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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 showing the association of CD4 (a and b, autoradiographic grains) and transferrin  receptors (b, gold particles) with CCP (arrowheads) in CEM T  lymphoid cells. CD4 and transferrin receptor association with  CCP were concomitantly detected in CEM T cells. CD4 was  traced with 125I–anti-CD4 with the Tf-R was labeled with an anti– Tf-R primary antibody followed by a secondary antibody coupled  with colloidal gold. Endocytosis of the radiolabeled antibody– CD4 complex and gold-conjugated transferrin receptor was the  allowed to occur by raising the temperature to 37°C for 5 min. After cell processing for EM autoradiography, quantification was  carried out as described previously (Carpentier et al., 1978, 1981,  1992; Fan et al., 1982). Autoradiographic grains located on the  plasma membrane were considered associated with CCP if their  centers were <250 nm from these surface domains. Gold particles were considered associated with CCP when they were observed immediately adjacent (at a distance <20 nm) to the clathrin coat or totally enclosed in CCP.
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Figure 6: Representative electron micrographs showing the association of CD4 (a and b, autoradiographic grains) and transferrin receptors (b, gold particles) with CCP (arrowheads) in CEM T lymphoid cells. CD4 and transferrin receptor association with CCP were concomitantly detected in CEM T cells. CD4 was traced with 125I–anti-CD4 with the Tf-R was labeled with an anti– Tf-R primary antibody followed by a secondary antibody coupled with colloidal gold. Endocytosis of the radiolabeled antibody– CD4 complex and gold-conjugated transferrin receptor was the allowed to occur by raising the temperature to 37°C for 5 min. After cell processing for EM autoradiography, quantification was carried out as described previously (Carpentier et al., 1978, 1981, 1992; Fan et al., 1982). Autoradiographic grains located on the plasma membrane were considered associated with CCP if their centers were <250 nm from these surface domains. Gold particles were considered associated with CCP when they were observed immediately adjacent (at a distance <20 nm) to the clathrin coat or totally enclosed in CCP.

Mentions: The observation that Nef triggered the formation of cell surface CCP while concomitantly and specifically inducing CD4 endocytosis suggested that the viral protein was mediating the generation of CD4-enriched CCP. This hypothesis was tested by the simultaneous morphological tracking of CD4 and Tf-Rs. Surface CD4 molecules were traced with 125I–anti-CD4 while Tf-Rs were labeled with an anti– Tf-R primary antibody followed by a secondary antibody coupled with colloidal gold (10 nm) (Fig. 6). In the presence of Nef, the per cell number of CCP containing exclusively CD4 tripled, while the corresponding number of CCP incorporating solely the transferrin receptor only increased by ∼7% (Table I). The increase in the number of CCP labeled with both markers showed that additional CCP appearing in Nef-expressing cells do not incorporate exclusively CD4 but may trap, to a low extent, other receptors. This low amount of receptors other than CD4 present in additionally formed CCP is possibly responsible for the small increase in transferrin internalization observed in Nef-expressing cells (Fig. 5 A). The number of unlabeled CCP was also significantly increased by Nef expression. Given that the analysis was performed on thin sections, which does not allow visualization of the entire clathrin-coated pits, and that 125I–anti-CD4 binding was not saturating, results collected led to an underestimation of CD4 potentially present in these structures and to an overestimation of unlabeled CCP. This was verified in the course of an immunogold CD4 labeling detected on adherent plasma membranes preparations (Fig. 3 C). After 5 min of incubation at 37°C of CEM lymphocytes surface- labeled with anti-CD4 immunocomplexes, 13% of the total CCP counted on the adherent membranes incorporated the immunogold probe (data not shown) as compared with the 6.6% observed in the case of 125I–anti-CD4 binding (see above). As compared to the increase in the total number of clathrin-coated pits, these values remain low, which leaves the possibility open that some of the newly formed CCP are not functional, although the preferential Nef- induced formation of CCP versus flat clathrin lattices is in favor of the functionality of the generated structures. Moreover, Nef might generate additional CCP in CEM lymphocytes incorporating some unidentified receptors, the endocytosis of which could also be increased by the viral protein. Of note, in CEM T lymphocytes as well as in Namalwa B lymphocytes, little Nef-induced MHCI downregulation was observed (data not shown). Together, these morphological analyses strongly suggest that at least part of the Nef-induced CCP preferentially contain CD4.


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 showing the association of CD4 (a and b, autoradiographic grains) and transferrin  receptors (b, gold particles) with CCP (arrowheads) in CEM T  lymphoid cells. CD4 and transferrin receptor association with  CCP were concomitantly detected in CEM T cells. CD4 was  traced with 125I–anti-CD4 with the Tf-R was labeled with an anti– Tf-R primary antibody followed by a secondary antibody coupled  with colloidal gold. Endocytosis of the radiolabeled antibody– CD4 complex and gold-conjugated transferrin receptor was the  allowed to occur by raising the temperature to 37°C for 5 min. After cell processing for EM autoradiography, quantification was  carried out as described previously (Carpentier et al., 1978, 1981,  1992; Fan et al., 1982). Autoradiographic grains located on the  plasma membrane were considered associated with CCP if their  centers were <250 nm from these surface domains. Gold particles were considered associated with CCP when they were observed immediately adjacent (at a distance <20 nm) to the clathrin coat or totally enclosed in CCP.
© Copyright Policy
Related In: Results  -  Collection

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Figure 6: Representative electron micrographs showing the association of CD4 (a and b, autoradiographic grains) and transferrin receptors (b, gold particles) with CCP (arrowheads) in CEM T lymphoid cells. CD4 and transferrin receptor association with CCP were concomitantly detected in CEM T cells. CD4 was traced with 125I–anti-CD4 with the Tf-R was labeled with an anti– Tf-R primary antibody followed by a secondary antibody coupled with colloidal gold. Endocytosis of the radiolabeled antibody– CD4 complex and gold-conjugated transferrin receptor was the allowed to occur by raising the temperature to 37°C for 5 min. After cell processing for EM autoradiography, quantification was carried out as described previously (Carpentier et al., 1978, 1981, 1992; Fan et al., 1982). Autoradiographic grains located on the plasma membrane were considered associated with CCP if their centers were <250 nm from these surface domains. Gold particles were considered associated with CCP when they were observed immediately adjacent (at a distance <20 nm) to the clathrin coat or totally enclosed in CCP.
Mentions: The observation that Nef triggered the formation of cell surface CCP while concomitantly and specifically inducing CD4 endocytosis suggested that the viral protein was mediating the generation of CD4-enriched CCP. This hypothesis was tested by the simultaneous morphological tracking of CD4 and Tf-Rs. Surface CD4 molecules were traced with 125I–anti-CD4 while Tf-Rs were labeled with an anti– Tf-R primary antibody followed by a secondary antibody coupled with colloidal gold (10 nm) (Fig. 6). In the presence of Nef, the per cell number of CCP containing exclusively CD4 tripled, while the corresponding number of CCP incorporating solely the transferrin receptor only increased by ∼7% (Table I). The increase in the number of CCP labeled with both markers showed that additional CCP appearing in Nef-expressing cells do not incorporate exclusively CD4 but may trap, to a low extent, other receptors. This low amount of receptors other than CD4 present in additionally formed CCP is possibly responsible for the small increase in transferrin internalization observed in Nef-expressing cells (Fig. 5 A). The number of unlabeled CCP was also significantly increased by Nef expression. Given that the analysis was performed on thin sections, which does not allow visualization of the entire clathrin-coated pits, and that 125I–anti-CD4 binding was not saturating, results collected led to an underestimation of CD4 potentially present in these structures and to an overestimation of unlabeled CCP. This was verified in the course of an immunogold CD4 labeling detected on adherent plasma membranes preparations (Fig. 3 C). After 5 min of incubation at 37°C of CEM lymphocytes surface- labeled with anti-CD4 immunocomplexes, 13% of the total CCP counted on the adherent membranes incorporated the immunogold probe (data not shown) as compared with the 6.6% observed in the case of 125I–anti-CD4 binding (see above). As compared to the increase in the total number of clathrin-coated pits, these values remain low, which leaves the possibility open that some of the newly formed CCP are not functional, although the preferential Nef- induced formation of CCP versus flat clathrin lattices is in favor of the functionality of the generated structures. Moreover, Nef might generate additional CCP in CEM lymphocytes incorporating some unidentified receptors, the endocytosis of which could also be increased by the viral protein. Of note, in CEM T lymphocytes as well as in Namalwa B lymphocytes, little Nef-induced MHCI downregulation was observed (data not shown). Together, these morphological analyses strongly suggest that at least part of the Nef-induced CCP preferentially contain CD4.

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