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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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Representative electron micrographs of autoradiographic grains (CD4 radiolabeling, a and b) and colloidal gold  particles (CD4 immunogold labeling, c) associated with CCP (arrowheads) in CEM T lymphoid cells. Cells were incubated for 2 h  at 4°C with 125I-anti–CD4 antibody or a primary anti-CD4 antibody followed by a secondary colloidal gold–conjugated antibody  and endocytosis of the immune complex was then allowed to occur by raising the temperature to 37°C for 5 min.
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Figure 1: Representative electron micrographs of autoradiographic grains (CD4 radiolabeling, a and b) and colloidal gold particles (CD4 immunogold labeling, c) associated with CCP (arrowheads) in CEM T lymphoid cells. Cells were incubated for 2 h at 4°C with 125I-anti–CD4 antibody or a primary anti-CD4 antibody followed by a secondary colloidal gold–conjugated antibody and endocytosis of the immune complex was then allowed to occur by raising the temperature to 37°C for 5 min.

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)

Representative electron micrographs of autoradiographic grains (CD4 radiolabeling, a and b) and colloidal gold  particles (CD4 immunogold labeling, c) associated with CCP (arrowheads) in CEM T lymphoid cells. Cells were incubated for 2 h  at 4°C with 125I-anti–CD4 antibody or a primary anti-CD4 antibody followed by a secondary colloidal gold–conjugated antibody  and endocytosis of the immune complex was then allowed to occur by raising the temperature to 37°C for 5 min.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139808&req=5

Figure 1: Representative electron micrographs of autoradiographic grains (CD4 radiolabeling, a and b) and colloidal gold particles (CD4 immunogold labeling, c) associated with CCP (arrowheads) in CEM T lymphoid cells. Cells were incubated for 2 h at 4°C with 125I-anti–CD4 antibody or a primary anti-CD4 antibody followed by a secondary colloidal gold–conjugated antibody and endocytosis of the immune complex was then allowed to occur by raising the temperature to 37°C for 5 min.
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