RESUMO
Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes. Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer. Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute) was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature.
RESUMO
The coat of Trypanosoma brucei consists mainly of glycosylphosphatidylinositol-anchored proteins that are present in several million copies and are characteristic of defined stages of the life cycle. While these major components of the coats of bloodstream forms and procyclic (insect midgut) forms are well characterised, very little is known about less abundant stage-regulated surface proteins and their roles in infection and transmission. By creating epitope-tagged versions of procyclic-specific surface antigen 2 (PSSA-2) we demonstrated that it is a membrane-spanning protein that is expressed by several different life cycle stages in tsetse flies, but not by parasites in the mammalian bloodstream. In common with other membrane-spanning proteins in T. brucei, PSSA-2 requires its cytoplasmic domain in order to exit the endoplasmic reticulum. Correct localisation of PSSA-2 requires phosphorylation of a cytoplasmic threonine residue (T(305)), a modification that depends on the presence of TbMAPK4. Mutation of T(305) to alanine (T(305)A) has no effect on the localisation of the protein in cells that express wild type PSSA-2. In contrast, this protein is largely intracellular when expressed in a null mutant background. A variant with a T(305)D mutation gives strong surface expression in both the wild type and null mutant, but slows growth of the cells, suggesting that it may function as a dominant negative mutant. The PSSA-2 null mutant exhibits no perceptible phenotype in culture and is fully competent at establishing midgut infections in tsetse, but is defective in colonising the salivary glands and the production of infectious metacyclic forms. Given the protein's structure and the effects of mutation of T(305) on proliferation and localisation, we postulate that PSSA-2 might sense and transmit signals that contribute to the parasite's decision to divide, differentiate or migrate.
Assuntos
Antígenos de Protozoários/metabolismo , Glicosilfosfatidilinositóis/química , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Animais , Antígenos de Protozoários/química , Ácido Aspártico/química , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Microscopia de Fluorescência/métodos , Mutação , Fosforilação , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Moscas Tsé-TséRESUMO
Procyclic forms of Trypanosoma brucei reside in the midgut of tsetse flies where they are covered by several million copies of glycosylphosphatidylinositol-anchored proteins known as procyclins. It has been proposed that procyclins protect parasites against proteases and/or participate in tropism, directing them from the midgut to the salivary glands. There are four different procyclin genes, each subject to elaborate levels of regulation. To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo. When co-cultured in vitro, the null mutant and wild type trypanosomes (tagged with cyan fluorescent protein) maintained a near-constant equilibrium. In contrast, when flies were infected with the same mixture, the null mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo. Although the null mutant is patently defective in competition with procyclin-positive parasites, on its own it can complete the life cycle and generate infectious metacyclic forms. The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.
Assuntos
Glicoproteínas de Membrana/fisiologia , Proteínas de Protozoários/fisiologia , Trypanosoma brucei brucei/química , Tripanossomíase Africana/transmissão , Moscas Tsé-Tsé/parasitologia , Animais , Animais Geneticamente Modificados , Técnicas de Cocultura , Glicoproteínas de Membrana/genética , Proteínas de Protozoários/genéticaAssuntos
Sangue/parasitologia , Núcleo Celular/metabolismo , Transfecção/métodos , Transformação Genética , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/genética , Animais , Linhagem Celular , Células Cultivadas , Meios de Cultura , DNA/metabolismo , Eletroporação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Trypanosoma brucei brucei/metabolismoRESUMO
EP and GPEET procyclins are the major surface glycoproteins of Trypanosoma brucei in the midgut of tsetse flies (Glossina spp.). The procyclin genes are located at the beginning of polycistronic transcription units and are followed by at least one procyclin-associated gene (PAG). The EP/PAG1 locus on one copy of chromosome X begins with the three genes EP1, EP2 and PAG1; the end of this unit has not been characterized previously. The EP/PAG2 locus on the other copy of chromosome X contains the same procyclin genes followed by PAG2 and PAG4. Here we show that the EP/PAG1 locus in AnTat1.1 has to be extended by three more PAGs, which we named PAG5, PAG2* and PAG4. The EP/PAG2 locus most likely evolved from the EP/PAG1 locus by deletion of a fragment from within PAG1 to PAG2*. The procyclin loci on the two copies of chromosome VI are indistinguishable, and contain the genes GPEET, EP3, PAG3 and GRESAG2.1. The mRNA levels of PAG1, PAG2 and PAG3 are transiently increased during differentiation of bloodstream forms to procyclic forms. Unexpectedly, procyclic forms of a PAG knockout clone lacking all eight PAGs in the procyclin loci were transmissible by Glossina morsitans. Furthermore, the deletion mutant could still establish midgut infections when competing with a tagged clone with the full complement of PAGs. Cyclical transmission was also possible when tsetse flies were infected with bloodstream forms of the deletion mutant, demonstrating that the PAGs are not essential for the differentiation of bloodstream to procyclic forms in vivo.
Assuntos
Genes de Protozoários , Insetos Vetores/parasitologia , Glicoproteínas de Membrana/genética , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Tripanossomíase/parasitologia , Moscas Tsé-Tsé/parasitologia , Animais , Sequência de Bases , Mapeamento Cromossômico , Feminino , Biblioteca Gênica , Genoma de Protozoário , Estágios do Ciclo de Vida , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência , Trypanosoma brucei brucei/crescimento & desenvolvimento , Tripanossomíase/transmissãoRESUMO
Activation of the phosphoinositide 3-kinases (PI 3-kinases) has been implicated in multiple cellular responses such as proliferation and survival, membrane and cytoskeletal reorganization, and intracellular vesicular trafficking. The activities and subcellular localization of PI 3-kinases were shown to be regulated by phosphorylation. Previously we demonstrated that class II HsPIK3-C2alpha becomes phosphorylated upon inhibition of RNA pol II-dependent transcription (Didichenko, S. A., and Thelen, M. (2001) J. Biol. Chem. 276, 48135-48142). In this study we investigated cell cycle-dependent and genotoxic stress-induced phosphorylation of HsPIK3-C2alpha. We find that the kinase becomes phosphorylated upon exposure of cells to UV irradiation and in proliferating cells at the G2/M transition of the cell cycle. Stress-dependent and mitotic phosphorylation of HsPIK3-C2alpha occurs on the same serine residue (Ser259) within a recognition motif for proline-directed kinases. Mitotic phosphorylation of HsPIK3-C2alpha can be attributed to Cdc2 activity, and stress-induced phosphorylation of HsPIK3-C2alpha is mediated by JNK/SAPK. The protein level of HsPIK3-C2alpha is regulated by proteolysis in a cell cycle-dependent manner and in response of cells to stress. Phosphorylation appears to be a prerequisite for proteasome-dependent degradation of HsPIK3-C2alpha and may therefore contribute indirectly to the regulation of the activity of the kinase.