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 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 Clathrin-Dependent Targeting of Receptors to the Flagellar Pocket of Procyclic-Form Trypanosoma brucei

2004 ◽  
Vol 3 (4) ◽  
pp. 1004-1014 ◽  
Author(s):  
Chien-Hui Hung ◽  
Xugang Qiao ◽  
Pei-Tseng Lee ◽  
Mary Gwo-Shu Lee
Keyword(s):  
Rna Interference ◽  
Trypanosoma Brucei ◽  
Secretory Pathway ◽  
Morphological Changes ◽  
Cell Receptor ◽  
Flagellar Pocket ◽  
Procyclic Form ◽  
Receptor Mediated Endocytosis ◽  
Distinct Structure ◽  
Endocytic Vesicles

ABSTRACT In trypanosomatids, endocytosis and exocytosis occur exclusively at the flagellar pocket, which represents about 0.43% of the pellicle membrane and is a deep invagination of the plasma membrane where the flagellum extends from the cell. Receptor molecules are selectively retained at the flagellar pocket. We studied the function of clathrin heavy chain (TbCLH) in the trafficking of the flagellar pocket receptors in Trypanosoma brucei by using the double-stranded RNA interference approach. It appears that TbCLH is essential for the survival of both the procyclic form and the bloodstream form of T. brucei, even though structures resembling large coated endocytic vesicles are absent in procyclic-form trypanosomes. Down-regulation of TbCLH by RNA interference (RNAi) for 24 h rapidly and drastically reduced the uptake of macromolecules via receptor-mediated endocytosis in procyclic-form trypanosomes. This result suggested the importance of TbCLH in receptor-mediated endocytosis of the procyclic-form trypanosome, in which the formation of large coated endocytic vesicles may not be required. Surprisingly, induction of TbCLH RNAi in the procyclic T. brucei for a period of 48 h prohibited the export of the flagellar pocket-associated transmembrane receptor CRAM from the endoplasmic reticulum to the flagellar pocket, while trafficking of the glycosylphosphatidylinositol-anchored procyclin coat was not significantly affected. After 72 h of induction of TbCLH RNAi, procyclics exhibited morphological changes to an apolar round shape without a distinct structure of the flagellar pocket and flagellum. Although trypanosomes, like other eukaryotes, use similar organelles and machinery for protein sorting and transport, our studies reveal a novel role for clathrin in the secretory pathway of trypanosomes. We speculate that the clathrin-dependent trafficking of proteins to the flagellar pocket may be essential for the biogenesis and maintenance of the flagellar pocket in trypanosomes.

scholarly journals Synchronous expression of individual metacyclic variant surface glycoprotein genes in Trypanosoma brucei

2015 ◽  
Vol 200 (1-2) ◽  
pp. 1-4 ◽  
Author(s):  
Kiantra Ramey-Butler ◽  
Elisabetta Ullu ◽  
Nikolay G. Kolev ◽  
Christian Tschudi
Keyword(s):  
Trypanosoma Brucei ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein

Stability and reconstitution of the soluble variant surface glycoprotein (sVSG) from Trypanosoma brucei

Biochemistry ◽  
1990 ◽  
Vol 29 (36) ◽  
pp. 8217-8223 ◽  
Author(s):  
Paul Rehaber ◽  
Norbert Staudacher ◽  
Robert Seckler ◽  
Roland Buelow ◽  
Peter Overath ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein

Frequent independent duplicative transpositions activate a single VSG gene

1987 ◽  
Vol 7 (1) ◽  
pp. 357-364
Author(s):  
M G Lee ◽  
L H Van der Ploeg
Keyword(s):  
Gene Conversion ◽  
Trypanosoma Brucei ◽  
Surface Antigen ◽  
Strong Evidence ◽  
Base Pair ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Gene Variants ◽  
Repeat Array ◽  
Expression Site

The expression of several surface antigen genes in Trypanosoma brucei is mediated by the duplicative transposition of a basic-copy variant surface glycoprotein (VSG) gene into an expression site. We determined that the appearance of variant 118, in a parasitemia, resulted from at least four independent duplicative transpositions of the same VSG 118 gene. Variants 117 and 118 both appeared at specific periods but resulted from multiple independent activations. Antigenic variants thus occur in an ordered manner. We show that in the duplicative transpositions of VSG genes, the ends of the transposed segments were homologous between the basic copy and the expression site. Sequences other than the previously reported 70-base-pair (bp) repeats could be involved. In one variant, 118 clone 1, the homology was between a sequence previously transposed into the expression site and a sequence located 6 kilobases upstream of the VSG 118 gene. In variant 118b the homology was presumably in 70-bp repeat arrays, while in a third 118 variant yet another sequence was involved. The possibility that the 70-bp repeats are important in the initial steps of the recombinational events was illustrated by a rearrangement involving a 70-bp repeat array. The data provide strong evidence for the notion that gene conversion mediates the duplicative transposition of VSG genes. We discuss a model that explains how the process of duplicative transposition can occur at random and still produce an ordered appearance of variants.

Transient activity assays of the Trypanosoma brucei variant surface glycoprotein gene promoter: control of gene expression at the posttranscriptional level

1991 ◽  
Vol 11 (1) ◽  
pp. 338-343
Author(s):  
D Jefferies ◽  
P Tebabi ◽  
E Pays
Keyword(s):  
Trypanosoma Brucei ◽  
Gene Promoter ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Gene Promoters ◽  
Control Of Gene Expression ◽  
Transient Activity ◽  
Expression Site ◽  
Cat Gene ◽  
Cat Expression

The putative promoter of the variant surface glycoprotein (VSG) gene of Trypanosoma brucei was cloned into a plasmid containing the chloramphenicol acetyltransferase (CAT) gene. After electroporation into trypanosomes, this construct directed the expression of the CAT reporter gene. The essential region for promoter activity was found to reside within 88 bp upstream of the putative transcription start site. Transcription of the CAT construct occurred at approximately the same level in both bloodstream and procyclic forms and was resistant to alpha-amanitin. However, CAT expression appeared to be modulated in the two forms of the parasite. Sequences 3' to the gene seemed to be important in this respect, as CAT activity in bloodstream forms was readily detectable only when the 3' region of a VSG cDNA was placed downstream of the CAT gene. Two separate VSG gene promoter sequences, both cloned from T. brucei AnTat 1.3A, were equally able to direct CAT expression, which suggests that there are a number of potential VSG gene promoters in the genome, although usually only one expression site is fully active at any one time.

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