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Fig. 5 | Molecular Neurodegeneration

Fig. 5

From: SOD1 protein aggregates stimulate macropinocytosis in neurons to facilitate their propagation

Fig. 5

Aggregated SOD1 induces ruffles and blebs, dextran uptake and activation of RAC1. a Field emission SEM of cells treated with SOD1 aggregates or controls, non-aggregated SOD1 (sol SOD1), PMA or PBS (untreated). Increases in membrane perturbations can be observed, such as ruffles (black arrow) and blebs (black arrow head). Bars represent 2 μm. (b) Laser scanning confocal micrographs of treated cells stained with the membrane dye FM1-43FX to measure membrane perturbation and fluorescence intensity per cell was quantified using ImageJ. Scale bars represent 20 μm. A minimum of 200 cells were scored per treatment. Results shown are means ± SD of three experiments, * p < 0.05 ** p < 0.01. (c) The induction of fluid phase uptake was measured using fluorescently labelled dextran. Laser scanning confocal micrographs of dextran-Alexa647 uptake in treated NSC-34 cells. Outline of cells are indicated with white dashed lines. Scale bars represent 20 μm. (e) Flow cytometry quantification of dextran uptake in the treated NSC-34 cells. Results shown as means ± SD of 6 experiments * p <0.05 ** p < 0.01. (d) Rac1 activation in treated NSC-34 cell lysates was measured using a Rac1 activation ELISA assay that probes for Rac1-GDP. Results are mean ± SD of 6 experiments, ** p < 0.01. (e) Addition of a Rac1 inhibitor reduces SOD1 uptake. Laser scanning confocal micrographs of SOD1 aggregate uptake in the presence of absence of W56, mean fluorescence per cell was calculated using ImageJ. Data are mean fluorescence intensity per cell of a minimum of 100 cells ± SD, p < 0.001

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