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Fig. 2 | Molecular Brain

Fig. 2

From: Arf6 controls beta-amyloid production by regulating macropinocytosis of the Amyloid Precursor Protein to lysosomes

Fig. 2

Rapid lysosomal transport of APP occurs by Macropinocytosis. a SN56 cells were transfected with untagged full length APP695 and Lamp1-mRFP (red) and surface labeled the antibody 6E10 labeled with Alexa Fluor 647 (green) on ice. Cells were placed in HBSS containing fluorescent dextran (blue) and allowed to internalize at 37 °C for 15 min, washed in HBSS and fixed. The approximate outline of each cell is superimposed for clarity. Colocalized pixels identified by Imaris software are overlayed in white. These images show that Surface labeled APP and dextran are both rapidly co-internalized together to a LAMP1 positive compartment. Scale bar = 5 microns. b SN56 cells were transfected with HA-βAPP-CFP and Lamp1-mRFP (red), surface labeled with Alexa Fluor 647 anti-HA antibodies (green) on ice and incubated at 37 °C for 15 min with or without 2.5 μM latrunculin B. c The percentage of green pixels (internalized surface-labeled APP) colocalized with red pixels (LAMP1) was calculated by Imaris Software and from at least 38 cells drawn from 3 separate experiments is graphed. * denotes p < 0.05 (t-test). d Mouse cortical neurons were transfected with untagged full length APP695 and Lamp1-mRFP (red) and surface labeled the antibody 6E10 labeled with Alexa Fluor 647 (green) on ice and then placed in HBSS containing fluorescent dextran (blue) at 37 °C for 15 min. Colocalized pixels are overlayed in white. APP and dextran are both rapidly internalize together to a LAMP1 positive compartment. Scale bar = 5 microns. e SN56 cells were transfected with HA-βAPP-CFP and Lamp1-mRFP (red), surface labeled with Alexa Fluor 647 anti-HA antibodies (green) on ice and incubated at 37 °C for 15 min with or without 2.5 μM latrunculin B. f Quantitation of the colocalization of internalized surface-labeled APP and LAMP1 was generated using Imaris software from at least 30 cells drawn from 4 independent experiments. * denotes p < 0.05 (t-test)

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