Chan D , Thomas CJ , Taylor VJ , Burke RD .

	FIGURE 1:. Eggs 90 min after fertilization reveal differences in the distribution of FAK and pY397FAK. (A) Egg prepared with anti-FAK shows that immunoreactivity is granular and found throughout the cytoplasm. (B) Egg prepared with anti- pY397FAK reveals that the phosphorylated form of FAK is restricted to the cell surface. (C, D) Intensity profiles along an arbitrary radius of the egg show differences in intensity throughout the cytoplasm and that most of the fluorescence at the membrane can be accounted for by pY397FAK. Bar, 20 μm.
	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.
	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.
	FIGURE 4:. Fusion of pronuclei is affected by FAK inhibitors. (A, B) Confocal images of eggs prepared with DAPI, anti-tubulin, and anti- pY397FAK to show a typical location of male (arrow) and female (circle) pronuclei at 60 and 90 min in eggs treated with the FAK inhibitor PF573 228. Bar, 20 μm. (C) Results of scoring pronuclear fusion in eggs (expressed as percentage) treated with either PF573 228 or Y11; time is minutes postfertilization. (D) To determine the effects of inhibitors subsequent to pronuclear fusion, inhibitors were added to eggs 30 min after fertilization. Although a zygotic nucleus formed, eggs did not cleave normally.
	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.
	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.
	FIGURE 7:. Treatment with the FAK inhibitor PF573 228 interferes with the accumulation of nuclear pERK in fertilized eggs. (A) Immuno­fluorescence preparations showing confocal optical sections of nuclei through the first 30 min after fertilization. (B) Quantification of preparations by the total fluorescence per pixel of treated and control nuclei. (C, D) Whole egg lysates of treated and control preparations as immunoblots that were quantified by measuring band density.
	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.
	FIGURE 9:. Summary of the roles of FAK after fertilization in sea urchin eggs. FAK is present in the cytoplasm and is phosphorylated immediately after fertilization. This activation appears to be necessary for the fusion of male and female pronuclei, and FAK has inputs into the MAP kinase regulation of the reinitiation of the cell cycle. Activation of FAK appears to be necessary for nuclearization of ERK and down-regulation of cyclin E. About 15 min after fertilization, βC integrins are expressed and bind Sp-Del in the extracellular hyaline layer. pFAK is recruited to an integrin-containing complex at the cell surface, where it appears necessary for the elaboration of the cytoplasmic arrays of cortical actin and regulation of phosphorylation of myosin.

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