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Nef-mediated clathrin-coated pit formation.

Foti M, Mangasarian A, Piguet V, Lew DP, Krause KH, Trono D, Carpentier JL - J. Cell Biol. (1997)

Bottom Line: Results collected showed that: (a) Nef promotes CD4 internalization via an increased association of CD4 with CCP; (b) the Nef-mediated increase of CD4 association with CCP is related to a doubling of the plasma membrane area occupied by clathrin-coated structures; (c) this increased CCP number at the plasma membrane has functional consequences preferentially on CD4 uptake and does not significantly affect transferrin receptor internalization or fluid-phase endocytosis; (d) the presence of a CD4 cytoplasmic tail including a critical dileucine motif is required to induce CCP formation via Nef; and (e) when directly anchored to the cytoplasmic side of the plasma membrane, Nef itself can promote CCP formation.In this model, Nef interacts on one side with CD4 through a dileucine-based motif present on CD4 cytoplasmic tail and on the other side with components of clathrin-coated surface domain (i.e., adaptins).These Nef-generated complexes would then initiate the nucleation of CCP.

View Article: PubMed Central - PubMed

Affiliation: Department of Morphology, Centre Médical Universitaire, University of Geneva, Switzerland.

ABSTRACT
The sequence of events leading to clathrin-coated pit (CCP) nucleation on the cell surface and to the incorporation of receptors into these endocytic structures is still imperfectly understood. In particular, the question remains as to whether receptor tails initiate the assembly of the coat proteins or whether receptors migrate into preformed CCP. This question was approached through a dissection of the mechanisms implemented by Nef, an early protein of human and simian immunodeficiency virus (HIV and SIV, respectively), to accelerate the endocytosis of cluster of differentiation antigen type 4 (CD4), the major receptor for these viruses. Results collected showed that: (a) Nef promotes CD4 internalization via an increased association of CD4 with CCP; (b) the Nef-mediated increase of CD4 association with CCP is related to a doubling of the plasma membrane area occupied by clathrin-coated structures; (c) this increased CCP number at the plasma membrane has functional consequences preferentially on CD4 uptake and does not significantly affect transferrin receptor internalization or fluid-phase endocytosis; (d) the presence of a CD4 cytoplasmic tail including a critical dileucine motif is required to induce CCP formation via Nef; and (e) when directly anchored to the cytoplasmic side of the plasma membrane, Nef itself can promote CCP formation. Taken together, these observations lead us to propose that CD4 can promote CCP generation via the connector molecule Nef. In this model, Nef interacts on one side with CD4 through a dileucine-based motif present on CD4 cytoplasmic tail and on the other side with components of clathrin-coated surface domain (i.e., adaptins). These Nef-generated complexes would then initiate the nucleation of CCP.

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Nef-induced CD4 internalization and association with  CCP. (A) Kinetics of 125I-antibody–CD4 complex internalization  in CEM T lymphoid cells expressing or not expressing Nef. (B)  Kinetics of 125I–antibody–CD4 complex association with clathrin-coated structures in CEM T lymphoid cells expressing or not expressing Nef. (C) Kinetics of CD4–gold complex association with  clathrin-coated structures in CEM T lymphoid cells expressing or  not expressing Nef. (D) CD4 internalization as a function of CD4  association with CCP in EM autoradiography. The relationship  between CD4 internalization and CD4 association with CCP, in  cells expressing or not expressing Nef, were fitted by a linear regression (R = 0.76). Cells were incubated 2 h at 4°C with 125I– anti-CD4 antibody (or a primary anti-CD4 antibody followed by  a secondary gold–conjugated antibody), and endocytosis of the  radiolabeled antibody–CD4 complex (or CD4–immunogold complex) was allowed to occur by raising the temperature to 37°C for  different periods of time. After cell processing for EM autoradiography or gold detection, quantification was carried out as described previously (Salpeter et al., 1977; Carpentier et al., 1978,  1981, 1991, 1992; Fan et al., 1982). As control, cells harboring an  empty plasmid were used. For each time point studied and for  each cell line, ∼950–1,150 autoradiographic grains (or 700–2,250  gold particles) were analyzed from cells judged to be morphologically well preserved. Autoradiographic grains within a distance  of ±250 nm from the plasma membrane were considered associated with the cell surface; grains overlying the cytoplasm and  >250 nm from the plasma membrane were considered internalized. Grains associated with the plasma membrane were considered associated with CCP if their centers were <250 nm from  these surface domains. Gold particles were considered associated  with clathrin-coated structures when they were observed immediately adjacent (at a distance <20 nm) to the clathrin coat or totally enclosed in clathrin-coated pits/vesicles. Data are mean ±  SEM of three experiments.
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Figure 2: Nef-induced CD4 internalization and association with CCP. (A) Kinetics of 125I-antibody–CD4 complex internalization in CEM T lymphoid cells expressing or not expressing Nef. (B) Kinetics of 125I–antibody–CD4 complex association with clathrin-coated structures in CEM T lymphoid cells expressing or not expressing Nef. (C) Kinetics of CD4–gold complex association with clathrin-coated structures in CEM T lymphoid cells expressing or not expressing Nef. (D) CD4 internalization as a function of CD4 association with CCP in EM autoradiography. The relationship between CD4 internalization and CD4 association with CCP, in cells expressing or not expressing Nef, were fitted by a linear regression (R = 0.76). Cells were incubated 2 h at 4°C with 125I– anti-CD4 antibody (or a primary anti-CD4 antibody followed by a secondary gold–conjugated antibody), and endocytosis of the radiolabeled antibody–CD4 complex (or CD4–immunogold complex) was allowed to occur by raising the temperature to 37°C for different periods of time. After cell processing for EM autoradiography or gold detection, quantification was carried out as described previously (Salpeter et al., 1977; Carpentier et al., 1978, 1981, 1991, 1992; Fan et al., 1982). As control, cells harboring an empty plasmid were used. For each time point studied and for each cell line, ∼950–1,150 autoradiographic grains (or 700–2,250 gold particles) were analyzed from cells judged to be morphologically well preserved. Autoradiographic grains within a distance of ±250 nm from the plasma membrane were considered associated with the cell surface; grains overlying the cytoplasm and >250 nm from the plasma membrane were considered internalized. Grains associated with the plasma membrane were considered associated with CCP if their centers were <250 nm from these surface domains. Gold particles were considered associated with clathrin-coated structures when they were observed immediately adjacent (at a distance <20 nm) to the clathrin coat or totally enclosed in clathrin-coated pits/vesicles. Data are mean ± SEM of three experiments.

Mentions: To understand the mechanisms governing Nef-induced CD4 downregulation, CD4 tagged with 125I–anti-CD4 was tracked morphologically by a quantitative EM autoradiographic analysis in CEM T lymphocytes stably expressing the viral protein (Fig. 1). Confirming previous biochemical observations (Aiken et al., 1994; Rhee and Marsh, 1994), the EM analysis showed that at the permissive temperature of 37°C, endocytosis of 125I–anti-CD4 bound to plasma membrane of Nef-expressing CEM T cells was significantly increased as compared to control cells (stably expressing an empty vector) (Fig. 2 A). After a 2-h incubation at 4°C in the presence of 125I–anti-CD4, ∼3% of plasma membrane–bound CD4 was found associated with CCP in control cells. This value slightly increased with time and temperature to reach 5% after 30 min of incubation at 37°C. By contrast, in cells expressing Nef, the association of 125I–anti-CD4 with CCP peaked after 5 min of incubation at 37°C and plateaued at ∼8% (Fig. 2 B). CD4 association with CCP was alternatively determined by EM immunogold labeling visualized on ultrathin sections. This method allows a more precise localization of anti-CD4 on the cell surface and a better definition of its association with specialized membrane domains such as CCP (Fig. 1 C). Similar conclusions to the one reached using 125I–anti-CD4 as a ligand were collected through immunogold labeling: in the presence of Nef at 37°C, anti-CD4 association with CCP increased at each time point studied by a factor between two and three (Fig. 2 C). The amount of cell surface 125I–anti-CD4 internalized, in both control and Nef-expressing cells, was closely related to the propensity of CD4 to associate with CCP, as shown by the linear relationship (R = 0.76) connecting the two events (Fig. 2 D). Thus, in the presence of Nef, the stimulated rate of CD4 internalization was related, at least in part, to an increased association of the receptor with CCP.


Nef-mediated clathrin-coated pit formation.

Foti M, Mangasarian A, Piguet V, Lew DP, Krause KH, Trono D, Carpentier JL - J. Cell Biol. (1997)

Nef-induced CD4 internalization and association with  CCP. (A) Kinetics of 125I-antibody–CD4 complex internalization  in CEM T lymphoid cells expressing or not expressing Nef. (B)  Kinetics of 125I–antibody–CD4 complex association with clathrin-coated structures in CEM T lymphoid cells expressing or not expressing Nef. (C) Kinetics of CD4–gold complex association with  clathrin-coated structures in CEM T lymphoid cells expressing or  not expressing Nef. (D) CD4 internalization as a function of CD4  association with CCP in EM autoradiography. The relationship  between CD4 internalization and CD4 association with CCP, in  cells expressing or not expressing Nef, were fitted by a linear regression (R = 0.76). Cells were incubated 2 h at 4°C with 125I– anti-CD4 antibody (or a primary anti-CD4 antibody followed by  a secondary gold–conjugated antibody), and endocytosis of the  radiolabeled antibody–CD4 complex (or CD4–immunogold complex) was allowed to occur by raising the temperature to 37°C for  different periods of time. After cell processing for EM autoradiography or gold detection, quantification was carried out as described previously (Salpeter et al., 1977; Carpentier et al., 1978,  1981, 1991, 1992; Fan et al., 1982). As control, cells harboring an  empty plasmid were used. For each time point studied and for  each cell line, ∼950–1,150 autoradiographic grains (or 700–2,250  gold particles) were analyzed from cells judged to be morphologically well preserved. Autoradiographic grains within a distance  of ±250 nm from the plasma membrane were considered associated with the cell surface; grains overlying the cytoplasm and  >250 nm from the plasma membrane were considered internalized. Grains associated with the plasma membrane were considered associated with CCP if their centers were <250 nm from  these surface domains. Gold particles were considered associated  with clathrin-coated structures when they were observed immediately adjacent (at a distance <20 nm) to the clathrin coat or totally enclosed in clathrin-coated pits/vesicles. Data are mean ±  SEM of three experiments.
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Related In: Results  -  Collection

Show All Figures
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Figure 2: Nef-induced CD4 internalization and association with CCP. (A) Kinetics of 125I-antibody–CD4 complex internalization in CEM T lymphoid cells expressing or not expressing Nef. (B) Kinetics of 125I–antibody–CD4 complex association with clathrin-coated structures in CEM T lymphoid cells expressing or not expressing Nef. (C) Kinetics of CD4–gold complex association with clathrin-coated structures in CEM T lymphoid cells expressing or not expressing Nef. (D) CD4 internalization as a function of CD4 association with CCP in EM autoradiography. The relationship between CD4 internalization and CD4 association with CCP, in cells expressing or not expressing Nef, were fitted by a linear regression (R = 0.76). Cells were incubated 2 h at 4°C with 125I– anti-CD4 antibody (or a primary anti-CD4 antibody followed by a secondary gold–conjugated antibody), and endocytosis of the radiolabeled antibody–CD4 complex (or CD4–immunogold complex) was allowed to occur by raising the temperature to 37°C for different periods of time. After cell processing for EM autoradiography or gold detection, quantification was carried out as described previously (Salpeter et al., 1977; Carpentier et al., 1978, 1981, 1991, 1992; Fan et al., 1982). As control, cells harboring an empty plasmid were used. For each time point studied and for each cell line, ∼950–1,150 autoradiographic grains (or 700–2,250 gold particles) were analyzed from cells judged to be morphologically well preserved. Autoradiographic grains within a distance of ±250 nm from the plasma membrane were considered associated with the cell surface; grains overlying the cytoplasm and >250 nm from the plasma membrane were considered internalized. Grains associated with the plasma membrane were considered associated with CCP if their centers were <250 nm from these surface domains. Gold particles were considered associated with clathrin-coated structures when they were observed immediately adjacent (at a distance <20 nm) to the clathrin coat or totally enclosed in clathrin-coated pits/vesicles. Data are mean ± SEM of three experiments.
Mentions: To understand the mechanisms governing Nef-induced CD4 downregulation, CD4 tagged with 125I–anti-CD4 was tracked morphologically by a quantitative EM autoradiographic analysis in CEM T lymphocytes stably expressing the viral protein (Fig. 1). Confirming previous biochemical observations (Aiken et al., 1994; Rhee and Marsh, 1994), the EM analysis showed that at the permissive temperature of 37°C, endocytosis of 125I–anti-CD4 bound to plasma membrane of Nef-expressing CEM T cells was significantly increased as compared to control cells (stably expressing an empty vector) (Fig. 2 A). After a 2-h incubation at 4°C in the presence of 125I–anti-CD4, ∼3% of plasma membrane–bound CD4 was found associated with CCP in control cells. This value slightly increased with time and temperature to reach 5% after 30 min of incubation at 37°C. By contrast, in cells expressing Nef, the association of 125I–anti-CD4 with CCP peaked after 5 min of incubation at 37°C and plateaued at ∼8% (Fig. 2 B). CD4 association with CCP was alternatively determined by EM immunogold labeling visualized on ultrathin sections. This method allows a more precise localization of anti-CD4 on the cell surface and a better definition of its association with specialized membrane domains such as CCP (Fig. 1 C). Similar conclusions to the one reached using 125I–anti-CD4 as a ligand were collected through immunogold labeling: in the presence of Nef at 37°C, anti-CD4 association with CCP increased at each time point studied by a factor between two and three (Fig. 2 C). The amount of cell surface 125I–anti-CD4 internalized, in both control and Nef-expressing cells, was closely related to the propensity of CD4 to associate with CCP, as shown by the linear relationship (R = 0.76) connecting the two events (Fig. 2 D). Thus, in the presence of Nef, the stimulated rate of CD4 internalization was related, at least in part, to an increased association of the receptor with CCP.

Bottom Line: Results collected showed that: (a) Nef promotes CD4 internalization via an increased association of CD4 with CCP; (b) the Nef-mediated increase of CD4 association with CCP is related to a doubling of the plasma membrane area occupied by clathrin-coated structures; (c) this increased CCP number at the plasma membrane has functional consequences preferentially on CD4 uptake and does not significantly affect transferrin receptor internalization or fluid-phase endocytosis; (d) the presence of a CD4 cytoplasmic tail including a critical dileucine motif is required to induce CCP formation via Nef; and (e) when directly anchored to the cytoplasmic side of the plasma membrane, Nef itself can promote CCP formation.In this model, Nef interacts on one side with CD4 through a dileucine-based motif present on CD4 cytoplasmic tail and on the other side with components of clathrin-coated surface domain (i.e., adaptins).These Nef-generated complexes would then initiate the nucleation of CCP.

View Article: PubMed Central - PubMed

Affiliation: Department of Morphology, Centre Médical Universitaire, University of Geneva, Switzerland.

ABSTRACT
The sequence of events leading to clathrin-coated pit (CCP) nucleation on the cell surface and to the incorporation of receptors into these endocytic structures is still imperfectly understood. In particular, the question remains as to whether receptor tails initiate the assembly of the coat proteins or whether receptors migrate into preformed CCP. This question was approached through a dissection of the mechanisms implemented by Nef, an early protein of human and simian immunodeficiency virus (HIV and SIV, respectively), to accelerate the endocytosis of cluster of differentiation antigen type 4 (CD4), the major receptor for these viruses. Results collected showed that: (a) Nef promotes CD4 internalization via an increased association of CD4 with CCP; (b) the Nef-mediated increase of CD4 association with CCP is related to a doubling of the plasma membrane area occupied by clathrin-coated structures; (c) this increased CCP number at the plasma membrane has functional consequences preferentially on CD4 uptake and does not significantly affect transferrin receptor internalization or fluid-phase endocytosis; (d) the presence of a CD4 cytoplasmic tail including a critical dileucine motif is required to induce CCP formation via Nef; and (e) when directly anchored to the cytoplasmic side of the plasma membrane, Nef itself can promote CCP formation. Taken together, these observations lead us to propose that CD4 can promote CCP generation via the connector molecule Nef. In this model, Nef interacts on one side with CD4 through a dileucine-based motif present on CD4 cytoplasmic tail and on the other side with components of clathrin-coated surface domain (i.e., adaptins). These Nef-generated complexes would then initiate the nucleation of CCP.

Show MeSH
Related in: MedlinePlus