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Integrins on eggs: focal adhesion kinase is activated at fertilization, forms a complex with integrins, and is necessary for cortex formation and cell cycle initiation.

Chan D, Thomas CJ, Taylor VJ, Burke RD - Mol. Biol. Cell (2013)

Bottom Line: Cyclin E normally accumulates in the nucleus 15 min after fertilization, then returns to background levels.PF573 228- or Y11-treated eggs accumulate cyclin E in the nucleus; however, levels remain high.In addition, PF573 228 interferes with the accumulation of pERK1/2 in the nucleus and in eggs initiating mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8P 5C2, Canada.

ABSTRACT
We investigate the proposal that integrins and focal adhesion kinase (FAK) form a complex that has structural and signaling functions in eggs. FAK protein is present in eggs and is phosphorylated at fertilization. pY(397)FAK localizes to the membrane 30 min after fertilization, which correlates with the expression of βC integrins and egg cortex development. The βC integrin and pY(397)FAK coimmunoprecipitate from egg cortex lysates. PF573 228 and Y11, inhibitors of FAK, interfere with pronuclear fusion and reduce the abundance of pY(397)FAK and cortical actin without affecting microvillar actin. Cyclin E normally accumulates in the nucleus 15 min after fertilization, then returns to background levels. PF573 228- or Y11-treated eggs accumulate cyclin E in the nucleus; however, levels remain high. In addition, PF573 228 interferes with the accumulation of pERK1/2 in the nucleus and in eggs initiating mitosis. Injection of eggs with a fusion protein consisting of the focal adhesion-targeting domain of FAK fused to green fluorescent protein interferes with cortex formation and produces abnormal nuclei. These data indicate that an integrin-FAK adhesion complex forms at the egg surface that functions in formation of actin arrays in the egg cortex and provides signaling inputs for cell cycle initiation.

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Treatment with the FAK inhibitor PF573 228 interferes with the accumulation of nuclear pERK in fertilized eggs. (A) Immuno­fluorescence preparations showing confocal optical sections of nuclei through the first 30 min after fertilization. (B) Quantification of preparations by the total fluorescence per pixel of treated and control nuclei. (C, D) Whole egg lysates of treated and control preparations as immunoblots that were quantified by measuring band density.
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Figure 7: Treatment with the FAK inhibitor PF573 228 interferes with the accumulation of nuclear pERK in fertilized eggs. (A) Immuno­fluorescence preparations showing confocal optical sections of nuclei through the first 30 min after fertilization. (B) Quantification of preparations by the total fluorescence per pixel of treated and control nuclei. (C, D) Whole egg lysates of treated and control preparations as immunoblots that were quantified by measuring band density.

Mentions: The transient increase in cytoplasmic Ca2+ at fertilization is believed to activate ERK1, which accumulates in the nucleus 4–5 min after fertilization (Philipova et al., 2005; Zhang et al., 2005). To determine whether inhibition of FAK activity is critical to ERK1 activation, we measured nuclear pERK through the first 30 min after fertilization. In untreated control eggs, we were able to measure brief accumulation of pERK1/2 in the nucleus 5 min after fertilization, which fades to background levels by 15 min after fertilization (Figure 7, A and B). In eggs treated with PF573 228, the levels of nuclear pERK are about half of those in the control eggs. With immunoblots in which whole egg lysates were probed with anti-pERK1/2, we find a similar pattern; PF573 228–treated eggs have less detectable pERK1/2. These experiments indicate that inhibition of FAK kinase activity with PF573 228 interferes with the activation of pERK.


Integrins on eggs: focal adhesion kinase is activated at fertilization, forms a complex with integrins, and is necessary for cortex formation and cell cycle initiation.

Chan D, Thomas CJ, Taylor VJ, Burke RD - Mol. Biol. Cell (2013)

Treatment with the FAK inhibitor PF573 228 interferes with the accumulation of nuclear pERK in fertilized eggs. (A) Immuno­fluorescence preparations showing confocal optical sections of nuclei through the first 30 min after fertilization. (B) Quantification of preparations by the total fluorescence per pixel of treated and control nuclei. (C, D) Whole egg lysates of treated and control preparations as immunoblots that were quantified by measuring band density.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814141&req=5

Figure 7: Treatment with the FAK inhibitor PF573 228 interferes with the accumulation of nuclear pERK in fertilized eggs. (A) Immuno­fluorescence preparations showing confocal optical sections of nuclei through the first 30 min after fertilization. (B) Quantification of preparations by the total fluorescence per pixel of treated and control nuclei. (C, D) Whole egg lysates of treated and control preparations as immunoblots that were quantified by measuring band density.
Mentions: The transient increase in cytoplasmic Ca2+ at fertilization is believed to activate ERK1, which accumulates in the nucleus 4–5 min after fertilization (Philipova et al., 2005; Zhang et al., 2005). To determine whether inhibition of FAK activity is critical to ERK1 activation, we measured nuclear pERK through the first 30 min after fertilization. In untreated control eggs, we were able to measure brief accumulation of pERK1/2 in the nucleus 5 min after fertilization, which fades to background levels by 15 min after fertilization (Figure 7, A and B). In eggs treated with PF573 228, the levels of nuclear pERK are about half of those in the control eggs. With immunoblots in which whole egg lysates were probed with anti-pERK1/2, we find a similar pattern; PF573 228–treated eggs have less detectable pERK1/2. These experiments indicate that inhibition of FAK kinase activity with PF573 228 interferes with the activation of pERK.

Bottom Line: Cyclin E normally accumulates in the nucleus 15 min after fertilization, then returns to background levels.PF573 228- or Y11-treated eggs accumulate cyclin E in the nucleus; however, levels remain high.In addition, PF573 228 interferes with the accumulation of pERK1/2 in the nucleus and in eggs initiating mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8P 5C2, Canada.

ABSTRACT
We investigate the proposal that integrins and focal adhesion kinase (FAK) form a complex that has structural and signaling functions in eggs. FAK protein is present in eggs and is phosphorylated at fertilization. pY(397)FAK localizes to the membrane 30 min after fertilization, which correlates with the expression of βC integrins and egg cortex development. The βC integrin and pY(397)FAK coimmunoprecipitate from egg cortex lysates. PF573 228 and Y11, inhibitors of FAK, interfere with pronuclear fusion and reduce the abundance of pY(397)FAK and cortical actin without affecting microvillar actin. Cyclin E normally accumulates in the nucleus 15 min after fertilization, then returns to background levels. PF573 228- or Y11-treated eggs accumulate cyclin E in the nucleus; however, levels remain high. In addition, PF573 228 interferes with the accumulation of pERK1/2 in the nucleus and in eggs initiating mitosis. Injection of eggs with a fusion protein consisting of the focal adhesion-targeting domain of FAK fused to green fluorescent protein interferes with cortex formation and produces abnormal nuclei. These data indicate that an integrin-FAK adhesion complex forms at the egg surface that functions in formation of actin arrays in the egg cortex and provides signaling inputs for cell cycle initiation.

Show MeSH
Related in: MedlinePlus