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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 (green) and dynamin (red) in primary hippocampal neurons by confocal microscopy. (a and a′) Abp1 strongly accumulated at defined sites in neurons kept in culture for 9 d (arrowheads). (c and c′) Besides cytosolic dynamin, sites of moderate dynamin accumulation proximal to the cell bodies were seen (arrowheads). These sites were immunopositive for Abp1, as seen in the merged images (b and b′). Note that subpools of both proteins did not spatially overlap (see neurites for Abp1 and cytoplasm for dynamin). a′–c′ are 2.5-fold enlargements of the center-right areas in a–c. Bar, 15 μm.
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Figure 3: Colocalization of Abp1 (green) and dynamin (red) in primary hippocampal neurons by confocal microscopy. (a and a′) Abp1 strongly accumulated at defined sites in neurons kept in culture for 9 d (arrowheads). (c and c′) Besides cytosolic dynamin, sites of moderate dynamin accumulation proximal to the cell bodies were seen (arrowheads). These sites were immunopositive for Abp1, as seen in the merged images (b and b′). Note that subpools of both proteins did not spatially overlap (see neurites for Abp1 and cytoplasm for dynamin). a′–c′ are 2.5-fold enlargements of the center-right areas in a–c. Bar, 15 μm.

Mentions: It was next important to ask whether a spatial overlap of Abp1 and F-actin with dynamin could be detected. We therefore performed Abp1/dynamin colocalization studies using primary neuronal cells at different stages of differentiation. At all times points analyzed, Abp1 and dynamin immunoreactivity showed some overlap at the light microscopical level (data not shown). Most prominently, in cells kept in culture for 9 d (Fig. 3), Abp1 showed strong accumulations near the cell body (Fig. 3, a and a′), similar to those seen in our Abp1/F-actin double labeling studies (Fig. 2). In these cells, dynamin was still cytosolic but also showed some enrichment at sites at the periphery of the cell bodies (Fig. 3c and Fig. c′), where it colocalized with Abp1, as seen by the yellow color in the merged images (Fig. 3b and Fig. b′). It should, however, be stressed that the spatial overlap of both proteins was restricted to these sites. Little dynamin was present in the extended neuritic network at this time point, and relatively low amounts of Abp1 were present in the cytoplasm, where dynamin was still readily detectable.


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 (green) and dynamin (red) in primary hippocampal neurons by confocal microscopy. (a and a′) Abp1 strongly accumulated at defined sites in neurons kept in culture for 9 d (arrowheads). (c and c′) Besides cytosolic dynamin, sites of moderate dynamin accumulation proximal to the cell bodies were seen (arrowheads). These sites were immunopositive for Abp1, as seen in the merged images (b and b′). Note that subpools of both proteins did not spatially overlap (see neurites for Abp1 and cytoplasm for dynamin). a′–c′ are 2.5-fold enlargements of the center-right areas in a–c. Bar, 15 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169459&req=5

Figure 3: Colocalization of Abp1 (green) and dynamin (red) in primary hippocampal neurons by confocal microscopy. (a and a′) Abp1 strongly accumulated at defined sites in neurons kept in culture for 9 d (arrowheads). (c and c′) Besides cytosolic dynamin, sites of moderate dynamin accumulation proximal to the cell bodies were seen (arrowheads). These sites were immunopositive for Abp1, as seen in the merged images (b and b′). Note that subpools of both proteins did not spatially overlap (see neurites for Abp1 and cytoplasm for dynamin). a′–c′ are 2.5-fold enlargements of the center-right areas in a–c. Bar, 15 μm.
Mentions: It was next important to ask whether a spatial overlap of Abp1 and F-actin with dynamin could be detected. We therefore performed Abp1/dynamin colocalization studies using primary neuronal cells at different stages of differentiation. At all times points analyzed, Abp1 and dynamin immunoreactivity showed some overlap at the light microscopical level (data not shown). Most prominently, in cells kept in culture for 9 d (Fig. 3), Abp1 showed strong accumulations near the cell body (Fig. 3, a and a′), similar to those seen in our Abp1/F-actin double labeling studies (Fig. 2). In these cells, dynamin was still cytosolic but also showed some enrichment at sites at the periphery of the cell bodies (Fig. 3c and Fig. c′), where it colocalized with Abp1, as seen by the yellow color in the merged images (Fig. 3b and Fig. b′). It should, however, be stressed that the spatial overlap of both proteins was restricted to these sites. Little dynamin was present in the extended neuritic network at this time point, and relatively low amounts of Abp1 were present in the cytoplasm, where dynamin was still readily detectable.

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