Fluorescent imaging of ER Protein Bodies induced by elastin like protein expression in Nicotiana benthamiana leaves

OCAMPO, CAROLINA GABRIELA; MAZZINI, FLAVIA NOELIA; MARIN VIEGAS, VANESA SOLEDAD; PETRUCCELLI, SILVANA

La PLATA

Workshop; Imaging Techniques for Biotechnology and Biomedical Applications ?Workshop; 2016

Centro Científico Tecnológico del CONICET, -La Plata

Protein bodies (PBs) are endoplasmic reticulum (ER) derived organelles found monocotyledonous seeds specialized in accumulation of storage proteins (Herman and Lakins, 1999). They are considered an interesting target for stable accumulation of foreign valuable proteins (Arcalis et al., 2004; Takaiwa et al., 2009; Takagi et al., 2010). Naturally, PBs are not found in leaves, however, over-expression of certain heterologous proteins or fusions to insoluble fusion tags like elastin-like protein (ELP) can induce its formation (Gutiérrez et al., 2013; Saberianfar et al., 2015). ELPs are artificial polypeptides derived of animal tropoelastin compose of repeats of the pentapeptide motif (VPGXG)n where X is any amino acid other than proline and n is the number of pentapeptide repeats (Urry and Parker, 2002). The aim of this work was evaluate the capability of an ELP to induce PB formation and passively sequestrate an ER-targeted protein. To reach this goal, a GFP-HDEL encoding construct was transiently expressed in Nicotiana benthamiana leaves in the present and absence of RFP-ELP construct and changes in ER structure and colocalization was analyzed by confocal laser scanning microscopy (CLSM) using a CLSM LEICA TCS SP5AOBS (Advanced Microscopy Facility, FCE, UNLP). GFP-HDEL was selected as the ER-retained protein and ELP was fused to RFP with the finality to detect if the two proteins are integrated in the same PB. The selection of GFP and mRFP cherry for multilabelling was based in their maximum emission peaks, which are sufficiently separated to perform simultaneous observation of the two proteins with low interference between them. GFP was excited at 488 nm (Argon 100 mW Laser) and detected in the 496?532 nm range. RFP was excited at 543 nm (HeNe 1.5 mW Laser) and detected in the 570?630 nm range. Simultaneous detection of GFP and RFP was performed by combining the settings indicated above in a sequential scanning set-up, as instructed by the manufacturer. All images shown were acquired using the same photomultiplier gain and offset settings. Taking into account that chlorophyll autofluorescence may challenge the detection of RFP that emit/absorb in the red region, its fluorescence was detected between 601 to 708 nm after excitation with the 594-nm (HeNe 1.5 mW Laser). The obtained images were analyzed and deconvolutionated using ImageJ using Diffraction PSF 3D plugging to obtain the theorical PSF function, with the following parameters: numerical aperture (objective HCX PL APO CS 20.0x0.70 IMM UV) 0.70 and refraction index 1.52. Deconvolution was made with parallel spectral deconvolution plugging using generalized Tikhonov (reflexive) method. When ER-GFP and RFP-ELP were co-expressed large ER-PB were observed predominantly close to the nuclei and in cortical regions (Figure 1B vs 1A). Some of the ELP induced PBs were larger than the nucleolus. PBs, in the cortical region are smaller than in the nuclear sections and had heterogeneous size and composition distribution since some of them had only RFP-ELP and other only ER-GFP. In contrast, in the nuclear region, a complete co-localization of green ER-GFP PBs and red ER-RFP-ELP PBs was observed. These results indicates that RFP-ELP induced a generalized PB ER formation in which other ER proteins such as ER-GFP are sequestered.