Journal: eLife

Article Title: Pathway-specific dysregulation of striatal excitatory synapses by LRRK2 mutations

doi: 10.7554/eLife.58997

Figure Lengend Snippet: ( A ) Schematic depicting experimental design. ( B ) Structured Illumination super-resolution microscopy (SIM) image of dendritic spines on + /+, +/RC , and +/GS Drd1-Tomato expressing SPNs, labeled with antibodies to GluA1 (purple), and PSD95 (green). Open arrowheads, GluA1 nanodomains; arrowheads, PSD95 nanodomains. ( C ) Schematic diagram and object masks depicting GluA1, PSD95, and overlap nanodomains within a dendritic spine. Minimum distance between GluA1-PSD95 is measured from the closest edge of the two nanodomains, as shown. ( D, E ) Summary graphs and cumulative distribution of the minimum distance between GluA1 and PSD95 nanodomains. ( F, G ) Summary data and cumulative frequency for the overlap area of GluA1 and PSD95 nanodomains within dendritic spines. Asterisk in D and F reflect statistical significance for Tukey’s multiple comparison tests after one-way ANOVA, whereas asterisks in E and G show statistical significance for Bonferroni post-hoc comparisons after Kolmogorov-Smirnov tests. ( H ) Bar graphs showing the ratio of GluA1-PSD95 overlap area in E relative to PSD95 area, across genotypes. Data are represented as mean ± SEM. ( I-K ) Correlation plots of overlap in GluA1-PSD95 area versus PSD95 area for + /+, +/RC , and +/GS Drd1-Tomato expressing SPNs. *p<0.05, **p<0.01, ***p<0.001. Figure 2—source data 1. Raw data plotted in graphs in .

Article Snippet: Multichannel SIM images were obtained with a Nikon Structured Illumination super-resolution microscope using a 100x, 1.4 NA objective as previously described ( ).

Techniques: Super-Resolution Microscopy, Expressing, Labeling, Comparison

Journal: Autophagy

Article Title: Binding of Avibirnavirus VP3 to the PIK3C3-PDPK1 complex inhibits autophagy by activating the AKT-MTOR pathway

doi: 10.1080/15548627.2019.1704118

Figure Lengend Snippet: VP3 enhances the interaction between AKT and PDPK1 and the phosphorylation of AKT and MTOR. (A) Three-dimensional image analysis of the colocalization between PDPK1, AKT, the ER, and VP3 using a super resolution microscope. HEK293T cells were transiently co-transfected with four vectors DsRed-ER, GFP-PDPK1, FLAG-AKT, and pCI-neo-VP3 or empty vector pCI-neo for 24 h. The cells were then immunostained with anti-FLAG and anti-VP3 mAbs and observed under a structured illumination microscope. Scale bar: 1 μm. (B) VP3 expression promoted interaction between PDPK1 and AKT. HEK293T cells were transiently co-transfected with vectors expressing FLAG-AKT, MYC-PDPK1, and pCI-neo-VP3 or empty vector pCI-neo for 48 h. The cell lysates were subjected to anti-FLAG IP and immunoblotting analysis of AKT, PDPK1, and VP3 using the indicated antibodies. (C) PIK3C3 binding to PDPK1 was decreased at 3 h post-infection. HEK293T cells were co-transfected with FLAG-PIK3C3 and MYC-PDPK1 for 24 h. The resultant cells were then infected with Avibirnavirus at MOI = 10 for 0, 1, 3, and 6 h and subjected to IP and immunoblotting assays with the indicated antibodies. (D) PDPK1 binding to AKT was enhanced at 3 h post-infection. The HEK293T cells were co-transfected with FLAG-AKT and MYC-PDPK1 for 24 h. The resultant cells were then infected with Avibirnavirus at MOI = 10 for 0, 1, 3, and 6 h and subjected to IP and immunoblotting assays using the indicated antibodies. (E and F) HEK293T cells were transiently transfected with VP3 and vector. After 20 h, the cells were separately treated with DMSO, LY294002 (10 μg/ml), or rapamycin (5 μM) for 4 h. Immunoassays of extracts of HEK293T cells were then performed using the indicated antibodies. (G) CC3 domain is required for VP3 mediating activation of AKT-MTOR pathway. HEK293T cells were transfected with VP3, VP3∆CC1, VP3∆CC2, VP3∆CC3, and vector, respectively. At 20 h post-transfection, HEK293T cells were starved for 4 h. The cellular extracts were then subjected to immunoblotting assay using the indicated antibodies. Error bars: Mean ± SD of 3 independent tests. One-way ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001 compared to control.

Article Snippet: The cells were washed with PBS and then incubated with FITC, Dylight 405, Alexa Fluor 488, Alexa Fluor 546, or Alexa Fluor 647- labeled IgG antibodies for 1 h. After washing three times with PBST, the cells were observed under a Nikon A1R/A1 laser scanning confocal microscope (Nikon, Tokyo, JPN) or under an N-structured illumination microscopy Super-Resolution microscope (Nikon, Tokyo, JPN).

Techniques: Microscopy, Transfection, Plasmid Preparation, Expressing, Western Blot, Binding Assay, Infection, Activation Assay