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Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin.

Kessels MM, Engqvist-Goldstein AE, Drubin DG, Qualmann B - J. Cell Biol. (2001)

Bottom Line: While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake.The endocytosis block was rescued by cooverexpression of dynamin.Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, D-39008 Magdeburg, Germany. kessels@ifn-magdeburg.de

ABSTRACT
The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin-binding protein Abp1 (SH3P7/HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.

Show MeSH
Cooverexpression of dynamin rescues the endocytosis block caused by overexpression of the Abp1 SH3 domain. Cos-7 cells were double transfected with HA-dynamin1 and myc-Abp1 constructs. Quantification of transferrin uptake, as in Fig. 5 and Fig. 9, dark grey, no transferrin uptake, light grey, significantly reduced levels of uptake, and hatched cells show a transferrin uptake comparable with wild-type cells. Myc-(Abp1-)Flex/SH3–overexpressing cells were rescued up to a level similar to that caused by the flexible domain alone (a). Myc-(Abp1-)SH3–overexpressing cells were rescued up to a level similar to wild-type or HA-dynamin–overexpressing cells (b). Myc-flex/SH3: 19.5 ± 1.5% normal, 20.5 ± 1.5% reduced, 60.0 ± 3.0% block, n = 447; myc-flex/SH3 + HA-dynamin1: 68.7 ± 3.2%, 16.5 ± 2.5%, 14.75 ± 0.75%, n = 237; myc-flex/SH3mut: 53.0 ± 2.9%, 27.7 ± 3.3%, 19.3 ± 5.6%, n = 701; HA-dynamin1: 83.5 ± 3.7%, 13.7 ± 3.6%, 2.85 ± 0.05%, n = 213; hatched dark grey column: rescue effect, 81.0 ± 3.75 % (cotransfection rate of 96.5% was taken into consideration). This value represents 104.7 ± 8.7% of theoretically achievable maximal rescue (the fact that effect of SH3 domain but not that of the flexible domain can be rescued was taken into consideration; hatched light grey column). Myc-SH3: 16.5 ± 2%, 29.5 ± 0.5%, 54.5 ± 1.5%, n = 371; myc-SH3 + HA-dynamin1: 82.6 ± 2.3%, 12.7 ± 1.8%, 4.65 ± 0.45%; HA-dynamin1: 83.5 ± 3.7%, 13.7 ± 3.6%, 2.85 ± 0.05%, n = 213; hatched dark grey column: rescue effect (in case of myc-SH3 overexpression, identical to the achievable maximal rescue of SH3 domain effect), 96.5 ± 3.8% (cotransfection rate of 92.0% was taken into consideration).
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Figure 6: Cooverexpression of dynamin rescues the endocytosis block caused by overexpression of the Abp1 SH3 domain. Cos-7 cells were double transfected with HA-dynamin1 and myc-Abp1 constructs. Quantification of transferrin uptake, as in Fig. 5 and Fig. 9, dark grey, no transferrin uptake, light grey, significantly reduced levels of uptake, and hatched cells show a transferrin uptake comparable with wild-type cells. Myc-(Abp1-)Flex/SH3–overexpressing cells were rescued up to a level similar to that caused by the flexible domain alone (a). Myc-(Abp1-)SH3–overexpressing cells were rescued up to a level similar to wild-type or HA-dynamin–overexpressing cells (b). Myc-flex/SH3: 19.5 ± 1.5% normal, 20.5 ± 1.5% reduced, 60.0 ± 3.0% block, n = 447; myc-flex/SH3 + HA-dynamin1: 68.7 ± 3.2%, 16.5 ± 2.5%, 14.75 ± 0.75%, n = 237; myc-flex/SH3mut: 53.0 ± 2.9%, 27.7 ± 3.3%, 19.3 ± 5.6%, n = 701; HA-dynamin1: 83.5 ± 3.7%, 13.7 ± 3.6%, 2.85 ± 0.05%, n = 213; hatched dark grey column: rescue effect, 81.0 ± 3.75 % (cotransfection rate of 96.5% was taken into consideration). This value represents 104.7 ± 8.7% of theoretically achievable maximal rescue (the fact that effect of SH3 domain but not that of the flexible domain can be rescued was taken into consideration; hatched light grey column). Myc-SH3: 16.5 ± 2%, 29.5 ± 0.5%, 54.5 ± 1.5%, n = 371; myc-SH3 + HA-dynamin1: 82.6 ± 2.3%, 12.7 ± 1.8%, 4.65 ± 0.45%; HA-dynamin1: 83.5 ± 3.7%, 13.7 ± 3.6%, 2.85 ± 0.05%, n = 213; hatched dark grey column: rescue effect (in case of myc-SH3 overexpression, identical to the achievable maximal rescue of SH3 domain effect), 96.5 ± 3.8% (cotransfection rate of 92.0% was taken into consideration).

Mentions: We have shown that Abp1 interacts with dynamin in vitro and in vivo through its SH3 domain (Fig. 1 and Fig. 4). The overexpression phenotype of the SH3 domain implicates Abp1 protein interactions in endocytic functions (Fig. 5). To prove that the endocytosis impairment observed in Abp1-SH3 domain–overexpressing cells is indeed due to the Abp1/dynamin interaction, we cotransfected Cos-7 cells with an HA-dynamin1 plasmid together with constructs encoding for the Abp1-SH3 domain and for the entire COOH-terminal half of Abp1 (myc-flex/SH3). Quantifications of transferrin uptake-positive cells demonstrated that endocytic function was restored upon co-overexpression of wild-type dynamin (Fig. 6). Cells overexpressing dynamin and the COOH-terminal half of Abp1 took up transferrin with a rate similar to cells overexpressing the mutated COOH-terminal half of Abp1; i.e., a protein with an inactivated SH3 domain (rescue 81.0 ± 3.7%). The remaining slight inhibitory effect of both of the latter constructs represents the effect of the overexpression of the flexible, Src kinase target domain (Fig. 6 a). The influence of this domain on endocytosis was thus independent of dynamin function. When both the extend of double transfection (quantified by double immunofluorescence staining; data not shown) and the not rescuable effect of the flexible domain were considered (theoretically achievable rescue) the rescue was 104.7 ± 8.7%.


Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin.

Kessels MM, Engqvist-Goldstein AE, Drubin DG, Qualmann B - J. Cell Biol. (2001)

Cooverexpression of dynamin rescues the endocytosis block caused by overexpression of the Abp1 SH3 domain. Cos-7 cells were double transfected with HA-dynamin1 and myc-Abp1 constructs. Quantification of transferrin uptake, as in Fig. 5 and Fig. 9, dark grey, no transferrin uptake, light grey, significantly reduced levels of uptake, and hatched cells show a transferrin uptake comparable with wild-type cells. Myc-(Abp1-)Flex/SH3–overexpressing cells were rescued up to a level similar to that caused by the flexible domain alone (a). Myc-(Abp1-)SH3–overexpressing cells were rescued up to a level similar to wild-type or HA-dynamin–overexpressing cells (b). Myc-flex/SH3: 19.5 ± 1.5% normal, 20.5 ± 1.5% reduced, 60.0 ± 3.0% block, n = 447; myc-flex/SH3 + HA-dynamin1: 68.7 ± 3.2%, 16.5 ± 2.5%, 14.75 ± 0.75%, n = 237; myc-flex/SH3mut: 53.0 ± 2.9%, 27.7 ± 3.3%, 19.3 ± 5.6%, n = 701; HA-dynamin1: 83.5 ± 3.7%, 13.7 ± 3.6%, 2.85 ± 0.05%, n = 213; hatched dark grey column: rescue effect, 81.0 ± 3.75 % (cotransfection rate of 96.5% was taken into consideration). This value represents 104.7 ± 8.7% of theoretically achievable maximal rescue (the fact that effect of SH3 domain but not that of the flexible domain can be rescued was taken into consideration; hatched light grey column). Myc-SH3: 16.5 ± 2%, 29.5 ± 0.5%, 54.5 ± 1.5%, n = 371; myc-SH3 + HA-dynamin1: 82.6 ± 2.3%, 12.7 ± 1.8%, 4.65 ± 0.45%; HA-dynamin1: 83.5 ± 3.7%, 13.7 ± 3.6%, 2.85 ± 0.05%, n = 213; hatched dark grey column: rescue effect (in case of myc-SH3 overexpression, identical to the achievable maximal rescue of SH3 domain effect), 96.5 ± 3.8% (cotransfection rate of 92.0% was taken into consideration).
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Figure 6: Cooverexpression of dynamin rescues the endocytosis block caused by overexpression of the Abp1 SH3 domain. Cos-7 cells were double transfected with HA-dynamin1 and myc-Abp1 constructs. Quantification of transferrin uptake, as in Fig. 5 and Fig. 9, dark grey, no transferrin uptake, light grey, significantly reduced levels of uptake, and hatched cells show a transferrin uptake comparable with wild-type cells. Myc-(Abp1-)Flex/SH3–overexpressing cells were rescued up to a level similar to that caused by the flexible domain alone (a). Myc-(Abp1-)SH3–overexpressing cells were rescued up to a level similar to wild-type or HA-dynamin–overexpressing cells (b). Myc-flex/SH3: 19.5 ± 1.5% normal, 20.5 ± 1.5% reduced, 60.0 ± 3.0% block, n = 447; myc-flex/SH3 + HA-dynamin1: 68.7 ± 3.2%, 16.5 ± 2.5%, 14.75 ± 0.75%, n = 237; myc-flex/SH3mut: 53.0 ± 2.9%, 27.7 ± 3.3%, 19.3 ± 5.6%, n = 701; HA-dynamin1: 83.5 ± 3.7%, 13.7 ± 3.6%, 2.85 ± 0.05%, n = 213; hatched dark grey column: rescue effect, 81.0 ± 3.75 % (cotransfection rate of 96.5% was taken into consideration). This value represents 104.7 ± 8.7% of theoretically achievable maximal rescue (the fact that effect of SH3 domain but not that of the flexible domain can be rescued was taken into consideration; hatched light grey column). Myc-SH3: 16.5 ± 2%, 29.5 ± 0.5%, 54.5 ± 1.5%, n = 371; myc-SH3 + HA-dynamin1: 82.6 ± 2.3%, 12.7 ± 1.8%, 4.65 ± 0.45%; HA-dynamin1: 83.5 ± 3.7%, 13.7 ± 3.6%, 2.85 ± 0.05%, n = 213; hatched dark grey column: rescue effect (in case of myc-SH3 overexpression, identical to the achievable maximal rescue of SH3 domain effect), 96.5 ± 3.8% (cotransfection rate of 92.0% was taken into consideration).
Mentions: We have shown that Abp1 interacts with dynamin in vitro and in vivo through its SH3 domain (Fig. 1 and Fig. 4). The overexpression phenotype of the SH3 domain implicates Abp1 protein interactions in endocytic functions (Fig. 5). To prove that the endocytosis impairment observed in Abp1-SH3 domain–overexpressing cells is indeed due to the Abp1/dynamin interaction, we cotransfected Cos-7 cells with an HA-dynamin1 plasmid together with constructs encoding for the Abp1-SH3 domain and for the entire COOH-terminal half of Abp1 (myc-flex/SH3). Quantifications of transferrin uptake-positive cells demonstrated that endocytic function was restored upon co-overexpression of wild-type dynamin (Fig. 6). Cells overexpressing dynamin and the COOH-terminal half of Abp1 took up transferrin with a rate similar to cells overexpressing the mutated COOH-terminal half of Abp1; i.e., a protein with an inactivated SH3 domain (rescue 81.0 ± 3.7%). The remaining slight inhibitory effect of both of the latter constructs represents the effect of the overexpression of the flexible, Src kinase target domain (Fig. 6 a). The influence of this domain on endocytosis was thus independent of dynamin function. When both the extend of double transfection (quantified by double immunofluorescence staining; data not shown) and the not rescuable effect of the flexible domain were considered (theoretically achievable rescue) the rescue was 104.7 ± 8.7%.

Bottom Line: While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake.The endocytosis block was rescued by cooverexpression of dynamin.Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, D-39008 Magdeburg, Germany. kessels@ifn-magdeburg.de

ABSTRACT
The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin-binding protein Abp1 (SH3P7/HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.

Show MeSH