Iioka H et al. (2004), Essential role of MARCKS in cortical actin dyna...

XB-ART-4133

J Cell Biol 2004 Jan 19;1642:169-74. doi: 10.1083/jcb.200310027.

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Essential role of MARCKS in cortical actin dynamics during gastrulation movements.

Iioka H , Ueno N , Kinoshita N .

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Myristoylated alanine-rich C kinase substrate (MARCKS) is an actin-binding, membrane-associated protein expressed during Xenopus embryogenesis. We analyzed its function in cytoskeletal regulation during gastrulation. Here, we show that blockade of its function impaired morphogenetic movements, including convergent extension. MARCKS was required for control of cell morphology, motility, adhesion, protrusive activity, and cortical actin formation in embryonic cells. We also demonstrate that the noncanonical Wnt pathway promotes the formation of lamellipodia- and filopodia-like protrusions and that MARCKS is necessary for this activity. These findings show that MARCKS regulates the cortical actin formation that is requisite for dynamic morphogenetic movements.

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Species referenced: Xenopus
Genes referenced: actl6a chrd dvl1 fzd7 gsc marcks wnt11b
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	Figure 1. MARCKS is essential for gastrulation movements. (A) Both 500 pg of MARCKS mRNA and 5 pmol of MARCKS Mo impaired gastrulation movements, when either was injected into the dorsal marginal region. (B) Statistical data of the gastrulation-defective phenotype caused by MARCKS mRNA and Mo. (C) Expression of chordin at the gastrula stage, detected by in situ hybridization. (D) Somites (left) and notochord (right) were immunostained with 12/101 and MZ15 antibodies, respectively. (E) 5 pmol of MARCKS Mo was injected into the two dorsal blastomeres at the four-cell stage; the DMZ explants were isolated, and the expression of mesodermal markers was detected by RT-PCR. gsc, goosecoid. (F) 2 pmol of MARCKS Mo inhibited the activin mRNA-induced elongation of animal caps. This inhibition was rescued by coinjection of 200 pg of MARCKS mRNA.
	Figure 2. MARCKS is essential for controlling cell polarity, motility, and adhesion. (A) 5 pmol of MARCKS Mo, Rhodamine dextran, and the mRNA for 100 pg of mb-Venus were coinjected into one of the two dorsal blastomeres at the four-cell stage. mb-Venus mRNA alone was injected into the other dorsal blastomere. DMZ explants were cultured on a cover glass coated with FN, and convergent extension movements were observed. (B) Control or 5 pmol of MARCKS Mo was coinjected with 100 pg of Venus mRNA into two blastomeres of four-cell embryos. DMZ explants were cultured on an FN-coated dish until sibling embryos reached the late neurula stage. Arrows indicate the direction of mesendoderm migration. Arrowheads indicate the leading edge. (C) MARCKS Mo inhibited the adhesion on FN. MARCKS Mo, control Mo, Venus mRNAs (green), and Rhodamine dextran (red) were coinjected dorsally as indicated. Cells were dissociated from the DMZ explants. Cells from the control- and MARCKS-Mo–injected explants were mixed, plated on FN-coated dishes, incubated for 6 h, and fixed in formaldehyde. Cells that did not adhere to the dish were removed by washing five times with PBS. (D) 3 pmol of MARCKS Mo inhibited the protrusive activity of cells in DMZ explants. MARCKS Mo or control Mo was coinjected dorsally with mb-Venus mRNA. DMZ explants were cultured on an FN-coated dish until sibling embryos reached the early neurula stage. The effect of MARCKS Mo was rescued by 200 pg of MARCKS mRNA. Bar, 50 μm. The graph shows statistical data obtained by analyzing 15 cells for each sample. The error bars represent statistical significance (p < 0.05).
	Figure 3. MARCKS regulates cortical actin formation. (A) Cells expressing 200 pg of MARCKS-Venus mRNA were dissociated from DMZ explants and plated on a FN-coated cover glass. Arrows indicate protrusions where both MARCKS and F-actin were enriched. (B) Wild-type, GA, and SD mutants were expressed in the DMZ explants and observed. RMA, RFP fused with the actin-binding domain of moesin. The explants were treated with 200 μM of cytochalasin B for 30 min. Bar, 50 μm. The graph shows percentages of the gastrulation-defective phenotype. (C) The actin-binding domain of Xenopus moesin was fused with Venus (Venus-actin BD) and expressed in CHO cells. (D) Mo was injected into one blastomere of two-cell embryo with mb-RFP mRNA as a tracer. Animal cap explants were fixed and stained with phalloidin. Bar, 50 μm. (E) Western blot with an antiactin antibody (left) and Coomassie Blue (CBB) staining (right). Mo was injected into both of the blastomeres of two-cell embryos. Lysates were prepared from the animal caps. (F) MARCKS Mo and mb-RFP were coinjected and animal cap cells were immunostained with antiactin antibody.
	Figure 4. MARCKS is required for the cortical actin dynamics regulated by the noncanonical Wnt signaling pathway. (A) 250 pg of Xdsh-Venus mRNA was expressed in DMZ explants. Cells were dissociated and plated on an FN-coated dish. Xdsh-Venus was colocalized with the cortical actin. (B) 100 pg of XdshΔDIX-Venus mRNA was expressed in DMZ explants. The explants were cultured on an FN-coated dish. F-actin was probed with RMA. XdshΔDIX and RMA were colocalized (arrows). (Right) Treatment with 30 μM Latrunculin A for 30 min. (C) Xwnt11 and Xfz7 mRNAs (200 pg each) were coexpressed in animal cap explants with mb-RFP. The coexpression of Xwnt11 and Xfz7 promoted the protrusive activity. 5 pmol of MARCKS Mo and Xdd1 inhibited it. The effect of MARCKS Mo was rescued by coinjection of 200 pg of MARCKS mRNA. Bar, 50 μm. (D) mb-RFP was injected with or without mRNA encoding dominant-negative Xwnt-11 (2 ng). Bar, 50 μm.

References [+] :

Ali, Identification and characterization of MARCKS in Xenopus laevis. 1997, Pubmed, Xenbase