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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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p104 targeted to mitochondria results in EB1 mislocalization.(A) Images of COS-7 cells expressing either p104-CT-V5 (top panel) or p104-CTSKNN-V5 (lower panel) fused to the mitochondrial targeting sequence of vaccinia virus protein F1L. Mitochondria were visualized using Mitotracker (red) 24 h post transfection prior to fixation in ice-cold methanol and staining with rat anti-EB1 (green) and mouse anti-V5 (white). (B) Image showing 2 transfected cells expressing p104-CT-V5 targeted to mitochondria and one untransfected cell (top); transfected cells were fixed with ice-cold methanol and stained with rat anti-EB1 (green) and mouse anti-V5 (red). DNA is stained with DAPI (blue); Scale bar = 10 µm. Note the presence of MT-associated EB1 in the untransfected cell.
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ppat-1003346-g007: p104 targeted to mitochondria results in EB1 mislocalization.(A) Images of COS-7 cells expressing either p104-CT-V5 (top panel) or p104-CTSKNN-V5 (lower panel) fused to the mitochondrial targeting sequence of vaccinia virus protein F1L. Mitochondria were visualized using Mitotracker (red) 24 h post transfection prior to fixation in ice-cold methanol and staining with rat anti-EB1 (green) and mouse anti-V5 (white). (B) Image showing 2 transfected cells expressing p104-CT-V5 targeted to mitochondria and one untransfected cell (top); transfected cells were fixed with ice-cold methanol and stained with rat anti-EB1 (green) and mouse anti-V5 (red). DNA is stained with DAPI (blue); Scale bar = 10 µm. Note the presence of MT-associated EB1 in the untransfected cell.

Mentions: Next we aimed to establish whether membrane-anchored p104 is capable of recruiting endogenous EB1 independently of other parasite proteins. To that extent, p104-CT was fused to the the C-terminal tail of the vaccinia virus F1L protein which targets the outer membrane of mitochondria [46]. V5-tagged p104-CT was targeted to COS-7 cell mitochondria and EB1 localization monitored by IFM. In transfected cells in which p104-CT was clearly visible at the mitochondria, EB1 was also notably re-distributed to the mitochondria (Figure 7A) Interestingly, in most instances, EB1 mislocalization was accompanied by reduced MT plus end binding. Importantly, whereas p104-CT-SKNN was also targeted to the mitochondria, it failed to recruit EB1 and, in such cells, EB1 localized correctly to MTs (Figure 7A lower panel). Figure 7B shows an area containing two transfected cells and one untransfected cell for comparision, demonstrating that in untransfected cells, EB1 was normally distributed to the plus ends of MTs. These data show that membrane-bound p104 functions as an EB1-recruiting protein, independently of other parasite proteins.


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)

p104 targeted to mitochondria results in EB1 mislocalization.(A) Images of COS-7 cells expressing either p104-CT-V5 (top panel) or p104-CTSKNN-V5 (lower panel) fused to the mitochondrial targeting sequence of vaccinia virus protein F1L. Mitochondria were visualized using Mitotracker (red) 24 h post transfection prior to fixation in ice-cold methanol and staining with rat anti-EB1 (green) and mouse anti-V5 (white). (B) Image showing 2 transfected cells expressing p104-CT-V5 targeted to mitochondria and one untransfected cell (top); transfected cells were fixed with ice-cold methanol and stained with rat anti-EB1 (green) and mouse anti-V5 (red). DNA is stained with DAPI (blue); Scale bar = 10 µm. Note the presence of MT-associated EB1 in the untransfected cell.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003346-g007: p104 targeted to mitochondria results in EB1 mislocalization.(A) Images of COS-7 cells expressing either p104-CT-V5 (top panel) or p104-CTSKNN-V5 (lower panel) fused to the mitochondrial targeting sequence of vaccinia virus protein F1L. Mitochondria were visualized using Mitotracker (red) 24 h post transfection prior to fixation in ice-cold methanol and staining with rat anti-EB1 (green) and mouse anti-V5 (white). (B) Image showing 2 transfected cells expressing p104-CT-V5 targeted to mitochondria and one untransfected cell (top); transfected cells were fixed with ice-cold methanol and stained with rat anti-EB1 (green) and mouse anti-V5 (red). DNA is stained with DAPI (blue); Scale bar = 10 µm. Note the presence of MT-associated EB1 in the untransfected cell.
Mentions: Next we aimed to establish whether membrane-anchored p104 is capable of recruiting endogenous EB1 independently of other parasite proteins. To that extent, p104-CT was fused to the the C-terminal tail of the vaccinia virus F1L protein which targets the outer membrane of mitochondria [46]. V5-tagged p104-CT was targeted to COS-7 cell mitochondria and EB1 localization monitored by IFM. In transfected cells in which p104-CT was clearly visible at the mitochondria, EB1 was also notably re-distributed to the mitochondria (Figure 7A) Interestingly, in most instances, EB1 mislocalization was accompanied by reduced MT plus end binding. Importantly, whereas p104-CT-SKNN was also targeted to the mitochondria, it failed to recruit EB1 and, in such cells, EB1 localized correctly to MTs (Figure 7A lower panel). Figure 7B shows an area containing two transfected cells and one untransfected cell for comparision, demonstrating that in untransfected cells, EB1 was normally distributed to the plus ends of MTs. These data show that membrane-bound p104 functions as an EB1-recruiting protein, independently of other parasite proteins.

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