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Flagellar membranes are rich in raft-forming phospholipids.

Serricchio M, Schmid AW, Steinmann ME, Sigel E, Rauch M, Julkowska D, Bonnefoy S, Fort C, Bastin P, Bütikofer P - Biol Open (2015)

Bottom Line: Our analyses revealed that phosphatidylethanolamine, phosphatidylserine, ceramide and the sphingolipids inositol phosphorylceramide and sphingomyelin are enriched in flagella relative to whole cells.Within individual glycerophospholipid classes, we observed a preference for ether-type over diacyl-type molecular species in membranes of flagella.Our study provides direct evidence for a preferential presence of raft-forming phospholipids in flagellar membranes of T. brucei.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry & Molecular Medicine, University of Bern, Bern 3012, Switzerland peter.buetikofer@ibmm.unibe.ch mauro.serricchio@utoronto.ca.

No MeSH data available.


Related in: MedlinePlus

Isolation of pure flagella. (A) Growth curve of trypanosomes upon tetracycline-induction of double-stranded RNA targeting Tb927.10.2880. Knockdown of Tb927.10.2880 results in a growth defect after 2 days of induction. The inset shows the Northern blot analysis confirming the disappearance of the corresponding mRNA upon induction (top) and the ethidium bromide stained rRNA as loading control (bottom). (B) Differential interference contrast (DIC) micrographs of uninduced (− tet) and RNAi induced (+ tet) parasites. After 1 day of induction, flagella appear detached from the cell bodies. (C) DIC micrograph of isolated flagella. Mitochondrial and nuclear DNA was stained with DAPI, imaged by fluorescence microscopy and is shown in blue. Scale bar: 10 µm. (D) Flagella were spread on slides, fixed and processed for double immunofluorescence with the axoneme marker Mab25 (top panel) and with anti-calflagin as a membrane marker (middle panel). Merged images are shown at the bottom as indicated. Flagella commonly tend to curve under these conditions. Scale bars: 5 µm. (E) Flagellar preparations were fixed, sectioned and examined by transmission electron microscopy. Recognisable elements were overwhelmingly flagella and half of them possessed a membrane (black arrows). Axonemes apparently deprived of their membrane are indicated with a white arrow. Scale bar: 500 nm.
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BIO011957F1: Isolation of pure flagella. (A) Growth curve of trypanosomes upon tetracycline-induction of double-stranded RNA targeting Tb927.10.2880. Knockdown of Tb927.10.2880 results in a growth defect after 2 days of induction. The inset shows the Northern blot analysis confirming the disappearance of the corresponding mRNA upon induction (top) and the ethidium bromide stained rRNA as loading control (bottom). (B) Differential interference contrast (DIC) micrographs of uninduced (− tet) and RNAi induced (+ tet) parasites. After 1 day of induction, flagella appear detached from the cell bodies. (C) DIC micrograph of isolated flagella. Mitochondrial and nuclear DNA was stained with DAPI, imaged by fluorescence microscopy and is shown in blue. Scale bar: 10 µm. (D) Flagella were spread on slides, fixed and processed for double immunofluorescence with the axoneme marker Mab25 (top panel) and with anti-calflagin as a membrane marker (middle panel). Merged images are shown at the bottom as indicated. Flagella commonly tend to curve under these conditions. Scale bars: 5 µm. (E) Flagellar preparations were fixed, sectioned and examined by transmission electron microscopy. Recognisable elements were overwhelmingly flagella and half of them possessed a membrane (black arrows). Axonemes apparently deprived of their membrane are indicated with a white arrow. Scale bar: 500 nm.

Mentions: In T. brucei, the flagellum extends along and is closely attached to the cell body (Kohl and Bastin, 2005). It has previously been shown that RNAi-mediated down-regulation of expression of a putative calcium channel protein (Tb927.10.2880) in T. brucei bloodstream forms resulted in detachment of the flagellum from the cell body (Oberholzer et al., 2011). We now show that a similar phenotype is also observed in procyclic forms. Tetracycline-inducible expression of a hairpin RNA targeting Tb927.10.2880 mRNA led to its strong depletion as confirmed by Northern blotting (Fig. 1A, inset), resulting in a strong growth defect after two days of culture (Fig. 1A), a feature noted for most mutants where flagellum adhesion is compromised (LaCount et al., 2002; Rotureau et al., 2014; Sun et al., 2013; Sunter et al., 2015; Zhou et al., 2010, 2015). Down-regulation of Tb927.10.2880 expression resulted in detachment of the flagellum (Fig. 1B), allowing its release from the parasite body by use of mechanical forces and subsequent isolation of detached flagella using previously published procedures (Oberholzer et al., 2011; Subota et al., 2014). Examination by light microscopy demonstrated that the preparation contains pure flagella, with no visible contamination by intact trypanosomes or remnant cell bodies (Fig. 1C). Approximately 30–50% of purified flagella contained kinetoplasts (mitochondrial DNA) attached to the basal bodies of flagella. In trypanosomes, the mitochondrial DNA is physically linked to the basal body by specific cytoskeletal structures (Ogbadoyi et al., 2003; Robinson and Gull, 1991). To evaluate the quality of the preparation, samples were double stained with antibody markers for the axoneme (MAP6-related protein) (Dacheux et al., 2012) and for the flagellar membrane (calflagins) (Engman et al., 1989). All flagella appear positive for the anti-axoneme marker whereas some but not all were strongly positive for the membrane marker (Fig. 1D). The rest of the population produced weaker, yet positive signals. Next, the preparations were fixed and analysed by transmission electron microscopy (Fig. 1E). The vast majority of the sections were across flagella and very few contaminants were observed, confirming the high enrichment in flagella. Closer examination revealed that 54% of the axonemes (black arrows) were wrapped by a membrane (n=81). We regularly noticed the presence of membrane “remnants” that may have come off the flagella during the purification process. Overall, these results demonstrate the quality of the flagella preparations, with more than half of the flagella possessing an intact membrane.Fig. 1.


Flagellar membranes are rich in raft-forming phospholipids.

Serricchio M, Schmid AW, Steinmann ME, Sigel E, Rauch M, Julkowska D, Bonnefoy S, Fort C, Bastin P, Bütikofer P - Biol Open (2015)

Isolation of pure flagella. (A) Growth curve of trypanosomes upon tetracycline-induction of double-stranded RNA targeting Tb927.10.2880. Knockdown of Tb927.10.2880 results in a growth defect after 2 days of induction. The inset shows the Northern blot analysis confirming the disappearance of the corresponding mRNA upon induction (top) and the ethidium bromide stained rRNA as loading control (bottom). (B) Differential interference contrast (DIC) micrographs of uninduced (− tet) and RNAi induced (+ tet) parasites. After 1 day of induction, flagella appear detached from the cell bodies. (C) DIC micrograph of isolated flagella. Mitochondrial and nuclear DNA was stained with DAPI, imaged by fluorescence microscopy and is shown in blue. Scale bar: 10 µm. (D) Flagella were spread on slides, fixed and processed for double immunofluorescence with the axoneme marker Mab25 (top panel) and with anti-calflagin as a membrane marker (middle panel). Merged images are shown at the bottom as indicated. Flagella commonly tend to curve under these conditions. Scale bars: 5 µm. (E) Flagellar preparations were fixed, sectioned and examined by transmission electron microscopy. Recognisable elements were overwhelmingly flagella and half of them possessed a membrane (black arrows). Axonemes apparently deprived of their membrane are indicated with a white arrow. Scale bar: 500 nm.
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Related In: Results  -  Collection

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BIO011957F1: Isolation of pure flagella. (A) Growth curve of trypanosomes upon tetracycline-induction of double-stranded RNA targeting Tb927.10.2880. Knockdown of Tb927.10.2880 results in a growth defect after 2 days of induction. The inset shows the Northern blot analysis confirming the disappearance of the corresponding mRNA upon induction (top) and the ethidium bromide stained rRNA as loading control (bottom). (B) Differential interference contrast (DIC) micrographs of uninduced (− tet) and RNAi induced (+ tet) parasites. After 1 day of induction, flagella appear detached from the cell bodies. (C) DIC micrograph of isolated flagella. Mitochondrial and nuclear DNA was stained with DAPI, imaged by fluorescence microscopy and is shown in blue. Scale bar: 10 µm. (D) Flagella were spread on slides, fixed and processed for double immunofluorescence with the axoneme marker Mab25 (top panel) and with anti-calflagin as a membrane marker (middle panel). Merged images are shown at the bottom as indicated. Flagella commonly tend to curve under these conditions. Scale bars: 5 µm. (E) Flagellar preparations were fixed, sectioned and examined by transmission electron microscopy. Recognisable elements were overwhelmingly flagella and half of them possessed a membrane (black arrows). Axonemes apparently deprived of their membrane are indicated with a white arrow. Scale bar: 500 nm.
Mentions: In T. brucei, the flagellum extends along and is closely attached to the cell body (Kohl and Bastin, 2005). It has previously been shown that RNAi-mediated down-regulation of expression of a putative calcium channel protein (Tb927.10.2880) in T. brucei bloodstream forms resulted in detachment of the flagellum from the cell body (Oberholzer et al., 2011). We now show that a similar phenotype is also observed in procyclic forms. Tetracycline-inducible expression of a hairpin RNA targeting Tb927.10.2880 mRNA led to its strong depletion as confirmed by Northern blotting (Fig. 1A, inset), resulting in a strong growth defect after two days of culture (Fig. 1A), a feature noted for most mutants where flagellum adhesion is compromised (LaCount et al., 2002; Rotureau et al., 2014; Sun et al., 2013; Sunter et al., 2015; Zhou et al., 2010, 2015). Down-regulation of Tb927.10.2880 expression resulted in detachment of the flagellum (Fig. 1B), allowing its release from the parasite body by use of mechanical forces and subsequent isolation of detached flagella using previously published procedures (Oberholzer et al., 2011; Subota et al., 2014). Examination by light microscopy demonstrated that the preparation contains pure flagella, with no visible contamination by intact trypanosomes or remnant cell bodies (Fig. 1C). Approximately 30–50% of purified flagella contained kinetoplasts (mitochondrial DNA) attached to the basal bodies of flagella. In trypanosomes, the mitochondrial DNA is physically linked to the basal body by specific cytoskeletal structures (Ogbadoyi et al., 2003; Robinson and Gull, 1991). To evaluate the quality of the preparation, samples were double stained with antibody markers for the axoneme (MAP6-related protein) (Dacheux et al., 2012) and for the flagellar membrane (calflagins) (Engman et al., 1989). All flagella appear positive for the anti-axoneme marker whereas some but not all were strongly positive for the membrane marker (Fig. 1D). The rest of the population produced weaker, yet positive signals. Next, the preparations were fixed and analysed by transmission electron microscopy (Fig. 1E). The vast majority of the sections were across flagella and very few contaminants were observed, confirming the high enrichment in flagella. Closer examination revealed that 54% of the axonemes (black arrows) were wrapped by a membrane (n=81). We regularly noticed the presence of membrane “remnants” that may have come off the flagella during the purification process. Overall, these results demonstrate the quality of the flagella preparations, with more than half of the flagella possessing an intact membrane.Fig. 1.

Bottom Line: Our analyses revealed that phosphatidylethanolamine, phosphatidylserine, ceramide and the sphingolipids inositol phosphorylceramide and sphingomyelin are enriched in flagella relative to whole cells.Within individual glycerophospholipid classes, we observed a preference for ether-type over diacyl-type molecular species in membranes of flagella.Our study provides direct evidence for a preferential presence of raft-forming phospholipids in flagellar membranes of T. brucei.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry & Molecular Medicine, University of Bern, Bern 3012, Switzerland peter.buetikofer@ibmm.unibe.ch mauro.serricchio@utoronto.ca.

No MeSH data available.


Related in: MedlinePlus