Generation of Rab-based transgenic lines for in vivo studies of endosome biology in zebrafish

Dev Dyn. 2011 Nov;240(11):2452-65. doi: 10.1002/dvdy.22758. Epub 2011 Oct 4.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Validation Study

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

Substances

  • Crb2a protein, zebrafish
  • Membrane Proteins
  • Zebrafish Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • rab7 protein
  • Rab5c protein, zebrafish
  • rab11 protein
  • rab GTP-Binding Proteins
  • rab5 GTP-Binding Proteins