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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.

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Colocalization of Abp1 and F-actin by confocal immunofluorescence microscopy. Primary hippocampal neurons kept in culture for 9 d displayed Abp1 accumulations in the extended peripheries of cell bodies as well as a cytosolic and neuritic immunostaining (a). F-actin was localized similarly (c), also seen in the merged image (b) and in the enlargements of the central areas of the images (a′–c′). (d–f) Neurons kept in culture for 20 d. At synapses, Abp1 (d) and F-actin (f) colocalize, as seen in the merged image (e) and in the enlargements of an area in the upper center of d–f (d′–f′). Stars in a–c and a′–c′ mark positions of cell bodies and arrows in d′–f′ mark selected examples of actin- and Abp1-rich postsynaptic structures. Bar: (c) 20 μm, (f) 10 μm.
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Figure 2: Colocalization of Abp1 and F-actin by confocal immunofluorescence microscopy. Primary hippocampal neurons kept in culture for 9 d displayed Abp1 accumulations in the extended peripheries of cell bodies as well as a cytosolic and neuritic immunostaining (a). F-actin was localized similarly (c), also seen in the merged image (b) and in the enlargements of the central areas of the images (a′–c′). (d–f) Neurons kept in culture for 20 d. At synapses, Abp1 (d) and F-actin (f) colocalize, as seen in the merged image (e) and in the enlargements of an area in the upper center of d–f (d′–f′). Stars in a–c and a′–c′ mark positions of cell bodies and arrows in d′–f′ mark selected examples of actin- and Abp1-rich postsynaptic structures. Bar: (c) 20 μm, (f) 10 μm.

Mentions: Most of the binding partners of the Abp1 SH3 domain are brain-specific or brain-enriched proteins. This prompted us to analyze the Abp1 localization in the neuronal context. Primary hippocampal neurons at different stages of development were immunolabeled for Abp1. Abp1 displayed a relatively uniform localization in young primary neuronal cultures (2 and 6 d; data not shown). Abp1 was slightly enriched in growth cones, reminiscent of its accumulation in lamellipodial areas in nonneuronal cells (data not shown; Kessels et al. 2000). This situation changed with the onset of synaptogenesis. Cells kept in culture for 9 d contained spatially well-defined areas of Abp1 accumulation at the extended periphery of the cell bodies. Costaining with Texas red-phalloidin revealed that the sites of Abp1 accumulation were actin-rich (Fig. 2). Actin was strongly restricted to sites proximal to the cell bodies. Abp1 also exhibited a readily detectable neuritic staining (Fig. 2, a–c). After synaptogenesis was complete and the synapses were functional, Abp1 and F-actin still colocalized in many cells, but now showed a broader distribution reflecting the extremely high number of synapses present in the culture (Fig. 2, d–f). In mature neurons, F-actin is enriched in dendritic spines and postsynaptic densities (Matus et al. 1982). At the light microscopic level, Abp1 seems to localize similarly to actin at both stages of neuronal development examined (9 and 20 d), as well seen in the enlarged images (Fig. 2, a′–c′ and d′–f′). In the cases where Abp1 showed a staining, which appeared synaptic, it seemed to be more restricted spatially to these sites compared with F-actin, which appeared a little more diffuse (Fig. 2d′–f′).


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)

Colocalization of Abp1 and F-actin by confocal immunofluorescence microscopy. Primary hippocampal neurons kept in culture for 9 d displayed Abp1 accumulations in the extended peripheries of cell bodies as well as a cytosolic and neuritic immunostaining (a). F-actin was localized similarly (c), also seen in the merged image (b) and in the enlargements of the central areas of the images (a′–c′). (d–f) Neurons kept in culture for 20 d. At synapses, Abp1 (d) and F-actin (f) colocalize, as seen in the merged image (e) and in the enlargements of an area in the upper center of d–f (d′–f′). Stars in a–c and a′–c′ mark positions of cell bodies and arrows in d′–f′ mark selected examples of actin- and Abp1-rich postsynaptic structures. Bar: (c) 20 μm, (f) 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169459&req=5

Figure 2: Colocalization of Abp1 and F-actin by confocal immunofluorescence microscopy. Primary hippocampal neurons kept in culture for 9 d displayed Abp1 accumulations in the extended peripheries of cell bodies as well as a cytosolic and neuritic immunostaining (a). F-actin was localized similarly (c), also seen in the merged image (b) and in the enlargements of the central areas of the images (a′–c′). (d–f) Neurons kept in culture for 20 d. At synapses, Abp1 (d) and F-actin (f) colocalize, as seen in the merged image (e) and in the enlargements of an area in the upper center of d–f (d′–f′). Stars in a–c and a′–c′ mark positions of cell bodies and arrows in d′–f′ mark selected examples of actin- and Abp1-rich postsynaptic structures. Bar: (c) 20 μm, (f) 10 μm.
Mentions: Most of the binding partners of the Abp1 SH3 domain are brain-specific or brain-enriched proteins. This prompted us to analyze the Abp1 localization in the neuronal context. Primary hippocampal neurons at different stages of development were immunolabeled for Abp1. Abp1 displayed a relatively uniform localization in young primary neuronal cultures (2 and 6 d; data not shown). Abp1 was slightly enriched in growth cones, reminiscent of its accumulation in lamellipodial areas in nonneuronal cells (data not shown; Kessels et al. 2000). This situation changed with the onset of synaptogenesis. Cells kept in culture for 9 d contained spatially well-defined areas of Abp1 accumulation at the extended periphery of the cell bodies. Costaining with Texas red-phalloidin revealed that the sites of Abp1 accumulation were actin-rich (Fig. 2). Actin was strongly restricted to sites proximal to the cell bodies. Abp1 also exhibited a readily detectable neuritic staining (Fig. 2, a–c). After synaptogenesis was complete and the synapses were functional, Abp1 and F-actin still colocalized in many cells, but now showed a broader distribution reflecting the extremely high number of synapses present in the culture (Fig. 2, d–f). In mature neurons, F-actin is enriched in dendritic spines and postsynaptic densities (Matus et al. 1982). At the light microscopic level, Abp1 seems to localize similarly to actin at both stages of neuronal development examined (9 and 20 d), as well seen in the enlarged images (Fig. 2, a′–c′ and d′–f′). In the cases where Abp1 showed a staining, which appeared synaptic, it seemed to be more restricted spatially to these sites compared with F-actin, which appeared a little more diffuse (Fig. 2d′–f′).

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
Related in: MedlinePlus