Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

Abstract

Sphingolipids and their metabolites have been thought crucial for cell growth and cell cycle progression, membrane and protein trafficking, signal transduction, and formation of lipid rafts; however, recent studies in trypanosomes point to the dispensability of sphingolipids in some of these processes. In this study, we explore the requirements for de novo sphingolipid biosynthesis in the insect life cycle stage of the African trypanosome Trypanosoma brucei by inhibiting the enzyme serine palmitoyltransferase (SPT2) by using RNA interference or treatment with a potent SPT2 inhibitor myriocin. Mass spectrometry revealed that upon SPT2 inhibition, the parasites contained substantially reduced levels of inositolphosphorylceramide. Although phosphatidylcholine and cholesterol levels were increased to compensate for this loss, the cells were ultimately not viable. The most striking result of sphingolipid reduction in procyclic T. brucei was aberrant cytokinesis, characterized by incomplete cleavage-furrow formation, delayed kinetoplast segregation and emergence of cells with abnormal DNA content. Organelle replication continued despite sphingolipid depletion, indicating that sphingolipids act as second messengers regulating cellular proliferation and completion of cytokinesis. Distention of the mitochondrial membrane, formation of multilamellar structures within the mitochondrion and near the nucleus, accumulation of lipid bodies and, less commonly, disruption of the Golgi complex were observed after prolonged sphingolipid depletion. These findings suggest that some aspects of vesicular trafficking may be compromised. However, flagellar membrane targeting and the association of the flagellar membrane protein calflagin with detergent-resistant membranes were not affected, indicating that the vesicular trafficking defects were mild. Our studies indicate that sphingolipid biosynthesis is vital for cell cycle progression and cell survival, but not essential for the normal trafficking of flagellar membrane-associated proteins or lipid raft formation in procyclic T. brucei.

Flagellar membrane localization via association with lipid rafts

“…In the second, depletion of sphingolipids in T. brucei bloodstream forms disrupted lipid rafts and abolished the buoyancy of calflagin on a sucrose gradient (Fridberg et al, 2008). Although our results pertain primarily to the trypanosome flagellum and its unique biochemistry, the adjacent flagellar pocket has a unique biochemistry of its own, is sterol rich, contains uniquely localizing glycolipids and is functionally disrupted by ablation of sphingolipids (Fridberg et al, 2008;Tetley, 1986). Although the flagellum arises from the flagellar pocket, membranes of the two surface domains are physically partitioned by a unique structure, a collar that may be a physical barrier to diffusion of some membrane macromolecules (Tetley, 1986).…”

“…In the first, Leishmania adaptor protein 1 was implicated in a variety of lipid raft-associated activities, including flagellar biogenesis, with sphingolipid and sterol inhibitors exacerbating flagellar biogenesis defects observed in defective mutants (Vince et al, 2008). In the second, depletion of sphingolipids in T. brucei bloodstream forms disrupted lipid rafts and abolished the buoyancy of calflagin on a sucrose gradient (Fridberg et al, 2008). Although our results pertain primarily to the trypanosome flagellum and its unique biochemistry, the adjacent flagellar pocket has a unique biochemistry of its own, is sterol rich, contains uniquely localizing glycolipids and is functionally disrupted by ablation of sphingolipids (Fridberg et al, 2008;Tetley, 1986).…”

“…On western blots, antiserum to Tb24 reacts with calflagin paralogs Tb24, Tb44 and Tb17 (>95%), which are almost identical in sequence (data not shown). In the procyclic form, Tb24 is the dominant paralog and appears as a singlet; in the bloodstream form, Tb17 is expressed at roughly similar levels to Tb24 and the two proteins appear as a doublet (Fridberg et al, 2008). Antiserum specific for galactocerebroside was purchased from Sigma-Aldrich (St Louis, MO) and EP procyclin (Cedar Lane Laboratories, Ontario, Canada).…”

Rab28 function in trypanosomes: interactions with retromer and ESCRT pathways

“…Disruption of sphingolipid biosynthesis through inhibition of serine palmitoyltransferase has a similar effect on the Golgi complex, but the effect is relatively mild (Fridberg et al, 2008). However, disruption of Golgi morphology upon depletion of T. brucei Rab28 parallels Vps26 knockdown in mammalian cells and trypanosomes (Seaman, 2004;Koumandou et al, 2011).…”

Match-making for posaconazole through systems thinking

“…Endogenous sterols and sphingolipids are required for proliferation of trypanosomes [38][39][40]. Interestingly, reduced inositolphosphoceramide (IPC) levels due to inhibition of serine palmitoyltransferase (Spt2) in T. brucei have been shown to be compensated for by increased levels of phosphatidylcholine and cholesterol, demonstrating a tight interaction of sterol and sphingolipid homeostasis [41]. As in yeast, IPC rather than glycerophospholipids is utilized as lipid anchor constituent of glycoproteins and free glycosylinositolphospholipids (GIPLs) in T. cruzi [42].…”