A developmentally regulated Rab11 homologue in Trypanosoma brucei is involved in recycling processes

2001 ◽  
Vol 114 (14) ◽  
pp. 2617-2626 ◽  
Author(s):  
Tim R. Jeffries ◽  
Gareth W. Morgan ◽  
Mark C. Field
Keyword(s):  
Trypanosoma Brucei ◽  
Basal Body ◽  
Transferrin Receptor ◽  
Developmental Regulation ◽  
Early Endosome ◽  
Surface Glycoprotein ◽  
Flagellar Pocket ◽  
Developmentally Regulated ◽  
Regulated Expression ◽  
Collecting Tubules

Endocytosis in the parasitic protozoan Trypanosoma brucei, a deeply divergent eukaryote, is implicated as important in both general cellular function and virulence, and is strongly developmentally regulated. We report the characterisation of a previously undefined endosomal compartment in T. brucei based on identification of a new trypanosome gene (TbRAB11) homologous to Rab11/Ypt31. Northern and western analyses indicated that TbRAB11 expression was significantly upregulated in the bloodstream stage of the parasite, the first trypanosome Rab to be identified with a developmentally regulated expression profile. In procyclic form parasites TbRAB11 localised to a compartment positioned close to the basal body, similar to mammalian Rab11. By contrast, in bloodstream form parasites, TbRAB11-containing structures were more extensive and the TbRAB11 compartment extended towards the posterior face of the nucleus, was more elaborate and was not always adjacent to the basal body. Colocalisation studies by light and confocal microscopy demonstrated that TbRAB11 was located on a compartment that did not correspond to other established trypanosomal organelles or markers. Using concanavalin A internalisation and temperature block procedures, TbRAB11 was observed on endomembranes anterior to the flagellar pocket that are juxtaposed to the collecting tubules. TbRAB11 colocalised with the trypanosomal transferrin receptor and internalised antivariant surface glycoprotein. Further, we show that the collecting tubules contain TbRAB5A, suggesting that they are the trypanosomatid early endosome. Hence, TbRAB11 is present on endosomal structures that contain recycling cargo molecules and is under developmental regulation, suggesting a role in stage-dependent endocytic processes.

scholarly journals Developmental Variation in Rab11-Dependent Trafficking in Trypanosoma brucei

2005 ◽  
Vol 4 (5) ◽  
pp. 971-980 ◽  
Author(s):  
Belinda S. Hall ◽  
Emma Smith ◽  
Wolfram Langer ◽  
Louisa A. Jacobs ◽  
David Goulding ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Fluid Phase ◽  
Small Gtpase ◽  
Surface Proteins ◽  
Surface Glycoprotein ◽  
Primary Role ◽  
Flagellar Pocket ◽  
Developmentally Regulated ◽  
Developmental Variation ◽  
Fluid Phase Endocytosis

ABSTRACT In Trypanosoma brucei, endocytosis is developmentally regulated and is substantially more active in the mammalian infective stage, where it likely plays a role in immune evasion. The small GTPase TbRAB11 is highly expressed in the mammalian stage and mediates recycling of glycosylphosphatidylinositol-anchored proteins, including the variant surface glycoprotein (VSG) and the transferrin receptor, plus trafficking of internalized anti-VSG antibody and transferrin. No function has been assigned to TbRAB11 in the procyclic (insect) stage trypanosome. The importance of TbRAB11 to both bloodstream and procyclic form viability was assessed by RNA interference (RNAi). Suppression of TbRAB11 in the bloodstream form was rapidly lethal and led to cells with round morphology and an enlarged flagellar pocket. TbRAB11 RNAi was also lethal in procyclic forms, which also became rounded, but progression to cell death was significantly slower and the flagellar pocket remained normal. In bloodstream forms, silencing of TbRAB11 had no effect on exocytosis of newly synthesized VSG, fluid-phase endocytosis, or transferrin uptake, but export of internalized transferrin was inhibited. Lectin endocytosis assays revealed a block to postendosomal transport mediated by suppressing TbRAB11. By contrast, in procyclic forms, depletion of TbRAB11 blocks both fluid-phase endocytosis and internalization of surface proteins. In normal bloodstream forms, most VSG is recycled, but in procyclics, internalized surface proteins accumulated in the lysosome. These data demonstrate that TbRAB11 controls recycling and is essential in both life stages of T. brucei but that its primary role is subject to developmental variation.

Developmental regulation of a novel repetitive protein of Trypanosoma brucei

1987 ◽  
Vol 7 (8) ◽  
pp. 2838-2844
Author(s):  
M R Mowatt ◽  
C E Clayton
Keyword(s):  
Trypanosoma Brucei ◽  
Tandem Repeats ◽  
Signal Sequence ◽  
Developmental Regulation ◽  
Developmentally Regulated ◽  
Hydrophobic Domain ◽  
Reading Frame ◽  
Amino Terminal ◽  
Membrane Bound

Trypanosoma brucei undergoes many morphological and biochemical changes during transformation from the bloodstream trypomastigote to the insect procyclic trypomastigote form. We cloned and determined the complete nucleotide sequence of a developmentally regulated cDNA. The corresponding mRNA was abundant in in vitro-cultivated procyclics but absent in bloodstream forms. The trypanosome genome contains eight genes homologous to this cDNA, arranged as four unlinked pairs of tandem repeats. The longest open reading frame of the cDNA predicts a protein of 15 kilodaltons, the central portion of which consists of 29 tandem glutamate-proline dipeptides. The repetitive region is preceded by an amino-terminal signal sequence and followed by a hydrophobic domain that could serve as a membrane anchor; the mRNA was found on membrane-bound polyribosomes. These results suggest that the protein is membrane associated.

scholarly journals Developmental regulation of edited CYb and COIII mitochondrial mRNAs is achieved by distinct mechanisms in Trypanosoma brucei

2020 ◽  
Vol 48 (15) ◽  
pp. 8704-8723
Author(s):  
Joseph T Smith Jr. ◽  
Eva Doleželová ◽  
Brianna Tylec ◽  
Jonathan E Bard ◽  
Runpu Chen ◽  
...  
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
High Throughput Sequencing ◽  
Environmental Changes ◽  
Developmental Regulation ◽  
Complex Life Cycle ◽  
Mrna Levels ◽  
Developmentally Regulated ◽  
Life Cycle Stages

Abstract Trypanosoma brucei is a parasitic protozoan that undergoes a complex life cycle involving insect and mammalian hosts that present dramatically different nutritional environments. Mitochondrial metabolism and gene expression are highly regulated to accommodate these environmental changes, including regulation of mRNAs that require extensive uridine insertion/deletion (U-indel) editing for their maturation. Here, we use high throughput sequencing and a method for promoting life cycle changes in vitro to assess the mechanisms and timing of developmentally regulated edited mRNA expression. We show that edited CYb mRNA is downregulated in mammalian bloodstream forms (BSF) at the level of editing initiation and/or edited mRNA stability. In contrast, edited COIII mRNAs are depleted in BSF by inhibition of editing progression. We identify cell line-specific differences in the mechanisms abrogating COIII mRNA editing, including the possible utilization of terminator gRNAs that preclude the 3′ to 5′ progression of editing. By examining the developmental timing of altered mitochondrial mRNA levels, we also reveal transcript-specific developmental checkpoints in epimastigote (EMF), metacyclic (MCF), and BSF. These studies represent the first analysis of the mechanisms governing edited mRNA levels during T. brucei development and the first to interrogate U-indel editing in EMF and MCF life cycle stages.

Maxicircle CR1 transcripts of Trypanosoma brucei are edited and developmentally regulated and encode a putative iron-sulfur protein homologous to an NADH dehydrogenase subunit

1992 ◽  
Vol 12 (5) ◽  
pp. 2100-2107
Author(s):  
A E Souza ◽  
P J Myler ◽  
K Stuart
Keyword(s):  
Trypanosoma Brucei ◽  
Nadh Dehydrogenase ◽  
Developmental Regulation ◽  
Developmentally Regulated ◽  
Reading Frame ◽  
Guide Rnas ◽  
Iron Sulfur ◽  
Mitochondrial Dnas ◽  
A Subunit ◽  
Sulfur Protein

The maxicircle of Trypanosoma brucei encodes components of the mitochondrial oxidative phosphorylation system, as do other mitochondrial DNAs, but maxicircle gene identification is complicated by extensive editing of some transcripts. We found that transcripts from the CR1 region were extensively edited, as are other transcripts from maxicircle regions which exhibit strong G versus C strand bias. Editing added 259 uridines and removed 46 uridines to produce an approximately 574-nucleotide mature mRNA. Partially edited cDNAs and potential guide RNAs were also characterized. Initiation and termination codons were created, and they defined an open reading frame encoding a predicted protein of 145 amino acids. This protein contains two iron-sulfur cysteine motifs and is homologous to a subunit of NADH dehydrogenase and to other electron-carrier proteins. Higher levels of both edited and unedited CR1 transcripts accumulated in bloodstream forms of the parasite than in procyclic forms, suggesting developmental regulation of CR1 gene expression.

scholarly journals Endocytosis of a Glycosylphosphatidylinositol-anchored Protein via Clathrin-coated Vesicles, Sorting by Default in Endosomes, and Exocytosis via RAB11-positive Carriers

2003 ◽  
Vol 14 (5) ◽  
pp. 2029-2040 ◽  
Author(s):  
Christoph G. Grünfelder ◽  
Markus Engstler ◽  
Frank Weise ◽  
Heinz Schwarz ◽  
York-Dieter Stierhof ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Trypanosoma Brucei ◽  
Coated Vesicles ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Flagellar Pocket ◽  
Protozoan Parasites ◽  
Basic Features ◽  
Membrane Concentration

Recently, proteins linked to glycosylphosphatidylinositol (GPI) residues have received considerable attention both for their association with lipid microdomains and for their specific transport between cellular membranes. Basic features of trafficking of GPI-anchored proteins or glycolipids may be explored in flagellated protozoan parasites, which offer the advantage that their surface is dominated by these components. In Trypanosoma brucei, the GPI-anchored variant surface glycoprotein (VSG) is efficiently sorted at multiple intracellular levels, leading to a 50-fold higher membrane concentration at the cell surface compared with the endoplasmic reticulum. We have studied the membrane and VSG flow at an invagination of the plasma membrane, the flagellar pocket, the sole region for endo- and exocytosis in this organism. VSG enters trypanosomes in large clathrin-coated vesicles (135 nm in diameter), which deliver their cargo to endosomes. In the lumen of cisternal endosomes, VSG is concentrated by default, because a distinct class of small clathrin-coated vesicles (50–60 nm in diameter) budding from the cisternae is depleted in VSG. TbRAB11-positive cisternal endosomes, containing VSG, fragment by an unknown process giving rise to intensely TbRAB11- as well as VSG-positive, disk-like carriers (154 nm in diameter, 34 nm in thickness), which are shown to fuse with the flagellar pocket membrane, thereby recycling VSG back to the cell surface.

scholarly journals Trypanosoma brucei FKBP12 Differentially Controls Motility and Cytokinesis in Procyclic and Bloodstream Forms

2012 ◽  
Vol 12 (2) ◽  
pp. 168-181 ◽  
Author(s):  
Anaïs Brasseur ◽  
Brice Rotureau ◽  
Marjorie Vermeersch ◽  
Thierry Blisnick ◽  
Didier Salmon ◽  
...  
Keyword(s):  
Rna Interference ◽  
Trypanosoma Brucei ◽  
Normal Cell ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Flagellar Pocket ◽  
Content Type ◽  
Procyclic Form ◽  
Specific Localization ◽  
Inside Out

ABSTRACT FKBP12 proteins are able to inhibit TOR kinases or calcineurin phosphatases upon binding of rapamycin or FK506 drugs, respectively. The Trypanosoma brucei FKBP12 homologue (TbFKBP12) was found to be a cytoskeleton-associated protein with specific localization in the flagellar pocket area of the bloodstream form. In the insect procyclic form, RNA interference-mediated knockdown of TbFKBP12 affected motility. In bloodstream cells, depletion of TbFKBP12 affected cytokinesis and cytoskeleton architecture. These last effects were associated with the presence of internal translucent cavities limited by an inside-out configuration of the normal cell surface, with a luminal variant surface glycoprotein coat lined up by microtubules. These cavities, which recreated the streamlined shape of the normal trypanosome cytoskeleton, might represent unsuccessful attempts for cell abscission. We propose that TbFKBP12 differentially affects stage-specific processes through association with the cytoskeleton.

scholarly journals Developmental regulation of a novel repetitive protein of Trypanosoma brucei.

1987 ◽  
Vol 7 (8) ◽  
pp. 2838-2844 ◽  
Author(s):  
M R Mowatt ◽  
C E Clayton
Keyword(s):  
Trypanosoma Brucei ◽  
Tandem Repeats ◽  
Signal Sequence ◽  
Developmental Regulation ◽  
Developmentally Regulated ◽  
Hydrophobic Domain ◽  
Reading Frame ◽  
Amino Terminal ◽  
Membrane Bound

Trypanosoma brucei undergoes many morphological and biochemical changes during transformation from the bloodstream trypomastigote to the insect procyclic trypomastigote form. We cloned and determined the complete nucleotide sequence of a developmentally regulated cDNA. The corresponding mRNA was abundant in in vitro-cultivated procyclics but absent in bloodstream forms. The trypanosome genome contains eight genes homologous to this cDNA, arranged as four unlinked pairs of tandem repeats. The longest open reading frame of the cDNA predicts a protein of 15 kilodaltons, the central portion of which consists of 29 tandem glutamate-proline dipeptides. The repetitive region is preceded by an amino-terminal signal sequence and followed by a hydrophobic domain that could serve as a membrane anchor; the mRNA was found on membrane-bound polyribosomes. These results suggest that the protein is membrane associated.

Trypanosoma brucei mitochondrial ribosomal RNA synthesis, processing and developmentally regulated expression

1992 ◽  
Vol 54 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Emil F. Michelotti ◽  
Michael E. Harris ◽  
Brian Adler ◽  
Al F. Torri ◽  
Stephen L. Hajduk
Keyword(s):  
Trypanosoma Brucei ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Developmentally Regulated ◽  
Regulated Expression

scholarly journals To the Surface and Back: Exo- and Endocytic Pathways in Trypanosoma brucei

2021 ◽  
Vol 9 ◽  
Author(s):  
Fabian Link ◽  
Alyssa R. Borges ◽  
Nicola G. Jones ◽  
Markus Engstler
Keyword(s):  
Cell Surface ◽  
Trypanosoma Brucei ◽  
Immune Evasion ◽  
Antigenic Variation ◽  
Critical Role ◽  
Surface Glycoprotein ◽  
Immune Recognition ◽  
Flagellar Pocket ◽  
Novel Technologies ◽  
Near Future

Trypanosoma brucei is one of only a few unicellular pathogens that thrives extracellularly in the vertebrate host. Consequently, the cell surface plays a critical role in both immune recognition and immune evasion. The variant surface glycoprotein (VSG) coats the entire surface of the parasite and acts as a flexible shield to protect invariant proteins against immune recognition. Antigenic variation of the VSG coat is the major virulence mechanism of trypanosomes. In addition, incessant motility of the parasite contributes to its immune evasion, as the resulting fluid flow on the cell surface drags immunocomplexes toward the flagellar pocket, where they are internalized. The flagellar pocket is the sole site of endo- and exocytosis in this organism. After internalization, VSG is rapidly recycled back to the surface, whereas host antibodies are thought to be transported to the lysosome for degradation. For this essential step to work, effective machineries for both sorting and recycling of VSGs must have evolved in trypanosomes. Our understanding of the mechanisms behind VSG recycling and VSG secretion, is by far not complete. This review provides an overview of the trypanosome secretory and endosomal pathways. Longstanding questions are pinpointed that, with the advent of novel technologies, might be answered in the near future.

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