Glycolipid trafficking in Drosophila undergoes pathway switching in response to aberrant cholesterol levels

Abstract

In lipid storage diseases, the intracellular trafficking of sphingolipids is altered by conditions of aberrant cholesterol accumulation. Drosophila has been used recently to model lipid storage diseases, but the effects of sterol accumulation on sphingolipid trafficking are not known in the fly, and the trafficking of sphingolipids in general has not been studied in this model organism. Here, we examined the uptake and intracellular distribution of a fluorescent glycolipid analog, BODIPY-lactosyl-ceramide, in Drosophila neurons. The uptake mechanism and intracellular trafficking route of this simple glycolipid are largely conserved. Our principle finding is that cholesterol steers trafficking of the glycolipid between Golgi, lysosome, and recycling compartments. Our analyses support the idea that cholesterol storage in Drosophila triggers a switch in glycolipid trafficking from the biosynthetic to the degradative endolysosomal pathway, whereas cholesterol depletion eliminates recycling of the glycolipid. Unexpectedly, we observe a novel phenomenon we term "hijacking," whereby lactosyl-ceramide diverts the trafficking pathway of an endocytic cargo, dextran, completely away from its lysosomal target. This work establishes that glycolipid trafficking in Drosophila undergoes changes similar to those seen in mammalian cells under conditions of cholesterol storage and therefore validates Drosophila as a suitable model organism in which to study lipid storage diseases.

Figure 1.

BODIPY-labeled lac-Cer analogs are taken up into endocytic vesicles in Drosophila neurons (A) BODIPY–lac-Cer is incorporated into the plasma membrane and endocytosed in Drosophila neurons incubated with a mixture of BODIPY FR-lac-Cer and FL-lac-Cer. The attached fluorophore (BODIPY 505-511 FL vs. BODIPY 650-665 FR) does not influence the uptake or subcellular trafficking of lac-Cer; the two labels traffic identically as can be seen by nearly complete overlap of the FL (green) and FR (red) signals. BODIPY–lac-Cer carrying compartments are highly mobile, typical for endosomes (see Supplemental Video1). Scale bar, 8 μm. (B) BODIPY-FR–lac-Cer (red) colocalization, with transfected lysosomal marker LAMP-GFP (green) after 30-min incubation at 4°C and subsequent chase at 25°C. Image shown is after 90 min. Scale bar, 5 μm. (C) Transfected LAMP-GFP and pulsed BODIPY-FR–lac-Cer colocalization (black trace) shows a peak in presumptive sorting endosomes at ∼30 min after uptake, falling to ∼15% at the sorting to late endosome transition (60–90 min). Red dextran was incubated on the cells for 5 min and chased for 15 h to label late endosomes and lysosomes. Note that colocalization with dextran (red trace) is not identical to that with LAMP-GFP. There is a transient peak in BODIPY-FR–lac-Cer colocalization with LAMP-GFP, versus the gradual increase in colocalization with dextran-positive lysosomes, presumably due to additional presence of the LAMP-GFP marker in sorting endosomes. (D) A nonglycosylated sphingolipid TR-ceramide (red) also colocalizes to a limited extent with the lysosomal markers dextran-Alexa488 (green) and Lysotracker-green that were incubated on the cells 3 h into the chase and imaged after 2 additional hours of chase (total chase time for TR-ceramide = 5 h; colocalization was not quantified).

Figure 2.

BODIPY–lac-Cer uptake is attenuated by inhibitors of both raft and nonraft endocytic mechanisms in fly neurons. (A–D) BODIPY-FR–lac-Cer (red), shown in far left column, compared with control probes (right column; blue) incubated with Drosophila c6 neurons treated with the given inhibitors: CPZ (chlorpromazine) inhibits clathrin-mediated uptake; Clostridium toxin B inhibits rho-family GTPases; dynasore inhibits dynamin; and nystatin inhibits cholesterol-dependent uptake. After imaging, cells were fixed and labeled with Hoechst dye (blue) to obtain images. (E) Uptake of the different labels in untreated control cells, for comparison (not quantified). (F) Quantification of uptake by spot count (see Materals and Methods) at various time points after labeling with FR–lac-Cer. nystatin, dynasore, and chlorpromazine (white, gray, and cross-hatched bars) exerted more long-lasting inhibition on uptake than Clostridium toxin B (lighter gray), which gave close to complete recovery by 60 min. Significant differences; ***p < 0.01).

Figure 3.

BODIPY–lac-Cer traffics primarily through recycling endosomes and is only partially Golgi-associated. (A and B) BODIPY–lac-Cer (red) colocalization with transfected MannII-GFP (green). After 60 min, BODIPY-FR–lac-Cer traffics only partially (∼15–20%) to cis and medial Golgi compartments, labeled by MannII-GFP. Bars, 5 μm. (C and D) BODIPY–lac-Cer– (red) colocalization with rab11-GFP– (green) positive recycling endosomes. Trafficking of BODIPY-FR–lac-Cer (35–40%) to recycling endosomes is substantially higher than that to Golgi. Scale bars, 4 μm.

Figure 4.

BODIPY–lac-Cer and dextran traffic identically when present simultaneously on cells. (A and B) Simultaneous labeling of BODIPY-FL–lac-Cer (green) with Alexa647 (FR)-dextran (red) resulted in very high colocalization. In contrast, sequential labeling of FL–lac-Cer and 3 h later with dextran results in essentially no colocalization between the two dyes, even given sufficient chasing time that each label by itself would have reached late endosomes or lysosomes. Bars, 8 μm. (C) Colocalization values are continuously high over time after simultaneous incubation of BODIPY–lac-Cer and dextran (black trace), even compared with that seen between dextran and another endolysosomal marker, LAMP-GFP (red trace; see Figure 1); “simultaneous” refers to addition of dextran immediately after brief washing of the BODIPY-FL–lac-Cer. (D) Colocalization after 3 h is high (>60%) when dextran and BODIPY–lac-Cer are simultaneously incubated (sim) and significantly lower when sequentially incubated, independent of the order of addition (dark and light gray bars). A failure to colocalize even after several hours indicates that BODIPY–lac-Cer and dextran are trafficked to different targets. Student's t test was used to compare the datasets from simultaneous versus sequential incubation; *p < 0.05. (E) BODIPY–lac-Cer influences trafficking of dextran independent of the relative concentrations of both dyes, indicating that the altered transport is not due to direct binding. Cells were incubated with 5 μM BODIPY-FL–lac-Cer and 0.5 mg/ml Alexa647-dextran (5:5), 5 μM BODIPY-FL–lac-Cer and 0.1 mg/ml dextran (5:1) and 1 μM BODIPY-FL–lac-Cer and 0.5 mg/ml dextran (1:5) simultaneously and colocalization values were calculated after 180 min.

Figure 5.

BODIPY–lac-Cer uptake in the presence of dextran alters trafficking itineraries of both tracers. Endolysosomes, Golgi, and lysosomes were labeled with LAMP-GFP (A), Alexa546-dextran (B), and MannII-GFP, chased for 15 h (C), and subsequently labeled with BODIPY-FL–lac-Cer and Alexa647-dextran simultaneously to determine whether the trafficking route of either label changed in the presence of the other. (A) After simultaneous incubation, both labels accumulate slowly in LAMP-positive compartments (black trace), more than would be expected for BODIPY–lac-Cer (red line), but less than dextran by itself (green line). (B) Trafficking of FR-dextran to lysosomes labeled by 15-h incubation of Alexa546-dextran (green line) was completely inhibited in the presence of BODIPY–lac-Cer (black trace). (C) Trafficking of BODIPY–lac-Cer to Golgi (normally ∼20%; red line) is also inhibited by the presence of dextran (black trace). dextran does not normally traverse the Golgi in the absence of BODIPY–lac-Cer (green line). Significant differences in colocalization at given time points; *p < 0.05. (D) Quantification of colocalization of BODIPY-FR–lac-Cer with dextran added simultaneously, in the presence or absence of the inhibitor Clostridium toxin B (black), which affects dextran uptake (see Figure 1), and dynasore (light gray), which affects BODIPY–lac-Cer uptake. Colocalization remains nearly as high as after the control simultaneous incubation (dark gray) and much higher than after sequential incubation (hatched). (E, left) Quantification of dextran-Alexa488 uptake efficiency after 90 min, either alone (white) or together with BODIPY-FR–lac-Cer (black) in untreated cells, versus lower uptake in Clostridium toxin B–treated cells (gray). Uptake inhibition of dextran is rescued to an intermediate level by the presence of BODIPY–lac-Cer (dark gray hatched). Uptake of dextran alone is not significantly inhibited by dynasore (light gray). (E, right) Quantification of BODIPY-FR–lac-Cer uptake efficiency after 90 min, either alone in untreated cells (white) or together with dextran (black), versus lower uptake in dynasore-treated cells (gray). Uptake inhibition is not rescued by the presence of dextran (dark gray hatched), in contrast to the situation on the left. Uptake of BODIPY-FR–lac-Cer alone is not inhibited by Clostridium toxin B (light gray). (F) Images of cells showing that the presence of inhibitors does not prevent colocalization of the two labels: BODIPY-FR–lac-Cer (red) and dextran (green).

Figure 6.

BODIPY–lac-Cer trafficking between Golgi, recycling endosomes, and the endolysosomal pathway is controlled by a cholesterol switch in fly neurons. (A, D, and G) Cholesterol depletion (red trace in all graphs). Loss of cholesterol leads to increased traffic through a LAMP-positive sorting compartment at 60 min (D) and then a significant but transient accumulation in Golgi labeled by MannII-GFP (A) at 180 min. BODIPY–lac-Cer finally accumulates in LAMP-positive late endolysosomes. Passage of BODIPY–lac-Cer through recycling endosomes in contrast is much reduced (G). The drop in Golgi at 240 min is probably not due to production of noncolocalizing metabolites of BODPIY-lac-Cer, because labeled lac-Cer remains stable for at least 6 h (see Supplemental Figure 3). Cholesterol excess (blue traces in all graphs): Cholesterol overloading strongly suppresses Golgi-targeting (A), in contrast to the enhancement of Golgi targeting seen under cholesterol depletion (red trace, A), and leads to aberrant accumulation in enlarged LAMP-positive (D) compartments, but with a later time course than depletion (compare blue and red traces, D). Colocalization is expressed as tM_lac-Cer, converted to %. Significance of the differences in colocalization; *p < 0.05. (B, C, E, F, H, and I) Images of c6 neurons transfected with markers of the different compartments and depleted or loaded with cholesterol: Golgi (MannII-GFP in B and C), endolysosomes (LAMP-GFP in E and F), and recycling endosomes (rab11-GFP in H and I; all green), and labeled with BODIPY-FR–lac-Cer (red). Cholesterol depletion and excess are shown for each compartment. LAMP-GFP–positive endolysosomes are visibly enlarged under cholesterol excess (cf. E and F) and FR–lac-Cer–carrying vesicles appear more diffuse (C, F, and I). Bars, 4 μm.

Figure 7.

Model for cholesterol regulation of Golgi, endo-lysosomal, and recycling endosomal traffic of a glycolipid. Under normal conditions (left), the bulk of the BODIPY–lac-Cer transits from sorting endosomes (SE) to recycling endosomes (RE), whereas a smaller portion goes to Golgi (G) and late endosomes/lysosomes (LE and LY). When the cells are depleted of sterols (middle), uptake into sorting endosomes and Golgi is slower (heavy broken arrow), whereas recycling endosomes are completely bypassed. After a delay, higher than normal amounts of BODIPY–lac-Cer are found transiently in Golgi, and then endolysosomes. Question mark indicates that BODIPY–lac-Cer may be transported out of the Golgi to endolysosomes. Under cholesterol overload (right), the Golgi compartment is completely bypassed, whereas endo/lysosomes receive much more than normal BODIPY–lac-Cer and recycling endosomes somewhat less. Question mark indicates that BODIPY–lac-Cer may be transported from recycling to endolysosomes. In summary, abnormally low amounts of sterol in Drosophila neurons seem to favor a Golgi and lysosomal pathway at the expense of recycling, whereas excess sterol favors a lysosomal pathway mainly at the expense of Golgi.