Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)

Souza, Ligia Cristina Kalb; Pinho, Rosana Elisa Gonçalves Gonçalves; Lima, Carla Vanessa de Paula; Fragoso, Stênio Perdigão; Soares, Maurílio J.

doi:10.1590/0074-0276108052013015

Cited by 10 publications

(4 citation statements)

References 20 publications

(46 reference statements)

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“…In trypomastigotes, TcAct1 was uniformly distributed with a low level of staining ( Cevallos et al, 2011 ), and in the cell-derived amastigotes, a heterogeneous TcAct1 localization with sometimes no apparent expression was observed ( Cevallos et al, 2011 ). Similar protein expression levels and intracellular TcAct1 distributions in epimastigotes, amastigotes and metacyclic trypomastigotes were also observed by using mouse polyclonal anti-recombinant TcAct1 antibodies ( Souza et al, 2013 ). In addition to TcAct1, TcAct2 has also been characterized.…”

Section: Trypanosomatid Actins and Actin Binding Proteinssupporting

confidence: 55%

Emerging Functions of Actins and Actin Binding Proteins in Trypanosomatids

Gupta

Ambaru

Bajaj

2020

Front. Cell Dev. Biol.

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Actin is the major protein constituent of the cytoskeleton that performs wide range of cellular functions. It exists in monomeric and filamentous forms, dynamics of which is regulated by a large repertoire of actin binding proteins. However, not much was known about existence of these proteins in trypanosomatids, till the genome sequence data of three important organisms of this class, viz. Trypanosoma brucei , Trypanosoma cruzi and Leishmania major , became available. Here, we have reviewed most of the findings reported to date on the intracellular distribution, structure and functions of these proteins and based on them, we have hypothesized some of their functions. The major findings are as follows: (1) All the three organisms encode at least a set of ten actin binding proteins (profilin, twinfilin, ADF/cofilin, CAP/srv2, CAPz, coronin, two myosins, two formins) and one isoform of actin, except that T. cruzi encodes for three formins and several myosins along with four actins. (2) Actin 1 and a few actin binding proteins (ADF/cofilin, profilin, twinfilin, coronin and myosin13 in L. donovani ; ADF/cofilin, profilin and myosin1 in T. brucei ; profilin and myosin-F in T.cruzi ) have been identified and characterized. (3) In all the three organisms, actin cytoskeleton has been shown to regulate endocytosis and intracellular trafficking. (4) Leishmania actin1 has been the most characterized protein among trypanosomatid actins. (5) This protein is localized to the cytoplasm as well as in the flagellum, nucleus and kinetoplast, and in vitro , it binds to DNA and displays scDNA relaxing and kDNA nicking activities. (6) The pure protein prefers to form bundles instead of thin filaments, and does not bind DNase1 or phalloidin. (7) Myosin13, myosin1 and myosin-F regulate endocytosis and intracellular trafficking, respectively, in Leishmania , T. brucei and T. cruzi . (8) Actin-dependent myosin13 motor is involved in dynamics and assembly of Leishmania flagellum. (9) Leishmania twinfilin localizes mostly to the nucleolus and coordinates karyokinesis by effecting splindle elongation and DNA synthesis. (10) Leishmania coronin binds and promotes actin filament formation and exists in tetrameric form rather than trimeric form, like other coronins. (11) Trypanosomatid profilins are essential for survival of all the three parasites.

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Section: Trypanosomatid Actins and Actin Binding Proteinssupporting

confidence: 55%

Emerging Functions of Actins and Actin Binding Proteins in Trypanosomatids

Gupta

Ambaru

Bajaj

2020

Front. Cell Dev. Biol.

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“…Following protein transfer, the membranes were blocked with 5% non-fat milk/0.05% Tween-20 in PBS and incubated for 1 h with blocking buffer containing either the antiserum raised against the TcCLC protein (1:200 dilution) or the monoclonal antibody against TcCHC. A polyclonal anti-TcActin antibody [ 37 ] was used as a loading control. After 3 washes with 0.05% Tween-20/PBS, the membranes were incubated for 1 h with alkaline phosphatase-conjugated rabbit anti-mouse IgG (1:10,000 dilution), washed three times with 0.05% Tween-20/PBS and bands then visualized using BCIP-NBT (Promega, USA) solution.…”

Section: Methodsmentioning

confidence: 99%

Clathrin expression in Trypanosoma cruzi

et al. 2014

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BackgroundClathrin-mediated vesicular trafficking, the mechanism by which proteins and lipids are transported between membrane-bound organelles, accounts for a large proportion of import from the plasma membrane (endocytosis) and transport from the trans-Golgi network towards the endosomal system. Clathrin-mediated events are still poorly understood in the protozoan Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. In this study, clathrin heavy (TcCHC) and light (TcCLC) chain gene expression and protein localization were investigated in different developmental forms of T. cruzi (epimastigotes, trypomastigotes and amastigotes), using both polyclonal and monoclonal antibodies raised against T. cruzi recombinant proteins.ResultsAnalysis by confocal microscopy revealed an accumulation of TcCHC and TcCLC at the cell anterior, where the flagellar pocket and Golgi complex are located. TcCLC partially colocalized with the Golgi marker TcRAB7-GFP and with ingested albumin, but did not colocalize with transferrin, a protein mostly ingested via uncoated vesicles at the cytostome/cytopharynx complex.ConclusionClathrin heavy and light chains are expressed in T. cruzi. Both proteins typically localize anterior to the kinetoplast, at the flagellar pocket and Golgi complex region. Our data also indicate that in T. cruzi epimastigotes clathrin-mediated endocytosis of albumin occurs at the flagellar pocket, while clathrin-independent endocytosis of transferrin occurs at the cytostome/cytopharynx complex.

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“…The membrane was washed three times in 0.05% Tween-20/PBS and then incubated for 1 h with AP-conjugated rabbit anti-mouse IgG (diluted 1 : 10,000). A polyclonal antirecombinant actin (TcActin; diluted at 1 : 200) mouse serum [ 23 ] was used for normalization. The membrane was then washed three times with 0.05% Tween-20/PBS and the reactive bands were visualized with BCIP-NBT solution, as described by the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

mAb CZP-315.D9: An Antirecombinant Cruzipain Monoclonal Antibody That Specifically Labels the Reservosomes ofTrypanosoma cruziEpimastigotes

Batista

Medeiros

Eger

et al. 2014

BioMed Research International

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Reservosomes are large round vesicles located at the posterior end of epimastigote forms of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. They are the specific end organelles of the endocytosis pathway of T. cruzi, and they play key roles in nutrient uptake and cell differentiation. These lysosome-like organelles accumulate ingested macromolecules and contain large amounts of a major cysteine proteinase (cruzipain or GP57/51 protein). Aim of this study was to produce a monoclonal antibody (mAb) against a recombinant T. cruzi cruzipain (TcCruzipain) that specifically labels the reservosomes. BALB/c mice were immunized with purified recombinant TcCruzipain to obtain the mAb. After fusion of isolated splenocytes with myeloma cells and screening, a mAb was obtained by limiting dilution and characterized by capture ELISA. We report here the production of a kappa-positive monoclonal IgG antibody (mAb CZP-315.D9) that recognizes recombinant TcCruzipain. This mAb binds preferentially to a protein with a molecular weight of about 50 kDa on western blots and specifically labels reservosomes by immunofluorescence and transmission electron microscopy. The monoclonal CZP-315.D9 constitutes a potentially powerful marker for use in studies on the function of reservosomes of T. cruzi.

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Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)

Cited by 10 publications

References 20 publications

Emerging Functions of Actins and Actin Binding Proteins in Trypanosomatids

Emerging Functions of Actins and Actin Binding Proteins in Trypanosomatids

Clathrin expression in Trypanosoma cruzi

mAb CZP-315.D9: An Antirecombinant Cruzipain Monoclonal Antibody That Specifically Labels the Reservosomes ofTrypanosoma cruziEpimastigotes

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