Journal:

Article Title: Convergence of Multiple Autophagy and Cytoplasm to Vacuole Targeting Components to a Perivacuolar Membrane Compartment Prior to de Novo Vesicle Formation *

doi: 10.1074/jbc.M109134200

Figure Lengend Snippet: Wild type (SEY6210) (A and B) or aut7Δ (WPHYD7) (C) cells were co-transformed with the following pairs of copper-inducible plasmids. A, YFP-Aut7 (pCuYFPAUT7(426)) and CFP-Cvt9 (pCuCFPCVT9(424)); B, YFP-Apg12 (pCuYFPAPG12(426)) and CFP-Cvt9; C, Apg5-YFP (pAPG5YFP(426)) and CFP-Cvt9. The transformed cells were grown to midlog stage in SMD, induced with 30 μm CuSO4 for 2 h (A and B) or with 5 μm CuSO4 for 30 min (C), and examined with a Nikon E-800 fluorescence microscope equipped with a Hamamatsu Orca 2 digital camera and processed with Openlab software. The YFP fusions to the vesicle component Aut7 and the conjugation components Apg12 and Apg5 co-localize with CFP-Cvt9. The DIC panels are images obtained with differential interference contrast optics.

Article Snippet: The transformed cells were grown to midlog stage in SMD, induced with 10 μ m CuSO 4 for 2 h, and examined with a Nikon E-800 fluorescence microscope as described in and under “Experimental Procedures.” A fluorescent protein fusion of Cvt19 co-localizes with both Cvt9 and Aut7 at the perivacuolar compartment.

Techniques: Transformation Assay, Fluorescence, Microscopy, Software, Conjugation Assay

Journal:

Article Title: Convergence of Multiple Autophagy and Cytoplasm to Vacuole Targeting Components to a Perivacuolar Membrane Compartment Prior to de Novo Vesicle Formation *

doi: 10.1074/jbc.M109134200

Figure Lengend Snippet: Wild type cells (SEY6210) were co-transformed with the following pairs of plasmids. A, copper-inducible YFP-Cvt9 (pCuYFP-CVT9(426)) and Cvt19-CFP under endogenous promoter control (pCVT19CFP(414)); B, copper-inducible YFP-Aut7 (pCuYFPAUT7(426)) and Cvt19-CFP under endogenous promoter control (pCVT19CFP(414)). The transformed cells were grown to midlog stage in SMD, induced with 10 μm CuSO4 for 2 h, and examined with a Nikon E-800 fluorescence microscope as described in Fig. 1 and under “Experimental Procedures.” A fluorescent protein fusion of Cvt19 co-localizes with both Cvt9 and Aut7 at the perivacuolar compartment. DIC, differential interference contrast.

Article Snippet: The transformed cells were grown to midlog stage in SMD, induced with 10 μ m CuSO 4 for 2 h, and examined with a Nikon E-800 fluorescence microscope as described in and under “Experimental Procedures.” A fluorescent protein fusion of Cvt19 co-localizes with both Cvt9 and Aut7 at the perivacuolar compartment.

Techniques: Transformation Assay, Fluorescence, Microscopy

Journal:

Article Title: Convergence of Multiple Autophagy and Cytoplasm to Vacuole Targeting Components to a Perivacuolar Membrane Compartment Prior to de Novo Vesicle Formation *

doi: 10.1074/jbc.M109134200

Figure Lengend Snippet: Wild type cells (SEY6210) were co-transformed with plasmids, under CUP1 copper-inducible control, expressing either YFP-Apg9 (pCuYFPAPG9(426)) and CFP-Cvt9 (pCuCFPCVT9(424)) (A), YFP-Apg9 and Cvt19-CFP (pCVT19CFP(414)) (B), or YFP-Aut7 (pCuYFPAUT7(426)) and CFP-Apg9 (pCuCFPAPG9(424)) (C). Transformed cells were grown to midlog stage in SMD and induced for 1–2 h with 30 μm CuSO4. Images were taken and examined with a Nikon E-800 fluorescence microscope as described in Fig. 1 and under “Experimental Procedures.” Apg9 displays multiple punctate dots when co-expressed with Cvt19 and Aut7 but only a single dot when co-expressed with Cvt9. The YFP-Apg9 and CFP-Cvt9 dots co-localize. DIC, differential interference contrast.

Article Snippet: The transformed cells were grown to midlog stage in SMD, induced with 10 μ m CuSO 4 for 2 h, and examined with a Nikon E-800 fluorescence microscope as described in and under “Experimental Procedures.” A fluorescent protein fusion of Cvt19 co-localizes with both Cvt9 and Aut7 at the perivacuolar compartment.

Techniques: Transformation Assay, Expressing, Fluorescence, Microscopy

Journal: Nature Communications

Article Title: Reversible photoregulation of cell-cell adhesions with opto-E-cadherin

doi: 10.1038/s41467-023-41932-0

Figure Lengend Snippet: a Schematic representation of opto-E-cad-dependent cell signalling. In the dark, the opto-E-cads on adjacent cells homophilically bind to each other, and the catenins (p120, α- and β) are recruited to the intracellular tail and link it to F-actin. Under blue light, opto-E-cads do not form cell–cell adhesions, no catenins are recruited and there is no link to the actin cytoskeleton. b Confocal fluorescence microscopy images of opto-E-cad-MDA cells in the dark or under blue light (MDA-MB-231 and MCF-7 cells in 2D cultures). F-actin is shown in red, nuclei are shown in blue and p120 is shown in yellow. Scale bars are 50 µm and 15 µm for the inset. c Cell spreading area of opto-E-cad cells forming cell–cell adhesions in cluster compared to single cells. n = 35, 46, 14, 46, 46. Boxplots show median, 25th, and 75th percentile. The lower and upper boundaries of whiskers indicate the minima and maxima, respectively. All comparisons are performed using Fisher’s One Way ANOVA test and p < 0.05 was treated as the significance threshold. d 3D reconstruction and maximal intensity projection for p120 (yellow) and nuclei (blue) of opto-E-cad-MDA cell clusters after 120 min in the dark. Scale bar is 50 µm ( n = 3). e Western blot of E-cadherin, p120 and β-actin (loading control) for opto-E-cad-MDA in the dark or under blue light and E-cad-MDA and MDA-MB-231 cells as positive and negative control, respectively ( n = 4). f RT-PCR results for opto-E-cad-MDA in the dark and under blue light for EMT markers. (two-tailed t-test, n = 12 and 10). Data are represented as individual values ± SD. Source data are provided as a Source Data file.

Article Snippet: Subsequently, the cells were washed three times with PBS, blocked with 1% BSA (Sigma-Aldrich # A7030-100G) in PBS for 1 h, and incubated with the primary rabbit-anti-delta-catenin (p120 catenin, cell signalling #59854, diluted 1:800) or the primary mouse-anti-E-cadherin antibody (cell signalling #14472, diluted 1:200) in 1% BSA in PBS overnight at 4 °C.

Techniques: Fluorescence, Microscopy, Western Blot, Control, Negative Control, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test