Kim MH et al. (2014), Cell adhesion in zebrafish embryos is modulated...

XB-ART-48906

PLoS One 2014 Jan 01;94:e94873. doi: 10.1371/journal.pone.0094873.

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Cell adhesion in zebrafish embryos is modulated by March 8.

Kim MH , Rebbert ML , Ro H , Won M , Dawid IB .

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March 8 is a member of a family of transmembrane E3 ubiquitin ligases that have been studied mostly for their role in the immune system. We find that March 8 is expressed in the zebrafish egg and early embryo, suggesting a role in development. Both knock-down and overexpression of March 8 leads to abnormal development. The phenotype of zebrafish embryos and Xenopus animal explants overexpressing March 8 implicates impairment of cell adhesion as a cause of the effect. In zebrafish embryos and in cultured cells, overexpression of March 8 leads to a reduction in the surface levels of E-cadherin, a major cell-cell adhesion molecule. Experiments in cell culture further show that E-cadherin can be ubiquitinated by March 8. On the basis of these observations we suggest that March 8 functions in the embryo to modulate the strength of cell adhesion by regulating the localization of E-cadherin.

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Species referenced: Xenopus
Genes referenced: cdh1 evl marchf8

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	Figure 2. Overexpression of march8 mRNA induces early embryonic death.(A–C) Uninjected embryos (A), and embryos injected with WT march8 mRNA (B) or RING domain mutant (W109A) march8 mRNA (C), at shield to 60% epiboly stage. Overexpression of March8 results in loss of cell adhesion, abnormal cell migration, and cell death (B); individual embryos showing disaggregation of embryonic cells are shown in B′. Embryos injected with W109A mutant mRNA were normal (C). (D) Self ubiquitination of March8. HEK293T cells were transfected with the plasmids indicated and activated by addition of tebufenozide (see Materials and Methods). Lysates were immunoprecipitated (IP) with anti-March8 antibody and then immunoblotted (IB) with anti-HA antibody to detect ubiquitinated proteins (upper panel). Expression of March8 and tubulin as control are shown in bottom panels.
	Figure 3. The RING domain of March8 is critical for induction of embryonic defects.The classification of morphological defects at 26 hpf after march8 mRNA injection is shown at the left. Quantification shows that defects were induced in a dose-dependent manner by WT, but very weakly by W109A mutant march8 mRNA. Number of embryos is listed on top of bars. UI, uninjected; GFP, embryos injected with 50 pg GFP mRNA.
	Figure 4. March8 induces cell dissociation in Xenopus animal caps.Xenopus embryos were injected with 400LacZ, Xenopus march8 or zebrafish march8 mRNA. Animal caps were dissected and incubated as described in Materials and Methods. Caps were photographed at the indicated times after being placed in dissociation media.
	Figure 5. March8 down-regulates cell surface levels of E-cadherin.Control and march8 mRNA (25 pg) injected embryos were fixed at germ ring stage and stained with anti-March8 (red) and anti-E-cadherin (green) antibodies. Confocal images of EVL and deep cells are shown in animal pole view. March8 overexpression is visualized by higher levels of March8 in intracellular vesicles, and leads to reduced cell surface localization of E-cadherin.
	Figure 6. March8 overexpression reduces cell surface expression of E-cadherin in cultured cells.(A) HEK293T cells were transfected with plasmids encoding E-cadherin-GFP, and inducible March8 WT or W109A mutant. After 12 h, cells were treated for 8 h with tebufenozide to induce March8 expression or maintained without the drug, as indicated (see Figure 7 for induction efficiency). For membrane E-cadherin staining, non-permeablized cells were stained using an antibody to an extracellular epitope (red). Cells were analyzed by flow cytometry in red (Y axis; surface E-cadherin) and green channels (X axis; total E-cadherin). Thus the upper right quadrant represents cells with E-cadherin at the surface, while the lower right quadrant represents non-surface (intracellular) E-cadherin. (B, C) Fraction of total E-cadherin that is intracellular. The data from the experiment in (A) are shown in (B). Panel (C) shows the average intracellular E-cadherin measured in 20 separate flow cytometry experiments. Induction of WT March8 led to an increase in intracellular E-cadherin that is highly significant, whereas induction of the W109A mutant did not lead to a significant change (student's T test).
	Figure 7. E-cadherin is ubiquitinated by March 8.HEK293T cells were transfected with HA-Ubiquitin, E-cadherin-GFP, and WT or W109A mutant March8, as indicated, and March8 expression was induced with tebufenozide (see Materials and Methods). E-cadherin was immunoprecipitated (IP) from lysates and immunoblotted (IB) with anti-HA antibody to detect ubiquitinated E-cadherin. Expression of the different proteins was checked by IB of lysates. Three additional independent experiments, and quantification of E-cadherin ubiquitination, are shown in Figure S5.

References [+] :

Babb, Zebrafish E-cadherin: expression during early embryogenesis and regulation during brain development. 2001, Pubmed