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.

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 A MORN Repeat Protein Facilitates Protein Entry into the Flagellar Pocket of Trypanosoma brucei

2015 ◽  
Vol 14 (11) ◽  
pp. 1081-1093 ◽  
Author(s):  
Brooke Morriswood ◽  
Katy Schmidt
Keyword(s):  
Trypanosoma Brucei ◽  
Concanavalin A ◽  
Adaptive Immune System ◽  
Fluid Phase ◽  
High Rate ◽  
Flagellar Pocket ◽  
Content Type ◽  
Rapid Enlargement ◽  
Fluid Phase Endocytosis ◽  
Protein Entry

ABSTRACTThe parasiteTrypanosoma bruceilives in the bloodstream of infected mammalian hosts, fully exposed to the adaptive immune system. It relies on a very high rate of endocytosis to clear bound antibodies from its cell surface. All endo- and exocytosis occurs at a single site on its plasma membrane, an intracellular invagination termed the flagellar pocket. Coiled around the neck of the flagellar pocket is a multiprotein complex containing the repeat motif proteinT. bruceiMORN1 (TbMORN1). In this study, the phenotypic effects of TbMORN1 depletion in the mammalian-infective form ofT. bruceiwere analyzed. Depletion of TbMORN1 resulted in a rapid enlargement of the flagellar pocket. Dextran, a polysaccharide marker for fluid phase endocytosis, accumulated inside the enlarged flagellar pocket. Unexpectedly, however, the proteins concanavalin A and bovine serum albumin did not do so, and concanavalin A was instead found to concentrate outside it. This suggests that TbMORN1 may have a role in facilitating the entry of proteins into the flagellar pocket.

Ligand-stimulated beta 2-adrenergic receptor internalization via the constitutive endocytic pathway into rab5-containing endosomes

1995 ◽  
Vol 108 (9) ◽  
pp. 2983-2991 ◽  
Author(s):  
R.H. Moore ◽  
N. Sadovnikoff ◽  
S. Hoffenberg ◽  
S. Liu ◽  
P. Woodford ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Adrenergic Receptor ◽  
Fluid Phase ◽  
Small Gtpase ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Beta Agonist ◽  
Hek293 Cells ◽  
Fluid Phase Endocytosis ◽  
Beta 2

The small GTPase rab5 appears to be rate-limiting for the constitutive internalization of transferrin receptor and for fluid-phase endocytosis. However, it is unknown whether rab5 regulates receptors whose internalization is stimulated by the binding of ligand, and whether such receptors change the underlying rate of the endocytic pathways they utilize. As a model for ligand-stimulated endocytosis, we used transfected HEK293 cells expressing high levels of an epitope-tagged human beta 2-adrenergic receptor. Nearly all receptors were on the cell surface in the absence of agonist, but within ten minutes of agonist addition > 50% of receptors internalized and colocalized extensively with rab5. Hypertonic sucrose blocked beta 2-adrenergic receptor internalization, as well as that of transferrin receptor, suggesting a clathrin-mediated process. In contrast, an inhibitor of potocytosis had little effect upon beta 2-adrenergic receptor internalization, suggesting that this process did not require active caveolae. Consistent with this finding, caveolin was not detectable in the 12 beta 6 line, as assessed by western blotting with a polyclonal anti-caveolin antibody. Stimulated receptor internalization did not affect the rate or capacity of the constitutive endocytic pathway since there was no detectable increase in fluid-phase endocytosis after addition of beta-agonist, nor was there a significant change in the amount of surface transferrin receptor. Altogether, these data suggest that beta 2-adrenergic receptors internalize by a clathrin-mediated and rab5-regulated constitutive endocytic pathway. Further, agonist-stimulated receptor internalization has no detectable effect upon the function of this pathway.

scholarly journals Variation of tandem repeats in the developmentally regulated procyclic acidic repetitive proteins of Trypanosoma brucei.

1989 ◽  
Vol 9 (3) ◽  
pp. 1332-1335 ◽  
Author(s):  
M R Mowatt ◽  
G S Wisdom ◽  
C E Clayton
Keyword(s):  
Gene Family ◽  
Trypanosoma Brucei ◽  
Tandem Repeats ◽  
Surface Proteins ◽  
Northern Hybridization ◽  
Developmentally Regulated ◽  
Coordinate Regulation ◽  
Polymorphic Genes ◽  
Alpha Gene ◽  
Carboxy Terminal

The procyclic acidic repetitive proteins (PARPs) of Trypanosoma brucei are developmentally regulated surface proteins encoded by a family of polymorphic genes. We have determined the complete nucleotide sequence of a novel member of the PARP gene family and investigated its expression. The amino acid sequence deduced from the parpA alpha gene showed a marked conservation of both the amino- and carboxy-terminal regions compared with other PARPs but revealed the substitution of a pentapeptide for the dipeptide repeating unit that is characteristic of all other PARPs. Northern hybridization analysis indicated that expression of the parpA alpha gene, like that of other members of this gene family, is confined to the procyclic stage of the T. brucei life cycle. This result implies coordinate regulation of the unlinked genetic loci that encode PARPs.

Transcription of the procyclic acidic repetitive protein genes of Trypanosoma brucei

1990 ◽  
Vol 10 (6) ◽  
pp. 3036-3047
Author(s):  
C E Clayton ◽  
J P Fueri ◽  
J E Itzhaki ◽  
V Bellofatto ◽  
D R Sherman ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Chloramphenicol Acetyltransferase ◽  
Surface Proteins ◽  
Base Pairs ◽  
Developmentally Regulated ◽  
Putative Promoter Region ◽  
Transcriptional Initiation ◽  
Chloramphenicol Acetyltransferase Gene ◽  
Alpha Amanitin ◽  
Repetitive Protein

The procyclic acidic repetitive protein (parp) genes of Trypanosoma brucei encode a small family of abundant surface proteins whose expression is restricted to the procyclic form of the parasite. They are found at two unlinked loci, parpA and parpB; transcription of both loci is developmentally regulated. The region of homology upstream of the A and B parp genes is only 640 base pairs long and may contain sequences responsible for transcriptional initiation and regulation. Transcription upstream of this putative promoter region is not developmentally regulated and is much less active than that of the parp genes; the polymerase responsible is inhibited by alpha-amanitin, whereas that transcribing the parp genes is not. Transcription of the parp genes is strongly stimulated by low levels of UV irradiation. The putative parp promoter, when placed upstream of the chloramphenicol acetyltransferase gene, is sufficient to cause production of chloramphenicol acetyltransferase in a T. brucei DNA transformation assay. Taken together, these results suggest that a promoter for an alpha-amanitin-resistant RNA polymerase lies less than 600 nucleotides upstream of the parp genes.

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 The crystal structure and localization of Trypanosoma brucei invariant surface glycoproteins suggest a more permissive VSG coat in the tsetse-transmitted metacyclic stage

2018 ◽  
Author(s):  
Aitor Casas-Sánchez ◽  
Samïrah Perally ◽  
Raghavendran Ramaswamy ◽  
Lee R. Haines ◽  
Clair Rose ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Trypanosoma Brucei ◽  
Fluorescent Microscopy ◽  
Surface Antigens ◽  
Surface Proteins ◽  
Surface Glycoprotein ◽  
Invariant Surface ◽  
Helical Bundle ◽  
N Terminus ◽  
Key Features

AbstractTrypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside the tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, nothing is known about expression of invariant surface antigens by the metacyclic stage. Proteomic analysis of saliva from T. brucei-infected flies revealed a novel family of hypothetical GPI-anchored surface proteins herein named Metacyclic Invariant Surface Proteins (MISP). MISP are encoded by five homolog genes and share ~80% protein identity. The crystal structure of MISP N-terminus at 1.82 Å resolution revealed a triple helical bundle that shares key features with other trypanosome surface proteins. However, molecular modelling combined with live fluorescent microscopy suggest that MISP N-termini are extended above the metacyclic VSG coat, exposing immunogenic epitopes. Collectively, we suggest that the metacyclic cell surface architecture appears more permissive than bloodstream forms in terms of expression of invariant GPI-anchored glycoproteins, which could be exploited for the development of novel vaccines against African trypanosomiases.

Phorbol ester promotes endocytosis by activating a factor involved in endosome fusion

1999 ◽  
Vol 112 (15) ◽  
pp. 2549-2557 ◽  
Author(s):  
A. Aballay ◽  
P.D. Stahl ◽  
L.S. Mayorga
Keyword(s):  
Phorbol Ester ◽  
Fluid Phase ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Early Endosomes ◽  
Zinc Depletion ◽  
Fluid Phase Endocytosis ◽  
Endocytic Vesicles

Previous studies indicate that a zinc- and phorbol ester-binding factor is necessary for in vitro endosome fusion and for the effect of Rab5 on endosome fusion. Rab5 is a small GTPase that regulates membrane fusion between early endosomes derived from either receptor-mediated endocytosis or fluid-phase endocytosis. In its GTP-bound form, Rab5 promotes endocytosis and enhances fusion among early endosomes. To determine if PMA stimulates endocytosis by activating a factor required for endosome fusion, we overexpressed wild-type Rab5, a dominant negative mutant (Rab5:S34N), and a GTPase deficient mutant (Rab5:Q79L) in BHK-21 cells. The phorbol ester PMA stimulates endocytosis and increases the number and the size of endocytic vesicles, even in the presence of Rab5:S34N. Zinc depletion with N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and addition of calphostin C (CPC), an inhibitor of PKC that interacts with zinc and phorbol ester binding motifs, inhibited both basal and Rab5-stimulated fluid phase endocytosis. These two reagents also inhibited the size and number of endocytic vesicles promoted by Rab5. These results suggest that PMA stimulates endocytosis by regulating the dynamics of the early endosome compartment.

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.

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