ABSTRACT
To dissect the molecular mechanisms underlying convergent extension (CE), a prominent set of cell movements during Xenopus gastrulation, we performed a functional expression screen and identified a GTPase-activating protein for ADP ribosylation factors (ArfGAP), which we termed XGAP. We demonstrated that XGAP is required to confine or restrict the cellular protrusive activity to the mediolateral ends of cells, where XGAP is normally localized, and therefore for the proper intercalation of cells participating in CE. We also demonstrated that a C-terminal conserved domain of XGAP, but not its GAP activity, is required and sufficient for this intracellular localization and function. We further showed that XGAP physically interacts with the known polarity proteins 14-3-3epsilon, aPKC, and PAR-6 and directs them to the mediolateral ends of dorsal mesoderm cells during gastrulation. We propose that XGAP controls CE through the restriction and maintenance of partitioning-defective (PAR) proteins in the regions that harbor protrusive activity.
Subject(s)
Carrier Proteins/metabolism , Cell Polarity/physiology , GTPase-Activating Proteins/metabolism , Gastrula/metabolism , Xenopus Proteins/metabolism , Xenopus/embryology , Xenopus/metabolism , 14-3-3 Proteins/metabolism , Animals , Base Sequence , Carrier Proteins/genetics , Cell Movement , GTPase-Activating Proteins/antagonists & inhibitors , GTPase-Activating Proteins/chemistry , GTPase-Activating Proteins/genetics , Gastrula/cytology , HeLa Cells , Humans , Models, Biological , Molecular Sequence Data , Oligodeoxyribonucleotides, Antisense/genetics , Oligodeoxyribonucleotides, Antisense/pharmacology , Phosphorylation , Protein Binding , Protein Kinase C/metabolism , Protein Structure, Tertiary , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Xenopus/genetics , Xenopus Proteins/geneticsABSTRACT
To easily monitor living cells and organisms, we have created a transgenic Xenopus line expressing Venus, a brighter variant of yellow fluorescent protein, under the control of the CMV enhancer/chicken beta-actin (CAG) promoter. The established line exhibited high fluorescent intensity not only in most tissues of tadpoles to adult frogs but also in germ cells of both sexes, which enabled three-dimensional imaging of fluorescing organs from images of the serial slices of the transgenic animals. Furthermore, by using this transgenic line, we generated chimeric animals by brain implantation and importantly, we found that the brain grafts survived and expressed Venus in recipients after development, highlighting the boundary between fluorescent and nonfluorescent areas in live animals. Thus, Venus-expressing transgenic frogs, tadpoles, and embryos would facilitate their use in many applications, including the tracing of the fluorescent cells after tissue/organ transplantation.
