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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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GFP::PIE-1 exhibits unrestricted mobility within the zygote. (a and b) At pronuclear meeting, 4.5-µm-diameter circular regions (arrow) were photobleached in the posterior cytoplasm of embryos expressing GFP::PIE-1. Mean fluorescence intensity in the photobleached regions recovered to within 95% of their initial value (n = 7). (c and d) Repeated photobleaching of a 15-µm circle adjacent to the posterior cortex causes anterior fluorescence to decrease to a value of 55 ± 5% compared with controls (P = 0.00011, n = 4). (e and f) Similarly, repeated photobleaching of a 30-µm circle in the anterior cytoplasm caused a decrease of posterior fluorescence to a value of 40 ± 6% compared with controls (P = 0.000032, n = 5). Time 0 represents the time of photobleaching in each experiment, which was chosen to be coincident with pronuclear meeting. The difference in the size of the areas chosen for photobleaching is due to the difference in relative volume of the anterior and posterior cytoplasm. Error bars represent SEM. ***, P < 0.001. Bars, 10 µm.
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fig2: GFP::PIE-1 exhibits unrestricted mobility within the zygote. (a and b) At pronuclear meeting, 4.5-µm-diameter circular regions (arrow) were photobleached in the posterior cytoplasm of embryos expressing GFP::PIE-1. Mean fluorescence intensity in the photobleached regions recovered to within 95% of their initial value (n = 7). (c and d) Repeated photobleaching of a 15-µm circle adjacent to the posterior cortex causes anterior fluorescence to decrease to a value of 55 ± 5% compared with controls (P = 0.00011, n = 4). (e and f) Similarly, repeated photobleaching of a 30-µm circle in the anterior cytoplasm caused a decrease of posterior fluorescence to a value of 40 ± 6% compared with controls (P = 0.000032, n = 5). Time 0 represents the time of photobleaching in each experiment, which was chosen to be coincident with pronuclear meeting. The difference in the size of the areas chosen for photobleaching is due to the difference in relative volume of the anterior and posterior cytoplasm. Error bars represent SEM. ***, P < 0.001. Bars, 10 µm.

Mentions: Another hypothetical explanation for PIE-1 enrichment is asymmetrical protein immobilization or “protein trapping.” To investigate this possibility, we used FRAP (Axelrod et al., 1976; Lippincott-Schwartz et al., 2001; Reits and Neefjes, 2001) to characterize GFP::PIE-1 dynamics in the germplasm (Fig. 2, a and b). We photobleached 4.5-µm-diameter circular regions in the posterior cytoplasm of zygotes at pronuclear meeting and measured the subsequent fluorescence intensity recovery within the photobleached region. Because photobleaching can be considered irreversible over our experimental time scales (Axelrod et al., 1976; Lippincott-Schwartz et al., 2001; Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200809077/DC1), fluorescence recovery can be used to measure the mobility of GFP::PIE-1. The photobleached regions underwent essentially full recovery (immobile fraction <5%), demonstrating that GFP::PIE-1 remains freely mobile in the germplasm during the enrichment process.


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)

GFP::PIE-1 exhibits unrestricted mobility within the zygote. (a and b) At pronuclear meeting, 4.5-µm-diameter circular regions (arrow) were photobleached in the posterior cytoplasm of embryos expressing GFP::PIE-1. Mean fluorescence intensity in the photobleached regions recovered to within 95% of their initial value (n = 7). (c and d) Repeated photobleaching of a 15-µm circle adjacent to the posterior cortex causes anterior fluorescence to decrease to a value of 55 ± 5% compared with controls (P = 0.00011, n = 4). (e and f) Similarly, repeated photobleaching of a 30-µm circle in the anterior cytoplasm caused a decrease of posterior fluorescence to a value of 40 ± 6% compared with controls (P = 0.000032, n = 5). Time 0 represents the time of photobleaching in each experiment, which was chosen to be coincident with pronuclear meeting. The difference in the size of the areas chosen for photobleaching is due to the difference in relative volume of the anterior and posterior cytoplasm. Error bars represent SEM. ***, P < 0.001. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: GFP::PIE-1 exhibits unrestricted mobility within the zygote. (a and b) At pronuclear meeting, 4.5-µm-diameter circular regions (arrow) were photobleached in the posterior cytoplasm of embryos expressing GFP::PIE-1. Mean fluorescence intensity in the photobleached regions recovered to within 95% of their initial value (n = 7). (c and d) Repeated photobleaching of a 15-µm circle adjacent to the posterior cortex causes anterior fluorescence to decrease to a value of 55 ± 5% compared with controls (P = 0.00011, n = 4). (e and f) Similarly, repeated photobleaching of a 30-µm circle in the anterior cytoplasm caused a decrease of posterior fluorescence to a value of 40 ± 6% compared with controls (P = 0.000032, n = 5). Time 0 represents the time of photobleaching in each experiment, which was chosen to be coincident with pronuclear meeting. The difference in the size of the areas chosen for photobleaching is due to the difference in relative volume of the anterior and posterior cytoplasm. Error bars represent SEM. ***, P < 0.001. Bars, 10 µm.
Mentions: Another hypothetical explanation for PIE-1 enrichment is asymmetrical protein immobilization or “protein trapping.” To investigate this possibility, we used FRAP (Axelrod et al., 1976; Lippincott-Schwartz et al., 2001; Reits and Neefjes, 2001) to characterize GFP::PIE-1 dynamics in the germplasm (Fig. 2, a and b). We photobleached 4.5-µm-diameter circular regions in the posterior cytoplasm of zygotes at pronuclear meeting and measured the subsequent fluorescence intensity recovery within the photobleached region. Because photobleaching can be considered irreversible over our experimental time scales (Axelrod et al., 1976; Lippincott-Schwartz et al., 2001; Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200809077/DC1), fluorescence recovery can be used to measure the mobility of GFP::PIE-1. The photobleached regions underwent essentially full recovery (immobile fraction <5%), demonstrating that GFP::PIE-1 remains freely mobile in the germplasm during the enrichment process.

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