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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 increases the extent of plasma membrane coated  by clathrin-lined structures (CCP and clathrin lattices) in CEM  lymphoid T cells. Adherent plasma membranes 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 negatively stained for EM. Membranes considered well conserved were then randomly photographed and the extent of  membrane surfaces coated by clathrin was quantitated on electron micrographs as described in Materials and Methods. Data  are means ± SEM of quantitative analysis performed on 110  cells/487.8 μm2 of plasma membrane segments, and 104 cells/447  μm2 of plasma membrane segments for CEM control (transduced  with a control retroviral vector) and Nef-expressing cells, respectively.
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Figure 4: Nef increases the extent of plasma membrane coated by clathrin-lined structures (CCP and clathrin lattices) in CEM lymphoid T cells. Adherent plasma membranes 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 negatively stained for EM. Membranes considered well conserved were then randomly photographed and the extent of membrane surfaces coated by clathrin was quantitated on electron micrographs as described in Materials and Methods. Data are means ± SEM of quantitative analysis performed on 110 cells/487.8 μm2 of plasma membrane segments, and 104 cells/447 μm2 of plasma membrane segments for CEM control (transduced with a control retroviral vector) and Nef-expressing cells, respectively.

Mentions: Surprisingly, the Nef-triggered CD4 association with CCP correlated with an increase in the plasma membrane surface coated with clathrin in CEM T cells. As determined on conventional EM thin sections, the plasma membrane surface decorated by a clathrin coat (2.0 ± 0.4% of the plasma membrane in control CEM T lymphocytes, 102 cell sections analyzed) increased by 63% in cells transfected with Nef (data not shown). These initial observations were verified on isolated plasma membranes adherent to EM grids and negatively stained (Sanan and Anderson, 1991). This technique allows: (a) an en face view of large surfaces of the inside of the membrane; (b) a distinction between clathrin-coated flat lattices and clathrin-coated invaginations, both easily identified by their typical honeycomb organization; and (c) an accurate quantification of these respective clathrin-coated structures (Fig. 3). Quantitative analysis confirmed that, in the presence of Nef, surfaces occupied by CCP and flat clathrin lattices on the inner leaflet of the plasma membrane increased by 95.4 ± 37.3% (Fig. 4). Breaking these structures into their two components showed that CCP and flat clathrin lattices were increased 2.2- and 1.7-fold, respectively, by Nef expression (representing two out of three and one out of three, of the total clathrin-coated membrane increase, respectively2) indicating that the increase in clathrin-coated plasma membrane was related primarily to an augmentation of invaginated clathrin-coated structures. Under these conditions, CCP size remained unchanged: 86.1 ± 0.9 and 90.8 ± 2.5 nm in control and Nef-expressing cells, respectively. Clathrin synthesis was also not significantly affected by Nef as determined by SDS-PAGE and quantitative analysis of clathrin-specific immunoblots (data not shown). Taken together, these data demonstrate that Nef-induced CD4 internalization is associated with a stimulation of the recruitment and assembly of clathrin coat constituents at the plasma membrane, giving rise to the production of a majority of invaginated 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 increases the extent of plasma membrane coated  by clathrin-lined structures (CCP and clathrin lattices) in CEM  lymphoid T cells. Adherent plasma membranes 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 negatively stained for EM. Membranes considered well conserved were then randomly photographed and the extent of  membrane surfaces coated by clathrin was quantitated on electron micrographs as described in Materials and Methods. Data  are means ± SEM of quantitative analysis performed on 110  cells/487.8 μm2 of plasma membrane segments, and 104 cells/447  μm2 of plasma membrane segments for CEM control (transduced  with a control retroviral vector) and Nef-expressing cells, respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139808&req=5

Figure 4: Nef increases the extent of plasma membrane coated by clathrin-lined structures (CCP and clathrin lattices) in CEM lymphoid T cells. Adherent plasma membranes 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 negatively stained for EM. Membranes considered well conserved were then randomly photographed and the extent of membrane surfaces coated by clathrin was quantitated on electron micrographs as described in Materials and Methods. Data are means ± SEM of quantitative analysis performed on 110 cells/487.8 μm2 of plasma membrane segments, and 104 cells/447 μm2 of plasma membrane segments for CEM control (transduced with a control retroviral vector) and Nef-expressing cells, respectively.
Mentions: Surprisingly, the Nef-triggered CD4 association with CCP correlated with an increase in the plasma membrane surface coated with clathrin in CEM T cells. As determined on conventional EM thin sections, the plasma membrane surface decorated by a clathrin coat (2.0 ± 0.4% of the plasma membrane in control CEM T lymphocytes, 102 cell sections analyzed) increased by 63% in cells transfected with Nef (data not shown). These initial observations were verified on isolated plasma membranes adherent to EM grids and negatively stained (Sanan and Anderson, 1991). This technique allows: (a) an en face view of large surfaces of the inside of the membrane; (b) a distinction between clathrin-coated flat lattices and clathrin-coated invaginations, both easily identified by their typical honeycomb organization; and (c) an accurate quantification of these respective clathrin-coated structures (Fig. 3). Quantitative analysis confirmed that, in the presence of Nef, surfaces occupied by CCP and flat clathrin lattices on the inner leaflet of the plasma membrane increased by 95.4 ± 37.3% (Fig. 4). Breaking these structures into their two components showed that CCP and flat clathrin lattices were increased 2.2- and 1.7-fold, respectively, by Nef expression (representing two out of three and one out of three, of the total clathrin-coated membrane increase, respectively2) indicating that the increase in clathrin-coated plasma membrane was related primarily to an augmentation of invaginated clathrin-coated structures. Under these conditions, CCP size remained unchanged: 86.1 ± 0.9 and 90.8 ± 2.5 nm in control and Nef-expressing cells, respectively. Clathrin synthesis was also not significantly affected by Nef as determined by SDS-PAGE and quantitative analysis of clathrin-specific immunoblots (data not shown). Taken together, these data demonstrate that Nef-induced CD4 internalization is associated with a stimulation of the recruitment and assembly of clathrin coat constituents at the plasma membrane, giving rise to the production of a majority of invaginated 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