Identification of paralogous life-cycle stage specific cytoskeletal proteins in the parasite Trypanosoma brucei

Abstract

The life cycle of the African trypanosome Trypanosoma brucei, is characterised by a transition between insect and mammalian hosts representing very different environments that present the parasite with very different challenges. These challenges are met by the expression of life-cycle stage-specific cohorts of proteins, which function in systems such as metabolism and immune evasion. These life-cycle transitions are also accompanied by morphological rearrangements orchestrated by microtubule dynamics and associated proteins of the subpellicular microtubule array. Here we employed a gel-based comparative proteomic technique, Difference Gel Electrophoresis, to identify cytoskeletal proteins that are expressed differentially in mammalian infective and insect form trypanosomes. From this analysis we identified a pair of novel, paralogous proteins, one of which is expressed in the procyclic form and the other in the bloodstream form. We show that these proteins, CAP51 and CAP51V, localise to the subpellicular corset of microtubules and are essential for correct organisation of the cytoskeleton and successful cytokinesis in their respective life cycle stages. We demonstrate for the first time redundancy of function between life-cycle stage specific paralogous sets in the cytoskeleton and reveal modification of cytoskeletal components in situ prior to their removal during differentiation from the bloodstream form to the insect form. These specific results emphasise a more generic concept that the trypanosome genome encodes a cohort of cytoskeletal components that are present in at least two forms with life-cycle stage-specific expression.

Figure 1. CAP51 and CAP51V are paralogous lifecycle stage-regulated cytoskeletal components.

(A) Sections from a 2D-DiGE comparison of detergent extracted Bloodstream forms and Procyclic forms (Full gels in Figure S1) showing views of the gel regions corresponding to CAP51 and CAP51V. The spot corresponding to CAP51 is present in samples from procyclic form only and the spot corresponding to CAP51V has greater density in bloodstream form samples. (B) Dotplot alignment of CAP51 (x axis) and CAP51V (y axis) protein sequences using a 4 residue window. CAP51V contains a 75 residue lysine rich insert that is not present in CAP51. (C) Western blot of detergent extracted bloodstream and procyclic form cells expressing YFP::Ty tagged CAP51V or CAP51. CAP51V::YFP::Ty is detected only in the bloodstream form and CAP51::YFP::Ty only in the procyclic form. A ponceau stain of the membrane showing the tubulin (Tub) region is included as an indication of relative loading. (D) Procyclic form cells expressing CAP51::YFP::Ty. CAP51 localises to the whole of the subpellicular corset apart from the extreme posterior end of the cell and is not present on the flagellum. (E) Bloodstream form cells expressing CAP51V::YFP::Ty. CAP51V also localises to the subpellicular corset with no signal detected on the flagellum. Red = whole cell body (WCB), blue = DAPI, bar = 5 µm.

Figure 2. CAP proteins during differentiation Monomorphic bloodstream form cells with CAP51V::YFP::Ty or CAP51::YFP::Ty expressed from an endogenous locus were induced to differentiate into procyclic forms by the addition of cis-aconitate and incubation at 28°c.

(A) CAP51V signal (green) disappears uniformly across the cell body during differentiation. (B) CAP51 signal (green) intrudes from the posterior end of the cell during the course of the differentiation and (C) cells with uniform fluorescence are detected by 48 hours after induction (white arrowhead). Red = WCB, blue = DAPI, bar = 5 µm. (D) Western blot of CAP51V::YTFP::Ty on detergent extracted cells using BB2. A single band is visible in bloodstream forms but over the time course a second band approximately 10 kDa larger than the first appears. Neither form of CAP51V is detectable by 72 hours after induction. Tubulin (ponceau) is shown as an indication of relative loading. (E) Western blot of CAP51::YFP::Ty on detergent extracted cells using BB2. CAP51 is undetectable in bloodstream form cells. A band of the correct predicted molecular mass appears by 24 hours, slightly later than the first detection of CAP5.5 (detected with the CAP5.5 antibody) at 16 hours. PFR2 (L8C4) is shown as an indication of relative loading.

Figure 3. Ablation of CAP51 and CAP51V leads to reduced growth rate and aberrant morphology.

(A, B) RNAi mediated ablation of CAP51 in procyclic forms (A) and CAP51V in bloodstream forms (B) results in reduced growth rate. Representative plots from three replicates are shown. Grey, RNAi induced; black non-induced. (C) Ablation of CAP51 in procyclic forms results in an accumulation of cells with aberrant nucleus/kinetoplast numbers including the production of 1K0N zoids. Cells with multiple nuclei (i.e. greater than 2) are classified as “Other”. Representative counts from three replicates are shown. (D) 72 hours after induction of RNAi against CAP51 in procyclic forms, multinucleate cells with multiple kinetoplasts and flagella and 1K0N zoids can readily be observed, green = PFR (L8C4). (E) RNAi against CAP51V in the bloodstream form. Cells undergo aberrant cytokinesis and cytoplasts that contain no nuclear or kinetoplast DNA (white asterisk) are also observed. Red = WCB. (F, G) RNAi against CAP51 in the procyclic form. CAP51 (green) is lost preferentially in the posterior portion of the cell which remains positive for other markers of the subpellicular corset (F) WCB (red) and (G) CAP5.5 (red). Cell morphology is distorted with the diameter of the midpoint of the cell being abnormally large compared to the anterior and posterior. (D–G) Blue = DAPI, bar = 5 µm.

Figure 4. Ablation of CAP proteins disrupts the organisation of the microtubule corset.

Thin section TEM of transverse sections through bloodstream form trypanosomes 24 hours after induction of RNAi against CAP51V. (A, B) The microtubules of the subpellicular array become disordered at the midpoint of the cell. Bar 800 nm (C, D) higher magnification view of part of the subpellicular array of A and B respectively. (E, F) Multiple layers of microtubules are present at the anterior end of the cell. Bar 400 nm (G, H) higher magnification view of part of the subpellicular array of E and F respectively. Similar disruptions to the organisation of the subpellicular corset can be seen in procyclic form sections 48 hours after induction of RNAi (not shown). N = Nucleus, F = flagellum.

Figure 5. Localisation of CAP proteins to the subpellicular microtubule array is not life-cycle stage dependant.

A) Ty::GFP::CAP51V and Ty::GFP::CAP51 expressed from inducible ectopic loci for 24 hours localise to the subpellicular array of procyclic form and bloodstream form cells respectively. Expression of the ectopic protein does not disrupt positioning of the nuclei and kinetoplasts of 2K2N cells in either life-cycle stage. Bar = 5 µm. (C, D) Simultaneous induction of RNAi against endogenous protein and expression of ectopic protein rescues the parental growth defect. (C) Ablation of CAP51 in procyclic forms reduces growth rate (grey) that is rescued by simultaneous expression of CAP51V (red). (D) Ablation of CAP51V in the bloodstream form reduces growth rate (grey) that can be rescued by simultaneous expression of CAP51 (red). Representative plots from three independent replicates are shown. Growth rates of non-induced cells (black) are comparable to that of rescued cells and expression of the endogenous protein with an N terminal GFP tag from an inducible ectopic locus did not rescue the growth defect (not shown).