Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells (original) (raw)
Internalized BODIPY-LacCer accumulates in the Golgi apparatus of normal HSFs. When HSFs are incubated with BO-DIPY-LacCer at 4°C, only the PM becomes labeled (11). However, shifting the temperature to 37°C results in internalization into endosomes (11), and by 15–45 minutes, fluorescence begins to accumulate at the Golgi complex. Double-label experiments using the LacCer analogue and antibodies against mannosidase II (Figure 1a) or TGN38 (data not shown) showed extensive colocalization with these Golgi markers, suggesting that BODIPY-LacCer accumulated predominantly at the Golgi apparatus. To further characterize LacCer transport to the Golgi, we used nocodazole (a microtubule-depolymerizing agent) and wortmannin (a PI3K inhibitor), which have previously been used to characterize differences in trafficking of furin and TGN38 from the PM to the Golgi apparatus (35). HSFs were treated with nocodazole or wortmannin and pulse-labeled with 2.5 μM BODIPY-LacCer. Both treatments blocked transport of BODIPY-LacCer to the Golgi (Figure 1b). Similar results to those shown for wortmannin were also obtained using another PI3K inhibitor, LY294002 (Figure 1, legend). Control experiments using BODIPY-Cer, a vital stain for the Golgi apparatus (33), indicated that the overall Golgi morphology was not affected by nocodazole, wortmannin (Figure 1b), or LY294002 (data not shown). These results suggest that, similar to furin, the transport of BODIPY-LacCer to the Golgi apparatus requires the activity of PI3Ks and that its transport occurs via microtubules.
Fluorescent LacCer is transported from the PM to the Golgi apparatus in normal HSFs and is dependent on microtubules and activity of PI3Ks. (a) Cells grown on etched grid coverslips were incubated with 2.5 μM BODIPY-LacCer for 30 minutes at 4°C in HMEM, washed, further incubated for 45 minutes at 37°C, and then back-exchanged at 10°C to remove any fluorescent lipid present at the PM. Live cell imaging (left panel) revealed a perinuclear distribution of the LacCer analogue. Cells were then fixed and immunolabeled with an antibody against the Golgi marker mannosidase II, and the same field was rephotographed for mannosidase II staining (MannII; right panel). (b) Cells were preincubated with wortmannin or nocodazole and subsequently pulse-labeled with BODIPY-LacCer as above. Golgi targeting of LacCer was inhibited in 80% (nocodazole), 50% (wortmannin), 70% (LY294002, not shown), or 5% (untreated) of the cells (n = 20 for each). No disruption of Golgi morphology was detected for the indicated inhibitors using BODIPY-Cer as a vital stain for this organelle. Bar, 10 μM.
Biochemical and functional evaluation of DsRed- and EGFP-Rab fusion proteins. We generated WT and DN constructs of Rab7, Rab9, and Rab11 in pDsRed1-C1 and pEGFP-C1 vectors (see Methods) and expressed them in HSFs or HeLa cells for 24–48 hours. Strong overexpression (∼20- to 30-fold compared with endogenous Rab’s after correction for transfection efficiency; data not shown) was observed. Cell lysates were prepared from transfected HSFs and analyzed by SDS-PAGE followed by GTP overlay and immunoblotting. As expected, the DN Rab’s showed no GTP binding, while the WT forms efficiently bound GTP (Figure 2a). Equivalent sample loading and expression of all the Rab fusion proteins was confirmed by immunoblotting using native Rab antibodies (Figure 2a) or anti-DsRed or anti-EGFP antibodies (data not shown).
Characterization of Rab–fluorescent protein constructs. (a) GTP overlay assay and immunoblot analysis of DsRed-Rab fusion proteins. HSFs were transfected with DsRed-Rab7, -Rab9, or -Rab11 (WT or DN) constructs, and 48 hours later cell lysates were prepared. Lysate samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. The blots were incubated for 2 hours with [α-32P]GTP and exposed to x-ray film. The GTP blots were then stripped of GTP and were independently incubated with rabbit anti-Rab7, -Rab9, or -Rab11 monoclonal antibodies. Bands were visualized after secondary antibody incubations by chemiluminescence. (b) Effect of Rab7 WT and mutant protein overexpression on [125I]EGF degradation in HeLa cells. Transfected or mock-transfected cells were incubated with [125I]EGF for 10 minutes at 37°C, washed, and incubated for another 30 minutes at 37°C. TCA-soluble counts in cell lysates and extracellular medium were used to calculate EGF degradation (see Methods). Data are expressed as mean ± SD of three independent experiments. (c) Intracellular localization of Tfn in HeLa cells. Cells overexpressing DsRed-Rab11 WT or DN fusion proteins were incubated with FITC-Tfn for 45 minutes at 37°C. Samples were then washed, acid-stripped to remove fluorescent Tfn from the cell surface, and observed under the fluorescence microscope. In cells overexpressing WT DsRed-Rab11, Tfn colocalized with Rab11 fluorescent protein in punctate vesicular structures, while in cells overexpressing the DN construct, Tfn was found in tubular structures. These distributions are readily seen in the higher-magnification insets (right panels). Bar, 10 μM.
We also evaluated the effect of WT and DN forms of Rab7 and Rab11, which were produced as DsRed or EGFP fusion proteins, on membrane transport through different endocytic compartments. To study the effect of the Rab7 constructs on trafficking from early to late endosomes and lysosomes, we monitored the degradation of radiolabeled EGF. HeLa cells were transfected with the indicated fluorescent Rab fusion protein construct, and 48 hours later they were incubated for 10 minutes at 37°C with [125I]EGF to load the early endosomes. The samples were then washed and incubated for 30 minutes at 37°C, and the TCA-soluble material (an indicator of EGF degradation) was quantified. As shown in Figure 2b, approximately 50% of the total cell-associated EGF was degraded in mock-transfected cells and cells transfected with WT DsRed- or EGFP-Rab7, while in cells overexpressing the corresponding DN Rab7 fusion proteins, degradation was reduced to about 25% of the total cell-associated EGF.
To study the effect of DsRed1-Rab11 WT and mutant proteins on recycling, we examined the intracellular distribution of endocytosed Tfn in HeLa cells overexpressing these proteins. In these experiments, cells were incubated with fluorescent Tfn for 45 minutes at 37°C. As expected, there was significant colocalization of the internalized Tfn with WT DsRed-Rab11–positive compartments (Figure 2c). In cells overexpressing DN DsRed-Rab11, Tfn was restricted to extended tubular structures (Figure 2c). A similar effect on Tfn distribution has previously been reported using an untagged Rab11S25N mutant (36). Results similar to those shown in Figure 2c were obtained using WT EGFP-Rab11 versus DN EGFP-Rab11 constructs (data not shown).
BODIPY-LacCer transport to the Golgi apparatus is Rab7- and Rab9-dependent. We first examined the effect of the DN Rab7 mutant on BODIPY-LacCer targeting in normal HSFs. Cells were transiently transfected with constructs for DsRed-Rab7 (DN or WT), and 48 hours later they were pulse-labeled with BODIPY-LacCer to evaluate transport to the Golgi apparatus. As previously reported (37, 38), cells expressing WT constructs of the Rab fusion proteins showed fluorescent vesicles that were present throughout the cytoplasm, whereas cells expressing the DN constructs exhibited diffuse fluorescence with little apparent concentration on vesicular structures (see Figure 2c, Figure 3, and Figure 4). In cells expressing the WT Rab7 construct, trafficking of BODIPY-LacCer to the Gol gi was not inhibited (data not shown). In contrast, LacCer targeting to the Golgi was blocked in cells overexpressing the DN Rab7 construct (Figure 3a, top panels), although LacCer was targeted to the Golgi apparatus in untransfected cells on the same coverslip (data not shown). Inhibition of LacCer targeting to the Golgi by DN Rab7 was also seen after microinjection of similar constructs lacking the DsRed1 tag (data not shown).
Golgi targeting of BODIPY-LacCer is blocked in HSFs expressing DN mutants of Rab7 or Rab9, but not Rab11. (a) Normal HSFs were transfected with plasmids encoding DsRed fusion proteins of DN Rab7, Rab9, or Rab11. Forty-eight hours later, the samples were pulse-labeled with BODIPY-LacCer, and Golgi targeting was assessed by fluorescence microscopy. Golgi morphology was not significantly altered by overexpression of the DN Rab proteins as assessed by BO-DIPY-Cer, seen in insets showing overlay images of DN Rab–expressing cells (red) and Cer (green). N, nucleus; G, Golgi apparatus. Bar, 10 μm. (b) Golgi targeting of BODIPY-LacCer in HSFs overexpressing DN DsRed-Rab7, -Rab9, or -Rab11 proteins. Transfected cells were identified by DsRed fluorescence and then scored as Golgi-positive (e.g., Figure 3a, bottom right panel) or Golgi-negative (e.g., Figure 3a, top panels). Bar labeled “Mock” represents cells transfected with empty vector. Values are expressed as mean of three independent experiments (n = 10) for each condition.
Overexpression of WT Rab7 or Rab9 (but not Rab11) restores BODIPY-LacCer and fluorescent CtxB targeting to the Golgi apparatus of NP-C fibroblasts. Cells were transfected with plasmids encoding (a) DsRed fusion proteins or (b) EGFP fusion proteins of WT Rab7, Rab9, or Rab11. Forty-eight hours later, the samples were pulse-labeled with (a) BODIPY-LacCer or (b) Rh-CtxB. Golgi targeting of LacCer and CtxB was assessed by fluorescence microscopy. (a and b) Top rows: Transfected cells overexpressing WT Rab7 showed intense Golgi staining of LacCer (green) and CtxB (red), with little punctate cytoplasmic staining. In contrast, untransfected cells showed punctate cytoplasmic staining with either LacCer or CtxB (see insets). Middle rows: Similar results to those obtained with WT Rab7 were seen in cells overexpressing WT Rab9. Bottom rows: Little or no Golgi targeting of fluorescent LacCer or CtxB was observed in transfected cells overexpressing WT Rab11. Bar, 10 μm. (c) Golgi-labeling by BODIPY-LacCer or CtxB in NP-C cells overexpressing WT Rab’s was quantified as described in Figure 3b legend.
We next examined the role of Rab9 in LacCer trafficking. As shown in Figure 3a (middle panels), DN Rab9 also blocked BODIPY-LacCer targeting to the Golgi. In control experiments we used BODIPY-Cer as a vital stain for the Golgi apparatus (33) and confirmed that Golgi morphology was not altered in cells transfected with DN Rab7 or Rab9 (Figure 3a, insets). Thus, DN Rab7 and Rab9 disrupted Golgi targeting of the LacCer analogue, but not the organization of this organelle. To identify other potential trafficking pathways (e.g., transport from the recycling compartment to the Golgi), HSFs were transfected with plasmids expressing the DN Rab11 fusion protein; however, no difference in LacCer trafficking was observed between transfected (Figure 3a, bottom panels) and untransfected cells (Figure 1). The results shown in Figure 3a were quantified by scoring the number of transfected cells that showed Golgi staining after pulse-labeling with BODIPY-LacCer. Approximately 80% of the cells transfected with DN Rab7 (n = 30) or Rab9 (n = 30) showed loss of LacCer targeting to the Golgi, compared with less than 10% (n = 30) of cells transfected with empty vector (Figure 3b) or with untransfected cells (data not shown).
Overexpression of Rab7 and Rab9 in NP-C cells restores BODIPY-LacCer and fluorescent CtxB trafficking to the Golgi apparatus. We previously demonstrated that BODIPY-LacCer is mistargeted to endosomes and lysosomes, rather than the Golgi apparatus in SLSD fibroblasts as a consequence of their high levels of intracellular cholesterol (24, 26). Furthermore, NP-C fibroblasts are reported to exhibit a severe reduction in vesicular traffic through late endosomes (32, 39, 40). To determine whether overexpression of certain Rab proteins could stimulate transport through this endosomal compartment, NP-C fibroblasts were transfected with WT Rab7, Rab9, or Rab11 constructs and then pulse-labeled with fluorescent LacCer. NP-C cells transfected with the WT Rab7 or Rab9 plasmids showed significantly greater transport of BODIPY-LacCer to the Golgi apparatus (Figure 4a) than did untransfected NP-C cells (Figure 4a, top panel inset). Overexpression of WT Rab11 in NP-C cells did not restore the Golgi targeting of BO-DIPY-LacCer (Figure 4a). These results were quantified by scoring transfected and mock-transfected cells (n = 30 in each case) for Golgi staining by the LacCer analogue. Approximately 70% of the transfected cells overexpressing WT DsRed-Rab7 or -Rab9 fusion proteins showed Golgi staining, while less than 10% of the cells overexpressing WT Rab11 or mock-transfected cells showed Golgi labeling (Figure 4c).
We also studied the internalization and subsequent intracellular targeting of Rh-CtxB in NP-C fibroblasts. CtxB binds to endogenous GM1 ganglioside at the cell surface, is internalized via caveolae (12), and is subsequently targeted to the Golgi complex of normal HSFs or to endosomal structures in SLSD cell types (11, 41). In untransfected NP-C cells, Rh-CtxB was targeted to punctate cytoplasmic structures (Figure 4b, top panel inset), while in cells transfected with WT constructs of Rab7 or Rab9, CtxB was transported to perinuclear Golgi-like structures (Figure 4b). Overexpression of WT Rab11 did not restore the normal Golgi targeting of CtxB (Figure 4b). Quantitative analysis of this experiment gave results for CtxB similar to those seen with BODIPY-LacCer in NP-C cells transfected with the various WT Rab constructs (Figure 4c).
Overexpression of Rab7 and Rab9 reduces cholesterol accumulation in NP-C cells. Since the defective transport of BODIPY-LacCer in NP-C cells (and other SLSD cell types) is due to elevated intracellular cholesterol (26), and since normal Golgi targeting was restored by overexpression of WT Rab7 or Rab9, we next investigated the effect of overexpressing these Rab proteins on the accumulation of intracellular cholesterol. NP-C fibroblasts were transfected with the WT constructs of EGFP-Rab7, -Rab9, or -Rab11, cultured for 48 hours, and then stained for intracellular cholesterol using filipin (32). Cells transfected with WT Rab7 or Rab9 constructs showed dramatically reduced filipin staining compared with untransfected cells or with cells overexpressing WT Rab11 (Figure 5a). DN constructs of Rab7 or Rab9 also had little or no effect on filipin staining (data not shown). Quantitative analysis showed that filipin fluorescence was consistently reduced more than 50% in cells transfected with the WT Rab7 or Rab9 constructs compared with adjacent untransfected cells in the same field (n = 50 for each condition). Overexpression of WT EGFP-Rab11 fusion protein showed less than 10% difference in filipin staining compared with untransfected cells. Similar results were observed when WT DsRed-Rab constructs were used instead of EGFP-tagged constructs (data not shown).
Overexpression of WT Rab7 or Rab9 (but not Rab11) in NP-C cells reduced the accumulation of intracellular cholesterol. (a) Cells were transfected with plasmids expressing EGFP fusion proteins of WT Rab7, Rab9, or Rab11 and cultured in EMEM plus 10% FBS. Forty-eight hours later, the distribution of free cholesterol was examined by filipin staining (blue fluorescence). Transfected cells identified by green fluorescence are outlined in the filipin images. Bar, 25 μm. (b) Quantitation of filipin fluorescence in NP-C cells overexpressing WT Rab7, Rab9, or Rab11 proteins. Total filipin fluorescence of individual transfected or adjacent untransfected cells was calculated by image analysis. Values shown are mean ± SD from a typical experiment (n = 50). Similar results were obtained in each of three independent experiments.
We also examined the intracellular distribution of the NPC1 protein (32) before and after overexpression of WT Rab7, Rab9, and Rab11; there were no obvious changes in either the distribution of NPC1 or the intensity of NPC1 immunofluorescence in transfected versus untransfected NP-C cells for any of the Rab proteins (data not shown). Finally, we used Nile Red in NP-C cells overexpressing WT Rab constructs to stain for cholesterol esters and other neutral lipids. Cells transfected with WT Rab7 or Rab9 showed an almost twofold increase in Nile Red fluorescence, whereas no such effect was seen using the WT Rab11 construct (Figure 6).
Overexpression of WT Rab7 or Rab9 (but not Rab11) in NP-C cells increased the accumulation of neutral lipids. (a) Cells were transfected with plasmids expressing EGFP fusion proteins of WT Rab7, Rab9, or Rab11 as in Figure 5. Forty-eight hours later, samples were fixed and stained for the presence of cholesterol esters and other neutral lipids using Nile Red (34). Transfected cells identified by green fluorescence are outlined on the Nile Red images. Bar, 25 μm. (b) Quantitation of Nile Red fluorescence in NP-C cells overexpressing WT Rab7, Rab9, or Rab11 proteins. Total Nile Red fluorescence of individual transfected or adjacent untransfected cells was calculated by image analysis. Values are mean ± SD from a typical experiment (n = 30). Similar results were obtained in each of three independent experiments.