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

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

Author information

1
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.

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.

PMID:
21976318
PMCID:
PMC3197968
DOI:
10.1002/dvdy.22758
[Indexed for MEDLINE]
Free PMC Article

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