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Journal of Biomedical Optics • Open Access

Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy
Author(s): Hewang Li; Peiying Yu; Yuansheng Sun; Robin A. Felder; Ammasi Periasamy; Pedro A. Jose

Paper Abstract

The dynamic regulation of the cellular trafficking of human angiotensin (Ang) type 1 receptor (AT1R) is not well understood. Therefore, we investigated the cellular trafficking of AT1R-enhanced green fluorescent protein (EGFP) (AT1R-EGFP) heterologously expressed in HEK293 cells by determining the change in donor lifetime (AT1R-EGFP) in the presence or absence of acceptor(s) using fluorescence lifetime imaging-fluorescence resonance energy transfer (FRET) microscopy. The average lifetime of AT1R-EGFP in our donor-alone samples was ~2.33 ns. The basal state lifetime was shortened slightly in the presence of Rab5 (2.01±0.10 ns) or Rab7 (2.11±0.11 ns) labeled with Alexa 555, as the acceptor fluorophore. A 5-min Ang II treatment markedly shortened the lifetime of AT1R-EGFP in the presence of Rab5-Alexa 555 (1.78±0.31 ns) but was affected minimally in the presence of Rab7-Alexa 555 (2.09±0.37 ns). A 30-min Ang II treatment further decreased the AT1R-EGFP lifetime in the presence of both Rab5- and Rab7-Alexa 555. Latrunculin A but not nocodazole pretreatment blocked the ability of Ang II to shorten the AT1R-EGFP lifetime. The occurrence of FRET between AT1R-EGFP (donor) and LAMP1-Alexa 555 (acceptor) with Ang II stimulation was impaired by photobleaching the acceptor. These studies demonstrate that Ang II-induced AT1R lysosomal degradation through its association with LAMP1 is regulated by Rab5/7 via mechanisms that are dependent on intact actin cytoskeletons.

Paper Details

Date Published: 1 September 2010
PDF: 9 pages
J. Biomed. Opt. 15(5) 056003 doi: 10.1117/1.3484751
Published in: Journal of Biomedical Optics Volume 15, Issue 5
Show Author Affiliations
Hewang Li, Children's National Medical Ctr. (United States)
Peiying Yu, Children's National Medical Ctr. (United States)
Yuansheng Sun, Univ. of Virginia (United States)
Robin A. Felder, Univ. of Virginia (United States)
Ammasi Periasamy, Univ. of Virginia (United States)
Pedro A. Jose, Children's National Medical Ctr. (United States)

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