Journal: bioRxiv

Article Title: Chromogranin A regulates the dynamics of neurosecretion through its interaction with phosphatidic acid

doi: 10.64898/2026.01.29.699889

Figure Lengend Snippet: (a) Schematic showing the experimental plan for CgA/PA interaction monitoring in living cells by the FLIM-FRET technique. Following the overexpression of CgA- or CgAΔPABD-mKate2 in COS-7 cells, their secretion was induced during 5 min using a BaCl 2 solution. Then cells were washed and incubated 5 min with PA-ATTO647N. (b) Principle of FLIM-FRET technique to track the lifetime of the fluorescence donor (mKate2) without or with CgA/PA-ATTO647N interaction. The more CgA-mKate2 interacts with PA, the more the lifetime of mKate2 decreases due to its energy transfer to the acceptor fluorophore (ATTO647N). (c) Confocal observations of COS-7 cells overexpressing CgA-mKate2 or CgAΔPABD-mKate2 (green) after secretion stimulation and incubation with the PA-ATTO647N probe (magenta). Scale bars: 10 µm. The regions delimited by a white square on left images are enlarged to show the distribution of each fluorescent molecule. Scale bars: 2 µm. (d) Confocal observations of COS-7 cells overexpressing CgA-mKate2 or CgAΔPABD-mKate2, after cell incubation with PA-ATTO647N, showing the intensity of mKate2 fluorescence (grey), and mKate2 lifetime between 3 and 1.8 ns (rainbow color bar). The regions delimited by a white square are enlarged to show mKate2 lifetime at the plasma membrane. Scale bars: 5 µm. (e) Representative phasor plots showing the CgA-mKate2 lifetime at the level of the whole image and at the plasma membrane zone. Green line corresponds to 2.4 ns and yellow line to 2 ns, used as visual landmarks. (f) Representative phasor plot showing the CgAΔPABD-mKate2 lifetime at the level of the whole image. Green line corresponds to 2.4 ns and yellow line to 2 ns, used as visual landmarks. (g) Quantification of CgA-mKate2 or CgAΔPABD-mKate2 lifetime at the level of the plasma membrane (PM) or secretory granule (SG) without (-) or with (+) PA-ATTO647N. nd: not detected. Kruskall-Wallis test * p<0,05, each point representing the mean mKate2 lifetime in a ROI at the PM or SG level of 2 to 3 cells.

Article Snippet: African green monkey kidney fibroblast-derived COS-7 cells (American Type Culture Collection; CRL 1651) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Thermo FisherScientific) supplemented with 5% fetal bovine serum (FBS, Sigma-Aldrich), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco, Thermo FisherScientific).

Techniques: Over Expression, Incubation, Fluorescence, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Chromogranin A regulates the dynamics of neurosecretion through its interaction with phosphatidic acid

doi: 10.64898/2026.01.29.699889

Figure Lengend Snippet: (a) TIRF-M imaging of COS-7 cells overexpressing and secreting CgA- and CgAΔPABD-pHluorin after 2 mM BaCl 2 stimulation. SG leading to exocytic events on whole cells are surrounded and indicated by yellow arrows. Scale bars: 10 µm. Below are represented typical images of CgA- or CgAΔPABD-pHluorin exocytosis events. (b) Quantification of the number of exocytosis events in COS-7 cells overexpressing CgA-pHluorin and CgAΔPABD-pHluorin after their stimulation. (n=4 independent experiments; 8 cells in CgA condition and 7 cells in CgAΔPABD condition). Data are represented as mean ± SEM. Each point is one analyzed cell and each color is an independent experiment. **p < 0.01, Mann-Whitney test. (c) Typical images of CgA-EGFP or CgAΔPABD-EGFP exocytosis events. Scale bar: 500 nm. (d) Curves represent the normalized variation of the fluorescence intensity of single exocytic events during 10 s from CgA-or CgAΔPABD-EGFP overexpressing COS-7 cells after 2 mM BaCl 2 stimulation. The mean ± SEM of 75 exocytosis of 5 different CgA-EGFP overexpressing cells in 3 independent experiments and the mean of 65 exocytic events of 7 different CgAΔPABD-EGFP overexpressing cells in 3 independent experiments. Data have been normalized between 0 (before exocytosis) and 1 (maximum intensity). (e) Plot representing the mean ± SEM of area under curves from CgA-EGFP or CgAΔPABD-EGFP secretion kinetic. Each point represents one exocytosis event, and one color represents one analyzed cell. ****p < 0.0001, Mann-Whitney test.

Article Snippet: African green monkey kidney fibroblast-derived COS-7 cells (American Type Culture Collection; CRL 1651) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Thermo FisherScientific) supplemented with 5% fetal bovine serum (FBS, Sigma-Aldrich), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco, Thermo FisherScientific).

Techniques: Imaging, MANN-WHITNEY, Fluorescence