Flagellar membrane localization via association with lipid rafts

Abstract

The eukaryotic flagellar membrane has a distinct composition from other domains of the plasmalemma. Our work shows that the specialized composition of the trypanosome flagellar membrane reflects increased concentrations of sterols and saturated fatty acids, correlating with direct observation of high liquid order by laurdan fluorescence microscopy. These findings indicate that the trypanosome flagellar membrane possesses high concentrations of lipid rafts: discrete regions of lateral heterogeneity in plasma membranes that serve to sequester and organize specialized protein complexes. Consistent with this, a dually acylated Ca(2+) sensor that is concentrated in the flagellum is found in detergent-resistant membranes and mislocalizes if the lipid rafts are disrupted. Detergent-extracted cells have discrete membrane patches localized on the surface of the flagellar axoneme, suggestive of intraflagellar transport particles. Together, these results provide biophysical and biochemical evidence to indicate that lipid rafts are enriched in the trypanosome flagellar membrane, providing a unique mechanism for flagellar protein localization and illustrating a novel means by which specialized cellular functions may be partitioned to discrete membrane domains.

Fig. 1.

Calflagin Tb24 is associated with detergent-resistant membranes. (A) To test temperature-dependent resistance to detergent, 10⁸ T. brucei procyclic forms were extracted in 1 ml of 1% Triton X-100 in PBS at 4°C or 37°C for 10 minute and centrifuged supernatants and pellets were analyzed by SDS-PAGE and western blotting with calflagin Tb24-specific antiserum. Of the proteins tested, only calflagin Tb24 was present in the pellet (P) at 4°C but the supernatant (S) at 37°C. (B) To test buoyancy, cells were extracted in 1% Triton X-100 in TBS at 4°C and floated through an OptiPrep (40%, 30%, 5%) step gradient. Sequential fractions (1-10) were collected and equal volumes (25 μl) were subjected to western blot analysis with the same antibodies used in A. Only calflagin Tb24 floated to the top of the step gradient, with the other proteins remaining close to the loading zone. In both assays, partitioning of Hsp70, PFR, EP procyclin and GP63 were assessed to control for cytosolic, flagellar cytoskeleton and diffuse surface distributions. W, whole-cell lysate.

Fig. 2.

The flagellum of T. brucei is enriched in molecules associated with lipid membrane rafts. (A) Paraformaldehyde-fixed procyclic forms were analyzed by fluorescence microscopy for the distribution of raft components. Ergosterol was visualized by staining with filipin, galactocerebroside was visualized by immunofluorescence microscopy, GM1 ganglioside was visualized by staining with FITC-conjugated cholera toxin, the dually acylated calflagin Tb24 was visualized by immunofluorescence microscopy and two GPI-anchored proteins (EP procyclin and GP63) were visualized by immunofluorescence microscopy. (B) Cellular distribution of cytoskeletal controls proteins used in fractionation. α-Tubulin is distributed in both flagellar and pellicular cytoskeleton, WCB is restricted to the pellicular cytoskeleton and PFR is confined to the flagellar cytoskeleton. (C) Purified flagella were evaluated by fluorescence microscopy for purity using cytoskeletal (α tubulin) and surface (EP procyclin) constituents, both of which are common to the flagellum and cell body. The preparation consisted of flagella only; there was no microscopic evidence of cell bodies or pellicular fragments, and kinetoplasts/basal bodies fractionated separately (data not shown). Bar, 5 μm.

Fig. 3.

The trypanosome flagellar membrane possesses increased liquid order. T. brucei procyclic forms were examined by two-photon microscopy after staining with the amphiphilic fluor laurdan. Both non-dividing (top) and dividing (bottom) cells are shown. Procyclic trypanosomes were washed and resuspended to 10⁸ cells per ml in PBS containing 1 μM laurdan, then settled on poly-L-lysine-coated slides for 10 minute at room temperature. (A) Laurdan fluorescence intensity relates to probe concentration and is scaled so that yellow corresponds to high intensity and red to low intensity. (B) The generalized polarity (GP) is a ratiometric depiction of fluorescent intensities from two wavelengths. The GP value is probe concentration independent, reflecting variation in the orderliness of a membrane. Areas appearing green are less ordered and those appearing blue are more ordered. (C) Line drawings are shown for orientation of the trypanosome cell: f, flagellum; pm, pellicular membrane; nf, new flagellum; of, old flagellum. Bar, 5 μm.

Fig. 4.

Calflagin Tb24 distribution reflects membrane raft stability. (A) The flagellum of bloodstream form T. brucei is enriched in cholesterol and calflagin Tb24. Filipin staining of untreated cells shows enrichment in the flagellum. The flagellar localization of calflagin Tb24 is resistant to 1% Triton X-100 at 4°C. The region of the flagellum connector (arrows, center panel) is particularly rich in staining for this DRM marker. If cholesterol is depleted by preincubation in 20 mM MBCD for 30 minutes, flagellar concentration of calflagins Tb24 is lost. (B) MBCD treatment leads to loss of buoyancy of calflagin Tb24. Bloodstream form T. brucei treated with MBCD and untreated control cells were analyzed by Optiprep gradient centrifugation and western blotting with calflagin Tb24-specific antiserum as described in Fig. 1. W, whole-cell lysate. (C) Chemical modulation of membrane fluidity. Washed procyclic form trypanosomes were treated with 10% DMSO for 1 minute to stabilize the liquid ordered phase or 1% diethyl ether for 30 seconds to fluidize the liquid ordered phase. Cells were then fixed and processed for immunofluorescence microscopy using anti-calflagin Tb24. Bar, 5 μm.

Fig. 5.

DRM patches localize to the flagellar axoneme. Cells washed with 1% Triton X-100 at 4°C retain patches of detergent-resistant membrane material along their flagellar axonemes, as visualized by scanning electron microscopy. (A) A more distal section and (B) a more proximal section of the single flagellum of a procyclic form parasite are shown. The patches of membrane (M) revealed are restricted to the flagellar axoneme (A) and absent from the paraflagellar rod (P). Some particles are also seen associated with subpellicular microtubules (T), but these are at much lower density than those distributed along the flagellar axoneme. (C) Micrograph of unextracted procyclic flagellum (no Triton X-100). Patches are most abundant at the flagellar tip and are larger in the ergosterol-containing procyclic form (D) than in the cholesterol-containing bloodstream form (E). Patches are absent from the flagellum and flagellar tip of bloodstream forms pretreated with 20 mM MBCD to deplete cholesterol (F). (G) Micrograph of unextracted bloodform flagellum (no Triton X-100). Bars, 200 nm.