Journal: Scientific Reports

Article Title: Concentration Dependent Ion-Protein Interaction Patterns Underlying Protein Oligomerization Behaviours

doi: 10.1038/srep24131

Figure Lengend Snippet: PC12 plasma membrane sheets incubated at 37 °C for 10 min with the indicated concentrations of calcium, strontium, barium or magnesium chloride, fixed, immunostained for SNAP25 and analysed with epifluorescence or superresolution STED microscopy. ( a ) Membrane sheets incubated with 0, 10, or 1000 mM CaCl 2 . Epifluorescence recordings from the dye TMA-DPH (left; documenting the integrity of the membranes) and the immunostaining (middle; overviews are shown at the same, magnified insets at arbitrary scaling). Right, STED micrographs of SNAP25 immunofluorescence to which the “red hot” look up table was applied which displays increasingly brighter pixel intensities applying a colour code from black to red to yellow to white. ( b ) SNAP25 clustering was quantified by calculating the relative standard deviation (rel. SD) of the immunostaining pattern, normalized to the baseline condition which contained no divalent cations. Values are means ± s.e.m. ( c ) SNAP25 cluster density (means ± s.e.m.) resolved by superresolution STED microscopy. Cluster size was similar under all conditions .

Article Snippet: For STED microscopy a 4-channel easy3D superresolution STED microscope (Abberior Instruments) equipped with an Olympus UPlanSApo 100x/NA 1.4 objective was used.

Techniques: Clinical Proteomics, Membrane, Incubation, Microscopy, Immunostaining, Immunofluorescence, Standard Deviation

Journal: PLoS Biology

Article Title: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia

doi: 10.1371/journal.pbio.3001480

Figure Lengend Snippet: (A) Representative confocal images of HEK293T cells transiently expressing FLAG-tagged WT, R1441C, or G2019S LRRK2 constructs, stained for FLAG LRRK2, endogenous Rab8a, and endogenous Lamp2. Cells were treated with 1 μM MLi-2 for 1 hour or DMSO prior to staining, 24 hours posttransfection. (B, C) Quantification of Manders colocalization coefficient between Rab8a, LRRK2, and Lamp2 (B, C: N > 20 cells for each group across 2 independent experiments, **** P < 0.0001, one-way ANOVA with Tukey post hoc test; B: F (5, 120) = 108.9; C: F (5, 120) = 35.85). (D) Superresolution confocal image of endogenous Rab8a and overexpressed G2019S LRRK2 localizing at the lysosomal membrane. (E) Mouse primary astrocytes were transfected with HaloTag-LRRK2(G2019S), GFP-Rab8a, and LAMP1-RFP (top panels). Cells were incubated with JFX650 (100 nM) for 1 hour, washed, and imaged using a Nikon SoRa spinning disk microscope utilizing 3D Landweber deconvolution, 48 hours later. To analyze Rab8a localization to centrosomes, fixed cells were stained for pericentrin, following transient expression of G2019S LRRK2 and GFP-Rab8a. Images were taken with Airyscan (bottom panels). (F) Quantitation of the percentage of LRRK2/Rab8a puncta per cell that colocalizes with either LAMP1 or Pericentrin ( N > 19 cells for each condition from 2 independent experiments; unpaired t test; P < 0.0001). The underlying data can be found in . LRRK2, leucine-rich repeat kinase 2; WT, wild-type.

Article Snippet: Following superresolution microscopy, the Imaris platform (Bitplane, Zürich, Switzerland) was used to analyze the localization of TfR and Tf in different experiments.

Techniques: Expressing, Construct, Staining, Membrane, Transfection, Incubation, Microscopy, Quantitation Assay

Journal: PLoS Biology

Article Title: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia

doi: 10.1371/journal.pbio.3001480

Figure Lengend Snippet: (A) HEK293T cells transiently expressing FLAG-tagged WT, R1441C, or G2019S LRRK2 for 24 hours were stained for FLAG LRRK2, Lamp2, and Cathepsin D and analyzed by confocal microscopy. Staining intensity profiles were generated on sections indicated by the dotted lines. (B, C) Manders colocalization coefficient of LRRK2 versus Lamp2 or Cathepsin D staining ( N > 20 cells per group across 2 independent experiments, **** P < 0.0001, one-way ANOVA with Tukey post hoc; Lamp2: F (5, 57) = 41.73; Cathepsin D: NS ). (C) Superresolution image of HEK293T cells stably expressing GFP G2019S LRRK2 costained for Cathepsin D. HEK293T cells stably expressing GFP WT LRRK2 were treated with 1 mM LLOMe for 4 hours and stained for endogenous Lamp1 (D) and Rab8a (E). (F) GFP WT and G2019S expressing cells were treated with LLOMe as in (E) and stained for Jip4 and Lamp1 prior to imaging by superresolution microscopy. The underlying data can be found in . LRRK2, leucine-rich repeat kinase 2; WT, wild-type.

Article Snippet: Following superresolution microscopy, the Imaris platform (Bitplane, Zürich, Switzerland) was used to analyze the localization of TfR and Tf in different experiments.

Techniques: Expressing, Staining, Confocal Microscopy, Generated, Stable Transfection, Imaging, Microscopy

Journal: PLoS Biology

Article Title: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia

doi: 10.1371/journal.pbio.3001480

Figure Lengend Snippet: (A) HEK293T cells exogenously expressing WT and mutant LRRK2 constructs were stained for TfR and visualized by superresolution microscopy. TfR vesicles were analyzed using the Imaris Spot Detection module, and the mean and minimum distances between spots were plotted (B, C). ( N > 2,000 vesicles were counted in at least 20 cells per construct from 2 independent experiments, * P < 0.05, **** P < 0.0001, one-way ANOVA with Tukey post hoc, B: F(4, 13334) = 46.14, C: F (4, 13336) = 194.1). (D) HEK293FT cells expressing LRRK2 genetic variants were incubated with Alexa Fluor 568–conjugated transferrin, fixed at 20 minutes of incubation and stained for endogenous Rab8a and FLAG LRRK2 (D). (E) Uptake of Alexa Fluor 568–conjugated transferrin was monitored by high-content imaging, and transferrin levels per cell were plotted at different time points (E: T = 20 minutes, N = 3 technical replicates per construct (>800 cells/well) one-way ANOVA, Tukey post hoc, * P < 0.05, *** P = 0.0005, **** P = < 0.0001). (F) HEK293T cells exogenously expressing FLAG WT or G2019S LRRK2 constructs were incubated in DMEM supplemented with 1 μM MLi-2 or DMSO for 45 minutes prior to addition of Alexa Fluor 568–conjugated transferrin in the same media, and high-content imaging was used to monitor Tf uptake at different time points (F: N = 3 technical replicates per construct (>800 cells/well), two-way ANOVA, genotype: P < 0.05, F (2,30) = 3.575; Time: P < 0.0001, F (4, 30) = 44.96). (G) Cells were treated with MLi-2 as in (F), and incubated with Alexa Fluor–conjugated transferrin for 30 minutes, prior to changing to fresh media containing MLi-2 and monitoring transferrin release by high-content imaging (G; at T10: N = 3 technical replicates per construct (>800 cells/well), one-way ANOVA, Tukey post hoc, P < 0.05, F (2, 30) = 3.575). (H) HEK293T cells transiently expressing WT or I2020T LRRK2 were stained for Tf, Lamp2, and LRRK2 (FLAG) and analyzed by confocal microscopy. (I, J) Cells were transfected with Rab8a siRNA or scrambled sequence constructs (control) and 24 hours later were transfected with I2020T LRRK2 that was expressed overnight, before fixation and staining for Tf, Rab8a, and LRRK2 (FLAG). ( N = 27 cells for NTC siRNA, N = 41 cells for Rab8a siRNA imaged across 2 independent experiments, two-tailed Mann–Whitney U test, **** P < 0.0001). Intracellular iron levels were analyzed by ICP-MS in cells stably expressing GFP WT or mutant LRRK2 constructs (K) ( N = 4 confluent plates of cells per construct, two-way ANOVA with Tukey post hoc, * P = 0.018, F (3, 11) = 6.225). [SD bars are shown]. The underlying data can be found in . ICP-MS, inductively coupled plasma mass spectrometry; LRRK2, leucine-rich repeat kinase 2; siRNA, small interfering RNA; TfR, transferrin receptor; WT, wild-type.

Article Snippet: Following superresolution microscopy, the Imaris platform (Bitplane, Zürich, Switzerland) was used to analyze the localization of TfR and Tf in different experiments.

Techniques: Expressing, Mutagenesis, Construct, Staining, Microscopy, Incubation, Imaging, Confocal Microscopy, Transfection, Sequencing, Control, Two Tailed Test, MANN-WHITNEY, Stable Transfection, Mass Spectrometry, Small Interfering RNA

Journal: PLoS Biology

Article Title: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia

doi: 10.1371/journal.pbio.3001480

Figure Lengend Snippet: (A) iPSC-derived human microglia from WT or G2019S LRRK2 carriers were treated with LPS, and the localization of endogenous Tf and Lamp2 was analyzed by superresolution microscopy and the Imaris Surface render module (A). Partial colocalization between Tf and Lamp2 was observed in control that was significantly decreased with LPS treatment in WT cells but not in G2019S LRRK2 cells that retained lysosomal association of Tf (B) ( N > 12 cells per group from 2 differentiations, one-way ANOVA Tukey post hoc, **** P < 0.0001, F(3,53) = 16.16). G2019S LRRK2 iPSC microglia exhibited larger Tf vesicles compared to WT while LPS treatment induced a decrease in average vesicle size in both cohorts (C) (minimum 3,000 vesicles were counted from 16 cells per group from 2 differentiations, two-way ANOVA, genotype: P < 0.0001, F(1,17753) = 16.38, treatment: P < 0.0001, F(1,17753) = 20.57). LPS treatment induced an increase in the average distance of Tf vesicles from the nucleus in WT cells but that was not significant in G2019S LRRK2 cells (D) (minimum 3,000 vesicles were counted from 16 cells per group from 2 differentiations, two-way ANOVA, genotype: P < 0.0001, F(1,14735) = 104.9, treatment: P < 0.0001, F (1, 14735) = 105.0). The frequency distributions of Tf vesicle proximity to the nucleus were plotted in E and F. The percentage of Tf vesicles proximal to the nucleus was significantly decreased with LPS treatment in WT but not in G2019S LRRK2 cells (E, F) (E: bin at 0.5 μm, two-tailed Student t test; Mann–Whitney U post hoc; ** P = 0.0022; F: bin at 0.5 μm, two-tailed Student t test; Mann–Whitney U post hoc; NS). The underlying data can be found in . iPSC, induced pluripotent stem cell; LPS, lipopolysaccharide; LRRK2, leucine-rich repeat kinase 2; Tf, transferrin; WT, wild-type.

Article Snippet: Following superresolution microscopy, the Imaris platform (Bitplane, Zürich, Switzerland) was used to analyze the localization of TfR and Tf in different experiments.

Techniques: Derivative Assay, Microscopy, Control, Two Tailed Test, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Multimodal on-chip nanoscopy and quantitative phase imaging reveals the nanoscale morphology of liver sinusoidal endothelial cells

doi: 10.1073/pnas.2115323118

Figure Lengend Snippet: Workflow of the integrated QPM and on-chip nanoscopy system. ( A ) Si 3 N 4 strip waveguides are cleaned thoroughly to perform all imaging experiments. ( B ) Cells are isolated on top of the chip within a restricted rectangular area created with PDMS. C and D show the data acquisitions and registration between quantitative phase imaging (QPI) and superresolution imaging to calculate the size of the fenestrations and the average thickness of the group of fenestrated areas in LSEC.

Article Snippet: B.S.A. is a cofounder of the company Chip NanoImaging AS, which commercializes on-chip superresolution microscopy systems.

Techniques: Stripping Membranes, Imaging, Isolation