We further extended these binding studies and confirmed the above Rab5-binding site in vivo in the cell, by monitoring Rab5-mediated APPL1 recruitment to early endosomes in the cell via confocal microscopy. In this case, the RFP (DsRed-monomer)−Rab5-Q79L fusion protein was expressed in PC12 cells, targeted to the early endosomes, and recruited effectively the coexpressed GFP (green fluorescence protein)−APPL1 to these early endosomes (Figure 5C). Importantly, APPL1 (5−385), that is, the BAR-PH domain, was sufficient to target to Rab5-Q79L containing early endosomes (Figure 5C). In contrast, one of the Rab5-binding defective mutants (A318D) failed to target the early endosomes and exhibited a diffused pattern throughout the cytoplasm in the cell (Figure 5C).
(C) Mutational effects on APPL1 targeting to Rab5-positive early endosomes in the cell. RFP−Rab5-Q79L was coexpressed with GFP−APPL1 (full length, FL; BAR-PH domain; or BAR-PH mutant) in PC12 cells as indicated, followed by confocal fluorescence microscopy. Shown are typical confocal microscopic images indicating the RFP−Rab5-Q79L labeled early endosomes (red) and the colocalization of GFP−APPL1 or mutants (green) in the same cells. Scale bar, 16 μm.Tobacco leaf epidermal cells from agroinfiltration were examined under a Zeiss LSM 510 inverted confocal laser-scanning microscope (Zeiss, Jena, Germany) following the settings described by Goodin et al. (2002) with minor modifications. Single optical sections were scanned as resulting images for each transient expression. For each plasmid construct, 10–15 cells were imaged with similar results. GFP fluorescence was excited at 488 nm, filtered through a primary dichroic (UV/488/543), a secondary dichroic of 545 nm, and subsequently through BP505–530 nm emission filters to the photomultiplier tube (PMT) detector. DsRed fluorescence was excited at 543 nm, the emission was passed through similar primary and secondary dichroic mirrors and finally through a BP560–615 nm emission filter to the PMT detector. Fluorescence resonance energy transfer (FRET) pairs GFP/DsRed were analysed using a confocal laser-scanning microscope (Zeiss LSM510 META). FRET measurements of DsRed emission with zero contribution from GFP, was accomplished as described by Erickson et al. (2003) using the following settings: excitation at 488 nm and emission filters, BP 505–530 nm for GFP and BP 600–637 nm for DsRed.
Following agroinfiltration of tobacco leaves with both ACBP4:DsRed and GFP:AtEBP, observations were carried out by confocal microscopy using a green filter to investigate the fluorescence pattern of GFP and a red filter to visualize the fluorescence of DsRed (Fig. 2). GFP:AtEBP was located mainly in the nucleus, with some signals at the cytosol and the plasma membrane (Fig. 2A). ACBP4:DsRed was localized predominantly to the cytosol, inclusive of signals detected in the cytosol surrounding the nucleus (Fig. 2B). Signals of both fusion proteins were common to the cytosol.
Confocal images indicating co-localization of ACBP4:DsRed and GFP:AtEBP fusion proteins transiently-expressed in tobacco leaves. Representative tobacco leaf epidermal cells are shown by laser-scanning confocal microscopy following agroinfiltration of plasmid pAT282 or pAT225.