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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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The SH3 domain of endophilin inhibits invagination and scission by reducing PtdInsP2 levels with a concomitant loss of coated pit components from the plasma membrane. (a) Permeabilized cell membranes which had been incubated with cytosol (2.5 mg/ml) and ATP for 30 min at 37°C in the presence or absence of GST–endoSH3 or GST–amph2 SH3D36R (25 μg/ml) were collected by centrifugation and assayed for PtdInsP2 levels. Results are expressed as the mean ± SD of two different experiments each performed in triplicate. (b) Western blots of the permeabilized cell membranes obtained from assay mixtures which had been incubated in the absence or presence of GST–endoSH3 or GST–amph2 SH3 D36R. The membranes were probed for α-adaptin, clathrin, dynamin, synaptojanin, and transferrin receptor as indicated. (c) Permeabilized cells which had been incubated with cytosol (2.5 mg/ml), ATP, and in the absence or presence of GST–endoSH3 or GST–amph2 SH3D36R were centrifuged onto coverslips and analyzed by indirect immunofluorescence for the presence of α-adaptin, clathrin, and dynamin.
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Figure 7: The SH3 domain of endophilin inhibits invagination and scission by reducing PtdInsP2 levels with a concomitant loss of coated pit components from the plasma membrane. (a) Permeabilized cell membranes which had been incubated with cytosol (2.5 mg/ml) and ATP for 30 min at 37°C in the presence or absence of GST–endoSH3 or GST–amph2 SH3D36R (25 μg/ml) were collected by centrifugation and assayed for PtdInsP2 levels. Results are expressed as the mean ± SD of two different experiments each performed in triplicate. (b) Western blots of the permeabilized cell membranes obtained from assay mixtures which had been incubated in the absence or presence of GST–endoSH3 or GST–amph2 SH3 D36R. The membranes were probed for α-adaptin, clathrin, dynamin, synaptojanin, and transferrin receptor as indicated. (c) Permeabilized cells which had been incubated with cytosol (2.5 mg/ml), ATP, and in the absence or presence of GST–endoSH3 or GST–amph2 SH3D36R were centrifuged onto coverslips and analyzed by indirect immunofluorescence for the presence of α-adaptin, clathrin, and dynamin.

Mentions: Synaptojanin is an inositol 5-phosphatase whose major in vivo substrates are likely to be PtdInsP2 and PtdInsP3 (Woscholski et al. 1997). Recently, a synaptojanin knockout mouse has been generated by de Camilli and colleagues (Cremona et al. 1999). Neurons isolated from these knockout mice exhibit elevated levels of PtdInsP2 and an accumulation of clathrin-coated vesicles, which implies a role for synaptojanin in uncoating clathrin-coated vesicles. To address whether the SH3 domain of endophilin might act by interfering with the biological function of synaptojanin, we investigated if the SH3 domain of endophilin affected PtdInsP2 or PtdInsP3 levels in the permeabilized cells. Treatment of the permeabilized cells with the SH3 domain of endophilin had no effect on the levels of PtdInsP3 (data not shown), but the levels of PtdInsP2 were reduced by >60% by the addition of the SH3 domain of endophilin while the SH3 domain of amphiphysin had no effect (Fig. 7 a). These results strongly suggest that the SH3 domain of endophilin may inhibit coated pit invagination and scission by mislocalizing synaptojanin or by activating synaptojanin at an inappropriate site resulting in a reduction of PtdInsP2 levels.


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)

The SH3 domain of endophilin inhibits invagination and scission by reducing PtdInsP2 levels with a concomitant loss of coated pit components from the plasma membrane. (a) Permeabilized cell membranes which had been incubated with cytosol (2.5 mg/ml) and ATP for 30 min at 37°C in the presence or absence of GST–endoSH3 or GST–amph2 SH3D36R (25 μg/ml) were collected by centrifugation and assayed for PtdInsP2 levels. Results are expressed as the mean ± SD of two different experiments each performed in triplicate. (b) Western blots of the permeabilized cell membranes obtained from assay mixtures which had been incubated in the absence or presence of GST–endoSH3 or GST–amph2 SH3 D36R. The membranes were probed for α-adaptin, clathrin, dynamin, synaptojanin, and transferrin receptor as indicated. (c) Permeabilized cells which had been incubated with cytosol (2.5 mg/ml), ATP, and in the absence or presence of GST–endoSH3 or GST–amph2 SH3D36R were centrifuged onto coverslips and analyzed by indirect immunofluorescence for the presence of α-adaptin, clathrin, and dynamin.
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Related In: Results  -  Collection

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Figure 7: The SH3 domain of endophilin inhibits invagination and scission by reducing PtdInsP2 levels with a concomitant loss of coated pit components from the plasma membrane. (a) Permeabilized cell membranes which had been incubated with cytosol (2.5 mg/ml) and ATP for 30 min at 37°C in the presence or absence of GST–endoSH3 or GST–amph2 SH3D36R (25 μg/ml) were collected by centrifugation and assayed for PtdInsP2 levels. Results are expressed as the mean ± SD of two different experiments each performed in triplicate. (b) Western blots of the permeabilized cell membranes obtained from assay mixtures which had been incubated in the absence or presence of GST–endoSH3 or GST–amph2 SH3 D36R. The membranes were probed for α-adaptin, clathrin, dynamin, synaptojanin, and transferrin receptor as indicated. (c) Permeabilized cells which had been incubated with cytosol (2.5 mg/ml), ATP, and in the absence or presence of GST–endoSH3 or GST–amph2 SH3D36R were centrifuged onto coverslips and analyzed by indirect immunofluorescence for the presence of α-adaptin, clathrin, and dynamin.
Mentions: Synaptojanin is an inositol 5-phosphatase whose major in vivo substrates are likely to be PtdInsP2 and PtdInsP3 (Woscholski et al. 1997). Recently, a synaptojanin knockout mouse has been generated by de Camilli and colleagues (Cremona et al. 1999). Neurons isolated from these knockout mice exhibit elevated levels of PtdInsP2 and an accumulation of clathrin-coated vesicles, which implies a role for synaptojanin in uncoating clathrin-coated vesicles. To address whether the SH3 domain of endophilin might act by interfering with the biological function of synaptojanin, we investigated if the SH3 domain of endophilin affected PtdInsP2 or PtdInsP3 levels in the permeabilized cells. Treatment of the permeabilized cells with the SH3 domain of endophilin had no effect on the levels of PtdInsP3 (data not shown), but the levels of PtdInsP2 were reduced by >60% by the addition of the SH3 domain of endophilin while the SH3 domain of amphiphysin had no effect (Fig. 7 a). These results strongly suggest that the SH3 domain of endophilin may inhibit coated pit invagination and scission by mislocalizing synaptojanin or by activating synaptojanin at an inappropriate site resulting in a reduction of PtdInsP2 levels.

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