p120’s Role in Regulating Cell Adhesion and Migration in Zebrafish Embryos in Vivo

Yue Xiang*,Dr. Merrill Hille, Anne Lyons

Abstract

Objectives

Methods P120 Catenin Phosphorylation Site Mutant

Possible Experimental Results

References

Cell migration and cell adhesion are the crucial process for embryonic development and cancer progression. Regulated changes in adhesion result in morphogenesis. Thus, examining specific regulators of the adhesion is important in understanding of the development and the disease progression. Our lab specifically studies p120 catenin’s involvement in csell migration in the zebrafish embryo. p120 catenin is a substrate for tyrosine and serine kinases. Its functions are:

•Make the Y228F mutant construct•Test the p120 catenin mutant•Understand the importance of p120

phosphorylation in term of cell migration during embryo gastrulation

The role of p120 catenin phosphorylation in early gastrulation of vertebrates is not known. We hypothesize tyrosine phosphorylation at 228 site of p120 is required for zebrafish gastrulation.• When p120 catenin can not phosphorylate at the

Y228 site, it stabilizes the cadherin, which increases cell to cell adhesion.

• When p120 catenin is phosphorylated at the Y228 site, it interacts with cytoplasmic downstream proteins. Thus, it increases cell motility.

Figure 2: p120 structure and function. The regulatory domain contains the tyrosine (red lollipops) and serine/threonine (circles) residues.

Final construct of Y228F p120ctn DNA

Figure 4 shows the final construct of the plasmid, which consists of the vector pCS2+, two pieces of inserts Tag RFP and p120 catenin (linked by the F linkers)

Figure 3 shows how the infusion enzyme fuses ends of the PCR fragment to the homologous ends of a linearized vector.

The Infusion protocol for tyrosine 228 mutation

Figure 5 S268A was made using point mutation. AGC translates as wild-type serine; GCC as alanine.

Figure 6 Y228F was made using infusion & point mutation. TAC translates as tyrosine and TTC as phenylalanine.

• Inject a splice Morpholino into one-celled embryos•Determine whether defects can be rescued by mRNAs of

p120 catenin mutant

Figure 7 mRNAs are transcribed and microinjected into the embryo. Embryos are observed by microscopy

Figure 9 Embryos injected with splice MO lack of cell to cell adhesion.(fig. 10-1) When coninjected morpholino and WT p120 catenin (fig 10-4), embryo rescue completely (fig. 10-3). High doses of p120 catenin mRNA did not cause abnormal morphology (fig. 10-2).

Result Continue

Future Experiment

 Table 1 Total # embryo

# of Death

# of Abnormality # of Normal

Uninjected 125 0 0 1255 pg p120 catenin 112 0 1 111

6 ng Morpholino 75 35 16  24

6 ng Morpholino +5pg p120 catenin

85 1 1 83

Figure 10 and table 1 show that coinjection of 5 pg of WT p120 catenin mRNA rescued the abnormality and deaths caused by splice morpholino. We expect to see no rescue with the Y228F p120 catenin mutant.

Predictions•We hypothesize that tyrosine phosphorylation at 228 site

of p120 is required for zebrafish gastrulation. •We predict the p120 catenin mutants will not successfully

rescue the cell migration defect. •Tyrosine phosphorylation site plays an important role in

maintaining catenin and cadherin interaction for the cell-cell adhesion as well as cell migration.

•Use fluorescent dyes to view p-120 distribution in WT versus p120catenin mutants at cell membrane and in cytosol • track interaction of p120

catenin with downstream protein.

1. Halbleib JM & Nelson WJ. (2006). Genes & Dev. 20: 3199-3214.2. Crawford BD, Henry CA, Clason TA, Becker AL & Hille MB. (2003). Molecular Biology of the Cell. 14: 3065-3081.3. Reynolds, AB. (2007). Biochimica et Biophysica Acta. 1773: 2–7.4. Chen X, Kojima S, Borisy G & Green KJ. (2003). JCB. 163: 547-557.6. Perez-Moreno M & Fuchs E. (2006). Developmental Cell. 11:601-612