Kofron M et al. (2002), Plakoglobin is required for maintenance of the ...

XB-ART-6665

J Cell Biol 2002 Aug 19;1584:695-708. doi: 10.1083/jcb.200202123.

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Plakoglobin is required for maintenance of the cortical actin skeleton in early Xenopus embryos and for cdc42-mediated wound healing.

Kofron M , Heasman J , Lang SA , Wylie CC .

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Early Xenopus embryos are large, and during the egg to gastrula stages, when there is little extracellular matrix, the cytoskeletons of the individual blastomeres are thought to maintain their spherical architecture and provide scaffolding for the cellular movements of gastrulation. We showed previously that depletion of plakoglobin protein during the egg to gastrula stages caused collapse of embryonic architecture. Here, we show that this is due to loss of the cortical actin skeleton after depletion of plakoglobin, whereas the microtubule and cytokeratin skeletons are still present. As a functional assay for the actin skeleton, we show that wound healing, an actin-based behavior in embryos, is also abrogated by plakoglobin depletion. Both wound healing and the amount of cortical actin are enhanced by overexpression of plakoglobin. To begin to identify links between plakoglobin and the cortical actin polymerization machinery, we show here that the Rho family GTPase cdc42, is required for wound healing in the Xenopus blastula. Myc-tagged cdc42 colocalizes with actin in purse-strings surrounding wounds. Overexpression of cdc42 dramatically enhances wound healing, whereas depletion of maternal cdc42 mRNA blocks it. In combinatorial experiments we show that cdc42 cannot rescue the effects of plakoglobin depletion, showing that plakoglobin is required for cdc42-mediated cortical actin assembly during wound healing. However, plakoglobin does rescue the effect of cdc42 depletion, suggesting that cdc42 somehow mediates the distribution or function of plakoglobin. Depletion of alpha-catenin does not remove the cortical actin skeleton, showing that plakoglobin does not mediate its effect by its known linkage through alpha-catenin to the actin skeleton. We conclude that in Xenopus, the actin skeleton is a major determinant of cell shape and overall architecture in the early embryo, and that plakoglobin plays an essential role in the assembly, maintenance, or organization of this cortical actin.

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Species referenced: Xenopus laevis
Genes referenced: actl6a cdc42 jup krt12.4 myc rho

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	Figure 1. Depletion of plakoglobin and effect on embryo cytoarchitecture. (Aâ€“C) The degree of depletion of plakoglobin mRNA detected by real-time PCR, normalized to the level of ornithine decarboxylase mRNA (A), using two doses of antisense oligo, expressed as a percentage of the uninjected level. (B and C) Protein detected by immunocytochemistry in optical sections through late blastulae either uninjected (B) or plakoglobin depleted (C). D and E are views from above and the side, respectively, to show the degree of flattening of the embryo after injection of 4 ng oligo into the oocyte. Top rows are control, bottom rows are plakoglobin depleted in D and E.
	Figure 2. Microtubule and cytokeratin filament systems are still present after plakoglobin depletion. (Aâ€“D) The microtubule (A and B) and cytokeratin (C and D) arrays in either uninjected (A and C), or plakoglobin depleted (B and D) blastulae. Microtubules are shown in the cells lining the roof of the blastocoel, whereas cytokeratin filaments are shown in the surface layer of animal cells. No significant differences were found after plakoglobin depletion.
	Figure 3. The actin skeleton of the late blastula visualized by phalloidin staining of whole animal caps. (A) The late blastulae used to dissect animal caps (B). These were allowed to heal for 5 min before fixing, stained with fluorescently coupled phalloidin, and the cells of the blastocoel roof were examined directly with the confocal microscope (C). (D) A low power image of the inside surface of the cap and the actin-rich purse-string around its circumference. The extensive actin network in the cortices of all blastomeres can be seen in cells immediately lining the blastocoel roof (E). This network extends round the cortex of each cell, seen best in animal caps cut in half before staining to allow the phalloidin access to the insides of the blastomeres (F, arrow).
	Figure 4. Wound healing and the cortical actin skeleton require plakoglobin. Depletion of plakoglobin prevents healing of both animal caps (A), and the embryos from which they were removed (B). Top rows (A and B) are untreated, bottom rows are plakoglobin depleted. (B) Bottom row shows that injection of Î²-catenin mRNA does not rescue healing after plakoglobin depletion. Photographs were taken 1 h after dissection. (C) The rescue of wound healing in plakoglobin-depleted caps by the injection of plakoglobin mRNA and RLDX into one cell at the two-cell stage. The injected half of the animal cap (red) is healing normally, whereas the unrescued half (green) is not. (Dâ€“I) Actin patterns, stained using phalloidin, and recorded using the same settings on the confocal microscope, in control (D and E) and plakoglobin depleted (F and G), and rescued by injection of plakoglobin mRNA (H and I). Purse-strings around the wounds and cortical actin in individual blastomeres both require plakoglobin.
	Figure 5. Overexpression ofplakoglobinmRNA by injection of mRNA into both cells at the 2-cell stage causes an increase in cortical actin and more rapid wound healing. The animal cap from the uninjected embryo (A) has significantly less cortical actin than that from an embryo injected with 400 pg mRNA (B). (C) Bases from which the caps were removed. The plakoglobin-overexpressing bases heal faster than those of the uninjected controls. Picture was taken 15 min after excision of the caps. Notice that increasing doses of mRNA cause increasingly more spherical embryos.
	Figure 6. Plakoglobin is not required for the formation of actin-rich contractile rings during cytokinesis. (A and B) The first cleavage furrow of an uninjected embryo (A), and an embryo depleted using 4 ng of plakoglobin oligo (B). The vitelline membranes were manually removed immediately before the first cleavage division, and the cleavage furrow allowed to form before fixation and staining with Alexa 488â€“coupled phalloidin. In the depleted embryo, the weight of the egg contents in the absence of the elastic vitelline membrane pulls apart the contractile ring of actin. However, inside the vitelline membranes, these eggs go on to cleave normally.
	Figure 7. cdc translated from injected mRNA colocalizes with the actin purse-string during wound healing, and potentiates the wound-healing response. (A and B) Distribution of cdc42 in the cortices of animal cap blastomeres (A) or in the purse-string of a healing cap (B) after injection of 50 pg of myc-tagged cdc42 mRNA. (A) Newly synthesized cdc42 is concentrated in the cell cortices. (B) The cdc42 colocalizes with the actin of the purse-string (yellow line). (C and D) Enhanced wound healing caused by expression of 50 pg (D) of cdc42 mRNA in the animal cap, compared to uninjected caps (C). In contrast, depletion of the maternal mRNA-encoding cdc42 causes animal caps and bases to heal more slowly. (E) Caps 1 h after excision. (F) Bases 30 min after excision. (E and F) Top rows are untreated, bottom rows are cdc42- depleted. (G and H) Caps were allowed to heal for 5 min, then fixed and stained with phalloidin from embryos depleted of cdc42 (H) or untreated (G). No purse-strings are seen in the cdc42-depleted caps.
	Figure 8. The effect of depletion ofcdc42mRNA on both wound-healing and cortical actin assembly in isolated animal caps, and the effects of injection of eithercdc42orplakoglobinmRNA on the effects of cdc42 depletion. (A) The effect of cdc42 depletion on healing of bases (top row) compared to controls (middle row). Injection of 400 pg cdc42 mRNA rescues the effect (bottom row). (B) Injection of 500 pg plakoglobin mRNA also rescues the effect (rows are the same as in A). (Câ€“G) Pairs of images of isolated animal caps from the bases shown in A and B. On the left in each case are image stacks treated identically under the confocal microscope from Alexa 488â€“coupled phalloidin-stained caps, and on the right are quantitative measurements of pixel intensity across the image stack. (C) Uninjected; (D) cdc42 depleted; (E) plakoglobin depleted; (F) cdc42 depleted and injected at the two-cell stage with cdc42 mRNA (400 pg); (G) cdc42 depleted, and injected at the two-cell stage with plakoglobin mRNA (500 pg). Purse-strings and overall cortical actin depletion caused by depletion of cdc42 are rescued both by cdc42 and plakoglobin mRNAs.
	Figure 9. Î±-Catenin depletion does not cause the dramatic loss in cortical actin seen in plakoglobin-depleted embryos, even when they are disaggregated. Panel A shows whole devitellined blastula; and that embryos are not flattened after Î±-catenin depletion. (B) Animal caps from the same embryos. Both 8 and 12 ng Î±-catenin oligo caused disaggregation of the caps (bottom two rows). (C) The degree of depletion by Western blotting. (Dâ€“F) The cortical actin skeletons of uninjected (D) and 12-ng oligo-depleted (E and F) caps. Blastomeres that are partially (E) or completely (F) disaggregated from each other both retain a cortical actin skeleton.

References [+] :

Amitay, Reduced expression of plakoglobin correlates with adverse outcome in patients with neuroblastoma. 2001, Pubmed