RBD11, a bioengineered Rab11-binding module for visualizing and analyzing endogenous Rab11

Futaba Osaki, Takahide Matsui, Shu Hiragi, Yuta Homma, Mitsunori Fukuda

Research output: Contribution to journalArticlepeer-review

Abstract

The small GTPase Rab11 (herein referring to the Rab11A and Rab11B isoforms) plays pivotal roles in diverse physiological phenomena, including the recycling of membrane proteins, cytokinesis, neurite outgrowth and epithelial morphogenesis. One effective method of analyzing the function of endogenous Rab11 is to overexpress a Rab11-binding domain from one of its effectors, for example, the C-terminal domain of Rab11-FIP2 (Rab11-FIP2-C), as a dominant-negative construct. However, the drawback of this method is the broader Rab-binding specificity of the effector domain, because Rab11-FIP2-C binds to Rabs other than Rab11, for example, to Rab14 and Rab25. In this study, we bioengineered an artificial Rab11-specific binding domain, named RBD11. Expression of RBD11 allowed visualization of endogenous Rab11 without affecting its localization or function, whereas expression of a tandem RBD11, named 2×RBD11, inhibited epithelial morphogenesis and induced a multi-lumen phenotype characteristic of Rab11-deficient cysts. We also developed two tools for temporally and reversibly analyzing Rab11-dependent membrane trafficking – tetracycline-inducible 2×RBD11 and an artificially oligomerized domain (FM)-tagged RBD11.

Original languageEnglish
Article numberjcs257311
JournalJournal of cell science
Volume134
Issue number7
DOIs
Publication statusPublished - 2021 Apr

Keywords

  • Effector
  • Membrane traffic
  • Rab11
  • Rab11-binding domain
  • Small GTPase Rab
  • Trapper

ASJC Scopus subject areas

  • Cell Biology

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