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Membrane mobility of aquaporin‐2 (AQP2) tagged with photoconvertible fluorescence protein in mpkCCD cells
Author(s) -
Yip kayPong,
Amin Jahanshah,
Tse ChungMing,
Cha Byeong
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1111.6
Subject(s) - fluorescence recovery after photobleaching , aquaporin 2 , chemistry , apical membrane , aquaporin , biophysics , membrane , fluorescence , forskolin , photobleaching , vesicle , water channel , biochemistry , biology , optics , in vitro , physics , mechanical engineering , engineering , inlet
mEos is a photoconvertible fluorescence protein. Its emission can be switched from green to red by an UV pulse. Tagging AQP2 with mEos allows tracking of a subset of AQP2 after photoconversion. Here, we examined whether, 1) AQP2 tagged with mEos to amino‐terminus (Eos‐AQP2) or carboxyl‐terminus (AQP2‐Eos) has similar membrane mobility, and 2) photoconversion alters their membrane mobility. Cells grown on glass‐bottom dishes were transfected with either Eos‐AQP2 or AQP2‐Eos and their membrane mobility were measured by fluorescence recovery after photobleaching (FRAP). A 488 nm laser pulse was used to photobleach a 4 μm spot for FRAP. Fluorescence recovery half‐time (t 1/2 ) was calculated with ImageJ. Eos‐AQP2 was found in cytosolic vesicles and plasma membrane. Apical accumulation of Eos‐AQP2 was stimulated by forskolin. AQP2‐Eos was localized in apical membrane. t 1/2 of Eos‐AQP2 and AQP2‐Eos were in the same range (42–50 s). Local photoconversion of green‐Eos to red‐Eos was performed with a 405 nm laser pulse on the apical surface. Photoconversion did not alter the membrane mobility of EOS‐AQP2 or AQP2‐EOS as measured by the diffusion time course of red‐Eos from the photoconversion site. However, forskolin stimulation increased t 1/2 of Eos‐AQP2 by 3 folds, and decreased the t 1/2 of AQP2‐Eos by 25%. These results indicated that photoconversion did not alter AQP2 membrane mobility.