Inorganic polyphosphate (PolyP) is a biological polymer that plays important roles in the cell physiology of both prokaryotic and eukaryotic organisms. Among the available methods for PolyP localization and quantification, a 4’,6-diamidino-2-phenylindole(DAPI)-based assay has been used for visualization of PolyP-rich organelles. Due to differences in DAPI permeability to different compartments and/or PolyP retention after fixation, a general protocol for DAPI-PolyP staining has not yet been established. Here, we tested different protocols for DAPI-PolyP detection in a range of samples with different levels of DAPI permeability, including subcellular fractions, free-living cells and cryosections of fixed tissues. Subcellular fractions of PolyP-rich organelles yielded DAPI-PolyP fluorescence, although those with a complex external layer usually required longer incubation times, previous aldehyde fixation and/or detergent permeabilization. DAPI-PolyP was also detected in cryosections of OCT-embedded tissues analyzed by multiphoton microscopy. In addition, a semi-quantitative fluorimetric analysis of DAPI-stained fractions showed PolyP mobilization in a similar fashion to what has been demonstrated with the use of enzyme-based quantitative protocols. Taken together, our results support the use of DAPI for both PolyP visualization and quantification, although specific steps are suggested as a general guideline for DAPI-PolyP staining in biological samples with different degrees of DAPI and PolyP permeability.

Target of rapamycin (TOR) kinases are highly conserved protein kinases that integrate signals from nutrients and growth factors to coordinate cell growth and cell cycle progression. It has been previously described that two TOR kinases control cell growth in the protozoan parasite Trypanosoma brucei, the causative agent of African trypanosomiasis. Here we studied an unusual TOR-like protein named TbTOR-like 1 containing a PDZ domain and found exclusively in kinetoplastids. TbTORlike 1 localizes to unique cytosolic granules. After hyperosmotic stress, the localization of the protein shifts to the cell periphery, different from other organelle markers. Ablation of TbTOR-like 1 causes a progressive inhibition of cell proliferation, producing parasites accumulating in the S/G 2 phase of the cell cycle. TbTOR-like 1 knocked down cells have an increased area occupied by acidic vacuoles, known as acidocalcisomes, and are enriched in polyphosphate and pyrophosphate. These results suggest that TbTOR-like 1 might be involved in the control of acidocalcisome and polyphosphate metabolism in T. brucei.African trypanosomiasis, or sleeping sickness, is a disease caused by the parasitic protist Trypanosoma brucei that affects a half-million people in sub-Saharan Africa. Transmitted by the tsetse fly vector (Glossina spp.), trypanosomiasis represents an important public health problem and has a strong impact in the economic development of that region. T. brucei has a complex life cycle involving different morphological and functional stages. These parasites alternate between insect and mammalian hosts. The adaptation to these diverse environments requires rapid changes in gene expression to fulfill metabolic or morphological changes (1). This parasite also has the ability to survive a wide range of environmental conditions as it progresses through its life cycle, including extreme fluctuations in external osmolarity (2).Target of rapamycin (TOR) 5 proteins are evolutionarily conserved protein kinases that integrate information from energy levels, mitogenic signals, and nutrient supplies regulating cell growth accordingly. Studies on mammalian cells and yeast signaling pathways have shown that nutrient starvation inhibits TOR activities, which results in G 1 cell cycle arrest, and triggers a stress response program leading to a blockade of translation initiation. Similar stress responses can be observed in cells treated with rapamycin, an immunosuppressant drug, which binds to FKBP12, a prolyl-isomerase, forming a complex with the TOR kinase (reviewed in Refs. 3 and 4).Two distinct aspects of cell growth are regulated by two functionally different TOR kinases in T. brucei, termed TbTOR1 and TbTOR2. TbTOR1 and TbTOR2 function in two distinct TOR-containing multiprotein complexes (TORC) conserved along eukaryote evolution, named TORC1 and TORC2. TbTOR1, located in the nucleus, controls the synthesis and accumulation of cell mass through TORC1 signaling, whereas TbTOR2, associated with the mitochondrion or endoplasmic reticulum, con...

The structural organization of Trypanosoma cruzi has been intensely investigated by different microscopy techniques. At the electron microscopy level, bi-dimensional analysis of thin sections of chemically fixed cells has been one of the most commonly used techniques, despite the known potential of generating artifacts during chemical fixation and the subsequent steps of sample preparation. In contrast, more sophisticated and elaborate techniques, such as cryofixation followed by freeze substitution that are known to preserve the samples in a more close-to-native state, have not been widely applied to T. cruzi. In addition, the 3D characterization of such cells has been carried out mostly using 3D reconstruction from serial sections, currently considered a low resolution technique when compared to electron tomography (ET). In this work, we re-visited the 3D ultrastructure of T. cruzi using a combination of two approaches: (1) analysis of both conventionally processed and cryofixed and freeze substituted cells and (2) 3D reconstruction of large volumes by serial electron tomography. The analysis of high-pressure frozen and freeze substituted parasites showed novel characteristics in a number of intracellular structures, both in their structure and content. Organelles generally showed a smooth and regular morphology in some cases presenting a characteristic electron dense content. Ribosomes and new microtubule sets showed an unexpected localization in the cell body. The improved preservation and imaging in 3D of T. cruzi cells using cryopreparation techniques has revealed some novel aspects of the ultrastructural organization of this parasite.

Trypanosoma cruzi, the etiological agent of Chagas disease, has the ability to respond to a variety of environmental changes during its life cycle both in the insect vector and in the vertebrate host. Because regulation of transcription initiation seems to be nonfunctional in this parasite, it is important to investigate other regulatory mechanisms of adaptation. Regulatory mechanisms at the level of signal transduction pathways involving phosphoinositides are good candidates for this purpose. Here we report the identification of the first phosphatidylinositol 3-kinase (PI3K) in T. cruzi, with similarity with its yeast counterpart, Vps34p. TcVps34 specifically phosphorylates phosphatidylinositol to produce phosphatidylinositol 3-phosphate, thus confirming that it belongs to class III PI3K family. Overexpression of TcVps34 resulted in morphological and functional alterations related to vesicular trafficking. Although inhibition of TcVps34 with specific PI3K inhibitors, such as wortmannin and LY294,000, resulted in reduced regulatory volume decrease after hyposmotic stress, cells overexpressing this enzyme were resistant to these inhibitors. Furthermore, these cells were able to recover their original volume faster than wild type cells when they were submitted to severe hyposmotic stress. In addition, in TcVps34-overexpressing cells, the activities of vacuolar-H ؉ -ATPase and vacuolar H ؉ -pyrophosphatase were altered, suggesting defects in the acidification of intracellular compartments. Furthermore, receptor-mediated endocytosis was partially blocked although fluid phase endocytosis was not affected, confirming a function for TcVps34 in membrane trafficking. Taken together, these results strongly support that TcVps34 plays a prominent role in vital processes for T. cruzi survival such as osmoregulation, acidification, and vesicular trafficking.Phosphatidylinositol 3-kinase (PI3K) 6 activities have been found in all eukaryotic cell types examined to date (1, 2) and are linked to a diverse set of key cellular functions, including cell growth, survival, and intracellular trafficking. PI3Ks belong to a large family of enzymes that has been divided into three functional classes on the basis of their protein domain structure, lipid substrate specificity, and regulatory properties. Class I PI3Ks were the first ones to be identified and are important components of the signaling pathways that regulate eukaryotic cell growth (3, 4). These PI3Ks have a 110-kDa catalytic subunit that exhibits a substrate preference for PI 4-phosphate and PI 4,5-bisphosphate (5). Class II PI3Ks are less well known but may also function in the regulation of cell growth (5, 6) and, additionally, in clathrin-mediated endocytosis (7, 8). These enzymes prefer phosphatidylinositol (PI) as substrate but may also utilize PI 4-phosphate. Finally, class III family of PI3Ks is related to the yeast vacuolar protein sorting 34, Vps34p, and their homologs from other eukaryotes. Vps34p-like kinases specifically phosphorylate PI to produce phosphatidylinosi...