Inhibition of nucleotide exchange on Rab11 in HD cells. One hundred micrograms of total membranes from primary control and HD fibroblasts was extracted with 1% Triton X-100 and centrifuged. The extracted supernatants were used to catalyze GDP release from [³H]GDP-GST-Rab11 and [³H]GDP-Rab5-His₆ at 30°C for 30 min (a) or [³H]GTP uptake into GDP-GST-Rab11 at 30°C for indicated times (b). [³H]GDP-GST-Rab11, [³H]GDP-Rab5-His₆, or [³H]GTP-GST-Rab11 on beads was collected by centrifugation, washed in cold washing buffer, and measured by scintillation counting. Data for panel a are represented as mean percentages ± SD for retaining [³H]GDP (n = 3; *, P < 0.01 by the Student t test). n/s, no significance. In panel b, the cpm value for each time point from each experiment was converted into picomoles, and the mean values in picomoles ± SD of [³H]GTP were graphed (n = 3; *, P < 0.01, determined by the Student t test).

Abnormal distribution of transferrin receptor in live HD fibroblasts viewed by TIRF. (a) Small vesicles are predominant carriers of recycling cargo transferrin and gradually disappear in control fibroblasts. (b) HD cells show transferrin concentrated in large vesicular structures. a and b show TIRF images recorded at 5:01, 10:00, and 13:30 min after washout of transferrin. Scale bar, 5 μm. (c) Density (mean ± SD) of transferrin (Tfn)-positive structures per μm² in control and HD cells (n = 5 cells for each; *, P < 0.001, determined by the Student t test). (d) Size distribution of transferrin-positive structures. The density and the size of Alexa568-transferrin labeled structures were quantified after chase for 6 min 40 s: chase for 5 min before imaging and another chase for 1 min 40 s after the start of imaging (image 100 was used for quantification). Paired cells (means ± SD) (HD and control cells imaged at the same TIRF angle setting) were analyzed using NIH ImageJ software (n = 5 pairs) (*, P < 0.001, determined by the Student t test).

The delay in recycling of biotin-transferrin is rescued by expressing dominant active Rab11 in HD fibroblasts, and long tubules are detectable in fixed HD cells. (a) Primary HD fibroblasts (GM04857) were infected with lentivirus expressing Rab11Q70L and EGFP under a bicistronic promoter or lentivirus expressing EGFP alone for 3 days and processed for biochemical assay of biotin-transferrin recycling as described in the legend of Fig. 1b. The data contributed by the same HD cell line from results shown in Fig. 1b were graphed for comparison to the no-virus infection condition (n = 4) (mean percentage of remaining transferrin ± SD) (*, P < 0.05 for Rab11Q70L relative to no virus, determined by the Student t test). At top is a representative blot. (b) Accumulation of Alexa568-transferrin in Rab11-positive tubular structures in the periphery of HD fibroblasts. After the synchronized uptake of Alexa568-transferrin was performed for 30 min at 37°C, cells were fixed for immunofluorescence labeling with an antibody against Rab11 (green). Shown are representative confocal images. Scale bar, 5 μm. Signals of Alexa568-transferrin are undetectable in control fibroblasts after a 30-min chase, as expected. HD fibroblasts show the presence of Alexa568-transferrin long tubules and large puncta in the cell periphery. Arrows in the insets at bottom point to the site where immunoreactive Rab11 is localized to the Alexa568-transferrin-positive tubule.

Mutant Htt does not affect vesicle formation at the plasma membrane but does inhibit receptor recycling. (a) Formation of sealed clathrin-coated vesicles at the plasma membrane occurs normally in HD cells. Single-round transferrin (Tfn) uptake was performed for the indicated times (0.5, 1, and 2 min). Noninternalized biotin-transferrin was stripped from cell surfaces using an acid buffer. 0 means no incubation at 37°C. 0+strip means no incubation at 37°C plus a wash with acid buffer. Cells were lysed in sample buffer for SDS-PAGE analysis and detection of biotin transferrin using an ABC kit. Note that the 0+strip condition shows a marked reduction in levels of biotin-transferrin, indicating a blockade of the entry of biotin-transferrin into sealed vesicles under cold conditions. The mean percentage of internalized (resistant to an acid wash) transferrin signal at each time was compared to the signal at 0 min (n = 6) (mean ± SD; *, P < 0.05; **, P < 0.01 [determined by the Student t test]). The top shows a representative blot. (b) Kinetics of transferrin recycling in control and HD fibroblasts. After the cumulative uptake of biotin-transferrin for 30 min at 37°C, cells were collected, washed, and incubated in the presence of unlabeled diferric transferrin for the indicated times. The percentage of retained biotin-transferrin after incubation for each time point was plotted, and the percentage at 0 min was set as 100%. Data shown were from three lines of control fibroblasts and three lines of HD fibroblasts (mean percentage of remaining transferrin ± SD; *, P < 0.01, determined by the Student t test). At the top is a representative blot. (c) Reduction of cell surface transferrin receptors at the basal state. After synchronized binding on ice, cells were washed and lysed for analysis. The ratio of bound biotin-transferrin to tubulin was graphed (n = 4) (mean ± SD; *, P < 0.05, determined by the Student t test). At the right is a representative blot.

Abnormality of Rab11-positive endosomal structures in HD fibroblasts. (a) Primary fibroblasts were cultured on coverslips overnight and processed for immunofluorescence labeling. Both wild-type and mutant Htt (red) are localized to Rab11 (green)-positive structures (yellow). Shown are representative confocal images of human fibroblasts. Arrows indicate Rab11-positive tubular structures, and arrowheads point to punctate structures. Scale bar, 10 μm. (b) Comparison of Rab11-positive structures in low-passage HD and age-matched control fibroblasts. The length of Rab11-positive structures colabeled with Htt was measured in confocal images using NIH ImageJ software. Data are represented as mean lengths of Rab11-positive structures analyzed in control (n = 12 cells) and HD (n = 12 cells) fibroblasts (*, P < 0.001, determined by a two-tailed Student's t test). (c) Comparison of levels of Rab11 and Htt in low-passage control and HD fibroblasts. Twenty-five micrograms of cell lysates from control (GM08399) and HD (GM04857) fibroblasts was analyzed by SDS-PAGE and Western blotting with antibodies against Htt, Rab11, and tubulin. Films were scanned, and the density of each band was measured using NIH ImageJ software. Data are represented as mean percentages of the control ± SD (n = 4) (significance determined by the Student t test). n/s, no significance. A Western blot from one of the experiments is shown at the right.

Long tubular intermediates formed in living HD fibroblasts viewed by TIRF. (a) Short tubular and small vesicular intermediates in control fibroblasts. White arrows point to a short tubule emerging and moving, whereas the oval surrounds two vesicles that fused with the plasma membrane and disappeared in the next image. The low signal is due to the small size of transferrin (Tfn)-positive structures. (b) Long tubules serve as transport intermediates in HD fibroblasts. Images are shown 4 s apart in order to show the change of tubular intermediates. Open arrows point to a tubule gradually fusing with the plasma membrane and becoming shorter and shorter, while the solid arrow indicates a growing tubule that becomes longer and longer. Scale bars, 10 μm. (c) Increase in the mean average length of tubules in HD fibroblasts ± SD (n = 72 tubules from five control cells and 72 tubules from five HD cells; *, P < 0.001, determined by the Student t test). (d) Quantification of the mean number of tubular intermediates per cell observed in the last 100 images from each cell ± standard error of the mean (10 control and 10 HD cells) imaged by TIRF microscopy (*, P < 0.05, determined by paired Student t test).

Schematic representations of Rab11 activation in control and HD cells and functional consequences on cellular homeostasis. (Left) Normal Rab11 activity on endosomal membranes and downstream consequences. In the cytoplasm, GDP-bound Rab11 partners with rabGDP dissociation inhibitor. To fulfill functions, Rab11GDP is recruited onto endosomal membranes for activation by a GEF and converted to Rab11GTP, which recruits a cohort of effectors. The GEF for Rab11 is not known, but Htt functions in this process. The consequence of normal Rab11 activity is the formation of small vesicles and short tubules at recycling endosomes and the maintenance of a normal level of receptors and transporters at cell surfaces. (Right) Proposed model in which mutant Htt interferes with Rab11 activity. The presence of mutant Htt inhibits the conversion of Rab11GDP into Rab11GTP, as indicated by the dashed arrow. The consequence of insufficient Rab11 activity is the formation of long tubules and large vesicles at recycling endosomes and a reduced level of receptors and transporters at cell surfaces, leading to cellular dysfunction and atrophy.