Identification and Characterization of an Unusual Class I Myosin Involved in Vesicle Traffic in Trypanosoma brucei

Abstract

Myosins are a multimember family of motor proteins with diverse functions in eukaryotic cells. African trypanosomes possess only two candidate myosins and thus represent a useful system for functional analysis of these motors. One of these candidates is an unusual class I myosin (TbMyo1) that is expressed at similar levels but organized differently during the life cycle of Trypanosoma brucei. This myosin localizes to the polarized endocytic pathway in bloodstream forms of the parasite. This organization is actin dependent. Knock down of TbMyo1 results in a significant reduction in endocytic activity, a cessation in cell division and eventually cell death. A striking morphological feature in these cells is an enlargement of the flagellar pocket, which is consistent with an imbalance in traffic to and from the surface. In contrast TbMyo1 is distributed throughout procyclic forms of the tsetse vector and a loss of ∼90% of the protein has no obvious effects on growth or morphology. These results reveal a life cycle stage specific requirement for this myosin in essential endocytic traffic and represent the first description of the involvement of a motor protein in vesicle traffic in these parasites.

Proteomic Analysis of Clathrin Interactions in Trypanosomes Reveals Dynamic Evolution of Endocytosis

“…Five TbCAPs, TbHsc70, TbMyo1, TbCAP100 TbCAP116 and TbCAP118 were up‐regulated in BSF (Table ). Upregulation of TbMyo1 is consistent with Spitznagel et al where a threefold increase in the BSF was also observed. TbCAP161, TbCAP292 and TbCAP336 are expressed at equivalent levels in both life stages, while TbCAP125 and TbCAP186 are downregulated in BSF by twofold.…”

“…TbCHC had the highest score and was recovered from all three experiments, as was the product of Tb11.01.3110 (TbHsc70), the ortholog of the clathrin uncoating ATPase of higher eukaryotes. The ORF Tb927.4.3380, which encodes myosin IB (TbMyo1) and is involved in endocytosis , as well as the ortholog of metazoan RME8 (Tb927.6.3500), which is involved in recycling of endocytic proteins , were both recovered from one of three independent experiments. No clathrin uncoating proteins or proteins homologous to auxilin for example were found; however for the latter there is no ortholog in the genome.…”

Nicotinamide Inhibits the Lysosomal Cathepsin b-like Protease and Kills African Trypanosomes

“…Endocytosis Assays-Endocytosis assays were performed with FITC-labeled tomato lectin as described previously (27). Briefly, nicotinamide-treated and untreated bloodstream trypanosomes (10 7 cells) were washed with PBS, centrifuged at 7000 rpm, and resuspended in 1 ml of free serum HMI-9 supplemented with 10 mg/ml of BSA containing tomato lectin-FITC conjugate (Sigma) at 20 g/ml.…”

“…clathrin and components of the actin cytoskeleton (26,27,34). However, the nicotinamide-induced morphological effects are apparent after 3 h, whereas in the case of RNAi cells, typically, inductions of 24 h or more are required for morphological pathologies to present themselves.…”

Functions of myosin motors tailored for parasitism

“…brucei possesses two myosins belonging to classes I (TbMyo1) and XXI (TbMyo2). TbMyo1 is an unusual class I myosin that localizes to the polarized endocytic pathway in the bloodstream form and is evenly distributed throughout the procyclic form (insect from) [17]. Knockdown of TbMyo1 impacts on cell growth and morphology in the bloodstream but not in the procyclic form ( Figure 1) [17,18].…”