Volume 7, Issue 8, Pages 1393-1396 (August 2014) FISSION1A, an Arabidopsis Tail-Anchored Protein, Is Localized to Three Subcellular Compartments  Cristina Ruberti, Alex Costa, Emanuela Pedrazzini, Fiorella Lo Schiavo, Michela Zottini  Molecular Plant  Volume 7, Issue 8, Pages 1393-1396 (August 2014) DOI: 10.1093/mp/ssu027 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 The TA-Protein AtFIS1A Is Localized to Three Subcellular Compartments and Protrusions Extending from Them. (A–C) Confocal images of hypocotyl cells of transgenic plants expressing the YFP–FIS1A (green), under the control of the endogenous FIS1A promoter, along with the mitochondrial matrix marker COX–RFP (red) (A) or the peroxisomal lumen marker RFP–KSRM (magenta) (B). Chloroplasts were evidenced by the chlorophyll autofluorescence (Chl, blue) (C). The arrows indicate organelle protrusions, while the arrowheads indicate the sites where YFP–FIS1A clearly surrounds the organelles. (D–F) Biochemical analysis of FIS1A localization in Arabidopsis wild-type (wt) plants. (D) Western blot analysis of FIS1A (18.7kDa) performed on the membrane fraction (Memb) and in the cytosolic fraction (Cyt). (E) Western blot analysis of FIS1A performed on different subcellular compartments. Each fraction was immunoblotted with the anti-FIS1A antibody and specific antibodies against organelle markers. (F) Subcellular compartment fractionation by ultracentrifugation of leaf homogenate on isopycnic sucrose gradient. Proteins in each fraction were analyzed by SDS–PAGE and immunoblotted with antibodies against FIS1A and against organelle markers. (G) CLSM images of leaf cells of transgenic plants expressing the YFP–FIS1A (green) along with the cytosolic marker cyto-RFP (red) and the chlorophyll fluorescence (blue). (H–J) CLSM time-lapse analyses of movements of organelle protrusions in Arabidopsis seedlings co-expressing the YFP–FIS1A and COX–RFP (H). Effect of latrunculin B (I) and oryzalin (J) on movements of organelle protrusions. Arrows indicate the organelle protrusions. (K–N) Subcellular localization of YFP–FIS1ACT and YFP–FIS1ANT in leaf mesophyll cells. (K) Confocal image of leaf mesophyll cells, stably expressing the YFP–FIS1ACT, agro-infiltrated with COX–RFP (blue). (L) Confocal image of leaf mesophyll cells, stably expressing the YFP–FIS1ACT, agro-infiltrated with RFP–KSRM (magenta). (M) Confocal images of transgenic plant cells expressing the YFP–FIS1ACT. (N) Confocal images of transgenic plant cells, stably expressing the YFP–FIS1ANT (green), agro-infiltrated with the free RFP (red), both localized in the cytosol and nucleus. Chloroplasts were evidenced by the chlorophyll autofluorescence (Chl, in blue). (O, P) Analyses of FIS1A mobility in pFIS1A::YFP–FIS1A and pFIS1A::YFP–FIS1ACTArabidopsis plants. (O) Quantification of YFP–FIS1A and YFP–FIS1ACT fluorescence recovery and statistical significance of FRAP experiments. (P) Representative FRAP experiment. Pre-bleached (t–2.6s), bleached (t0s), and recovered (t230s) confocal images were reported (bl, bleached chloroplast). Scale bars = 2 μm. (The full version of the figure legend is given in the Supplementary Data online.) Molecular Plant 2014 7, 1393-1396DOI: (10.1093/mp/ssu027) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions