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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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Injection of dominant-negative constructs of FAK and control proteins (FATD:GFP, FAK:GFP, GFP). (A, B) Unfertilized eggs injected with high concentrations of FATD:GFP and fixed 4 h postfertilization. Ectopic actin and aberrant nuclear morphology resulted. (C, D) Lower concentrations of protein were injected, and eggs had defects in formation of the cortex, membrane accumulation of pY397 FAK, and nuclear abnormalities. (E, H) Similar preparations of eggs injected with full-length FAK:GFP. (F, I) Preparations in which eggs were injected with GFP protein. (G, J) Preparations of eggs that developed for 60 min before injection with FATD:GFP protein, demonstrating that the dominant-negative effect is stage specific and appears to interfere with formation of the egg cortex. (K) Quantification of membrane-associated pFAK or membrane-associated actin in eggs injected with dominant-negative constructs.
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Figure 8: Injection of dominant-negative constructs of FAK and control proteins (FATD:GFP, FAK:GFP, GFP). (A, B) Unfertilized eggs injected with high concentrations of FATD:GFP and fixed 4 h postfertilization. Ectopic actin and aberrant nuclear morphology resulted. (C, D) Lower concentrations of protein were injected, and eggs had defects in formation of the cortex, membrane accumulation of pY397 FAK, and nuclear abnormalities. (E, H) Similar preparations of eggs injected with full-length FAK:GFP. (F, I) Preparations in which eggs were injected with GFP protein. (G, J) Preparations of eggs that developed for 60 min before injection with FATD:GFP protein, demonstrating that the dominant-negative effect is stage specific and appears to interfere with formation of the egg cortex. (K) Quantification of membrane-associated pFAK or membrane-associated actin in eggs injected with dominant-negative constructs.

Mentions: As an alternative approach to interfering with the function of FAK, we injected unfertilized eggs with a portion of FAK believed to act as a dominant negative (Hildebrand et al., 1993; Ilic´ et al., 1998) and then fertilized the eggs. When high levels of focal adhesion–targeting domain:GFP (FATD:GFP) fusion protein are injected, eggs failed to complete cleavage by 4 h (Figure 8, A and B). The eggs had numerous large aggregates of GFP-immunoreactive material dispersed throughout the cytoplasm. In injected eggs, there was no clear cortical actin and numerous regions of ectopic filamentous actin within the endoplasm. In several eggs, nuclei were fragmented or aberrantly shaped. In eggs injected with lower concentrations of FATD:GFP, GFP immunoreactivity was dispersed throughout the cytoplasm (Figure 8, C and D). The intensity of pY397FAK immunoreactivity was reduced to ∼50% of the levels of eggs injected with GFP alone, and there was discontinuous localization on the egg surface (Figure 8, C, D, and K). In addition, there was a reduction in the intensity of cortical fluorescent phalloidin compared with eggs injected with GFP alone (Figure 8, C, D, and K). 4′,6-Diamidino-2-phenylindole (DAPI) staining indicated aberrant nuclear shapes, and the arrangements of tubulin in mitotic asters was also abnormal in FATD:GFP–injected eggs (Figure 8, C and D). Eggs injected with full-length FAK:GFP protein completed cleavage and had GFP immunoreactivity dispersed throughout the cytoplasm; however, cortical GFP appeared to be enhanced (Figure 8, G and J). The measurements of pY397FAK immunoreactivity and fluorescent phalloidin indicate lower levels than measured for GFP-injected eggs but levels higher than measured in FATD:GFP–injected eggs (Figure 8, E, H, and K). An additional control was to fertilize eggs and inject them with FATD:GFP after 60 min. In these preparations, the eggs underwent normal cleavage and had normal asters and what appeared to be normal levels of cortical actin and pY397FAK immunoreactivity. From these experiments we conclude that injection of FATD:GFP and FAK:GFP interferes with elaboration of the actin cortex and FATD:GFP appears to interfere with aspects of mitosis.


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)

Injection of dominant-negative constructs of FAK and control proteins (FATD:GFP, FAK:GFP, GFP). (A, B) Unfertilized eggs injected with high concentrations of FATD:GFP and fixed 4 h postfertilization. Ectopic actin and aberrant nuclear morphology resulted. (C, D) Lower concentrations of protein were injected, and eggs had defects in formation of the cortex, membrane accumulation of pY397 FAK, and nuclear abnormalities. (E, H) Similar preparations of eggs injected with full-length FAK:GFP. (F, I) Preparations in which eggs were injected with GFP protein. (G, J) Preparations of eggs that developed for 60 min before injection with FATD:GFP protein, demonstrating that the dominant-negative effect is stage specific and appears to interfere with formation of the egg cortex. (K) Quantification of membrane-associated pFAK or membrane-associated actin in eggs injected with dominant-negative constructs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 8: Injection of dominant-negative constructs of FAK and control proteins (FATD:GFP, FAK:GFP, GFP). (A, B) Unfertilized eggs injected with high concentrations of FATD:GFP and fixed 4 h postfertilization. Ectopic actin and aberrant nuclear morphology resulted. (C, D) Lower concentrations of protein were injected, and eggs had defects in formation of the cortex, membrane accumulation of pY397 FAK, and nuclear abnormalities. (E, H) Similar preparations of eggs injected with full-length FAK:GFP. (F, I) Preparations in which eggs were injected with GFP protein. (G, J) Preparations of eggs that developed for 60 min before injection with FATD:GFP protein, demonstrating that the dominant-negative effect is stage specific and appears to interfere with formation of the egg cortex. (K) Quantification of membrane-associated pFAK or membrane-associated actin in eggs injected with dominant-negative constructs.
Mentions: As an alternative approach to interfering with the function of FAK, we injected unfertilized eggs with a portion of FAK believed to act as a dominant negative (Hildebrand et al., 1993; Ilic´ et al., 1998) and then fertilized the eggs. When high levels of focal adhesion–targeting domain:GFP (FATD:GFP) fusion protein are injected, eggs failed to complete cleavage by 4 h (Figure 8, A and B). The eggs had numerous large aggregates of GFP-immunoreactive material dispersed throughout the cytoplasm. In injected eggs, there was no clear cortical actin and numerous regions of ectopic filamentous actin within the endoplasm. In several eggs, nuclei were fragmented or aberrantly shaped. In eggs injected with lower concentrations of FATD:GFP, GFP immunoreactivity was dispersed throughout the cytoplasm (Figure 8, C and D). The intensity of pY397FAK immunoreactivity was reduced to ∼50% of the levels of eggs injected with GFP alone, and there was discontinuous localization on the egg surface (Figure 8, C, D, and K). In addition, there was a reduction in the intensity of cortical fluorescent phalloidin compared with eggs injected with GFP alone (Figure 8, C, D, and K). 4′,6-Diamidino-2-phenylindole (DAPI) staining indicated aberrant nuclear shapes, and the arrangements of tubulin in mitotic asters was also abnormal in FATD:GFP–injected eggs (Figure 8, C and D). Eggs injected with full-length FAK:GFP protein completed cleavage and had GFP immunoreactivity dispersed throughout the cytoplasm; however, cortical GFP appeared to be enhanced (Figure 8, G and J). The measurements of pY397FAK immunoreactivity and fluorescent phalloidin indicate lower levels than measured for GFP-injected eggs but levels higher than measured in FATD:GFP–injected eggs (Figure 8, E, H, and K). An additional control was to fertilize eggs and inject them with FATD:GFP after 60 min. In these preparations, the eggs underwent normal cleavage and had normal asters and what appeared to be normal levels of cortical actin and pY397FAK immunoreactivity. From these experiments we conclude that injection of FATD:GFP and FAK:GFP interferes with elaboration of the actin cortex and FATD:GFP appears to interfere with aspects of mitosis.

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