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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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Relative amount of CCP decorating the plasma membrane of transfected Namalwa B lymphocytes. Cells were transfected with CD4, Nef, a dileucine mutated form of CD4,  (CD4ΔLL), CD4+Nef, CD4ΔLL+Nef, and the chimera 44Nef. As  control, cells transfected with an empty plasmid vector were used.  CCP on the inner face of the plasma membrane (PM) were visualized as described by Sanan and Anderson (1991) with minor  modifications. Briefly, cells were allowed to sediment on poly-l-lysine coated grids for 1 h at 4°C and adherent plasma membranes were then obtained by incubating the whole cells with hypotonic medium followed by a sonication at a weak power. This  procedure disrupts the cells but allows a large portion of plasma  membranes, with conserved internal structures such as clathrin-coated membranes and cytoskeleton elements, to stay adherent  to the poly-l-lysine–coated grids. Adherent membranes were  next fixed and negative stained for EM. Membranes considered  well conserved were then randomly photographed and the  amount of CCP present on plasma membrane segments was  quantitated on electron micrographs as described in Materials  and Methods. Data are means ± SEM of quantitiatve analysis  performed on 155 cells/710.6 μm2, 229 cells/998.0 μm2, 100 cells/ 459.3 μm2, 106 cells/456.4 μm2, 112 cells/445.7 μm2, 158 cells/ 656.3 μm2, and 104 cells/474.6 μm2 of plasma membrane segments from Namalwa transfected with the empty plasmid, CD4,  Nef, CD4ΔLL, CD4+Nef, CD4ΔLL+ Nef, and 44Nef, respectively.
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Figure 7: Relative amount of CCP decorating the plasma membrane of transfected Namalwa B lymphocytes. Cells were transfected with CD4, Nef, a dileucine mutated form of CD4, (CD4ΔLL), CD4+Nef, CD4ΔLL+Nef, and the chimera 44Nef. As control, cells transfected with an empty plasmid vector were used. CCP on the inner face of the plasma membrane (PM) were visualized as described by Sanan and Anderson (1991) with minor modifications. Briefly, cells were allowed to sediment on poly-l-lysine coated grids for 1 h at 4°C and adherent plasma membranes were then obtained by incubating the whole cells with hypotonic medium followed by a sonication at a weak power. This procedure disrupts the cells but allows a large portion of plasma membranes, with conserved internal structures such as clathrin-coated membranes and cytoskeleton elements, to stay adherent to the poly-l-lysine–coated grids. Adherent membranes were next fixed and negative stained for EM. Membranes considered well conserved were then randomly photographed and the amount of CCP present on plasma membrane segments was quantitated on electron micrographs as described in Materials and Methods. Data are means ± SEM of quantitiatve analysis performed on 155 cells/710.6 μm2, 229 cells/998.0 μm2, 100 cells/ 459.3 μm2, 106 cells/456.4 μm2, 112 cells/445.7 μm2, 158 cells/ 656.3 μm2, and 104 cells/474.6 μm2 of plasma membrane segments from Namalwa transfected with the empty plasmid, CD4, Nef, CD4ΔLL, CD4+Nef, CD4ΔLL+ Nef, and 44Nef, respectively.

Mentions: Was CD4 concentrated in Nef-induced preformed CCP or was it the primum movens in the generation of these structures? To answer this question, Namalwa B cells, which do not naturally express CD4, were stably transfected with CD4 and/or Nef (Mangasarian et al., 1997). The presence of either CD4 or Nef alone did not result in any increase of CCP over basal values (control = cells transfected with an empty vector), while the concomitant expression of CD4 and Nef promoted CCP formation (Fig. 7), in agreement with the increased rate of CD4 internalization in these cells (Fig. 8). Thus, CD4 was not segregated in preformed Nef-induced CCP, but instead the receptor participated in CCP formation. A possible implication of the CD4 cytoplasmic dileucine motif in CCP generation was tested by replacing these residues by alanines (CD4ΔLL). As previously described (Aiken et al., 1994), this mutation completely abolished Nef-induced CD4 accelerated endocytosis (Fig. 8). The quantitative morphological analysis of CCP present at the plasma membrane of established cell lines coexpressing Nef together with either wild-type or dileucine-mutated CD4 was consistent with the internalization kinetics of these molecules. The amount of CCP decorating the plasma membrane was thus similar in cells expressing either of these proteins. However, Nef induced the formation of CCP only when coexpressed with wild-type CD4, not with the CD4ΔLL mutant (Fig. 7). Together, these data demonstrate that the generation of new CCP capable of triggering CD4 internalization not only required Nef, but also a receptor cytoplasmic tail capable of a functional interaction with the viral protein.


Nef-mediated clathrin-coated pit formation.

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

Relative amount of CCP decorating the plasma membrane of transfected Namalwa B lymphocytes. Cells were transfected with CD4, Nef, a dileucine mutated form of CD4,  (CD4ΔLL), CD4+Nef, CD4ΔLL+Nef, and the chimera 44Nef. As  control, cells transfected with an empty plasmid vector were used.  CCP on the inner face of the plasma membrane (PM) were visualized as described by Sanan and Anderson (1991) with minor  modifications. Briefly, cells were allowed to sediment on poly-l-lysine coated grids for 1 h at 4°C and adherent plasma membranes were then obtained by incubating the whole cells with hypotonic medium followed by a sonication at a weak power. This  procedure disrupts the cells but allows a large portion of plasma  membranes, with conserved internal structures such as clathrin-coated membranes and cytoskeleton elements, to stay adherent  to the poly-l-lysine–coated grids. Adherent membranes were  next fixed and negative stained for EM. Membranes considered  well conserved were then randomly photographed and the  amount of CCP present on plasma membrane segments was  quantitated on electron micrographs as described in Materials  and Methods. Data are means ± SEM of quantitiatve analysis  performed on 155 cells/710.6 μm2, 229 cells/998.0 μm2, 100 cells/ 459.3 μm2, 106 cells/456.4 μm2, 112 cells/445.7 μm2, 158 cells/ 656.3 μm2, and 104 cells/474.6 μm2 of plasma membrane segments from Namalwa transfected with the empty plasmid, CD4,  Nef, CD4ΔLL, CD4+Nef, CD4ΔLL+ Nef, and 44Nef, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139808&req=5

Figure 7: Relative amount of CCP decorating the plasma membrane of transfected Namalwa B lymphocytes. Cells were transfected with CD4, Nef, a dileucine mutated form of CD4, (CD4ΔLL), CD4+Nef, CD4ΔLL+Nef, and the chimera 44Nef. As control, cells transfected with an empty plasmid vector were used. CCP on the inner face of the plasma membrane (PM) were visualized as described by Sanan and Anderson (1991) with minor modifications. Briefly, cells were allowed to sediment on poly-l-lysine coated grids for 1 h at 4°C and adherent plasma membranes were then obtained by incubating the whole cells with hypotonic medium followed by a sonication at a weak power. This procedure disrupts the cells but allows a large portion of plasma membranes, with conserved internal structures such as clathrin-coated membranes and cytoskeleton elements, to stay adherent to the poly-l-lysine–coated grids. Adherent membranes were next fixed and negative stained for EM. Membranes considered well conserved were then randomly photographed and the amount of CCP present on plasma membrane segments was quantitated on electron micrographs as described in Materials and Methods. Data are means ± SEM of quantitiatve analysis performed on 155 cells/710.6 μm2, 229 cells/998.0 μm2, 100 cells/ 459.3 μm2, 106 cells/456.4 μm2, 112 cells/445.7 μm2, 158 cells/ 656.3 μm2, and 104 cells/474.6 μm2 of plasma membrane segments from Namalwa transfected with the empty plasmid, CD4, Nef, CD4ΔLL, CD4+Nef, CD4ΔLL+ Nef, and 44Nef, respectively.
Mentions: Was CD4 concentrated in Nef-induced preformed CCP or was it the primum movens in the generation of these structures? To answer this question, Namalwa B cells, which do not naturally express CD4, were stably transfected with CD4 and/or Nef (Mangasarian et al., 1997). The presence of either CD4 or Nef alone did not result in any increase of CCP over basal values (control = cells transfected with an empty vector), while the concomitant expression of CD4 and Nef promoted CCP formation (Fig. 7), in agreement with the increased rate of CD4 internalization in these cells (Fig. 8). Thus, CD4 was not segregated in preformed Nef-induced CCP, but instead the receptor participated in CCP formation. A possible implication of the CD4 cytoplasmic dileucine motif in CCP generation was tested by replacing these residues by alanines (CD4ΔLL). As previously described (Aiken et al., 1994), this mutation completely abolished Nef-induced CD4 accelerated endocytosis (Fig. 8). The quantitative morphological analysis of CCP present at the plasma membrane of established cell lines coexpressing Nef together with either wild-type or dileucine-mutated CD4 was consistent with the internalization kinetics of these molecules. The amount of CCP decorating the plasma membrane was thus similar in cells expressing either of these proteins. However, Nef induced the formation of CCP only when coexpressed with wild-type CD4, not with the CD4ΔLL mutant (Fig. 7). Together, these data demonstrate that the generation of new CCP capable of triggering CD4 internalization not only required Nef, but also a receptor cytoplasmic tail capable of a functional interaction with the viral protein.

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