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ZFIN ID: ZDB-PUB-080331-12
Probing e-cadherin endocytosis by morpholino-mediated rab5 knockdown in zebrafish
Ulrich, F., and Heisenberg, C.P.
Date: 2008
Source: Methods in molecular biology (Clifton, N.J.) 440: 371-387 (Chapter)
Registered Authors: Heisenberg, Carl-Philipp, Ulrich, Florian
Keywords: Cell migration, endocytosis, endosomes, gastrulation, mesendoderm, morpholino, small GTPase rab5, zebrafish
MeSH Terms:
  • Animals
  • Antibodies
  • Cadherins/immunology
  • Cadherins/metabolism*
  • Cell Adhesion
  • Cell Culture Techniques
  • Cell Movement
  • Endocytosis*
  • Endosomes/metabolism
  • Fluorescent Antibody Technique
  • Gastrula/metabolism
  • Gene Deletion*
  • In Situ Hybridization
  • Morpholines/metabolism*
  • Oligonucleotides, Antisense/metabolism*
  • Protein Transport
  • Zebrafish/embryology
  • Zebrafish/genetics
  • Zebrafish/metabolism*
  • Zebrafish Proteins/genetics
  • Zebrafish Proteins/metabolism*
  • rab5 GTP-Binding Proteins/genetics
  • rab5 GTP-Binding Proteins/metabolism*
PubMed: 18369959 Full text @ Meth. Mol. Biol.
The controlled internalization of membrane receptors and lipids is crucial for cells to control signaling pathways and interact with their environment. During clathrin-mediated endocytosis, membrane constituents are transported via endocytic vesicles into early endosomes, from which they are further distributed within the cell. The small guanosine triphosphatase (GTPase) Rab5 is both required and sufficient for the formation of these early endosomes and can be used to experimentally address endocytic processes. Recent evidence shows that endocytic turnover of E-cadherin regulates the migration of mesendodermal cells during zebrafish gastrulation by modulating their adhesive interactions with neighboring cells. This in turn leads to effective and synchronized movement within the embryo. In this review, we discuss techniques to manipulate E-cadherin endocytosis by morpholino-mediated knockdown of rab5 during zebrafish gastrulation. We describe the use of antibodies specifically directed against zebrafish E-cadherin to detect its intracellular localization and of in situ hybridization and primary cell culture to reveal patterns of cell migration and adhesion, respectively.