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1.
Dev Dyn ; 251(12): 1934-1951, 2022 12.
Article in English | MEDLINE | ID: mdl-35996230

ABSTRACT

BACKGROUND: The cadherin-associated protein p120 catenin regulates convergent extension through interactions with cadherin proteins, Cdc42, and Rac1, as we previously showed in zebrafish (Danio rerio). Phosphorylation of p120 catenin changes the nature of its activity in vitro but is virtually unexplored in embryos. We used our previously developed antisense RNA splice-site morpholino targeted to endogenous p120 catenin-δ1 to cause defects in axis elongation probing the functions of three p120 catenin tyrosine-phosphorylation sites in gastrulating zebrafish embryos. RESULTS: The morpholino-induced defects were rescued by co-injections with mouse p120 catenin-δ1-3A mRNAs mutated at residues Y228 and Y217 to a non-phosphorylatable phenylalanine (F) or mutated at residue Y335 to a phosphomimetic glutamic acid (E). Co-injection of the complementary mutations Y228E, Y217E, or Y335F mRNAs partially rescued embryos whereas dual mutation to Y228E-Y217E blocked rescue. Immunopurification showed Y228F mutant proteins preferentially interacted with Rac1, potentially promoting cell migration. In contrast, the phosphomimetic Y228E preferentially interacted with E-cadherin increasing adhesion. Both Y228F and Y335F strongly bind VAV2. CONCLUSIONS: p120 catenin serves dual roles during gastrulation of zebrafish. Phosphorylation and dephosphorylation of tyrosine residues Y217, Y228, and Y335 precisely balance cell adhesion and cell migration to facilitate somite compaction and axis elongation.


Subject(s)
Gastrulation , Zebrafish , Mice , Animals , Zebrafish/metabolism , Phosphorylation , Morpholinos/metabolism , Catenins/genetics , Catenins/metabolism , Cadherins/genetics , Cadherins/metabolism , Cell Adhesion/physiology , Tyrosine/genetics , Tyrosine/metabolism , Phosphoproteins/metabolism , beta Catenin/metabolism
2.
Dev Dyn ; 251(12): 1952-1967, 2022 12.
Article in English | MEDLINE | ID: mdl-35706088

ABSTRACT

BACKGROUND: Cadherin-associated protein p120 catenin regulates cell adhesion and migration in cell cultures and is required for axial elongation in embryos. Its roles in adhesion and cell migration are regulated by phosphorylation. We determined the effects of phosphorylation of six serine and three threonine residues in p120 catenin during zebrafish (Danio rerio) embryogenesis. RESULTS: We knocked down endogenous p120 catenin-δ1 with an antisense RNA-splice-site morpholino (Sp-MO) causing defects in axis elongation. These defects were rescued by co-injections of mRNAs for wildtype mouse p120 catenin-δ1-3A or various mutated forms. Several mRNAs containing serine or threonine codons singly or doubly mutated to phosphomimetic glutamic acid rescued, and some nonphosphorylatable mutants did not. CONCLUSIONS: We discovered that phosphorylation of serine residue S252 or S879 is required for convergent extension of zebrafish embryos, since rescue occurred only when these residues were mutated to glutamic acid. In addition, the phosphorylation of either S268 or S269 is required, not both, consistent with the presence of only a single one of these residues in two isoforms of zebrafish and Xenopus laevis. In summary, phosphorylation of multiple serine and threonine residues of p120 catenin activates migration of presomitic mesoderm of zebrafish embryos facilitating elongation of the dorsal axis.


Subject(s)
Serine , Zebrafish , Mice , Animals , Phosphorylation , Zebrafish/metabolism , Serine/metabolism , Glutamic Acid/metabolism , Catenins/genetics , Catenins/metabolism , Cadherins/genetics , Cadherins/metabolism , Mesoderm/metabolism , Threonine/metabolism
3.
Mech Dev ; 130(4-5): 290-303, 2013.
Article in English | MEDLINE | ID: mdl-23313141

ABSTRACT

Is focal adhesion kinase (FAK) needed for embryonic cleavage? We find that FAK is expressed during early cleavage divisions of sea urchin embryos as determined by polyclonal antibodies to the Lytechinus variegatus protein. FAK is absent in eggs and zygotes and then cycles in abundance during the first cleavages after fertilization. It is maximal at anaphase, similar to the destruction and synthesis of cyclin proteins. To investigate whether FAK is needed during early cleavage, we interfered with its function by microinjecting eggs with anti-FAK antibodies or with FAK antisense morpholino oligonucleotides. Both treatments led to regression of the cleavage furrow. FAK knockdown with antibodies or morpholino oligonucleotides also resulted in an over-accumulation of endocytic vesicles. Thus, FAK could be restricting endocytosis or increasing exocytosis in localized areas important for abscission. FAK appears to be necessary for successful cleavage. These results are the first to document a functional role for FAK during embryonic cleavage.


Subject(s)
Blastomeres/enzymology , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/enzymology , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Lytechinus/embryology , Lytechinus/enzymology , Transport Vesicles/metabolism , Anaphase/drug effects , Animals , Blastomeres/cytology , Blastomeres/drug effects , Blotting, Western , Embryo, Nonmammalian/drug effects , Endocytosis/drug effects , Gene Knockdown Techniques , Lytechinus/cytology , Morpholinos/pharmacology , Time Factors , Transport Vesicles/drug effects
4.
Dev Dyn ; 241(10): 1545-61, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22911626

ABSTRACT

BACKGROUND: We investigated the roles of p120 catenin, Cdc42, Rac1, and RhoA GTPases in regulating migration of presomitic mesoderm cells in zebrafish embryos. p120 catenin has dual roles: It binds the intracellular and juxtamembrane region of cadherins to stabilize cadherin-mediated adhesion with the aid of RhoA GTPase, and it activates Cdc42 GTPase and Rac1 GTPase in the cytosol to initiate cell motility. RESULTS: During gastrulation of zebrafish embryos, knockdown of the synthesis of zygotic p120 catenin δ1 mRNAs with a splice-site morpholino caused lateral widening and anterior-posterior shortening of the presomitic mesoderm and somites and a shortened anterior-posterior axis. These phenotypes indicate a cell-migration effect. Co-injection of low amounts of wild-type Cdc42 or wild-type Rac1 or dominant-negative RhoA mRNAs, but not constitutively-active Cdc42 mRNA, rescued these p120 catenin δ1-depleted embryos. CONCLUSIONS: These downstream small GTPases require appropriate spatiotemporal stimulation or cycling of GTP to guide mesodermal cell migration. A delicate balance of Rho GTPases and p120 catenin underlies normal development.


Subject(s)
Cell Movement/physiology , Gastrulation/physiology , Mesoderm/metabolism , Zebrafish/embryology , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Blotting, Western , Catenins/genetics , Catenins/metabolism , Cloning, Molecular , Gene Knockdown Techniques , Guanosine Triphosphate/metabolism , In Situ Hybridization , Mesoderm/cytology , Mesoderm/enzymology , Oligonucleotides, Antisense/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Delta Catenin
5.
Gene Expr Patterns ; 4(2): 223-34, 2004 Mar.
Article in English | MEDLINE | ID: mdl-15161103

ABSTRACT

We have cloned three cDNA isoforms of focal adhesion kinase (FAK) from the sea urchin, Lytechinus variegatus. The sea urchin FAK is more closely related to FAK from other deuterostomes than from invertebrate protostomes or to cell adhesion kinase beta (CAKbeta/Pyk2/FAK2). FAK is expressed in all cells of sea urchin embryos by the 120-cell stage and strongly in blastulae. Phospho-FAK concentrates on basal surfaces of epithelial cells in early blastulae and occurs in syncytial cables of primary mesenchyme cells (PMC). Inhibition of FAK by constructs of FAK-related non-kinase delays blastocoel expansion and early PMC ingression. These results suggest that FAK has roles in cell adhesion and in the shape and integrity of the epithelial cells in sea urchin embryos.


Subject(s)
Embryo, Nonmammalian/enzymology , Protein-Tyrosine Kinases/genetics , Amino Acid Sequence , Animals , Cell Adhesion/physiology , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Focal Adhesion Protein-Tyrosine Kinases , Gene Expression/physiology , Genes, Reporter , Immunohistochemistry , Molecular Sequence Data , Phosphorylation , Phylogeny , Protein-Tyrosine Kinases/metabolism , Sea Urchins
6.
Mol Biol Cell ; 14(8): 3065-81, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12925747

ABSTRACT

We investigated the focal adhesion proteins paxillin and Fak, and the cell-cell adhesion protein cadherin in developing zebrafish (Danio rerio) embryos. Cadherins are expressed in presomitic mesoderm where they delineate cells. The initiation of somite formation coincides with an increase in the phosphorylation of Fak, and the accumulation of Fak, phosphorylated Fak, paxillin, and fibronectin at nascent somite boundaries. In the notochord, cadherins are expressed on cells during intercalation, and phosphorylated Fak accumulates in circumferential rings where the notochord cells contact laminin in the perichordal sheath. Subsequently, changes in the orientations of collagen fibers in the sheath suggest that Fak-mediated adhesion allows longitudinal expansion of the notochord, but not lateral expansion, resulting in notochord elongation. Novel observations showed that focal adhesion kinase and paxillin concentrate at sites of cell-cell adhesion in the epithelial enveloping layer and may associate with actin cytoskeleton at epithelial junctions containing cadherins. Fak is phosphorylated at these epithelial junctions but is not phosphorylated on Tyr397, implicating a noncanonical mechanism of regulation. These data suggest that Fak and paxillin may function in the integration of cadherin-based and integrin-based cell adhesion during the morphogenesis of the early zebrafish embryo.


Subject(s)
Cadherins/metabolism , Cytoskeletal Proteins/metabolism , Phosphoproteins/metabolism , Protein-Tyrosine Kinases/metabolism , Zebrafish/metabolism , Amino Acid Sequence , Animals , Cell Adhesion/physiology , Cloning, Molecular , Cytoskeletal Proteins/genetics , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Fibronectins/metabolism , Focal Adhesion Protein-Tyrosine Kinases , Laminin/metabolism , Molecular Sequence Data , Morphogenesis/physiology , Notochord/embryology , Notochord/metabolism , Paxillin , Phosphoproteins/genetics , Phosphorylation , Protein-Tyrosine Kinases/genetics , Somites/cytology , Somites/metabolism , Zebrafish/embryology , Zebrafish Proteins
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