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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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Inhibitors of FAK kinase activity interfere with the formation of cortical actin and accumulation of pY397FAK (pFAK) but enhance accumulation of pS19myosin light chain (pMyosin). (A–I) Representative confocal optical sections of eggs at 90 min postfertilization, untreated or treated with FAK inhibitorsY11 or PF573 228. (J–M) Quantification of images based on measurements of phalloidin fluorescence (J), linear measure of actin filament length (K), pFAK immunofluorescence (L), or pMyosin immunofluorescence (M). (N–Q) Confocal images of untreated eggs (Control) or eggs treated with Ca2+, Mg2+–free seawater (CMFSW) and prepared for immunofluorescence. The loss of pY397FAK and reduction in actin abundance indicate that normal cortical development requires extracellular divalent cations, a common feature of integrin-mediated adhesion.
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Figure 5: Inhibitors of FAK kinase activity interfere with the formation of cortical actin and accumulation of pY397FAK (pFAK) but enhance accumulation of pS19myosin light chain (pMyosin). (A–I) Representative confocal optical sections of eggs at 90 min postfertilization, untreated or treated with FAK inhibitorsY11 or PF573 228. (J–M) Quantification of images based on measurements of phalloidin fluorescence (J), linear measure of actin filament length (K), pFAK immunofluorescence (L), or pMyosin immunofluorescence (M). (N–Q) Confocal images of untreated eggs (Control) or eggs treated with Ca2+, Mg2+–free seawater (CMFSW) and prepared for immunofluorescence. The loss of pY397FAK and reduction in actin abundance indicate that normal cortical development requires extracellular divalent cations, a common feature of integrin-mediated adhesion.

Mentions: We evaluated the reorganization of cortical actin in eggs treated with PF 573 228. In eggs fixed 90 min after fertilization, filamentous actin is in the microvilli that project 2–3 μm above the surface of the egg and in radially oriented arrays that project 5–6 μm into the cytoplasm (Figure 5). In eggs treated with PF573 228 or Y11, there is overall reduction in the abundance of cortical actin, as measured by fluorescent-phalloidin binding (Figure 5, A–C and J). Eggs treated with either inhibitor have microvillar actin that is similar to controls, but the cortical arrays of actin are significantly shorter in preparations of treated eggs (Figure 5, A–C and K). We also examined the effects of PF573 228 or Y11 treatment on the phosphorylation of FAK by comparing the levels of anti-pY397FAK binding in treated and control preparations. In treated eggs, the distribution of pY397FAK was identical to that in untreated eggs; however, there was a reduction in fluorescence intensity to 50–60% of control levels (Figure 5, D–F and L). The effect of PF573 228 on the abundance of pY397FAK is also apparent from quantification of immunoblots (Supplemental Figure S2). Treatment with either inhibitor did not alter the distribution of pS19MLC in eggs; however, there was an increase in the abundance of pS19MLC when eggs were treated with either FAK inhibitor. Because integrin ligation is dependent on divalent cations, we rinsed fertilized eggs in calcium and magnesium–free seawater and after 90 min examined the abundance and distribution of pY397FAK and actin (Figure 5, N–Q). Actin abundance is markedly reduced and restricted to the membrane, and pY397FAK immunoreactivity is reduced to background levels by the lack of extracellular divalent cations, which implies that integrin function is necessary. Treatment with PF573 228 or Y11 did not have an effect on the abundance and distribution of βC integrins, atypical protein kinase C, or Dishevelled (Dsh; Supplemental Figure S3). We conclude from these experiments that FAK inhibitors alter the abundance and distribution of cortical actin, pY397FAK, and pS19MLC and these components of the cortex depend on extracellular divalent cations.


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)

Inhibitors of FAK kinase activity interfere with the formation of cortical actin and accumulation of pY397FAK (pFAK) but enhance accumulation of pS19myosin light chain (pMyosin). (A–I) Representative confocal optical sections of eggs at 90 min postfertilization, untreated or treated with FAK inhibitorsY11 or PF573 228. (J–M) Quantification of images based on measurements of phalloidin fluorescence (J), linear measure of actin filament length (K), pFAK immunofluorescence (L), or pMyosin immunofluorescence (M). (N–Q) Confocal images of untreated eggs (Control) or eggs treated with Ca2+, Mg2+–free seawater (CMFSW) and prepared for immunofluorescence. The loss of pY397FAK and reduction in actin abundance indicate that normal cortical development requires extracellular divalent cations, a common feature of integrin-mediated adhesion.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Inhibitors of FAK kinase activity interfere with the formation of cortical actin and accumulation of pY397FAK (pFAK) but enhance accumulation of pS19myosin light chain (pMyosin). (A–I) Representative confocal optical sections of eggs at 90 min postfertilization, untreated or treated with FAK inhibitorsY11 or PF573 228. (J–M) Quantification of images based on measurements of phalloidin fluorescence (J), linear measure of actin filament length (K), pFAK immunofluorescence (L), or pMyosin immunofluorescence (M). (N–Q) Confocal images of untreated eggs (Control) or eggs treated with Ca2+, Mg2+–free seawater (CMFSW) and prepared for immunofluorescence. The loss of pY397FAK and reduction in actin abundance indicate that normal cortical development requires extracellular divalent cations, a common feature of integrin-mediated adhesion.
Mentions: We evaluated the reorganization of cortical actin in eggs treated with PF 573 228. In eggs fixed 90 min after fertilization, filamentous actin is in the microvilli that project 2–3 μm above the surface of the egg and in radially oriented arrays that project 5–6 μm into the cytoplasm (Figure 5). In eggs treated with PF573 228 or Y11, there is overall reduction in the abundance of cortical actin, as measured by fluorescent-phalloidin binding (Figure 5, A–C and J). Eggs treated with either inhibitor have microvillar actin that is similar to controls, but the cortical arrays of actin are significantly shorter in preparations of treated eggs (Figure 5, A–C and K). We also examined the effects of PF573 228 or Y11 treatment on the phosphorylation of FAK by comparing the levels of anti-pY397FAK binding in treated and control preparations. In treated eggs, the distribution of pY397FAK was identical to that in untreated eggs; however, there was a reduction in fluorescence intensity to 50–60% of control levels (Figure 5, D–F and L). The effect of PF573 228 on the abundance of pY397FAK is also apparent from quantification of immunoblots (Supplemental Figure S2). Treatment with either inhibitor did not alter the distribution of pS19MLC in eggs; however, there was an increase in the abundance of pS19MLC when eggs were treated with either FAK inhibitor. Because integrin ligation is dependent on divalent cations, we rinsed fertilized eggs in calcium and magnesium–free seawater and after 90 min examined the abundance and distribution of pY397FAK and actin (Figure 5, N–Q). Actin abundance is markedly reduced and restricted to the membrane, and pY397FAK immunoreactivity is reduced to background levels by the lack of extracellular divalent cations, which implies that integrin function is necessary. Treatment with PF573 228 or Y11 did not have an effect on the abundance and distribution of βC integrins, atypical protein kinase C, or Dishevelled (Dsh; Supplemental Figure S3). We conclude from these experiments that FAK inhibitors alter the abundance and distribution of cortical actin, pY397FAK, and pS19MLC and these components of the cortex depend on extracellular divalent cations.

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