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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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Association of Abp1 with dynamin-containing pits upon growth factor stimulations of 3T3 fibroblasts. NIH3T3 fibroblasts were replated onto fibronectin-coated coverslips, stimulated with 300 ng/ml PMA and 5 ng/ml PDGF for 10 min, perforated with 0.02% saponin and subsequently fixed and processed for immunofluorescence microscopy (a–f). The lamellipodial accumulation of Abp1 (Kessels et al. 2000) was observable under these conditions (a and d). In addition, a widely distributed, punctate Abp1 immunostaining was detected (a and d). (c and f) Immunostaining of dynamin using the monoclonal antibody Hudy1 at perinuclear and cortical sites. (b and e) Merged images show that dynamin-containing sites are in almost all cases also immunopositive for Abp1 as especially well seen in extended lamellipodial areas (d–f). In contrast, in serum-starved cells, Abp1 (g) is readily extracted and the remaining protein shows no colocalization with dynamin (g–i). Inserts are enlargements of the marked areas. Bar, 10 μm.
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Figure 7: Association of Abp1 with dynamin-containing pits upon growth factor stimulations of 3T3 fibroblasts. NIH3T3 fibroblasts were replated onto fibronectin-coated coverslips, stimulated with 300 ng/ml PMA and 5 ng/ml PDGF for 10 min, perforated with 0.02% saponin and subsequently fixed and processed for immunofluorescence microscopy (a–f). The lamellipodial accumulation of Abp1 (Kessels et al. 2000) was observable under these conditions (a and d). In addition, a widely distributed, punctate Abp1 immunostaining was detected (a and d). (c and f) Immunostaining of dynamin using the monoclonal antibody Hudy1 at perinuclear and cortical sites. (b and e) Merged images show that dynamin-containing sites are in almost all cases also immunopositive for Abp1 as especially well seen in extended lamellipodial areas (d–f). In contrast, in serum-starved cells, Abp1 (g) is readily extracted and the remaining protein shows no colocalization with dynamin (g–i). Inserts are enlargements of the marked areas. Bar, 10 μm.

Mentions: Our data indicate that Abp1 may, via its actin-binding modules and its SH3 domain, link actin and endocytosis. However, Abp1 does not appear to colocalize with endosomal compartments (i.e., with late states of endocytosis) (Kessels et al. 2000), nor does it seem to be a stable component of the endocytic coat because it was not found to be enriched on clathrin-coated vesicles (Engqvist-Goldstein et al. 1999). In resting cells, Abp1 is not associated with the actin cytoskeleton either, but we have discovered that the association of Abp1 with cortical actin structures is controlled by signaling pathways converging on this protein (Kessels et al. 2000). We therefore used conditions that caused a redistribution of Abp1 to sites of high actin dynamics (i.e., the leading edge of growth factor-treated NIH3T3 fibroblasts) to examine its participation in the endocytic process. We asked whether sites of endocytosis would contain Abp1 in stimulated cells. Short perforations of these growth factor–treated cells resulted in a retainment of Abp1 at the periphery of lamellipodia and at some puncta scattered throughout the cell cortex (Kessels et al. 2000). We arrested endocytosis by incubation at low temperature. Under these conditions, we were able to demonstrate that at the cortex of NIH3T3 cells, dynamin-containing sites labeled by the antibody Hudy1 were almost always also immunopositive for Abp1 detected by the antibody GP5 (Fig. 7). Coimmunolabelings with oppositely labeled secondary antibodies led to similar results (data not shown). The accumulation of Abp1 at the leading edge of lamellipodia was preserved under the conditions applied (Fig. 7, a and d). The Abp1-rich leading edge was, however, virtually free of any dynamin staining using the monoclonal antibody Hudy1. Thus, either dynamin seems not to be recruited to the leading edge by the subcellular shift of the majority of Abp1, or it is not detectable with Hudy1 at this location. In serum-starved cells, no colocalization was detected (Fig. 7, g–i). Under these conditions, most of the Abp1 is readily extracted and little signal remains. Also in resting cells, only little overlap of Abp1 and dynamin immunolocalization was observed after permeabilization (data not shown). Thus, Abp1's detection at dynamin-rich sites seems to depend on growth factor receptor activation.


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)

Association of Abp1 with dynamin-containing pits upon growth factor stimulations of 3T3 fibroblasts. NIH3T3 fibroblasts were replated onto fibronectin-coated coverslips, stimulated with 300 ng/ml PMA and 5 ng/ml PDGF for 10 min, perforated with 0.02% saponin and subsequently fixed and processed for immunofluorescence microscopy (a–f). The lamellipodial accumulation of Abp1 (Kessels et al. 2000) was observable under these conditions (a and d). In addition, a widely distributed, punctate Abp1 immunostaining was detected (a and d). (c and f) Immunostaining of dynamin using the monoclonal antibody Hudy1 at perinuclear and cortical sites. (b and e) Merged images show that dynamin-containing sites are in almost all cases also immunopositive for Abp1 as especially well seen in extended lamellipodial areas (d–f). In contrast, in serum-starved cells, Abp1 (g) is readily extracted and the remaining protein shows no colocalization with dynamin (g–i). Inserts are enlargements of the marked areas. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 7: Association of Abp1 with dynamin-containing pits upon growth factor stimulations of 3T3 fibroblasts. NIH3T3 fibroblasts were replated onto fibronectin-coated coverslips, stimulated with 300 ng/ml PMA and 5 ng/ml PDGF for 10 min, perforated with 0.02% saponin and subsequently fixed and processed for immunofluorescence microscopy (a–f). The lamellipodial accumulation of Abp1 (Kessels et al. 2000) was observable under these conditions (a and d). In addition, a widely distributed, punctate Abp1 immunostaining was detected (a and d). (c and f) Immunostaining of dynamin using the monoclonal antibody Hudy1 at perinuclear and cortical sites. (b and e) Merged images show that dynamin-containing sites are in almost all cases also immunopositive for Abp1 as especially well seen in extended lamellipodial areas (d–f). In contrast, in serum-starved cells, Abp1 (g) is readily extracted and the remaining protein shows no colocalization with dynamin (g–i). Inserts are enlargements of the marked areas. Bar, 10 μm.
Mentions: Our data indicate that Abp1 may, via its actin-binding modules and its SH3 domain, link actin and endocytosis. However, Abp1 does not appear to colocalize with endosomal compartments (i.e., with late states of endocytosis) (Kessels et al. 2000), nor does it seem to be a stable component of the endocytic coat because it was not found to be enriched on clathrin-coated vesicles (Engqvist-Goldstein et al. 1999). In resting cells, Abp1 is not associated with the actin cytoskeleton either, but we have discovered that the association of Abp1 with cortical actin structures is controlled by signaling pathways converging on this protein (Kessels et al. 2000). We therefore used conditions that caused a redistribution of Abp1 to sites of high actin dynamics (i.e., the leading edge of growth factor-treated NIH3T3 fibroblasts) to examine its participation in the endocytic process. We asked whether sites of endocytosis would contain Abp1 in stimulated cells. Short perforations of these growth factor–treated cells resulted in a retainment of Abp1 at the periphery of lamellipodia and at some puncta scattered throughout the cell cortex (Kessels et al. 2000). We arrested endocytosis by incubation at low temperature. Under these conditions, we were able to demonstrate that at the cortex of NIH3T3 cells, dynamin-containing sites labeled by the antibody Hudy1 were almost always also immunopositive for Abp1 detected by the antibody GP5 (Fig. 7). Coimmunolabelings with oppositely labeled secondary antibodies led to similar results (data not shown). The accumulation of Abp1 at the leading edge of lamellipodia was preserved under the conditions applied (Fig. 7, a and d). The Abp1-rich leading edge was, however, virtually free of any dynamin staining using the monoclonal antibody Hudy1. Thus, either dynamin seems not to be recruited to the leading edge by the subcellular shift of the majority of Abp1, or it is not detectable with Hudy1 at this location. In serum-starved cells, no colocalization was detected (Fig. 7, g–i). Under these conditions, most of the Abp1 is readily extracted and little signal remains. Also in resting cells, only little overlap of Abp1 and dynamin immunolocalization was observed after permeabilization (data not shown). Thus, Abp1's detection at dynamin-rich sites seems to depend on growth factor receptor activation.

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