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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 PIE-1 is driven by binary counterdiffusion. (a) We propose that free PIE-1 (P) undergoes a heterogeneous surface reaction in the posterior of the zygote that causes a reduction in its effective diffusion coefficient. This resultant “slow” species of PIE-1 (P*) homogenously reverts back to its initial form in the cytoplasm. (b) Analytical solutions of the concentration profiles of both species of PIE-1, x = 0 to x = L, reveal an overall concentration gradient along the A/P axis. The experimental concentration profiles were taken from the central 30 µm of the A/P axis (where the shape of the gradient is most pronounced) to avoid intensity spikes from P granules in the posterior and large plateaus in intensity in the anterior, which interfere with data fitting. (c) The half-times of recovery of GFP::PIE-1 intensity in 4.5-µm-diameter circular photobleached region experiments from the anterior and posterior cytoplasm were 0.7 ± 0.3 (n = 38) and 4.5 ± 0.3 s (n = 7), respectively. (d) Normalized autocorrelation curves of FCS fluctuation data showed differences between anterior and posterior diffusion of GFP::PIE-1. Theoretical diffusion curves suggest the presence of two species of PIE-1 in the anterior cytoplasm, with diffusion coefficients of 8.7 ± 0.6 and 0.6 ± 0.2 µm2/s, respectively. The posterior cytoplasm is well described by a three-component model with diffusion coefficients of 14.2 ± 7.8, 0.6 ± 0.3, and 0.006 ± 0.002 µm2/s. FRAP and FCS analysis is summarized in panel e and deviations from fit are provided in Fig. S3 (available at http://www.jcb.org/cgi/content/full/jcb.200809077/DC1). Error bars represent SEM.
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fig3: Posterior enrichment of PIE-1 is driven by binary counterdiffusion. (a) We propose that free PIE-1 (P) undergoes a heterogeneous surface reaction in the posterior of the zygote that causes a reduction in its effective diffusion coefficient. This resultant “slow” species of PIE-1 (P*) homogenously reverts back to its initial form in the cytoplasm. (b) Analytical solutions of the concentration profiles of both species of PIE-1, x = 0 to x = L, reveal an overall concentration gradient along the A/P axis. The experimental concentration profiles were taken from the central 30 µm of the A/P axis (where the shape of the gradient is most pronounced) to avoid intensity spikes from P granules in the posterior and large plateaus in intensity in the anterior, which interfere with data fitting. (c) The half-times of recovery of GFP::PIE-1 intensity in 4.5-µm-diameter circular photobleached region experiments from the anterior and posterior cytoplasm were 0.7 ± 0.3 (n = 38) and 4.5 ± 0.3 s (n = 7), respectively. (d) Normalized autocorrelation curves of FCS fluctuation data showed differences between anterior and posterior diffusion of GFP::PIE-1. Theoretical diffusion curves suggest the presence of two species of PIE-1 in the anterior cytoplasm, with diffusion coefficients of 8.7 ± 0.6 and 0.6 ± 0.2 µm2/s, respectively. The posterior cytoplasm is well described by a three-component model with diffusion coefficients of 14.2 ± 7.8, 0.6 ± 0.3, and 0.006 ± 0.002 µm2/s. FRAP and FCS analysis is summarized in panel e and deviations from fit are provided in Fig. S3 (available at http://www.jcb.org/cgi/content/full/jcb.200809077/DC1). Error bars represent SEM.

Mentions: Having eliminated the leading hypotheses for enrichment, we propose a diffusion-limited reaction/diffusion model involving two distinct species of PIE-1 with unequal diffusion coefficients. Our model involves a cyclic change in the effective diffusion coefficient of PIE-1 caused by a diffusion-limited heterogeneous reaction taking place in the posterior coupled with a homogeneous reverse conversion taking place in the bulk cytoplasm. Specifically, free PIE-1 (P) is catalyzed by a heterogeneous posterior component to undergo an instantaneous conversion to a “slow” form (P*) that has a diffusion coefficient less than that of the free protein. This slow form then diffuses through the cytoplasm where it undergoes homogeneous conversion back to the rapidly diffusing form as governed by first-order kinetics (Fig. 3 a).


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 PIE-1 is driven by binary counterdiffusion. (a) We propose that free PIE-1 (P) undergoes a heterogeneous surface reaction in the posterior of the zygote that causes a reduction in its effective diffusion coefficient. This resultant “slow” species of PIE-1 (P*) homogenously reverts back to its initial form in the cytoplasm. (b) Analytical solutions of the concentration profiles of both species of PIE-1, x = 0 to x = L, reveal an overall concentration gradient along the A/P axis. The experimental concentration profiles were taken from the central 30 µm of the A/P axis (where the shape of the gradient is most pronounced) to avoid intensity spikes from P granules in the posterior and large plateaus in intensity in the anterior, which interfere with data fitting. (c) The half-times of recovery of GFP::PIE-1 intensity in 4.5-µm-diameter circular photobleached region experiments from the anterior and posterior cytoplasm were 0.7 ± 0.3 (n = 38) and 4.5 ± 0.3 s (n = 7), respectively. (d) Normalized autocorrelation curves of FCS fluctuation data showed differences between anterior and posterior diffusion of GFP::PIE-1. Theoretical diffusion curves suggest the presence of two species of PIE-1 in the anterior cytoplasm, with diffusion coefficients of 8.7 ± 0.6 and 0.6 ± 0.2 µm2/s, respectively. The posterior cytoplasm is well described by a three-component model with diffusion coefficients of 14.2 ± 7.8, 0.6 ± 0.3, and 0.006 ± 0.002 µm2/s. FRAP and FCS analysis is summarized in panel e and deviations from fit are provided in Fig. S3 (available at http://www.jcb.org/cgi/content/full/jcb.200809077/DC1). Error bars represent SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig3: Posterior enrichment of PIE-1 is driven by binary counterdiffusion. (a) We propose that free PIE-1 (P) undergoes a heterogeneous surface reaction in the posterior of the zygote that causes a reduction in its effective diffusion coefficient. This resultant “slow” species of PIE-1 (P*) homogenously reverts back to its initial form in the cytoplasm. (b) Analytical solutions of the concentration profiles of both species of PIE-1, x = 0 to x = L, reveal an overall concentration gradient along the A/P axis. The experimental concentration profiles were taken from the central 30 µm of the A/P axis (where the shape of the gradient is most pronounced) to avoid intensity spikes from P granules in the posterior and large plateaus in intensity in the anterior, which interfere with data fitting. (c) The half-times of recovery of GFP::PIE-1 intensity in 4.5-µm-diameter circular photobleached region experiments from the anterior and posterior cytoplasm were 0.7 ± 0.3 (n = 38) and 4.5 ± 0.3 s (n = 7), respectively. (d) Normalized autocorrelation curves of FCS fluctuation data showed differences between anterior and posterior diffusion of GFP::PIE-1. Theoretical diffusion curves suggest the presence of two species of PIE-1 in the anterior cytoplasm, with diffusion coefficients of 8.7 ± 0.6 and 0.6 ± 0.2 µm2/s, respectively. The posterior cytoplasm is well described by a three-component model with diffusion coefficients of 14.2 ± 7.8, 0.6 ± 0.3, and 0.006 ± 0.002 µm2/s. FRAP and FCS analysis is summarized in panel e and deviations from fit are provided in Fig. S3 (available at http://www.jcb.org/cgi/content/full/jcb.200809077/DC1). Error bars represent SEM.
Mentions: Having eliminated the leading hypotheses for enrichment, we propose a diffusion-limited reaction/diffusion model involving two distinct species of PIE-1 with unequal diffusion coefficients. Our model involves a cyclic change in the effective diffusion coefficient of PIE-1 caused by a diffusion-limited heterogeneous reaction taking place in the posterior coupled with a homogeneous reverse conversion taking place in the bulk cytoplasm. Specifically, free PIE-1 (P) is catalyzed by a heterogeneous posterior component to undergo an instantaneous conversion to a “slow” form (P*) that has a diffusion coefficient less than that of the free protein. This slow form then diffuses through the cytoplasm where it undergoes homogeneous conversion back to the rapidly diffusing form as governed by first-order kinetics (Fig. 3 a).

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
Related in: MedlinePlus