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5. Immunogold-EM analysis confirms VSR labelling of enlarged ring-like structures (i.e. and the TGN, but that they were labelled by an MVB marker protein. In addition, live cell imaging and EM analysis showed these spherical structures to be derived largely from the homotypic fusion of MVBs. Therefore, ARA7(Q69L) expression appears to serve as an excellent tool for inducing MVB enlargement and for studying the relative localization of different proteins on MVBs. Materials and methods Preparation of constructs A two-step cloning procedure was used to generate the final construct, which contained the HSPCGFPCARA7(Q69L) for PSBD cells. First, the heat shock promoter (hsp18.2) was excised from the pHGT1 vector (a gift from Dr Karin Schumacher, Heidelberg University) and subcloned into the binary vector pBI121 (Chen (CaMV) 35S promoter and the nopaline synthase (NOS) terminator RGX-104 free Acid (Miao PSBD cell suspension cultures (ecotype Landsberg cells The HSPCGFPCARA7(Q69L)/pBI121 construct was used for cell lines were maintained in both liquid and solid cultures supplemented with a lower concentration of kanamycin (50 g mlC1). Suspension-cultured cells were transferred onto MS plates and cultured for an additional 7C10 d before being used. Transgenic cells were imaged by confocal microscopy 1 h after heat shock treatment at 37 C and CD164 3C4 h after incubation at 27 C, respectively. Dynamic study of GFP fusions in transgenic cells by spinning disc confocal microscopy Transgenic cells expressing GFPCARA7(Q69L) were subjected to either a brief heat shock treatment or standard incubation before being observed by confocal microscopy. Images were collected using a Revolution XD spinning disc laser confocal microscopy system (Andor Technology China) fitted with a 100 oil lens. Three-dimensional time-lapse images were obtained from stacks of 2-D images, which were collected at short intervals (Wang and wild-type (WT) cells were subjected to heat shock treatment for 1 h at 37 C before fixation in MS cell culture medium containing 0.5% glutaraldehyde for 15 min at room temperature. After a brief wash with MS medium three times, the cells were treated with MS containing 0.1% pectinase and 1% cellulase for 1 h at 28 C. Then the cells were washed with phosphate-buffered saline (PBS), and treated with PBS containing 0.1% sodium tetrahydridoborate RGX-104 free Acid (NaBH4) at 4 C overnight. For RGX-104 free Acid immunolabelling, polyclonal antibodies against the vacuolar sorting receptor (VSR) (Tse protoplasts To determine the subcellular localization of ARA7(Q69L) in cells, GFPCARA7 or GFPCARA7(Q69L) was transiently expressed in protoplasts derived from suspension-cultured PSBD cells. As shown in Fig. 1A, GFP-tagged WT ARA7 labelled punctate structures, whereas the constitutively active mutant GFPCARA7(Q69L) localized to ring-like structures. To investigate the membrane nature of these ring-like structures, GFPCARA7(Q69L) was transiently co-expressed with the mRFP-tagged MVB marker, VSR2, the TGN marker, SYP61, or the Golgi marker, ManI, in protoplasts. As shown in Fig. 1B, only mRFPCVSR2 co-localized with GFPCARA7(Q69L) on the membranes of enlarged spheres, which supports the MVB-derived nature of these ring-like structures. In contrast, there was no co-localization between GFP-labelled ring-like structures and either mRFPCSYP61 or mRFPCManl (Fig. 1C, ?,D),D), indicating that neither TGN nor Golgi membranes contribute to the enlarged spheres. Open in a separate window Fig. 1. GFPCARA7(Q69L)-induced ring-like structures co-localize with an MVB marker, but not with TGN or Golgi markers, in protoplasts. (A) The GFP fusion construct GFPCARA7 or the GTP-bound mutant GFPCARA7(Q69L) were transiently expressed in protoplasts followed by confocal imaging. (BCD) GFPCARA7(Q69L) was transiently co-expressed with the mRFP-tagged MVB marker, mRFPCVSR2, the TGN marker, mRFPCSYP61, or the Golgi marker, ManICmRFP, in protoplasts, followed by confocal imaging. Enlarged images of selected areas are also shown (ACD). Scale bar=50 m. Generation and characterization of transgenic PSBD cell lines expressing GFPCARA7(Q69L) under the control of a heat shock promoter To investigate the nature of these GFPCARA7(Q69L)-induced ring-like structures further, transgenic cell lines stably expressing GFPCARA7(Q69L) under the control of a HSP RGX-104 free Acid were generated via cells. (A, B) Transgenic GFPCARA7(Q69L) cells were subjected to heat shock treatment for different durations as indicated (+), followed by either confocal microscopy or western blot analysis using anti-GFP or anti-tubulin antibodies. Transgenic cells without heat shock treatment (C) were used as the corresponding control. Scale bar=25 m. (This figure is available in colour at online.) Taken together, these results demonstrate that expression of GFPCARA7(Q69L) in transgenic cells is controlled by heat shock treatment, which induces the formation of GFP-labelled ring-like structures. It should be noted that a 4 h incubation was used for all.