Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet: ( A ) Schematic representation of clathrin-coated pit induction. The system, triggered by rapamycin, consists of a membrane anchor (mCherry-FRB fused to a membrane targeting domain) and a clathrin hook (clathrin-binding protein fused to FKBP and GFP). Targeting the anchor to mitochondria using Tom70p in MitoTrap. ( B ) Stills from live cell imaging of a HeLa cell expressing FKBP-β2-GFP (green), and the anchor, MitoTrap (red), treated with 200 nM rapamycin as indicated (orange bar). Scale bar, 2 µm. See . ( C ) Representative confocal micrographs of HeLa cells before (light orange bar) and 2 min after 200 nM rapamycin treatment (dark orange bar). Cells expressing MitoTrap (red) with either our standard clathrin hook (FKBP-β2-GFP), clathrin-binding deficient mutant (FKBP-β2-Y815A/∆CBM-GFP), or GFP-FKBP. In B and C, mitochondria were also labeled with MitoTracker Deep Red (blue) and orange arrowheads indicate MitoPits, where present. Inset, ×5 zoom. Scale bar, 10 µm. ( D ) Typical confocal micrograph of cells expressing FKBP-β2-GFP (green) and MitoTrap (red), treated with rapamycin (200 nM), fixed and stained with anti-PDHE2/E3 (blue). Inset, ×3 zoom. Scale bar, 5 µm. ( E ) Analysis of the spatial organization of MitoPits. ( i ) Line profile through the MitoPit shown in D, aligned to the FKBP-β2-GFP peak at 0 µm. Each of three channels is shown. (ii) Spatially averaged line profiles, aligned to the FKBP-β2-GFP peak at 0 µm, mean ± standard deviation (SD) is shown. (iii) Width of profiles for each channel in the dataset. (iv) Relative distance from the peak of FKBP-β2-GFP to the peak of MitoTrap (red) or PDHE2/3 (blue) for each profile in the dataset. Box plots indicate median, IQR, 9th and 91st percentiles. Each dot represents a profile.

Article Snippet: Recombinant DNA reagent , Dyn1 K44A-mCherry , This paper , - , See Materials and methods - Molecular Biology section. Modification of Addgene plasmid #34681. GFP replaced by mCherry. Available from the corresponding author..

Techniques: Membrane, Binding Assay, Live Cell Imaging, Expressing, Mutagenesis, Labeling, Staining, Standard Deviation

Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet: Representative confocal micrographs of HeLa cells coexpressing the indicated anchor protein together with FKBP-β2-GFP, FKBP-β2(Y815A/∆CBM)-GFP, or GFP-FKBP and treated with 200 nM rapamycin. The following anchors were used: ( A ) FRB-mCherry-Sec61β (ER), ( B ) FRB-mCherry-Giantin(3131–3259) (Golgi), and ( C ) Lamp1-mCherry-FRB (lysosomes), Insets, ×5 zoom. Scale bar, 10 µm.

Article Snippet: Recombinant DNA reagent , Dyn1 K44A-mCherry , This paper , - , See Materials and methods - Molecular Biology section. Modification of Addgene plasmid #34681. GFP replaced by mCherry. Available from the corresponding author..

Techniques:

Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet: ( A–D ) Representative confocal micrographs of MitoPit-derived vesicle formation under different approaches to inhibit dynamin or Drp1 function. ( A ) Dominant-negative dynamin-1: HeLa cells expressing dark MitoTrap and FKBP-β2-GFP (green) alone (–) or in combination with either Dyn1 WT-mCherry WT or Dyn1 K44A-mCherry (red), treated with rapamycin (200 nM, 30 min), stained with anti-PDHE2/E3 (blue). ( B ) Chemical inhibition: HeLa cells expressing dark MitoTrap and FKBP-β2-GFP (green), treated with control compound (Dynole 31–2, 30 µM) or Dynole 34–2 (30 µM) for 25 min and rapamycin (200 nM) for the final 10 min. Fluorescent human transferrin (Tfn 647, blue) indicates endocytic activity, mitochondrial matrix was stained with anti-PDHE2/E3 (red). ( C ) Dynamin triple knockout: inducible dynamin TKO mouse embryonic fibroblasts expressing dark MitoTrap and FKBP-β2-GFP (green), treated with vehicle (Control) or 3 µM Tamoxifen (TKO) for 2 days prior to transfection and with rapamycin (200 nM, 10 min) for clathrin-coated pit (CCP) induction. Fluorescent human transferrin (Tfn 647, blue) indicates endocytic activity, mitochondrial matrix was stained with anti-PDHE2/E3 (red). ( D ) Dominant-negative Drp1: HeLa cells expressing dark MitoTrap and FKBP-β2-GFP (green) alone (–) or in combination with either mCherry-Drp1 WT or mCherry-Drp1 K38A (red), treated with rapamycin (200 nM, 30 min), stained with anti-PDHE2/E3 (blue). Insets, ×5 zoom. Scale bars, 10 µm. ( E–H ) SuperPlots showing the percentage of free spots for each condition. Colors represent replicates, dots represent cells, solid dots represent the mean of each replicate. Indicated p values from Dunnett’s post hoc test ( E, H ) or Student’s t -test ( F, G ).

Article Snippet: Recombinant DNA reagent , Dyn1 K44A-mCherry , This paper , - , See Materials and methods - Molecular Biology section. Modification of Addgene plasmid #34681. GFP replaced by mCherry. Available from the corresponding author..

Techniques: Derivative Assay, Dominant Negative Mutation, Expressing, Staining, Inhibition, Control, Activity Assay, Triple Knockout, Transfection

Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet: ( A ) Representative confocal micrographs of MitoPit-derived vesicle formation in cells expressing dark MitoTrap and FKBP-β2-GFP (green) alone (–) or in combination with either mCherry-Vps4a WT or E228Q mutant (red), treated with rapamycin (200 nM, 30 min), stained with anti-PDHE2/E3 (blue). ( C ) Representative confocal micrographs of MitoPit-derived vesicle formation in cells expressing dark MitoTrap and FKBP-β2-GFP (green) pre-treated with Latrunculin B (1 µM) or control as indicated. Cells were treated with rapamycin (200 nM, 30 min), and stained with Acti-stain 555 (red) and anti-PDHE2/E3 (blue). Insets, ×5 zoom. Scale bars, 10 µm. ( B, D ) SuperPlots showing the percentage of free spots for each condition in A and C. Colors represent replicates, dots represent cells, solid dots represent the mean of each replicate. Indicated p values from Student’s t-test.

Article Snippet: Recombinant DNA reagent , Dyn1 K44A-mCherry , This paper , - , See Materials and methods - Molecular Biology section. Modification of Addgene plasmid #34681. GFP replaced by mCherry. Available from the corresponding author..

Techniques: Derivative Assay, Expressing, Mutagenesis, Staining, Control

Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet: Representative confocal micrographs of HeLa cells expressing dark MitoTrap and mCherry- Epsin-2 ( A ) or mCherry-Fcho2 ( E ) (red) with either clathrin hook (FKBP-β2-GFP) or GFP-FKBP (green). Cells were treated with 200 nM rapamycin before staining for PDHE2/E3 (blue). Inset, ×5 zoom. Scale bars, 10 μm. Box plots to compare colocalization ( B, F ) and spot density ( C, G ). Each dot represents a cell. Box plots indicate median, IQR, 9th and 91st percentiles. Spots of GFP and mCherry were detected and quantified. Colocalization is shown as the percentage of GFP spots that coincided with Epsin-2 or Fcho2 spots (left), or the percentage of Epsin-2 or Fcho2 spots that coincided with GFP spots (right). ( D, H ) Waffle plots to visualize the median number of spots per 100 μm 2 that were positive for GFP only (green), clathrin only (red), or both (yellow).

Article Snippet: Recombinant DNA reagent , Dyn1 K44A-mCherry , This paper , - , See Materials and methods - Molecular Biology section. Modification of Addgene plasmid #34681. GFP replaced by mCherry. Available from the corresponding author..

Techniques: Expressing, Staining

Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet: Representative confocal micrographs of HeLa cells coexpressing a red fluorescent protein-tagged accessory protein together with dark MitoTrap and either FKBP-β2-GFP or GFP-FKBP control. Cells were treated with 200 nM rapamycin and immunostained with anti-PDHE2/E3/A647. Accessory proteins were sigma subunits of the AP1, AP2, and AP3 complexes (σ1-mCherry, σ2-mCherry, or σ3-mCherry, left) or mCherry-amphiphysin 1, Endophilin-FL-RFP, Hip1R-tDimer-RFP, or mCherry-SNX9, as indicated. Insets, ×5 zoom. Scale bars, 10 µm.

Article Snippet: Recombinant DNA reagent , Dyn1 K44A-mCherry , This paper , - , See Materials and methods - Molecular Biology section. Modification of Addgene plasmid #34681. GFP replaced by mCherry. Available from the corresponding author..

Techniques: Control

Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , Dyn1 K44A-mCherry , This paper , - , See Materials and methods - Molecular Biology section. Modification of Addgene plasmid #34681. GFP replaced by mCherry. Available from the corresponding author..

Techniques: Triple Knockout, Recombinant, Modification, Plasmid Preparation, Mutagenesis

Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet: ( A–D ) Representative confocal micrographs of MitoPit-derived vesicle formation under different approaches to inhibit dynamin or Drp1 function. ( A ) Dominant-negative dynamin-1: HeLa cells expressing dark MitoTrap and FKBP-β2-GFP (green) alone (–) or in combination with either Dyn1 WT-mCherry WT or Dyn1 K44A-mCherry (red), treated with rapamycin (200 nM, 30 min), stained with anti-PDHE2/E3 (blue). ( B ) Chemical inhibition: HeLa cells expressing dark MitoTrap and FKBP-β2-GFP (green), treated with control compound (Dynole 31–2, 30 µM) or Dynole 34–2 (30 µM) for 25 min and rapamycin (200 nM) for the final 10 min. Fluorescent human transferrin (Tfn 647, blue) indicates endocytic activity, mitochondrial matrix was stained with anti-PDHE2/E3 (red). ( C ) Dynamin triple knockout: inducible dynamin TKO mouse embryonic fibroblasts expressing dark MitoTrap and FKBP-β2-GFP (green), treated with vehicle (Control) or 3 µM Tamoxifen (TKO) for 2 days prior to transfection and with rapamycin (200 nM, 10 min) for clathrin-coated pit (CCP) induction. Fluorescent human transferrin (Tfn 647, blue) indicates endocytic activity, mitochondrial matrix was stained with anti-PDHE2/E3 (red). ( D ) Dominant-negative Drp1: HeLa cells expressing dark MitoTrap and FKBP-β2-GFP (green) alone (–) or in combination with either mCherry-Drp1 WT or mCherry-Drp1 K38A (red), treated with rapamycin (200 nM, 30 min), stained with anti-PDHE2/E3 (blue). Insets, ×5 zoom. Scale bars, 10 µm. ( E–H ) SuperPlots showing the percentage of free spots for each condition. Colors represent replicates, dots represent cells, solid dots represent the mean of each replicate. Indicated p values from Dunnett’s post hoc test ( E, H ) or Student’s t -test ( F, G ).

Article Snippet: Dynamin-1-mCherry and dynamin-1(K44A)-mCherry were made by replacing the GFP from WT Dyn1 pEGFP and K44A Dyn1 pEGFP (Addgene #34680 and #34681), respectively, with mCherry from pmCherry-N1 using AgeI and NotI.

Techniques: Derivative Assay, Dominant Negative Mutation, Expressing, Staining, Inhibition, Control, Activity Assay, Triple Knockout, Transfection

Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet:

Article Snippet: Dynamin-1-mCherry and dynamin-1(K44A)-mCherry were made by replacing the GFP from WT Dyn1 pEGFP and K44A Dyn1 pEGFP (Addgene #34680 and #34681), respectively, with mCherry from pmCherry-N1 using AgeI and NotI.

Techniques: Triple Knockout, Recombinant, Modification, Plasmid Preparation, Mutagenesis