Limits...
Characterization of the interaction between Toxoplasma gondii rhoptry neck protein 4 and host cellular β-tubulin.

Takemae H, Sugi T, Kobayashi K, Gong H, Ishiwa A, Recuenco FC, Murakoshi F, Iwanaga T, Inomata A, Horimoto T, Akashi H, Kato K - Sci Rep (2013)

Bottom Line: Toxoplasma rhoptry neck protein 4 (TgRON4) is a component of the moving junction macromolecular complex that plays a central role during invasion.Analysis of T. gondii-infected lysates from CHO cells expressing the TgRON4-binding region showed that the C-terminal region of β-tubulin interacted with TgRON4 at early invasion step.Our results provide evidence for a parasite-specific interaction between TgRON4 and the host cell cytoskeleton in parasite-infected cells.

View Article: PubMed Central - PubMed

Affiliation: 1] National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan [2] Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

ABSTRACT
Toxoplasma rhoptry neck protein 4 (TgRON4) is a component of the moving junction macromolecular complex that plays a central role during invasion. TgRON4 is exposed on the cytosolic side of the host cell during invasion, but its molecular interactions remain unclear. Here, we identified host cellular β-tubulin as a binding partner of TgRON4, but not Plasmodium RON4. Coimmunoprecipitation studies in mammalian cells demonstrated that the C-terminal 15-kDa region of β-tubulin was sufficient for binding to TgRON4, and that a 17-kDa region in the proximal C-terminus of TgRON4 was required for binding to the C-terminal region of β-tubulin. Analysis of T. gondii-infected lysates from CHO cells expressing the TgRON4-binding region showed that the C-terminal region of β-tubulin interacted with TgRON4 at early invasion step. Our results provide evidence for a parasite-specific interaction between TgRON4 and the host cell cytoskeleton in parasite-infected cells.

Show MeSH

Related in: MedlinePlus

Localization of the TUBB2C C-terminal region and TgRON4 in the moving junction during Toxoplasma invasion.CHO cells expressing HA-TUBB2C F3 were infected with T. gondii RH tachyzoites using a potassium buffer shift. At 2-min invasion, infected CHO monolayers were fixed and permeabilized. HA-TUBB2C F3 was stained with a rat anti-HA antibody and TgRON4 was stained with an anti-TgRON4 mouse polyclonal antibody. Alexa Fluor 350 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rat IgG served as secondary antibodies. The HA-TUBB2C F3 accumulated around (a) or near (b) the moving junction. The cartoon illustrates the direction of the parasite invasion. Scale bar, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3824165&req=5

f6: Localization of the TUBB2C C-terminal region and TgRON4 in the moving junction during Toxoplasma invasion.CHO cells expressing HA-TUBB2C F3 were infected with T. gondii RH tachyzoites using a potassium buffer shift. At 2-min invasion, infected CHO monolayers were fixed and permeabilized. HA-TUBB2C F3 was stained with a rat anti-HA antibody and TgRON4 was stained with an anti-TgRON4 mouse polyclonal antibody. Alexa Fluor 350 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rat IgG served as secondary antibodies. The HA-TUBB2C F3 accumulated around (a) or near (b) the moving junction. The cartoon illustrates the direction of the parasite invasion. Scale bar, 5 μm.

Mentions: It was recently reported that host cell microtubules localize to a tight junction, called the moving junction, of early invading T. gondii parasites31. To investigate whether the 15-kDa region at the C-terminus of TUBB2C is sufficient for localization to the moving junction, we infected monolayers of CHO cells expressing HA-tagged TUBB2C F3 with T. gondii RH parasites. Parasites were allowed to synchronously invade for 2 min, after which the infected CHO monolayers were fixed with formaldehyde and permeabilized. The anti-TgRON4 polyclonal antibody recognized the moving junction on the invading parasites (Fig. 6a and 6b), and the anti-HA antibody detected TUBB2C F3 accumulation around (Fig. 6a) the moving junction, corresponding to the previous report that host microtubules are asymmetrically concentrated on one side of the moving junction31. While TUBB2C F3 did not completely overlap with TgRON4, TUBB2C F3 and TgRON4 were closely localized in invading parasites (Fig. 6b). Although early invasion of T. gondii seemed unaffected by expressing HA-tagged TUBB2C F3 (Supplemental Fig. 2), these results indicate that the 15-kDa region at the C-terminus of TUBB2C may associate with TgRON4 in invading parasites.


Characterization of the interaction between Toxoplasma gondii rhoptry neck protein 4 and host cellular β-tubulin.

Takemae H, Sugi T, Kobayashi K, Gong H, Ishiwa A, Recuenco FC, Murakoshi F, Iwanaga T, Inomata A, Horimoto T, Akashi H, Kato K - Sci Rep (2013)

Localization of the TUBB2C C-terminal region and TgRON4 in the moving junction during Toxoplasma invasion.CHO cells expressing HA-TUBB2C F3 were infected with T. gondii RH tachyzoites using a potassium buffer shift. At 2-min invasion, infected CHO monolayers were fixed and permeabilized. HA-TUBB2C F3 was stained with a rat anti-HA antibody and TgRON4 was stained with an anti-TgRON4 mouse polyclonal antibody. Alexa Fluor 350 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rat IgG served as secondary antibodies. The HA-TUBB2C F3 accumulated around (a) or near (b) the moving junction. The cartoon illustrates the direction of the parasite invasion. Scale bar, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824165&req=5

f6: Localization of the TUBB2C C-terminal region and TgRON4 in the moving junction during Toxoplasma invasion.CHO cells expressing HA-TUBB2C F3 were infected with T. gondii RH tachyzoites using a potassium buffer shift. At 2-min invasion, infected CHO monolayers were fixed and permeabilized. HA-TUBB2C F3 was stained with a rat anti-HA antibody and TgRON4 was stained with an anti-TgRON4 mouse polyclonal antibody. Alexa Fluor 350 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rat IgG served as secondary antibodies. The HA-TUBB2C F3 accumulated around (a) or near (b) the moving junction. The cartoon illustrates the direction of the parasite invasion. Scale bar, 5 μm.
Mentions: It was recently reported that host cell microtubules localize to a tight junction, called the moving junction, of early invading T. gondii parasites31. To investigate whether the 15-kDa region at the C-terminus of TUBB2C is sufficient for localization to the moving junction, we infected monolayers of CHO cells expressing HA-tagged TUBB2C F3 with T. gondii RH parasites. Parasites were allowed to synchronously invade for 2 min, after which the infected CHO monolayers were fixed with formaldehyde and permeabilized. The anti-TgRON4 polyclonal antibody recognized the moving junction on the invading parasites (Fig. 6a and 6b), and the anti-HA antibody detected TUBB2C F3 accumulation around (Fig. 6a) the moving junction, corresponding to the previous report that host microtubules are asymmetrically concentrated on one side of the moving junction31. While TUBB2C F3 did not completely overlap with TgRON4, TUBB2C F3 and TgRON4 were closely localized in invading parasites (Fig. 6b). Although early invasion of T. gondii seemed unaffected by expressing HA-tagged TUBB2C F3 (Supplemental Fig. 2), these results indicate that the 15-kDa region at the C-terminus of TUBB2C may associate with TgRON4 in invading parasites.

Bottom Line: Toxoplasma rhoptry neck protein 4 (TgRON4) is a component of the moving junction macromolecular complex that plays a central role during invasion.Analysis of T. gondii-infected lysates from CHO cells expressing the TgRON4-binding region showed that the C-terminal region of β-tubulin interacted with TgRON4 at early invasion step.Our results provide evidence for a parasite-specific interaction between TgRON4 and the host cell cytoskeleton in parasite-infected cells.

View Article: PubMed Central - PubMed

Affiliation: 1] National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan [2] Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

ABSTRACT
Toxoplasma rhoptry neck protein 4 (TgRON4) is a component of the moving junction macromolecular complex that plays a central role during invasion. TgRON4 is exposed on the cytosolic side of the host cell during invasion, but its molecular interactions remain unclear. Here, we identified host cellular β-tubulin as a binding partner of TgRON4, but not Plasmodium RON4. Coimmunoprecipitation studies in mammalian cells demonstrated that the C-terminal 15-kDa region of β-tubulin was sufficient for binding to TgRON4, and that a 17-kDa region in the proximal C-terminus of TgRON4 was required for binding to the C-terminal region of β-tubulin. Analysis of T. gondii-infected lysates from CHO cells expressing the TgRON4-binding region showed that the C-terminal region of β-tubulin interacted with TgRON4 at early invasion step. Our results provide evidence for a parasite-specific interaction between TgRON4 and the host cell cytoskeleton in parasite-infected cells.

Show MeSH
Related in: MedlinePlus