Journal: Cell chemical biology

Article Title: Live-cell imaging to analyze intracellular aggregation of recombinant IgG in CHO cells.

doi: 10.1016/j.chembiol.2021.08.010

Figure Lengend Snippet: Figure 3. Fluorescence live imaging of intracellular IgG aggregates in CHO-IgG1 cells (A and B) CHO-IgG1 cells were transfected with 50 ng anti-human IgG antibody DL650 and 2 mg of AF.2A1-F520 (A) or 2 mg of fluorescein (B). After 24 h, nuclear staining was performed, and preparations were visualized under a confocal laser-scanning microscope. Scale bars represent 5 mm.

Article Snippet: Confocal images were acquired on an inverted confocal microscope (Nikon A1, Nikon Co., Tokyo, Japan) with a 603 objective lens, and were analyzed using the NIS-Elements C/NIS-Elements C-ER software.

Techniques: Fluorescence, Imaging, Transfection, Staining, Laser-Scanning Microscopy

Journal: Frontiers in Molecular Neuroscience

Article Title: PLC-γ-Ca 2+ pathway regulates axonal TrkB endocytosis and is required for long-distance propagation of BDNF signaling

doi: 10.3389/fnmol.2024.1009404

Figure Lengend Snippet: Axonal BDNF promotes an increase in intracellular Ca 2+ in a PLC-γ-dependent manner in rat cortical neurons. (A) Evaluation of Ca 2+ signaling induced by BDNF. The change in fluorescence intensity associated with Fluo4-AM (2 μM) was used to measure the concentration change in cytosolic Ca 2+ . Representative images of compartmentalized cultures loaded with Fluo4-AM in the AC treated with vehicle or BDNF (50 ng/ml) in the AC with or without U73122 (5 μM) pretreatment. Live-cell imaging of each axonal field in the AC recorded before BDNF treatment (0 s) and during 60 s of BDNF treatment. Scale bar, 10 μm. (B) Mean Fluo4-AM fluorescence intensity (±SEM) for each treatment at different snapshot times. Fab represents the average fluorescence of the baseline. We calculate the average of the calcium recordings for the vehicle or U17322 treatments, and subsequently standardize the data of the baseline, BDNF and BDNF + U17622 calcium recordings by these respective values. n = 8–12 axons from three independent compartmentalized cultures. Statistical analysis was performed by two-way ANOVA followed by the Bonferroni correction for multiple comparisons. ** p < 0.01. (C) Quantification of the velocity of Ca 2+ back-propagation of the fluorescence signal associated with Fluo4-AM under BDNF conditions.

Article Snippet: Ca 2+ imaging was performed by confocal microscopy using a Nikon Eclipse C2 confocal microscope equipped with a digital camera connected to a computer with NIS-Elements C software.

Techniques: Fluorescence, Concentration Assay, Live Cell Imaging

Journal: Frontiers in Molecular Neuroscience

Article Title: PLC-γ-Ca 2+ pathway regulates axonal TrkB endocytosis and is required for long-distance propagation of BDNF signaling

doi: 10.3389/fnmol.2024.1009404

Figure Lengend Snippet: Axonal PLC-γ activity and intracellular Ca 2+ are required for axonal BDNF-containing signaling endosome generation and retrograde transport to cell bodies in mouse cortical neurons. (A) DIV 7 compartmentalized cortical neurons were retrograde labeled with Ctb555 (red) overnight. At DIV 8, the cell body compartment was treated with TrkB-Fc, and the AC was treated with DyLight 488-labeled streptavidin (f-streptavidin, green) alone or with biotinylated BDNF conjugated to DyLight 488-labelled streptavidin (f-BDNF, green), as indicated in the methodology section, for 6 h in the absence or presence of vehicle or 5 μM U73122 (U73122) or 20 μM BAPTA-AM (BAPTA). Then, the cells were fixed, mounted in Mowiol containing Hoechst (blue) and prepared for confocal microscopy. Scale bar, 5 μm (B) Quantification of green-labelled vesicles in cell bodies of neurons containing Ctb555. Only vesicles larger than 200 nm 2 were considered for the analysis. Forty-five neurons from three independent compartmentalized cultures were considered. Statistical analysis was performed by one-way ANOVA followed by the Bonferroni correction for multiple comparisons. **** p < 0.0001. (C) Left panels, representative images of f-streptavidin or f-BDNF (green) associated fluorescence. White lines are selecting the region of the microgroove label with Ctb555 (red) shown in the right panels. White arrows indicate green-labelled vesicles. Right panels, Representative images of axons (labeled with Ctb555, red) in microgrooves of neurons treated as described in (A) . Scale bar, 5 μm. (D) Quantification of green-labelled vesicles in microgrooves of axons containing Ctb555. Forty-five microgrooves from three independent compartmentalized cultures were considered for the analysis. Statistical analysis was performed by one-way ANOVA followed by the Bonferroni correction for multiple comparisons. **** p < 0.0001.

Article Snippet: Ca 2+ imaging was performed by confocal microscopy using a Nikon Eclipse C2 confocal microscope equipped with a digital camera connected to a computer with NIS-Elements C software.

Techniques: Activity Assay, Labeling, Confocal Microscopy, Fluorescence

Journal: Frontiers in Molecular Neuroscience

Article Title: PLC-γ-Ca 2+ pathway regulates axonal TrkB endocytosis and is required for long-distance propagation of BDNF signaling

doi: 10.3389/fnmol.2024.1009404

Figure Lengend Snippet: Model summarizing the role of known BDNF–TrkB signaling pathways on long-distance BDNF signaling. The findings from our study suggest that BDNF in the axon activates TrkB receptors and PLC-γ, leading to an increase in intracellular calcium concentration (Ca 2+ ). This, in turn, promotes the endocytosis of the receptor and the formation of signaling endosomes. The first step in this process enables the retrograde transport of signaling endosomes to the cell body, contributing to an elevation in CREB phosphorylation and dendritic arborization. Notably, we observed that cell body PLC-γ activity was not essential for CREB phosphorylation induced by axonal BDNF. In previous research, we demonstrated that PI3K activity is not necessary for endocytosis or transport of signaling endosomes but plays a crucial role in mTOR-dependent translation of CREB target genes . This process was also needed for dendritic arborization induced by BDNF in the axons.

Article Snippet: Ca 2+ imaging was performed by confocal microscopy using a Nikon Eclipse C2 confocal microscope equipped with a digital camera connected to a computer with NIS-Elements C software.

Techniques: Protein-Protein interactions, Concentration Assay, Phospho-proteomics, Activity Assay