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Recruitment of EB1, a master regulator of microtubule dynamics, to the surface of the Theileria annulata schizont.

Woods KL, Theiler R, Mühlemann M, Segiser A, Huber S, Ansari HR, Pain A, Dobbelaere DA - PLoS Pathog. (2013)

Bottom Line: The latter does not egress to invade and transform other cells.Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures.Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathobiology, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

ABSTRACT
The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs) surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cell's astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability). Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1), a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.

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EB1 interacts with the schizont surface in a MT independent manner, via its End-binding homology (EBH) domain.(A) Image of TaC12 cell expressing EB1-GFP; the schizont was stained using 1C12 (red). DNA is stained with DAPI (blue). Scale bar = 5 µm. (B) EB1 binding to the schizont does not require MTs: TaC12 cells expressing EB1-myc were treated with 0.1 µg/ml nocodazole for 16 h, collected by mitotic shake-off, fixed with 4% PFA on ice, and stained with anti-myc (green) and anti-tubulin (red) antibodies. The last panel (CONTROL) shows a cell in prometaphase that was not treated with nocodazole, to point out the effect of nocodazole on depolymerizing MTs. DNA is stained with DAPI (blue). Scale bar = 5 µm. (C) Truncation analysis of EB1 binding to the schizont surface. TaC12 cells were transfected with plasmids encoding either full-length GFP-, V5- or myc-tagged EB1, or deletion constructs encoding different domains of EB1 tagged with copGFP.
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ppat-1003346-g003: EB1 interacts with the schizont surface in a MT independent manner, via its End-binding homology (EBH) domain.(A) Image of TaC12 cell expressing EB1-GFP; the schizont was stained using 1C12 (red). DNA is stained with DAPI (blue). Scale bar = 5 µm. (B) EB1 binding to the schizont does not require MTs: TaC12 cells expressing EB1-myc were treated with 0.1 µg/ml nocodazole for 16 h, collected by mitotic shake-off, fixed with 4% PFA on ice, and stained with anti-myc (green) and anti-tubulin (red) antibodies. The last panel (CONTROL) shows a cell in prometaphase that was not treated with nocodazole, to point out the effect of nocodazole on depolymerizing MTs. DNA is stained with DAPI (blue). Scale bar = 5 µm. (C) Truncation analysis of EB1 binding to the schizont surface. TaC12 cells were transfected with plasmids encoding either full-length GFP-, V5- or myc-tagged EB1, or deletion constructs encoding different domains of EB1 tagged with copGFP.

Mentions: To determine whether EB1 can interact with the parasite surface, we transiently expressed GFP-tagged EB1 and EB3 (a kind gift from Anna Akhmanova) in TaC12 cells. EB1-GFP labeled the entire surface of the schizont (Figure 3A). The same pattern could be observed using plasmids encoding EB1 containing a C-terminal myc or V5 tag (not shown) confirming that the observed localization of ectopically expressed EB1 is not an artifact caused by the GFP tag. In live imaging experiments, EB1-GFP-labeled ‘comets’ were readily detected moving from the centrosome to the periphery as reported [37], along with a striking association of EB1-GFP with the parasite surface (Movie S1), ruling out the possibility that the association of over-expressed EB1 with the schizont was a fixation artifact. Transfection experiments using EB3-GFP revealed that EB3, which shows overlapping functions with EB1 [12], [25], [40], also associates with the schizont surface (Figure S3).


Recruitment of EB1, a master regulator of microtubule dynamics, to the surface of the Theileria annulata schizont.

Woods KL, Theiler R, Mühlemann M, Segiser A, Huber S, Ansari HR, Pain A, Dobbelaere DA - PLoS Pathog. (2013)

EB1 interacts with the schizont surface in a MT independent manner, via its End-binding homology (EBH) domain.(A) Image of TaC12 cell expressing EB1-GFP; the schizont was stained using 1C12 (red). DNA is stained with DAPI (blue). Scale bar = 5 µm. (B) EB1 binding to the schizont does not require MTs: TaC12 cells expressing EB1-myc were treated with 0.1 µg/ml nocodazole for 16 h, collected by mitotic shake-off, fixed with 4% PFA on ice, and stained with anti-myc (green) and anti-tubulin (red) antibodies. The last panel (CONTROL) shows a cell in prometaphase that was not treated with nocodazole, to point out the effect of nocodazole on depolymerizing MTs. DNA is stained with DAPI (blue). Scale bar = 5 µm. (C) Truncation analysis of EB1 binding to the schizont surface. TaC12 cells were transfected with plasmids encoding either full-length GFP-, V5- or myc-tagged EB1, or deletion constructs encoding different domains of EB1 tagged with copGFP.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649978&req=5

ppat-1003346-g003: EB1 interacts with the schizont surface in a MT independent manner, via its End-binding homology (EBH) domain.(A) Image of TaC12 cell expressing EB1-GFP; the schizont was stained using 1C12 (red). DNA is stained with DAPI (blue). Scale bar = 5 µm. (B) EB1 binding to the schizont does not require MTs: TaC12 cells expressing EB1-myc were treated with 0.1 µg/ml nocodazole for 16 h, collected by mitotic shake-off, fixed with 4% PFA on ice, and stained with anti-myc (green) and anti-tubulin (red) antibodies. The last panel (CONTROL) shows a cell in prometaphase that was not treated with nocodazole, to point out the effect of nocodazole on depolymerizing MTs. DNA is stained with DAPI (blue). Scale bar = 5 µm. (C) Truncation analysis of EB1 binding to the schizont surface. TaC12 cells were transfected with plasmids encoding either full-length GFP-, V5- or myc-tagged EB1, or deletion constructs encoding different domains of EB1 tagged with copGFP.
Mentions: To determine whether EB1 can interact with the parasite surface, we transiently expressed GFP-tagged EB1 and EB3 (a kind gift from Anna Akhmanova) in TaC12 cells. EB1-GFP labeled the entire surface of the schizont (Figure 3A). The same pattern could be observed using plasmids encoding EB1 containing a C-terminal myc or V5 tag (not shown) confirming that the observed localization of ectopically expressed EB1 is not an artifact caused by the GFP tag. In live imaging experiments, EB1-GFP-labeled ‘comets’ were readily detected moving from the centrosome to the periphery as reported [37], along with a striking association of EB1-GFP with the parasite surface (Movie S1), ruling out the possibility that the association of over-expressed EB1 with the schizont was a fixation artifact. Transfection experiments using EB3-GFP revealed that EB3, which shows overlapping functions with EB1 [12], [25], [40], also associates with the schizont surface (Figure S3).

Bottom Line: The latter does not egress to invade and transform other cells.Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures.Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathobiology, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

ABSTRACT
The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs) surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cell's astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability). Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1), a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.

Show MeSH
Related in: MedlinePlus