Control of assembly of extra-axonemal structures: The paraflagellar rod of trypanosomes

Eukaryotic flagella are complex microtubule based organelles and in many organisms there are extra axonemal structures present, including the outer dense fibres of mammalian sperm and the paraflagellar rod (PFR) of trypanosomes. Flagellum assembly is a complex process occurring across three main compartments, the cytoplasm, the transition fibre-transition zone, and the flagellum. It begins with translation of protein components, followed by their sorting and trafficking into the flagellum, transport to the assembly site and then incorporation. Flagella are formed from over 500 proteins; the principles governing axonemal component assembly are relatively clear. However, the coordination and sites of extra-axonemal structure assembly processes are less clear. We have discovered two cytoplasmic proteins in T. brucei that are required for PFR formation, PFR assembly factors 1 and 2. Deletion of either PFR-AF1 or PFR-AF2 dramatically disrupted PFR formation and caused a reduction in the amount of major PFR proteins. The presence of cytoplasmic factors required for PFR formation aligns with the concept of processes occurring across multiple compartments to facilitate axoneme assembly and this is likely a common theme for extra-axonemal structure assembly

Two special organelles found in Trypanosoma cruzi

We review here two unique organelles from Trypanosoma cruzi. One of them is the acidocalcisome, cytoplasmic vacuoles containing a very high Ca2+ concentration and a Ca2+ - H+ translocating ATPase activity, present in all trypanosomatids. The other organelle is the reservosome, site of accumulation of endocytosed macromolecules, very rich in cysteine proteinase, that is present only in epimastigote forms of trypanosomes belonging to the Schyzotrypanum sub-genus

New insights into the morphology of Trypanosoma cruzi reservosome

Reservosomes are late endosomes present only in members of the Schizotrypanum subgenus of the Trypanosoma genus and are defined as the site of storage of endocytosed macromolecules and lysosomal enzymes. They have been extensively described in Trypanosoma cruzi epimastigote: are bounded by a membrane unit, present an electron-dense protein matrix with electron-lucent lipid inclusions, being devoid of inner membranes. Here we performed a detailed ultrastructural analysis of these organelles using a variety of electron microscopy techniques, including ultrathin sectioning, uranyl acetate stained preparations, and freeze fracture, either in intact epimastigotes or in isolated reservosomes. New informations were obtained. First, both isolated and in situ reservosomes presented small profiles of inner membranes that are morphologically similar to the membrane surrounding the organelle. In uranyl acetate stained preparations, internal membrane profiles turned out to be longer than they appeared in ultrathin section images and traversed the organelle diameter. Internal vesicles were also found. Second, endocytosed cargo are not associated with internal vesicles and reach reservosomes on board of vesicles that fuse with the boundary membrane, delivering cargo directly into reservosome lumen. Third, electron-lucent bodies with saturated lipid core surrounded by a membrane monolayer and with unusual rectangular shape were also observed. Fourth, it was possible to demonstrate the presence of intramembranous particles on the E face of both internal vesicles and the surrounding membrane. Collectively, these results indicate that reservosomes have a complex internal structure, which may correlate with their multiple functions

ACAT-related activity assay.

<p>The ability of <i>T</i>. <i>cruzi</i> epimastigotes to esterify cholesterol was tested using Sandoz 58-035-58-035 for 16–18 h (A) or Avasimibe for 24 h (B) in different concentrations, followed by addition of BSA-³H-palmitate (800,000 DPM; 1 mg/mL) for 24 h. Lipid analysis was performed by TLC; CHOE spots were scraped and the lipid eluted from silica. The lipid-associated radioactivity was measured by liquid scintillation counting. The results are expressed as the mean (±SD) per 1 mg of protein of two independent experiments in duplicate, and analysed by one-way ANOVA followed by the Bonferroni test (*<i>P</i><0.05).</p

<i>Trypanosoma cruzi</i> Epimastigotes Are Able to Manage Internal Cholesterol Levels under Nutritional Lipid Stress Conditions

<div><p><i>Trypanosoma cruzi</i> epimastigotes store high amounts of cholesterol and cholesteryl esters in reservosomes. These unique organelles are responsible for cellular digestion by providing substrates for homeostasis and parasite differentiation. Here we demonstrate that under nutritional lipid stress, epimastigotes preferentially mobilized reservosome lipid stocks, instead of lipid bodies, leading to the consumption of parasite cholesterol reservoirs and production of ergosterol. Starved epimastigotes acquired more LDL-NBD-cholesterol by endocytosis and distributed the exogenous cholesterol to their membranes faster than control parasites. Moreover, the parasites were able to manage internal cholesterol levels, alternating between consumption and accumulation. With normal lipid availability, parasites esterified cholesterol exhibiting an ACAT-like activity that was sensitive to Avasimibe in a dose-dependent manner. This result also implies that exogenous cholesterol has a role in lipid reservoirs in epimastigotes.</p></div

Free and total (after saponification) cholesterol and ergosterol contents in lipid starved <i>T</i>. <i>cruzi</i> epimastigotes, kept in LIT medium supplemented with 10% dFCS for 0, 24, 48 and 72h.

<p>All samples correspond to 5 μg/mL of protein.</p><p>Free and total (after saponification) cholesterol and ergosterol contents in lipid starved <i>T</i>. <i>cruzi</i> epimastigotes, kept in LIT medium supplemented with 10% dFCS for 0, 24, 48 and 72h.</p

Incorporation of ³H-cholesterol into cholesteryl esters in epimastigotes.

<p>Parasites were incubated in LIT medium with 10% FCS (control) or 10% dFCS (starved) in the presence of LDL-³H-cholesterol (1,000,000 DPM; 2 mg/mL) for 3 days at 28°C. Parasites were washed and their lipids were extracted and analysed by TLC. The cholesterol (CHO) and cholesteryl-ester (CHOE) spots were scraped and the lipid eluted from silica. The lipid-associated radioactivity was measured by liquid scintillation counting. The results are expressed as the mean (±SD) per 1 mg of protein of three independent experiments analysed by one-way ANOVA followed by the Bonferroni test (*<i>P</i><0.05).</p