Recovery curves were plotted, and mobility fractions (Mf) and diffusion half-life occasions () were calculated using SlideBook5 FRAP module. siRNAs to clathrin heavy chain and 2 subunit of clathrin adaptor complex AP-2 as well as a dynamin inhibitor Dyngo-4A significantly decreased PKC-dependent endocytosis of HA-DAT. Similarly, endocytosis and degradation of DAT that is not epitope-tagged were highly sensitive to the clathrin siRNAs and dynamin inhibition but were not affected by flotillin knockdown. Very little co-localization of DAT with flotillins was observed in cells ectopically expressing DAT and in cultured mouse dopaminergic neurons. Depletion of flotillins increased diffusion rates of HA-DAT in the plasma membrane, suggesting that flotillin-organized microdomains may regulate the lateral mobility of DAT. We propose that clathrin-mediated endocytosis is the major pathway of PKC-dependent internalization of DAT, and that flotillins may modulate functional association of DAT with plasma membrane rafts rather than mediate DAT endocytosis. and C), lysed in TGH ((DIV) 6C10. Antibody uptake endocytosis assay and immunofluorescence detection The endocytosis assay using HA11 antibody was performed similarly as explained in Sorkina, 2006. Briefly, the cells produced on glass coverslips were incubated with 2 g/ml HA11 in conditioned media (same media the cells were produced) for 30 min and then in DMEM with DMSO (vehicle) or PMA (1 M), all at 37C in 5% CO2 atmosphere, for the indicated occasions. The cells were washed with ice-cold HBSS (Invitrogen) and fixed with freshly prepared 4% paraformaldehyde for 15 min at room heat. The cells were incubated with secondary donkey anti-mouse antibody conjugated with FITC (fluorescein) or Cy5 (5 g/ml) in DPBS (Invitrogen) made up of 0.5% BSA at room temperature for 1 hr. to occupy surface HA11. After triple wash and additional 15-min fixation, the cells were permeabilized by 5-min incubation in DPBS made up of 0.1% Triton NBI-98782 X-100/0.5% BSA at room temperature, and then incubated with the same secondary antibody conjugated with Cy3 (1 g/ml) in DPBS/0.5% BSA for 45 min to label internalized HA11. Each antibody incubations were followed by a 2-min wash in DPBS/0.5% BSA, repeated three times. Both main NBI-98782 and secondary antibody solutions were precleared by centrifugation at 100,000 g for 20 min. Coverslips were mounted on slides in Mowiol (Calbiochem, La Jolla, CA). For standard immunofluorescence staining, the cells on coverslips were fixed with paraformaldehyde and permeabilized with Triton X-100 as above, incubated with appropriate main and secondary antibodies, each followed by triple washes, and mounted in Mowiol. In experiments requiring co-staining of rat and mouse-developed antibody, all main and secondary antibody incubations were performed sequentially, separated by additional fixation. Fluorescence microscopy To obtain high resolution three-dimensional (3D) images of the cells, a z-stack of confocal images was acquired using a spinning disk confocal imaging system based on a Zeiss Axio Observer Z1 inverted fluorescence microscope (with 63x Plan Apo PH NA 1.4), equipped with a computer-controlled Spherical Aberration Correction unit, Yokogawa CSU-X1, Vector photomanipulation module, Photometrics Evolve 16-bit EMCCD video camera, HQ2 cooled CCD video camera, HGF environmental chamber and piezo stage controller and lasers (405, 445, 488, 515, 561, and 640 nm) (Intelligent Imaging Innovations, Inc., Denver, CO), all controlled by SlideBook 5 software (Intelligent Imaging Development, Denver, CO). Typically, up to 50 serial two-dimensional confocal images were recorded at 200C300 nm intervals. All image acquisition settings were identical in each experiment. Quantification of the relative amount of Cy5 or FITC (surface) and Cy3 (internalized) fluorescence was performed using the statistics module of the SlideBook5. The background-subtracted 3D images were segmented using a minimal intensity of Cy5 or FITC (non-permeabilized cells staining) and Cy3 (permeabilized cells staining) as a low threshold to obtain segment masks #1 and 2, respectively, corresponding to the total amount of surface and intracellular HA11, correspondingly. Additionally, segment mask #3 of Cy3 fluorescence overlapping with Cy5/FITC positive pixels was generated to determine the amount of Cy3-labeled antibodies that bind to surface HA11 due to incomplete occupancy of surface HA11 with Cy5/FITC-labeled secondary antibodies before cell permeabilization. Mask #3 was subtracted from Mask #2 to obtain Mask #4 corresponding to the corrected Cy3 fluorescence (internalized HA11 complexes with YFP- or NBI-98782 CFP-HA-DAT). The integrated voxel intensity of Masks #1 and #4 were quantitated in each image made up of typically 5C15 cells, and the ratio of Mask#4 to Mask#1 integrated intensities were calculated to determine the extent of DAT internalization. Fluorescence Recovery after Photobleaching (FRAP) HEK/CFP-HA-DAT cells were grown on glass bottom MatTek dishes.FRAP measurements were carried out in growth medium at 37C. FRAP experiments were performed using a spinning disk confocal microscope system equipped with an environmental chamber ensuring a constant temperature, humidity and 5% CO2 atmosphere throughout the duration of the imaging. Time-lapse imaging was conducted via a 63X objective lens using a 445 nm laser collection before and after photobleaching.
Recovery curves were plotted, and mobility fractions (Mf) and diffusion half-life occasions () were calculated using SlideBook5 FRAP module