In-Depth Fluorescence Lifetime Imaging Analysis Revealing SNAP25A-Rabphilin 3A Interactions

Abstract

The high sensitivity and spatial resolution enabled by two-photon excitation fluorescence lifetime imaging microscopy/fluorescence resonance energy transfer (2PE-FLIM/FRET) provide an effective approach that reveals protein-protein interactions in a single cell during stimulated exocytosis. Enhanced green fluorescence protein (EGFP)-labeled synaptosomal associated protein of 25 kDa (SNAP25A) and red fluorescence protein (mRFP)-labeled Rabphillin 3A (RPH3A) were co-expressed in PC12 cells as the FRET donor and acceptor, respectively. The FLIM images of EGFP-SNAP25A suggested that SNAP25A/RPH3A interaction was increased during exocytosis. In addition, the multidimensional (three-dimensional with time) nature of the 2PE-FLIM image datasets can also resolve the protein interactions in the z direction, and we have compared several image analysis methods to extract more accurate and detailed information from the FLIM images. Fluorescence lifetime was fitted by using one and two component analysis. The lifetime FRET efficiency was calculated by the peak lifetime (taupeak) and the left side of the half-peak width (tau1/2), respectively. The results show that FRET efficiency increased at cell surface, which suggests that SNAP25A/RPH3A interactions take place at cell surface during stimulated exocytosis. In summary, we have demonstrated that the 2PE-FLIM/FRET technique is a powerful tool to reveal dynamic SNAP25A/RPH3A interactions in single neuroendocrine cells.

Changes in Plasma Membrane Surface Potential of PC12 Cells as Measured by Kelvin Probe Force Microscopy

“…The PC12 cells, derived from pheochromocytoma of the rat adrenal medulla, were generously provided by Dr. Lung-Sen Kao [18], [19]. The cells were cultured in DMEM containing 10% horse serum and 5% fetal bovine serum, and were then incubated at 37°C in an atmosphere containing 10% CO 2 .…”

Visualization of the Orai1 Homodimer and the Functional Coupling of Orai1-STIM1 by Live-Cell Fluorescence Lifetime Imaging

“…Using suitable FRET pairs of fluorescent proteins (FPs), such as cyan fluorescent protein (CFP) with yellow fluorescent protein (YFP), or enhanced green fluorescent protein (EGFP) with a monomer of red fluorescent protein (mRFP, mCherry, or mOrange), several fluorescent platforms can be used to detect and analyze FRET. These include the intensity-based method (Barr et al, 2008; Muik et al, 2008; Navarro-Borelly et al, 2008; Periasamy et al, 2008; Shyu et al, 2008; Calloway et al, 2009; Wang et al, 2009) and the lifetime-based method (Goedhart et al, 2007; Lee et al, 2008 a , 2008 b ; Padilla-Parra et al, 2008; Chiu et al, 2009).…”

Rabphilin 3A retains NMDA receptors at synaptic sites through interaction with GluN2A/PSD-95 complex

“…Rph3A directly binds to other proteins including the MAGUK protein CASK 22 , SNAP-25 (ref. 23 ) and MyoVa 24 , localized in the pre- or post-synaptic compartment. Mice that lack Rph3A are viable and fertile without obvious physiological impairments 25 .…”