Limits...
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.

Show MeSH

Related in: MedlinePlus

Model of the role of dynamin and some of its binding partners in clathrin-coated vesicle formation. In this model, dynamin is recruited to participate in both the late stages of invagination and scission. GTP binding and hydrolysis by dynamin are required to form a deeply invaginated coated pit. Dynamin may increase the invagination of a coated pit such that avidin is excluded in a manner analogous to a ratchet where the energy of GTP hydrolysis is used to promote movement. This is indicated by the red dynamin molecules ratchetting over the green ones and causing a constriction of the neck. During invagination and scission, dynamin may recruit/interact with other binding partners, e.g., endophilin. After scission, endophilin may undergo a conformational change which would activate synaptojanin. The insert shows that addition of the SH3 domain of endophilin causes an inhibition of endocytosis by reducing PtdInsP2 levels, which results in the dissociation of AP2 from the membrane. The drop in PtdInsP2 levels may result from a mistiming in the activation of synaptojanin.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199618&req=5

Figure 8: Model of the role of dynamin and some of its binding partners in clathrin-coated vesicle formation. In this model, dynamin is recruited to participate in both the late stages of invagination and scission. GTP binding and hydrolysis by dynamin are required to form a deeply invaginated coated pit. Dynamin may increase the invagination of a coated pit such that avidin is excluded in a manner analogous to a ratchet where the energy of GTP hydrolysis is used to promote movement. This is indicated by the red dynamin molecules ratchetting over the green ones and causing a constriction of the neck. During invagination and scission, dynamin may recruit/interact with other binding partners, e.g., endophilin. After scission, endophilin may undergo a conformational change which would activate synaptojanin. The insert shows that addition of the SH3 domain of endophilin causes an inhibition of endocytosis by reducing PtdInsP2 levels, which results in the dissociation of AP2 from the membrane. The drop in PtdInsP2 levels may result from a mistiming in the activation of synaptojanin.

Mentions: The rescue by dynamin is dependent on the ability of dynamin to bind GTP. In contrast to the ability of wild-type dynamin to rescue the inhibitory effects of the SH3 domain of amphiphysin, neither S45N nor K44A dynamin was capable of rescue. The S45N mutant is deficient in the ability to bind GTP. The K44A mutant, although considered as an hydrolysis mutant, also has reduced binding of GTP (van der Bliek et al. 1993). However, even in the presence of high concentrations of GTP (500 μM), the K44A mutant failed to rescue the inhibitory effects of the amphiphysin SH3 domain (data not shown). Surprisingly, mutant dynamin deficient in the ability to hydrolyze GTP (T65A) was also unable to rescue the inhibitory effects of the SH3 domain of amphiphysin. This implies that dynamin must bind and hydrolyze GTP in order to promote the formation of deeply invaginated coated pits in vitro which are inaccessible to avidin. Our in vitro results were confirmed by studies in HEK293 cells overexpressing wild-type and mutant forms of dynamin. Overexpression of both dynamin K44A and dynamin T65A resulted in an inhibition of B-SS-Tfn uptake as measured by both the avidin inaccessibility and MesNa resistance assays. It has previously been demonstrated that these assays can distinguish between the formation of deeply invaginated coated pits and pinched off coated vesicles in intact cells (Schmid and Carter 1990). These results are in contrast to recent results reporting an increase in the uptake of transferrin on overexpression of mutant dynamin deficient in GTP hydrolysis and assembly (Sever et al. 1999, Sever et al. 2000a). Using dynamin which has point mutations in the GED (GTPase effector domain), these workers concluded that dynamin functions as a classical GTPase, recruiting effectors when in the GTP conformation. Our results indicate, however, that GTP hydrolysis by dynamin is required for the late stages of invagination. Our data are therefore most consistent with a ratchet model of dynamin action (Smirnova et al. 1999) whereby dynamin molecules would interact with those molecules on an adjacent rung of the dynamin collar. The energy of GTP hydrolysis would then be harnessed to the movement of the dynamin molecules resulting in an increase in constriction which would result in an increase in avidin inaccessibility in our system (Fig. 8). The action of dynamin would thus be akin to that of kinesin and myosin (Vale 1996; Smirnova et al. 1999).


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)

Model of the role of dynamin and some of its binding partners in clathrin-coated vesicle formation. In this model, dynamin is recruited to participate in both the late stages of invagination and scission. GTP binding and hydrolysis by dynamin are required to form a deeply invaginated coated pit. Dynamin may increase the invagination of a coated pit such that avidin is excluded in a manner analogous to a ratchet where the energy of GTP hydrolysis is used to promote movement. This is indicated by the red dynamin molecules ratchetting over the green ones and causing a constriction of the neck. During invagination and scission, dynamin may recruit/interact with other binding partners, e.g., endophilin. After scission, endophilin may undergo a conformational change which would activate synaptojanin. The insert shows that addition of the SH3 domain of endophilin causes an inhibition of endocytosis by reducing PtdInsP2 levels, which results in the dissociation of AP2 from the membrane. The drop in PtdInsP2 levels may result from a mistiming in the activation of synaptojanin.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Model of the role of dynamin and some of its binding partners in clathrin-coated vesicle formation. In this model, dynamin is recruited to participate in both the late stages of invagination and scission. GTP binding and hydrolysis by dynamin are required to form a deeply invaginated coated pit. Dynamin may increase the invagination of a coated pit such that avidin is excluded in a manner analogous to a ratchet where the energy of GTP hydrolysis is used to promote movement. This is indicated by the red dynamin molecules ratchetting over the green ones and causing a constriction of the neck. During invagination and scission, dynamin may recruit/interact with other binding partners, e.g., endophilin. After scission, endophilin may undergo a conformational change which would activate synaptojanin. The insert shows that addition of the SH3 domain of endophilin causes an inhibition of endocytosis by reducing PtdInsP2 levels, which results in the dissociation of AP2 from the membrane. The drop in PtdInsP2 levels may result from a mistiming in the activation of synaptojanin.
Mentions: The rescue by dynamin is dependent on the ability of dynamin to bind GTP. In contrast to the ability of wild-type dynamin to rescue the inhibitory effects of the SH3 domain of amphiphysin, neither S45N nor K44A dynamin was capable of rescue. The S45N mutant is deficient in the ability to bind GTP. The K44A mutant, although considered as an hydrolysis mutant, also has reduced binding of GTP (van der Bliek et al. 1993). However, even in the presence of high concentrations of GTP (500 μM), the K44A mutant failed to rescue the inhibitory effects of the amphiphysin SH3 domain (data not shown). Surprisingly, mutant dynamin deficient in the ability to hydrolyze GTP (T65A) was also unable to rescue the inhibitory effects of the SH3 domain of amphiphysin. This implies that dynamin must bind and hydrolyze GTP in order to promote the formation of deeply invaginated coated pits in vitro which are inaccessible to avidin. Our in vitro results were confirmed by studies in HEK293 cells overexpressing wild-type and mutant forms of dynamin. Overexpression of both dynamin K44A and dynamin T65A resulted in an inhibition of B-SS-Tfn uptake as measured by both the avidin inaccessibility and MesNa resistance assays. It has previously been demonstrated that these assays can distinguish between the formation of deeply invaginated coated pits and pinched off coated vesicles in intact cells (Schmid and Carter 1990). These results are in contrast to recent results reporting an increase in the uptake of transferrin on overexpression of mutant dynamin deficient in GTP hydrolysis and assembly (Sever et al. 1999, Sever et al. 2000a). Using dynamin which has point mutations in the GED (GTPase effector domain), these workers concluded that dynamin functions as a classical GTPase, recruiting effectors when in the GTP conformation. Our results indicate, however, that GTP hydrolysis by dynamin is required for the late stages of invagination. Our data are therefore most consistent with a ratchet model of dynamin action (Smirnova et al. 1999) whereby dynamin molecules would interact with those molecules on an adjacent rung of the dynamin collar. The energy of GTP hydrolysis would then be harnessed to the movement of the dynamin molecules resulting in an increase in constriction which would result in an increase in avidin inaccessibility in our system (Fig. 8). The action of dynamin would thus be akin to that of kinesin and myosin (Vale 1996; Smirnova et al. 1999).

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