The Leishmania ARL-1 and Golgi Traffic

We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study

Metabolic status rather than cell cycle signals control quiescence entry and exit

The use of new candidate markers for yeast quiescence reveals that quiescence entry and exit primarily rely on cellular metabolic status and can be uncoupled from the cell cycle

Polarized Growth in the Absence of F-Actin in Saccharomyces cerevisiae Exiting Quiescence

Polarity establishment and maintenance are crucial for morphogenesis and development. In budding yeast, these two intricate processes involve the superposition of regulatory loops between polarity landmarks, RHO GTPases, actin-mediated vesicles transport and endocytosis. Deciphering the chronology and the significance of each molecular step of polarized growth is therefore very challenging.We have taken advantage of the fact that yeast quiescent cells display actin bodies, a non polarized actin structure, to evaluate the role of F-actin in bud emergence. Here we show that upon exit from quiescence, actin cables are not required for the first steps of polarized growth. We further show that polarized growth can occur in the absence of actin patch-mediated endocytosis. We finally establish, using latrunculin-A, that the first steps of polarized growth do not require any F-actin containing structures. Yet, these structures are required for the formation of a bona fide daughter cell and cell cycle completion. We propose that upon exit from quiescence in the absence of F-actin, secretory vesicles randomly reach the plasma membrane but preferentially dock and fuse where polarity cues are localized, this being sufficient to trigger polarized growth

A MAP6-Related Protein Is Present in Protozoa and Is Involved in Flagellum Motility

In vertebrates the microtubule-associated proteins MAP6 and MAP6d1 stabilize cold-resistant microtubules. Cilia and flagella have cold-stable microtubules but MAP6 proteins have not been identified in these organelles. Here, we describe TbSAXO as the first MAP6-related protein to be identified in a protozoan, Trypanosoma brucei. Using a heterologous expression system, we show that TbSAXO is a microtubule stabilizing protein. Furthermore we identify the domains of the protein responsible for microtubule binding and stabilizing and show that they share homologies with the microtubule-stabilizing Mn domains of the MAP6 proteins. We demonstrate, in the flagellated parasite, that TbSAXO is an axonemal protein that plays a role in flagellum motility. Lastly we provide evidence that TbSAXO belongs to a group of MAP6-related proteins (SAXO proteins) present only in ciliated or flagellated organisms ranging from protozoa to mammals. We discuss the potential roles of the SAXO proteins in cilia and flagella function

ADP-Ribosylation Factor Like (étude fonctionnelle chez Leishmania)

Les leishmanies sont des parasites transmis par des insectes diptères, responsables de pathologies graves chez l'homme. La protéine G monomérique LdARL-3A (ADP-Ribosylation factor Like) de Leishmania donovani a été identifiée précédemment, et son rôle dans la biogenèse du flagelle, un organelle indispensable à la survie du parasite chez l'insecte, mis en évidence. Nous avons surexprimé des chimères (LdARL-3A/LdARL-3B), ce qui nous a permis d'identifier une région effectrice de LdARL-3A ; par double-hybride chez la levure, nous avons isolé deux partenaires potentiels, la clathrine, l'autre inconnu (ARLette). Nous avons montré que la fonction de LdARL-3A est conservée chez d'autres Trypanosomatidae, ce qui suggère que cette protéine pourrait être une cible potentielle commune à plusieurs parasites dans les stratégies visant à bloquer leur transmission par les insectes. Nous avons également caractérisé une autre protéine de type ARL chez Leishmania : LdARL-1, une protéine du réseau Trans Golgien.Leishmania parasites are transmitted by a dipterous insect and are responsible of serious human diseases. The LdARL-3A monomeric G protein (ADP Ribosylation factor-Like) from Leishmania donovani was previously isolated, and its function in the flagellum biogenesis, wich is essential for the life cycle of the parasite in the insect, was proved. We have overexpressed chimera (LdARL-3B), what allowed us to identify an effector domain of LdARL-3A ; by two-hybrids from yeast, we have isolated two potential partners, clathrin and an unknown partner that we called ARLette. We have also shown that LdARL-3A function is preserved at others trypanosomatidae ; what suggests that this protein xould be a potential common target to several parasites in the strategies aiming at blocking their transmission by insect. We also characterized another ARL protein from Leishmania : LdARL-1, a Trans Golgi Network protein.BORDEAUX2-BU Santé (330632101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

The susceptibility of Trypanosoma congolense and Trypanosoma brucei to isometamidium chloride and its synthetic impurities.

International audienceSince the 1950s, the chemotherapy of animal African trypanosomosis in cattle has essentially relied on only two compounds: isometamidium chloride (ISM), a phenanthridine, and diminazene aceturate, an aromatic diamidine. The commercial formulations of ISM, including Veridium(®) and Samorin(®), are a mixture of different compounds: ISM is the major component, mixed with the red isomer, blue isomer and disubstituted compound. To investigate the pharmacological effects of these individual compounds ISM, the blue and red isomers and the disubstituted compound were synthesised and purified by HPLC. The activity of each compound was analysed both in vitro, and in mice in vivo. For the in vitro analysis, a drug sensitivity assay was developed in 96-well tissue culture plates to determine the effective concentration which killed 50% of trypanosome population within 48 h of drug exposure (IC50). All compounds tested in vitro possessed trypanocidal activity, and purified ISM was the most active. Veridium(®) and Samorin(®) had similar IC50 values to purified ISM for both Trypanosoma congolense and Trypanosoma brucei brucei. The disubstituted compound had the highest IC50 values whereas intermediate IC50 values were obtained for the blue and red isomers. In vivo, single-dose tests were used to evaluate the trypanocidal and prophylactic activity against T. congolense. Interestingly, the prophylactic effect two months post treatment was as efficient with ISM, Veridium(®), Samorin(®) and the disubstituted compound at the highest dose of 1mg/kg whereas the red and blue isomers both showed much lower prophylactic activity. This study on T. congolense implies that it is necessary to limit the quantity of the blue and red isomers in the commercial mixture. Finally, the in vitro sensitivity assay may be useful for screening new trypanocides but also for the testing and detection of resistant trypanosome isolates

Involvement of caveolin-1 and CD36 in native LDL endocytosis by endothelial cells

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