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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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Confocal optical sections showing the chronology of cortex development. Eggs were fixed at intervals after fertilization (0 min is unfertilized), and samples of eggs were processed for immunofluorescence with phalloidin, anti-tubulin, and anti-pY397FAK. A separate set was processed with anti–pS19 myosin light chain. Bar, 40 μm.
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Figure 2: Confocal optical sections showing the chronology of cortex development. Eggs were fixed at intervals after fertilization (0 min is unfertilized), and samples of eggs were processed for immunofluorescence with phalloidin, anti-tubulin, and anti-pY397FAK. A separate set was processed with anti–pS19 myosin light chain. Bar, 40 μm.

Mentions: Antibodies that recognize FAK without regard to its phosphorylation state indicate that eggs contain immunoreactive granules that are dispersed throughout the cytoplasm. In eggs fixed 90 min after fertilization, the cells are in early mitosis and FAK immunoreactive granules are in the cytoplasm and the cortex (Figure 1A). Cortical FAK is most clearly revealed as regions of higher fluorescence intensity in profile plots along a radius of the egg (Figure 1C). In contrast, in similar preparations with antibodies that recognize only FAK phosphorylated at a tyrosine residue in the activation loop (pY397FAK; the activation loop is structurally conserved in sea urchin but is located between amino acids 450 and 475; Garcia et al., 2004), immunoreactivity is restricted to the egg cortex (Figure 1B). In preparations with anti-pY397FAK, it is clear in images and radial intensity profiles that pY397FAK is restricted to the egg plasmalemma and surface microvilli (Figure 1, B and D). In unfertilized eggs, pY397FAK also localizes to thin, discontinuous patches of cortex (Figure 2). These data indicate that FAK protein is present in eggs and pY397FAK is restricted to the egg cortex.


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)

Confocal optical sections showing the chronology of cortex development. Eggs were fixed at intervals after fertilization (0 min is unfertilized), and samples of eggs were processed for immunofluorescence with phalloidin, anti-tubulin, and anti-pY397FAK. A separate set was processed with anti–pS19 myosin light chain. Bar, 40 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Confocal optical sections showing the chronology of cortex development. Eggs were fixed at intervals after fertilization (0 min is unfertilized), and samples of eggs were processed for immunofluorescence with phalloidin, anti-tubulin, and anti-pY397FAK. A separate set was processed with anti–pS19 myosin light chain. Bar, 40 μm.
Mentions: Antibodies that recognize FAK without regard to its phosphorylation state indicate that eggs contain immunoreactive granules that are dispersed throughout the cytoplasm. In eggs fixed 90 min after fertilization, the cells are in early mitosis and FAK immunoreactive granules are in the cytoplasm and the cortex (Figure 1A). Cortical FAK is most clearly revealed as regions of higher fluorescence intensity in profile plots along a radius of the egg (Figure 1C). In contrast, in similar preparations with antibodies that recognize only FAK phosphorylated at a tyrosine residue in the activation loop (pY397FAK; the activation loop is structurally conserved in sea urchin but is located between amino acids 450 and 475; Garcia et al., 2004), immunoreactivity is restricted to the egg cortex (Figure 1B). In preparations with anti-pY397FAK, it is clear in images and radial intensity profiles that pY397FAK is restricted to the egg plasmalemma and surface microvilli (Figure 1, B and D). In unfertilized eggs, pY397FAK also localizes to thin, discontinuous patches of cortex (Figure 2). These data indicate that FAK protein is present in eggs and pY397FAK is restricted to the egg cortex.

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