9 research outputs found
Identification of Contractile Vacuole Proteins in Trypanosoma cruzi
Contractile vacuole complexes are critical components of cell volume regulation
and have been shown to have other functional roles in several free-living
protists. However, very little is known about the functions of the contractile
vacuole complex of the parasite Trypanosoma cruzi, the
etiologic agent of Chagas disease, other than a role in osmoregulation.
Identification of the protein composition of these organelles is important for
understanding their physiological roles. We applied a combined proteomic and
bioinfomatic approach to identify proteins localized to the contractile vacuole.
Proteomic analysis of a T. cruzi fraction enriched for
contractile vacuoles and analyzed by one-dimensional gel electrophoresis and
LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of
expressed proteins of epimastigotes. We also identified different peptides that
map to at least 39 members of the dispersed gene family 1 (DGF-1) providing
evidence that many members of this family are simultaneously expressed in
epimastigotes. Of the proteins present in the fraction we selected several
homologues with known localizations in contractile vacuoles of other organisms
and others that we expected to be present in these vacuoles on the basis of
their potential roles. We determined the localization of each by expression as
GFP-fusion proteins or with specific antibodies. Six of these putative proteins
(Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter)
predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and
calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our
results demonstrate the utility of combining subcellular fractionation,
proteomic analysis, and bioinformatic approaches for localization of organellar
proteins that are difficult to detect with whole cell methodologies. The CV
localization of the proteins investigated revealed potential novel roles of
these organelles in phosphate metabolism and provided information on the
potential participation of adaptor protein complexes in their biogenesis
Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.
In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field
Methods to Investigate Signal Transduction Pathways in Trypanosoma cruzi: Cyclic Nucleotide Phosphodiesterases Assay Protocols
Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases (PDEs). Most of the different PDE variants play specific physiological functions; in fact, PDEs can associate with other proteins allowing them to be strategically anchored throughout the cell. In this regard, precise cellular expression and compartmentalization of these enzymes produce the specific control of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) gradients in cells and enable their integration with other signaling pathways. In trypanosomatids, some PDEs are essential for their survival and play fundamental roles in the adaptation of these parasites to different environmental stresses, as well as in the differentiation between their different life cycle forms. Given that these enzymes not only are similar to human PDEs but also have differential biochemical properties, and due to the great knowledge of drugs that target human PDEs, trypanosomatid PDEs could be postulated as important therapeutic targets through the repositioning of drugs. In this chapter, we describe a simple and sensitive radioisotope-based method to measure cyclic 3âČ,5âČ-nucleotide phosphodiesterase using [3H]cAMP.Fil: Schoijet, Alejandra Cecilia. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Instituto de Investigaciones en IngenierĂa GenĂ©tica y BiologĂa Molecular "Dr. HĂ©ctor N. Torres"; ArgentinaFil: Sternlieb, Tamara. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Instituto de Investigaciones en IngenierĂa GenĂ©tica y BiologĂa Molecular "Dr. HĂ©ctor N. Torres"; ArgentinaFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Instituto de Investigaciones en IngenierĂa GenĂ©tica y BiologĂa Molecular "Dr. HĂ©ctor N. Torres"; Argentin