Journal: The Journal of Neuroscience

Article Title: Nanoscale Phosphoinositide Distribution on Cell Membranes of Mouse Cerebellar Neurons

doi: 10.1523/JNEUROSCI.1514-22.2023

Figure Lengend Snippet: PI(4,5)P 2 labeling using SDS-FRL. A , Example transmission electron microscopic (TEM) images of liposome replicas containing 5% PtdIns or different stereoisomers of PIs labeled with GST-PH, anti-GST antibody, and 5-nm gold particle-conjugated secondary antibody. Scale bar = 200 nm. B , Bar graph indicating the gold particle density on the liposome replicas. The density of gold particles was the highest in the liposome containing PI(4,5)P 2 . C , Example TEM images of liposome replicas containing different concentration of PI(4,5)P 2 . PI(4,5)P 2 was labeled with 5-nm gold particles. Scale bar = 200 nm. D , Concentration-dependent changes of the PI(4,5)P 2 labeling. Left, The gold particle densities in the liposome replica. Middle, The labeling efficiency for PI(4,5)P 2 . Right, The mean NNDs of the observed gold particles (open) and theoretical mean NNDs of randomly distributed PI(4,5)P 2 molecules (black). Dashed line indicates the diameter of PLCδ1-PH. Error bars indicate SEM. E , Schema of chemically-inducible PI(4,5)P 2 depletion with FRB-FKBR system. Rapamycin induces heterodimerization of FRB and FKBR, causing translocation of 5-ptase on the cell membrane. F , Example image of HEK293 cells transfected with PM-FRB-mRFP-TA-FKBR-5-ptase (left, bright field; middle, mRFP fluorescent; right, merged). Scale bar = 50 µm. G , Example TEM images of 5-nm gold particle labeling for PI(4,5)P 2 on the P-face of the somatic membranes of HEK293 cells treated with rapamycin (5 µM) or vehicle (0.1% DMSO). The somatic membranes of the transfected cells were identified by immunogold labeling for RFP (15 nm). Scale bar = 200 nm. H , Statistical comparison of the PI(4,5)P 2 particle density on the somatic membranes of HEK293 cells. Closed and transparent circles indicate the means of PI(4,5)P 2 particle density in each cell and each image, respectively, with colors indicating different cells. Horizontal bars and error bars indicate estimated marginal means (emmeans) and 95% confidence intervals (CIs) of the density estimated by GLMM (see Materials and Methods). The density on the rapamycin-treated transfected cells was significantly lower than others. I , Comparison of nearest neighbor distances (NND) between real (Data NND, x -axis) and simulated (Sim NND, y -axis) PI(4,5)P 2 particles on the somatic membranes of the untransfected HEK293 cells treated with DMSO. Data-NNDs are significantly smaller than Sim NNDs [Data: 27.8 ± 0.6 nm, Sim: 33.2 ± 0.7 nm, n = 49 images/4 cells, p < 0.001, likelihood ratio χ 2 test (Chi-LRT)]. n.s., not significant.

Article Snippet: Gold particle-conjugated secondary antibodies were purchased from British Biocell International (BBI, goat anti-rabbit IgG, 5 nm; goat anti-guinea pig IgG, 10 nm; goat anti-mouse IgG, 15 nm) and Jackson ImmunoResearch (donkey anti-chicken IgY, 6 nm; donkey anti-guinea pig IgG, 12 nm).

Techniques: Labeling, Transmission Assay, Concentration Assay, Translocation Assay, Transfection

Journal: The Journal of Neuroscience

Article Title: Nanoscale Phosphoinositide Distribution on Cell Membranes of Mouse Cerebellar Neurons

doi: 10.1523/JNEUROSCI.1514-22.2023

Figure Lengend Snippet: SDS-FRL of PI(4,5)P2 on somatic membranes of Purkinje cells in mouse cerebellum. A , Acute cerebellar slice preparation for high-pressure freezing (HPF) and replica preparation. Left-top, An acute sagittal slice of the mouse cerebellum. The dashed line indicates the trimmed region for HPF. Right-top, A trimmed cerebellar slice on a copper carrier with double-sided tape for HPF. Left-bottom, Low-magnification TEM image of the mouse cerebellar replica containing granule cell layer (GCL), Purkinje cell layer (PCL), and molecular layer (ML). Scale bar = 20 µm. Right-bottom, Example TEM image of the somatic membrane of a PC. Scale bar = 5 µm. B , Example TEM image of 5-nm gold particle labeling for PI(4,5)P 2 with (left) or without (right) GST-PH on P- (top) and E-face (bottom) of the PC somatic membranes of cerebellar PC. Ca V 2.1 was co-labeled with 12-nm gold particles. Scale bar = 200 nm. C , Statistical comparison of the PI(4,5)P 2 particle density on the E-face and P-face of the PC somatic membranes. Closed and transparent circles indicate the means of the PI(4,5)P 2 particle density in each cell and each image, respectively, with colors indicating different cells. Black horizontal bars and error bars indicate the emmeans and 95% CIs of the density, respectively. The PI(4,5)P 2 density was significantly higher on the P-face than on the E-face of the PC somatic membranes (P-face: 51.2 ± 8.5 particles/µm 2 , E-face: 8.0 ± 1.2 particles/µm 2 , n = 213 images/12 cells/4 mice, p < 0.001, Chi-LRT). D , Comparison of NND between real (Data NND, x -axis) and simulated (Sim NND, y -axis) PI(4,5)P 2 particles on PC somatic membranes. Data-NNDs are significantly smaller than Sim NNDs (Data: 35.1 ± 3.6 nm, Sim: 73.3 ± 7.0 nm, n = 206 images/11 cells/4 mice, p < 0.001, Chi-LRT). E , Distribution of NNDs of the PI(4,5)P 2 particles obtained from a single PC somatic membrane ( n = 2,929 particles). Red and blue lines indicate the distinct components of the NND distribution estimated from the Gaussian mixture modeling. µ, means; σ, standard deviation. F , Statistical comparison of the number of 5-nm gold particles per cluster on the PC somatic membranes. The particle clusters were detected using Ward Linkage hierarchical clustering method. The linkage distance threshold was set as 50 nm, which is nearby the maximum distance for cluster detection on the PC somatic membrane using DBSCAN ( ; Materials and Methods). The particle number per cluster without GST-PH was significantly lower than that with GST-PH in both faces (multiple comparisons with BH method), suggesting that the particle clusters observed on the somatic membranes are mainly due to clustering of PI(4,5)P 2 but not to nonspecific aggregation of the primary and secondary antibodies. n.s., not significant.

Article Snippet: Gold particle-conjugated secondary antibodies were purchased from British Biocell International (BBI, goat anti-rabbit IgG, 5 nm; goat anti-guinea pig IgG, 10 nm; goat anti-mouse IgG, 15 nm) and Jackson ImmunoResearch (donkey anti-chicken IgY, 6 nm; donkey anti-guinea pig IgG, 12 nm).

Techniques: Slice Preparation, Labeling, Standard Deviation