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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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FAK inhibitors interfere with the normal pattern of accumulation of cyclin E in the nucleus of eggs. (A) Confocal optical sections of representative nuclei of eggs treated with FAK inhibitor PF573 228 and prepared for immunofluorescence with anti-cyclin E. (B) Quantification of cyclin E immunofluorescence expressed as a percentage of the mean fluorescence of unfertilized egg nuclei. Normally cyclin E accumulates in nuclei at ∼15 min and returns to background levels as the nucleus enters S phase. Inhibitors of FAK cause a prolonged phase of accumulation. (C) Eggs were treated with inhibitors of MEK and cdk and quantified to provide a comparison.
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Figure 6: FAK inhibitors interfere with the normal pattern of accumulation of cyclin E in the nucleus of eggs. (A) Confocal optical sections of representative nuclei of eggs treated with FAK inhibitor PF573 228 and prepared for immunofluorescence with anti-cyclin E. (B) Quantification of cyclin E immunofluorescence expressed as a percentage of the mean fluorescence of unfertilized egg nuclei. Normally cyclin E accumulates in nuclei at ∼15 min and returns to background levels as the nucleus enters S phase. Inhibitors of FAK cause a prolonged phase of accumulation. (C) Eggs were treated with inhibitors of MEK and cdk and quantified to provide a comparison.

Mentions: As part of the mechanism by which the cell cycle is reinitiated in urchin eggs, it was demonstrated that calcium-dependent activation of ERK1 promotes accumulation of cyclin E/cdk2 in male and female pronuclei and entry into S-phase of mitosis (Kisielewska et al., 2009). To determine whether signaling from cortical integrin–FAK complex is critical to this process, we used an antibody to cyclin E (Sumerel et al., 2001) to determine nuclear cyclin E abundance in the presence of a FAK inhibitor. Cyclin E immunoreactivity in the female pronucleus normally increases within the first 15 min after fertilization (Figure 6, A and B). By 35 min, these levels return to background and remain so throughout mitosis. In eggs treated with PF573 228, there is no increase in nuclear cyclin E immunoreactivity until 60 min after fertilization, and cyclin E immunoreactivity remains high until ∼90 min after fertilization (Figure 6, A and B). When eggs are treated with Y11, cyclin E immunoreactivity increases through the first 60 min after fertilization and remains higher than in controls after 90 min (Figure 6B). Kisielewska et al. (2009) also demonstrated that nuclear accumulation of cyclin E is sensitive to the MEK inhibitor U0126 and roscovitine, a cdk2 inhibitor. With our antibody-based assay, these inhibitors block the increase in nuclear cyclin E immunoreactivity in a manner that is distinct from the effects we observe when eggs are treated with inhibitors for FAK (Figure 6C). We conclude from these experiments that inhibition of FAK interferes with the processes that regulate the nuclear accumulation of cyclin E.


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)

FAK inhibitors interfere with the normal pattern of accumulation of cyclin E in the nucleus of eggs. (A) Confocal optical sections of representative nuclei of eggs treated with FAK inhibitor PF573 228 and prepared for immunofluorescence with anti-cyclin E. (B) Quantification of cyclin E immunofluorescence expressed as a percentage of the mean fluorescence of unfertilized egg nuclei. Normally cyclin E accumulates in nuclei at ∼15 min and returns to background levels as the nucleus enters S phase. Inhibitors of FAK cause a prolonged phase of accumulation. (C) Eggs were treated with inhibitors of MEK and cdk and quantified to provide a comparison.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 6: FAK inhibitors interfere with the normal pattern of accumulation of cyclin E in the nucleus of eggs. (A) Confocal optical sections of representative nuclei of eggs treated with FAK inhibitor PF573 228 and prepared for immunofluorescence with anti-cyclin E. (B) Quantification of cyclin E immunofluorescence expressed as a percentage of the mean fluorescence of unfertilized egg nuclei. Normally cyclin E accumulates in nuclei at ∼15 min and returns to background levels as the nucleus enters S phase. Inhibitors of FAK cause a prolonged phase of accumulation. (C) Eggs were treated with inhibitors of MEK and cdk and quantified to provide a comparison.
Mentions: As part of the mechanism by which the cell cycle is reinitiated in urchin eggs, it was demonstrated that calcium-dependent activation of ERK1 promotes accumulation of cyclin E/cdk2 in male and female pronuclei and entry into S-phase of mitosis (Kisielewska et al., 2009). To determine whether signaling from cortical integrin–FAK complex is critical to this process, we used an antibody to cyclin E (Sumerel et al., 2001) to determine nuclear cyclin E abundance in the presence of a FAK inhibitor. Cyclin E immunoreactivity in the female pronucleus normally increases within the first 15 min after fertilization (Figure 6, A and B). By 35 min, these levels return to background and remain so throughout mitosis. In eggs treated with PF573 228, there is no increase in nuclear cyclin E immunoreactivity until 60 min after fertilization, and cyclin E immunoreactivity remains high until ∼90 min after fertilization (Figure 6, A and B). When eggs are treated with Y11, cyclin E immunoreactivity increases through the first 60 min after fertilization and remains higher than in controls after 90 min (Figure 6B). Kisielewska et al. (2009) also demonstrated that nuclear accumulation of cyclin E is sensitive to the MEK inhibitor U0126 and roscovitine, a cdk2 inhibitor. With our antibody-based assay, these inhibitors block the increase in nuclear cyclin E immunoreactivity in a manner that is distinct from the effects we observe when eggs are treated with inhibitors for FAK (Figure 6C). We conclude from these experiments that inhibition of FAK interferes with the processes that regulate the nuclear accumulation of cyclin E.

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