Protein Targeting and Vesicular Trafficking
Griffing, Lawrence , Johnson, Carol .
FRET analysis of trans-membrane flipping of FM 4-64 in living root hairs: Is FM 4-64 a robust marker for endocytosis?
Although FM 4-64 is now used routinely to monitor endocytosis in plants, argument about its potential to cytoplasmically, non-endocytically relocate into a selective set of vesicular compartments persists. We have addressed this question with fluorescence resonance energy transfer (FRET) analysis using cytoplasmically-expressed short-wavelength excitation GFP as a FRET partner for FM 4-64 in Nicotiana benthaminana. Rapidly streaming, young growing root hairs from 6-9 day old seedlings grown in constant light were used. Root hair plasma membrane labeled with FM 4-64 dye and some near the tip was immediately internalized. Under these conditions, no FRET was seen. However, if cells were treated with a low concentration of saponin, a membrane permeabilization agent, the cells continued to stream and FRET was detected. This positive control is considered necessary to demonstrate that under conditions that do not severely compromise cell viability, FM 4-64 dye can indeed flip in the membrane and enter the cytosolic half of the plasma membrane, providing a suitable FRET partner for the cytoplasmically-localized GFP. The interpretation of these results is that under normal conditions and at short times, FM 4-64 dye is a robust marker for endocytosis. Future studies that show the endocytosis of macromolecules in the fluid phase as confirmed with FRET analysis are discussed.
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1 - Texas A&M University, Department of Biology, 3258 Tamu, Bsbw Rm 104, College Station, Texas, 77843, United States
2 - Texas A&M University, Department of Biology
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM