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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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Immunoblots of 60-min egg cortex lysates and immunoprecipitates of egg cortex lysates showing a physical association of βC-containing integrins and pY397FAK. Egg cortex lysates (top) contain immunoreactive bands at the predicted molecular weights (βC, 110 kDa; pY397FAK, 125 kDa). Immunoprecipitates with anti-βC (middle) are enriched with immunoreactive material at110 kDa and contain an anti-pY397FAK immunoreactive band. Similarly, blots of immunoprecipitates with anti-pY397FAK (bottom) contain a band that is immunoreactive with anti-βC. All images are from a single experiment, run on a single gel, and double labeled with anti-βC and anti-pY397FAK. Two levels of exposure were used for the pY397FAK channel.
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Figure 3: Immunoblots of 60-min egg cortex lysates and immunoprecipitates of egg cortex lysates showing a physical association of βC-containing integrins and pY397FAK. Egg cortex lysates (top) contain immunoreactive bands at the predicted molecular weights (βC, 110 kDa; pY397FAK, 125 kDa). Immunoprecipitates with anti-βC (middle) are enriched with immunoreactive material at110 kDa and contain an anti-pY397FAK immunoreactive band. Similarly, blots of immunoprecipitates with anti-pY397FAK (bottom) contain a band that is immunoreactive with anti-βC. All images are from a single experiment, run on a single gel, and double labeled with anti-βC and anti-pY397FAK. Two levels of exposure were used for the pY397FAK channel.

Mentions: It was previously demonstrated that βC-containing integrins are expressed within 30 min of fertilization and associate with the surface of the egg (Murray et al., 2000). Immunoprecipitates of detergent-solubilized egg cortices were used to determine whether pY397FAK is physically associated with βC integrins. Using 1B10, a monoclonal antibody that binds the cytoplasmic domain of the βC subunit (Murray et al., 2000), in immunoprecipitations and subsequently probing with the same antibody, we identified a single band migrating at ∼110 kDa in the egg cortex lysates and the bead fraction (Figure 3). When probed with anti-pY397FAK, a band at ∼125 kDa is revealed. In the reciprocal arrangement using anti-pY397FAK, egg cortex lysates have an immunoreactive band at 125 kDa and similar bands in samples of material bound to beads in immunoprecipitates where either anti-pY397FAK or anti-βC (1B10) was used as the primary antibody. Thus antibodies to βC integrins immunoprecipitate a complex containing pY397FAK and antibodies to pY397FAK immunoprecipitate a complex containing βC integrins. We conclude that pY397FAK is physically associated with a complex containing βC integrins in 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)

Immunoblots of 60-min egg cortex lysates and immunoprecipitates of egg cortex lysates showing a physical association of βC-containing integrins and pY397FAK. Egg cortex lysates (top) contain immunoreactive bands at the predicted molecular weights (βC, 110 kDa; pY397FAK, 125 kDa). Immunoprecipitates with anti-βC (middle) are enriched with immunoreactive material at110 kDa and contain an anti-pY397FAK immunoreactive band. Similarly, blots of immunoprecipitates with anti-pY397FAK (bottom) contain a band that is immunoreactive with anti-βC. All images are from a single experiment, run on a single gel, and double labeled with anti-βC and anti-pY397FAK. Two levels of exposure were used for the pY397FAK channel.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814141&req=5

Figure 3: Immunoblots of 60-min egg cortex lysates and immunoprecipitates of egg cortex lysates showing a physical association of βC-containing integrins and pY397FAK. Egg cortex lysates (top) contain immunoreactive bands at the predicted molecular weights (βC, 110 kDa; pY397FAK, 125 kDa). Immunoprecipitates with anti-βC (middle) are enriched with immunoreactive material at110 kDa and contain an anti-pY397FAK immunoreactive band. Similarly, blots of immunoprecipitates with anti-pY397FAK (bottom) contain a band that is immunoreactive with anti-βC. All images are from a single experiment, run on a single gel, and double labeled with anti-βC and anti-pY397FAK. Two levels of exposure were used for the pY397FAK channel.
Mentions: It was previously demonstrated that βC-containing integrins are expressed within 30 min of fertilization and associate with the surface of the egg (Murray et al., 2000). Immunoprecipitates of detergent-solubilized egg cortices were used to determine whether pY397FAK is physically associated with βC integrins. Using 1B10, a monoclonal antibody that binds the cytoplasmic domain of the βC subunit (Murray et al., 2000), in immunoprecipitations and subsequently probing with the same antibody, we identified a single band migrating at ∼110 kDa in the egg cortex lysates and the bead fraction (Figure 3). When probed with anti-pY397FAK, a band at ∼125 kDa is revealed. In the reciprocal arrangement using anti-pY397FAK, egg cortex lysates have an immunoreactive band at 125 kDa and similar bands in samples of material bound to beads in immunoprecipitates where either anti-pY397FAK or anti-βC (1B10) was used as the primary antibody. Thus antibodies to βC integrins immunoprecipitate a complex containing pY397FAK and antibodies to pY397FAK immunoprecipitate a complex containing βC integrins. We conclude that pY397FAK is physically associated with a complex containing βC integrins in 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