ZFIN ID: ZDB-PUB-111024-9
Generation of Rab-based transgenic lines for in vivo studies of endosome biology in zebrafish
Clark, B.S., Winter, M., Cohen, A.R., and Link, B.A.
Date: 2011
Source: Developmental dynamics : an official publication of the American Association of Anatomists   240(11): 2452-2465 (Journal)
Registered Authors: Clark, Brian, Link, Brian
Keywords: endosome dynamics, neuroepithelia, organelle polarization, zebrafish, transgenic lines
MeSH Terms:
  • Amino Acid Sequence
  • Animals
  • Animals, Genetically Modified
  • Biology/methods*
  • Endosomes/genetics
  • Endosomes/metabolism
  • Endosomes/physiology*
  • Gene Transfer Techniques*
  • Green Fluorescent Proteins/genetics
  • Green Fluorescent Proteins/metabolism
  • Membrane Proteins/metabolism
  • Models, Biological
  • Neuroepithelial Cells/metabolism
  • Sequence Homology, Amino Acid
  • Zebrafish/embryology
  • Zebrafish/genetics*
  • Zebrafish/metabolism
  • Zebrafish/physiology
  • Zebrafish Proteins/metabolism
  • rab GTP-Binding Proteins/genetics*
  • rab GTP-Binding Proteins/metabolism
  • rab5 GTP-Binding Proteins/genetics
  • rab5 GTP-Binding Proteins/metabolism
PubMed: 21976318 Full text @ Dev. Dyn.
The Rab family of small GTPases function as molecular switches regulating membrane and protein trafficking. Individual Rab isoforms define and are required for specific endosomal compartments. To facilitate in vivo investigation of specific Rab proteins, and endosome biology in general, we have generated transgenic zebrafish lines to mark and manipulate Rab proteins. We also developed software to track and quantify endosome dynamics within time-lapse movies. The established transgenic lines ubiquitously express EGFP fusions of Rab5c (early endosomes), Rab11a (recycling endosomes), and Rab7 (late endosomes) to study localization and dynamics during development. Additionally, we generated UAS-based transgenic lines expressing constitutive active (CA) and dominant-negative (DN) versions for each of these Rab proteins. Predicted localization and functional consequences for each line were verified through a variety of assays, including lipophilic dye uptake and Crumbs2a localization. In summary, we have established a toolset for in vivo analyses of endosome dynamics and functions.