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Invasion by Toxoplasma gondii establishes a moving junction that selectively excludes host cell plasma membrane proteins on the basis of their membrane anchoring.

Mordue DG, Desai N, Dustin M, Sibley LD - J. Exp. Med. (1999)

Bottom Line: In contrast, host cell transmembrane proteins, including CD44, Na(+)/K(+) ATPase, and beta1-integrin, were excluded from the vacuole.Wild-type ICAM-1, which contains a transmembrane domain, was excluded from the PV, whereas both GPI-anchored ICAM-1 and a mutant of ICAM-1 missing the cytoplasmic tail (ICAM-1-Cyt(-)) were readily incorporated into the PV membrane.Our results demonstrate that during host cell invasion, Toxoplasma selectively excludes host cell transmembrane proteins at the moving junction by a mechanism that depends on their anchoring in the membrane, thereby creating a nonfusigenic compartment.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The protozoan parasite Toxoplasma gondii actively penetrates its host cell by squeezing through a moving junction that forms between the host cell plasma membrane and the parasite. During invasion, this junction selectively controls internalization of host cell plasma membrane components into the parasite-containing vacuole. Membrane lipids flowed past the junction, as shown by the presence of the glycosphingolipid G(M1) and the cationic lipid label 1. 1'-dihexadecyl-3-3'-3-3'-tetramethylindocarbocyanine (DiIC(16)). Glycosylphosphatidylinositol (GPI)-anchored surface proteins, such as Sca-1 and CD55, were also readily incorporated into the parasitophorous vacuole (PV). In contrast, host cell transmembrane proteins, including CD44, Na(+)/K(+) ATPase, and beta1-integrin, were excluded from the vacuole. To eliminate potential differences in sorting due to the extracellular domains, parasite invasion was examined in host cells transfected with recombinant forms of intercellular adhesion molecule 1 (ICAM-1, CD54) that differed in their mechanism of membrane anchoring. Wild-type ICAM-1, which contains a transmembrane domain, was excluded from the PV, whereas both GPI-anchored ICAM-1 and a mutant of ICAM-1 missing the cytoplasmic tail (ICAM-1-Cyt(-)) were readily incorporated into the PV membrane. Our results demonstrate that during host cell invasion, Toxoplasma selectively excludes host cell transmembrane proteins at the moving junction by a mechanism that depends on their anchoring in the membrane, thereby creating a nonfusigenic compartment.

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Confocal localization of wild-type ICAM-1 versus ICAM-1–GPI, and ICAM-1–Cyt− in BHK cells infected with Toxoplasma (5-min pulse; 0.5-μm section). (A) Wild type ICAM-1 (top, green) was excluded from PVs, whereas ICAM-1–GPI (middle, green) and ICAM-1–Cyt− (bottom, green) were internalized into PVs. Parasites were detected with rabbit anti-p30, followed by Texas red–conjugated goat anti–rabbit IgG. ICAM-1 was detected with mAb RR1/1, followed by bodipy-conjugated goat anti–mouse IgG. Arrowheads mark the position of PVs. Red lines indicate the transects used for densitometry analysis as plotted to the right. The location of the PV along the line is shown by a cartoon of the parasite above the plot. (B) Confocal quantitative analysis of the percentage of PVs that internalized various forms of ICAM-1 after a 5-min challenge with parasites. Results shown are the mean and SE from two experiments. (C) Diagram of the ICAM-1, ICAM-1–GPI, and ICAM-1–Cyt− constructs anchored in the membrane.
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Figure 5: Confocal localization of wild-type ICAM-1 versus ICAM-1–GPI, and ICAM-1–Cyt− in BHK cells infected with Toxoplasma (5-min pulse; 0.5-μm section). (A) Wild type ICAM-1 (top, green) was excluded from PVs, whereas ICAM-1–GPI (middle, green) and ICAM-1–Cyt− (bottom, green) were internalized into PVs. Parasites were detected with rabbit anti-p30, followed by Texas red–conjugated goat anti–rabbit IgG. ICAM-1 was detected with mAb RR1/1, followed by bodipy-conjugated goat anti–mouse IgG. Arrowheads mark the position of PVs. Red lines indicate the transects used for densitometry analysis as plotted to the right. The location of the PV along the line is shown by a cartoon of the parasite above the plot. (B) Confocal quantitative analysis of the percentage of PVs that internalized various forms of ICAM-1 after a 5-min challenge with parasites. Results shown are the mean and SE from two experiments. (C) Diagram of the ICAM-1, ICAM-1–GPI, and ICAM-1–Cyt− constructs anchored in the membrane.

Mentions: The observation that GPI-anchored proteins are incorporated into the PV suggests that membrane anchoring determines internalization. However, to eliminate potential differences due to the size and configuration of the extracellular domain, parasite invasion was examined in BHK cells transiently transfected with recombinant forms of ICAM-1 (CD54) that were anchored into the host cell plasma membrane by different means, as diagrammed in Fig. 5 C. Wild-type ICAM-1 (CD54) is a 90-kD molecule that possesses a single transmembrane segment and short cytoplasmic domain 21. We compared the wild-type ICAM-1 to a lipid-anchored form (ICAM-1–GPI) and a cytoplasmic deletion form (ICAM-1–Cyt−) of the protein 2223. Like other transmembrane proteins, wild-type ICAM-1 was excluded from the PV, whereas ICAM-1–GPI and ICAM-1–Cyt− were both incorporated into PVs. All three forms of ICAM were abundantly expressed and visualized with the same antibody to the extracellular domain, ruling out possible differences due to detection. Exclusion was also not due to size constraints, since the molecular masses of wild-type ICAM-1 and ICAM-1–Cyt− only differ by 3 kD (90 and 87 kD, respectively [22]). To quantify the distribution of the three forms of ICAM-1 in the PV membrane, confocal images were scaled to 256 gray levels and analyzed using the line transect feature of NIH Image. The relative intensity of a line extending through the plasma membrane and across the PV was determined for each of the three forms (Fig. 5 C). These analyses revealed that ICAM-1–GPI and ICAM-1–Cyt− were enriched by two- to threefold in the PV membrane relative to the plasma membrane, whereas wild-type ICAM-1 was found at background levels (Fig. 5 A).


Invasion by Toxoplasma gondii establishes a moving junction that selectively excludes host cell plasma membrane proteins on the basis of their membrane anchoring.

Mordue DG, Desai N, Dustin M, Sibley LD - J. Exp. Med. (1999)

Confocal localization of wild-type ICAM-1 versus ICAM-1–GPI, and ICAM-1–Cyt− in BHK cells infected with Toxoplasma (5-min pulse; 0.5-μm section). (A) Wild type ICAM-1 (top, green) was excluded from PVs, whereas ICAM-1–GPI (middle, green) and ICAM-1–Cyt− (bottom, green) were internalized into PVs. Parasites were detected with rabbit anti-p30, followed by Texas red–conjugated goat anti–rabbit IgG. ICAM-1 was detected with mAb RR1/1, followed by bodipy-conjugated goat anti–mouse IgG. Arrowheads mark the position of PVs. Red lines indicate the transects used for densitometry analysis as plotted to the right. The location of the PV along the line is shown by a cartoon of the parasite above the plot. (B) Confocal quantitative analysis of the percentage of PVs that internalized various forms of ICAM-1 after a 5-min challenge with parasites. Results shown are the mean and SE from two experiments. (C) Diagram of the ICAM-1, ICAM-1–GPI, and ICAM-1–Cyt− constructs anchored in the membrane.
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Figure 5: Confocal localization of wild-type ICAM-1 versus ICAM-1–GPI, and ICAM-1–Cyt− in BHK cells infected with Toxoplasma (5-min pulse; 0.5-μm section). (A) Wild type ICAM-1 (top, green) was excluded from PVs, whereas ICAM-1–GPI (middle, green) and ICAM-1–Cyt− (bottom, green) were internalized into PVs. Parasites were detected with rabbit anti-p30, followed by Texas red–conjugated goat anti–rabbit IgG. ICAM-1 was detected with mAb RR1/1, followed by bodipy-conjugated goat anti–mouse IgG. Arrowheads mark the position of PVs. Red lines indicate the transects used for densitometry analysis as plotted to the right. The location of the PV along the line is shown by a cartoon of the parasite above the plot. (B) Confocal quantitative analysis of the percentage of PVs that internalized various forms of ICAM-1 after a 5-min challenge with parasites. Results shown are the mean and SE from two experiments. (C) Diagram of the ICAM-1, ICAM-1–GPI, and ICAM-1–Cyt− constructs anchored in the membrane.
Mentions: The observation that GPI-anchored proteins are incorporated into the PV suggests that membrane anchoring determines internalization. However, to eliminate potential differences due to the size and configuration of the extracellular domain, parasite invasion was examined in BHK cells transiently transfected with recombinant forms of ICAM-1 (CD54) that were anchored into the host cell plasma membrane by different means, as diagrammed in Fig. 5 C. Wild-type ICAM-1 (CD54) is a 90-kD molecule that possesses a single transmembrane segment and short cytoplasmic domain 21. We compared the wild-type ICAM-1 to a lipid-anchored form (ICAM-1–GPI) and a cytoplasmic deletion form (ICAM-1–Cyt−) of the protein 2223. Like other transmembrane proteins, wild-type ICAM-1 was excluded from the PV, whereas ICAM-1–GPI and ICAM-1–Cyt− were both incorporated into PVs. All three forms of ICAM were abundantly expressed and visualized with the same antibody to the extracellular domain, ruling out possible differences due to detection. Exclusion was also not due to size constraints, since the molecular masses of wild-type ICAM-1 and ICAM-1–Cyt− only differ by 3 kD (90 and 87 kD, respectively [22]). To quantify the distribution of the three forms of ICAM-1 in the PV membrane, confocal images were scaled to 256 gray levels and analyzed using the line transect feature of NIH Image. The relative intensity of a line extending through the plasma membrane and across the PV was determined for each of the three forms (Fig. 5 C). These analyses revealed that ICAM-1–GPI and ICAM-1–Cyt− were enriched by two- to threefold in the PV membrane relative to the plasma membrane, whereas wild-type ICAM-1 was found at background levels (Fig. 5 A).

Bottom Line: In contrast, host cell transmembrane proteins, including CD44, Na(+)/K(+) ATPase, and beta1-integrin, were excluded from the vacuole.Wild-type ICAM-1, which contains a transmembrane domain, was excluded from the PV, whereas both GPI-anchored ICAM-1 and a mutant of ICAM-1 missing the cytoplasmic tail (ICAM-1-Cyt(-)) were readily incorporated into the PV membrane.Our results demonstrate that during host cell invasion, Toxoplasma selectively excludes host cell transmembrane proteins at the moving junction by a mechanism that depends on their anchoring in the membrane, thereby creating a nonfusigenic compartment.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The protozoan parasite Toxoplasma gondii actively penetrates its host cell by squeezing through a moving junction that forms between the host cell plasma membrane and the parasite. During invasion, this junction selectively controls internalization of host cell plasma membrane components into the parasite-containing vacuole. Membrane lipids flowed past the junction, as shown by the presence of the glycosphingolipid G(M1) and the cationic lipid label 1. 1'-dihexadecyl-3-3'-3-3'-tetramethylindocarbocyanine (DiIC(16)). Glycosylphosphatidylinositol (GPI)-anchored surface proteins, such as Sca-1 and CD55, were also readily incorporated into the parasitophorous vacuole (PV). In contrast, host cell transmembrane proteins, including CD44, Na(+)/K(+) ATPase, and beta1-integrin, were excluded from the vacuole. To eliminate potential differences in sorting due to the extracellular domains, parasite invasion was examined in host cells transfected with recombinant forms of intercellular adhesion molecule 1 (ICAM-1, CD54) that differed in their mechanism of membrane anchoring. Wild-type ICAM-1, which contains a transmembrane domain, was excluded from the PV, whereas both GPI-anchored ICAM-1 and a mutant of ICAM-1 missing the cytoplasmic tail (ICAM-1-Cyt(-)) were readily incorporated into the PV membrane. Our results demonstrate that during host cell invasion, Toxoplasma selectively excludes host cell transmembrane proteins at the moving junction by a mechanism that depends on their anchoring in the membrane, thereby creating a nonfusigenic compartment.

Show MeSH
Related in: MedlinePlus