrate that the enrichment process does not involve protein immobilization, intracellular compartmentalization, or localized protein degradation. Instead, our results support a heterogeneous reaction/diffusion model for PIE-1 enrichment in which the diffusion coefficient of PIE-1 is reversibly reduced in the posterior, resulting in a stable protein gradient across the zygote at steady state.

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Posterior enrichment of GFP::PIE-1 does not involve degradation. Zygotes expressing GFP::PIE-1 under the pie-1 promoter and 3′-UTR (a) and the corresponding fluorescence intensity levels in distinct regions of the zygote (b; pronuclear meeting is denoted as t = 0 and n = 6). The increase in posterior fluorescence intensity is greater in magnitude than the decrease in anterior fluorescence intensity caused by the difference in the volumes of these two regions. (c) The presence of the PIE-1 ORF in GFP::PIE-1 (n = 6) does not affect overall levels of the protein product as compared with GFP alone (n = 6). mex-5/6(RNAi) prevents the posterior enrichment of GFP::PIE-1 in the zygote (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200809077/DC1), but does not affect overall protein levels in the zygote. Error bars represent SEM. Bar, 10 µm.
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fig1: Posterior enrichment of GFP::PIE-1 does not involve degradation. Zygotes expressing GFP::PIE-1 under the pie-1 promoter and 3′-UTR (a) and the corresponding fluorescence intensity levels in distinct regions of the zygote (b; pronuclear meeting is denoted as t = 0 and n = 6). The increase in posterior fluorescence intensity is greater in magnitude than the decrease in anterior fluorescence intensity caused by the difference in the volumes of these two regions. (c) The presence of the PIE-1 ORF in GFP::PIE-1 (n = 6) does not affect overall levels of the protein product as compared with GFP alone (n = 6). mex-5/6(RNAi) prevents the posterior enrichment of GFP::PIE-1 in the zygote (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200809077/DC1), but does not affect overall protein levels in the zygote. Error bars represent SEM. Bar, 10 µm.

Mentions: To characterize PIE-1 behavior in the early embryo, we quantitatively measured fluorescence levels within zygotes expressing a GFP::PIE-1 fusion gene under control of the pie-1 promoter and 3′-untranslated region (UTR; Fig. 1, a and b). GFP::PIE-1 becomes enriched in the posterior, on and around punctate structures known as P granules, which are RNA-rich components of the germplasm. Although GFP::PIE-1 also becomes enriched in pronuclei, the nuclear localization of PIE-1 has been shown to have little effect on cytoplasmic enrichment (Tenenhaus et al., 2001). Posterior fluorescence intensity reached a plateau value approximately three times the initial prefertilization level, whereas anterior fluorescence intensity decreased to approximately half its initial value. Overall fluorescence intensity in the zygote increased slightly during the first cell division, suggesting a low level of expression throughout the first division.


Asymmetric enrichment of PIE-1 in the Caenorhabditis elegans zygote mediated by binary counterdiffusion.

Daniels BR, Perkins EM, Dobrowsky TM, Sun SX, Wirtz D - J. Cell Biol. (2009)

Posterior enrichment of GFP::PIE-1 does not involve degradation. Zygotes expressing GFP::PIE-1 under the pie-1 promoter and 3′-UTR (a) and the corresponding fluorescence intensity levels in distinct regions of the zygote (b; pronuclear meeting is denoted as t = 0 and n = 6). The increase in posterior fluorescence intensity is greater in magnitude than the decrease in anterior fluorescence intensity caused by the difference in the volumes of these two regions. (c) The presence of the PIE-1 ORF in GFP::PIE-1 (n = 6) does not affect overall levels of the protein product as compared with GFP alone (n = 6). mex-5/6(RNAi) prevents the posterior enrichment of GFP::PIE-1 in the zygote (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200809077/DC1), but does not affect overall protein levels in the zygote. Error bars represent SEM. Bar, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654130&req=5

fig1: Posterior enrichment of GFP::PIE-1 does not involve degradation. Zygotes expressing GFP::PIE-1 under the pie-1 promoter and 3′-UTR (a) and the corresponding fluorescence intensity levels in distinct regions of the zygote (b; pronuclear meeting is denoted as t = 0 and n = 6). The increase in posterior fluorescence intensity is greater in magnitude than the decrease in anterior fluorescence intensity caused by the difference in the volumes of these two regions. (c) The presence of the PIE-1 ORF in GFP::PIE-1 (n = 6) does not affect overall levels of the protein product as compared with GFP alone (n = 6). mex-5/6(RNAi) prevents the posterior enrichment of GFP::PIE-1 in the zygote (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200809077/DC1), but does not affect overall protein levels in the zygote. Error bars represent SEM. Bar, 10 µm.
Mentions: To characterize PIE-1 behavior in the early embryo, we quantitatively measured fluorescence levels within zygotes expressing a GFP::PIE-1 fusion gene under control of the pie-1 promoter and 3′-untranslated region (UTR; Fig. 1, a and b). GFP::PIE-1 becomes enriched in the posterior, on and around punctate structures known as P granules, which are RNA-rich components of the germplasm. Although GFP::PIE-1 also becomes enriched in pronuclei, the nuclear localization of PIE-1 has been shown to have little effect on cytoplasmic enrichment (Tenenhaus et al., 2001). Posterior fluorescence intensity reached a plateau value approximately three times the initial prefertilization level, whereas anterior fluorescence intensity decreased to approximately half its initial value. Overall fluorescence intensity in the zygote increased slightly during the first cell division, suggesting a low level of expression throughout the first division.

Bottom Line: Despite its critical involvement in cell fate determination, the enrichment of germline determinants remains poorly understood.Here, combining live-cell fluorescence methods and kinetic modeling, we demonstrate that the enrichment process does not involve protein immobilization, intracellular compartmentalization, or localized protein degradation.Instead, our results support a heterogeneous reaction/diffusion model for PIE-1 enrichment in which the diffusion coefficient of PIE-1 is reversibly reduced in the posterior, resulting in a stable protein gradient across the zygote at steady state.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA.

ABSTRACT
To generate cellular diversity in developing organisms while simultaneously maintaining the developmental potential of the germline, germ cells must be able to preferentially endow germline daughter cells with a cytoplasmic portion containing specialized cell fate determinants not inherited by somatic cells. In Caenorhabditis elegans, germline inheritance of the protein PIE-1 is accomplished by first asymmetrically localizing the protein to the germplasm before cleavage and subsequently degrading residual levels of the protein in the somatic cytoplasm after cleavage. Despite its critical involvement in cell fate determination, the enrichment of germline determinants remains poorly understood. Here, combining live-cell fluorescence methods and kinetic modeling, we demonstrate that the enrichment process does not involve protein immobilization, intracellular compartmentalization, or localized protein degradation. Instead, our results support a heterogeneous reaction/diffusion model for PIE-1 enrichment in which the diffusion coefficient of PIE-1 is reversibly reduced in the posterior, resulting in a stable protein gradient across the zygote at steady state.

Show MeSH