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The role of dynamin and its binding partners in coated pit invagination and scission.

Hill E, van Der Kaay J, Downes CP, Smythe E - J. Cell Biol. (2001)

Bottom Line: Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits.We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro.This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Cell Biology, Wellcome Trust Biocentre, Dundee DD1 5EH, United Kingdom.

ABSTRACT
Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain-containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission. To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits. We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.

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Exogenously added dynamin stimulates the late stages of invagination but not scission in permeabilized A431 cells. (a) B-SS-Tfn sequestration and internalization were measured in the avidin inaccessibility assay in the presence of increasing amounts of cytosol as indicated, in the presence or absence of 5 μg of purified dynamin. Results are from a typical experiment where each assay point was carried out in duplicate which differed from each other <10%. (b) B-SS-Tfn sequestration and internalization measured by the avidin inaccessibility assay (filled squares) and internalization measured by the MesNa resistance assay (filled diamonds) were analyzed in the presence of cytosol (2.5 mg/ml) in the presence of increasing amounts of dynamin. Results are from a typical experiment where each assay point is the mean of duplicates which differed by <10%.
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Figure 4: Exogenously added dynamin stimulates the late stages of invagination but not scission in permeabilized A431 cells. (a) B-SS-Tfn sequestration and internalization were measured in the avidin inaccessibility assay in the presence of increasing amounts of cytosol as indicated, in the presence or absence of 5 μg of purified dynamin. Results are from a typical experiment where each assay point was carried out in duplicate which differed from each other <10%. (b) B-SS-Tfn sequestration and internalization measured by the avidin inaccessibility assay (filled squares) and internalization measured by the MesNa resistance assay (filled diamonds) were analyzed in the presence of cytosol (2.5 mg/ml) in the presence of increasing amounts of dynamin. Results are from a typical experiment where each assay point is the mean of duplicates which differed by <10%.

Mentions: Coated pits on membranes which have been preincubated with GST–amph2 SH3D36R were able to become deeply invaginated and sequester ligand in the presence of added dynamin. This indicated that it is possible to reconstitute the recruitment of dynamin from the cytosol onto the membranes of permeabilized cells. Given our result that the SH3 domain of amphiphysin can inhibit both the late stages of coated pit invagination (measured by the avidin inaccessibility assay) and clathrin-coated vesicle scission (measured by MesNa resistance), we were interested to know whether adding additional dynamin directly into assay mixes could stimulate B-SS-Tfn sequestration. Therefore, we measured the effects of purified dynamin in the endocytic assays. Fig. 4 a shows a cytosol titration in the presence of purified dynamin in the avidin inaccessibility assay. There is a significant stimulation of ligand sequestration. The maximum stimulation with dynamin in the avidin inaccessibility assay was observed at high cytosol concentrations and half-maximal stimulation was seen at ∼0.3 μM dynamin (Fig. 4 b). In contrast, addition of purified dynamin to the MesNa resistance assay had no significant effect on transferrin internalization into sealed coated vesicles (Fig. 4 b). Exogenously added dynamin can therefore stimulate the formation of deeply invaginated coated pits in a cytosol-dependent manner but has no effect on coated vesicle scission in permeabilized A431 cells. Thus, dynamin cannot rescue coated vesicle scission in GST–amph2 SH3D36R treated membranes nor can it stimulate this stage in untreated membranes.


The role of dynamin and its binding partners in coated pit invagination and scission.

Hill E, van Der Kaay J, Downes CP, Smythe E - J. Cell Biol. (2001)

Exogenously added dynamin stimulates the late stages of invagination but not scission in permeabilized A431 cells. (a) B-SS-Tfn sequestration and internalization were measured in the avidin inaccessibility assay in the presence of increasing amounts of cytosol as indicated, in the presence or absence of 5 μg of purified dynamin. Results are from a typical experiment where each assay point was carried out in duplicate which differed from each other <10%. (b) B-SS-Tfn sequestration and internalization measured by the avidin inaccessibility assay (filled squares) and internalization measured by the MesNa resistance assay (filled diamonds) were analyzed in the presence of cytosol (2.5 mg/ml) in the presence of increasing amounts of dynamin. Results are from a typical experiment where each assay point is the mean of duplicates which differed by <10%.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199618&req=5

Figure 4: Exogenously added dynamin stimulates the late stages of invagination but not scission in permeabilized A431 cells. (a) B-SS-Tfn sequestration and internalization were measured in the avidin inaccessibility assay in the presence of increasing amounts of cytosol as indicated, in the presence or absence of 5 μg of purified dynamin. Results are from a typical experiment where each assay point was carried out in duplicate which differed from each other <10%. (b) B-SS-Tfn sequestration and internalization measured by the avidin inaccessibility assay (filled squares) and internalization measured by the MesNa resistance assay (filled diamonds) were analyzed in the presence of cytosol (2.5 mg/ml) in the presence of increasing amounts of dynamin. Results are from a typical experiment where each assay point is the mean of duplicates which differed by <10%.
Mentions: Coated pits on membranes which have been preincubated with GST–amph2 SH3D36R were able to become deeply invaginated and sequester ligand in the presence of added dynamin. This indicated that it is possible to reconstitute the recruitment of dynamin from the cytosol onto the membranes of permeabilized cells. Given our result that the SH3 domain of amphiphysin can inhibit both the late stages of coated pit invagination (measured by the avidin inaccessibility assay) and clathrin-coated vesicle scission (measured by MesNa resistance), we were interested to know whether adding additional dynamin directly into assay mixes could stimulate B-SS-Tfn sequestration. Therefore, we measured the effects of purified dynamin in the endocytic assays. Fig. 4 a shows a cytosol titration in the presence of purified dynamin in the avidin inaccessibility assay. There is a significant stimulation of ligand sequestration. The maximum stimulation with dynamin in the avidin inaccessibility assay was observed at high cytosol concentrations and half-maximal stimulation was seen at ∼0.3 μM dynamin (Fig. 4 b). In contrast, addition of purified dynamin to the MesNa resistance assay had no significant effect on transferrin internalization into sealed coated vesicles (Fig. 4 b). Exogenously added dynamin can therefore stimulate the formation of deeply invaginated coated pits in a cytosol-dependent manner but has no effect on coated vesicle scission in permeabilized A431 cells. Thus, dynamin cannot rescue coated vesicle scission in GST–amph2 SH3D36R treated membranes nor can it stimulate this stage in untreated membranes.

Bottom Line: Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits.We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro.This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Cell Biology, Wellcome Trust Biocentre, Dundee DD1 5EH, United Kingdom.

ABSTRACT
Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain-containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission. To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits. We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.

Show MeSH
Related in: MedlinePlus