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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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Cortical dynamin-rich sites in NIH3T3 cells, which become Abp1 immunopositive after receptor activation, are sites of endocytosis. Dynamin (a) and the endocytic coat component Hip1R (c) colocalize (b, merge) in cells stimulated with growth factors, as observed by confocal microscopy. Colocalization of dynamin (d) and eps15 (f), as seen in the merged image (e). Colocalization of AP2 (g) and Abp1 (i) at puncta at the cell cortex, but not at the leading edge of the cell; as seen in the merged image (h). (j–l) In nonperforated activated cells, the cytosolic pool of Abp1 largely obscures the weak Abp1 immunostaining (l) at sites of endocytosis [here marked by anti–AP2 immunostaining (j)]. Inserts represent enlargements of the areas boxed in a–l. Bars, 10 μm.
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Figure 8: Cortical dynamin-rich sites in NIH3T3 cells, which become Abp1 immunopositive after receptor activation, are sites of endocytosis. Dynamin (a) and the endocytic coat component Hip1R (c) colocalize (b, merge) in cells stimulated with growth factors, as observed by confocal microscopy. Colocalization of dynamin (d) and eps15 (f), as seen in the merged image (e). Colocalization of AP2 (g) and Abp1 (i) at puncta at the cell cortex, but not at the leading edge of the cell; as seen in the merged image (h). (j–l) In nonperforated activated cells, the cytosolic pool of Abp1 largely obscures the weak Abp1 immunostaining (l) at sites of endocytosis [here marked by anti–AP2 immunostaining (j)]. Inserts represent enlargements of the areas boxed in a–l. Bars, 10 μm.

Mentions: Several observations have suggested that dynamins may play an as yet unidentified cytoskeletal role in addition to their endocytic function (for review, see McNiven et al. 2000). At least dynamin2aa was observed at actin-rich sites within cells, which seem independent of sites of endocytosis (Cao et al. 1998; Ochoa et al. 2000). It was thus important to examine whether the dynamin-rich sites detected by the antibody Hudy1, which were observed to be Abp1 immunopositive under conditions also leading to a recruitment of Abp1 to actin-rich lamellipodia, were sites of endocytosis, or whether they reflect a putative cytoskeletal role of dynamin. We therefore stained cells, which had been stimulated and permeabilized as before, with the monoclonal Hudy1 antibody (Fig. 8 a) and a polyclonal antibody against the clathrin coat component Hip1R (Engqvist-Goldstein et al. 1999) (Fig. 8 c). Besides some labeling in and/or near the nucleus, which seemed to be a result of our membrane perforations, both antibodies detected their antigen in cortical spots resistant to the perforation procedure (Fig. 8, a and c). Colocalization experiments demonstrated that the dynamin-rich sites detected by the monoclonal antibody Hudy1 were also positive for Hip1R (Fig. 8 b). We have previously also shown that Hip1R puncta at the cell cortex colocalize quantitatively with the adaptor protein AP2 detected with the monoclonal antibody AP6 (Engqvist-Goldstein et al. 1999). The perforation-resistant, dynamin-rich areas at the cell cortex also contain eps15 (Fig. 8, d–f), another component of clathrin coats (Tebar et al. 1996). Eps15 immunostaining is strongest at puncta at the periphery and decreases towards the center of the cells (Fig. 8 f), whereas dynamin labeling shows the opposite gradient (Fig. 8 d). However Eps15 and dynamin show clear colocalization (Fig. 8 e).


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)

Cortical dynamin-rich sites in NIH3T3 cells, which become Abp1 immunopositive after receptor activation, are sites of endocytosis. Dynamin (a) and the endocytic coat component Hip1R (c) colocalize (b, merge) in cells stimulated with growth factors, as observed by confocal microscopy. Colocalization of dynamin (d) and eps15 (f), as seen in the merged image (e). Colocalization of AP2 (g) and Abp1 (i) at puncta at the cell cortex, but not at the leading edge of the cell; as seen in the merged image (h). (j–l) In nonperforated activated cells, the cytosolic pool of Abp1 largely obscures the weak Abp1 immunostaining (l) at sites of endocytosis [here marked by anti–AP2 immunostaining (j)]. Inserts represent enlargements of the areas boxed in a–l. Bars, 10 μm.
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Figure 8: Cortical dynamin-rich sites in NIH3T3 cells, which become Abp1 immunopositive after receptor activation, are sites of endocytosis. Dynamin (a) and the endocytic coat component Hip1R (c) colocalize (b, merge) in cells stimulated with growth factors, as observed by confocal microscopy. Colocalization of dynamin (d) and eps15 (f), as seen in the merged image (e). Colocalization of AP2 (g) and Abp1 (i) at puncta at the cell cortex, but not at the leading edge of the cell; as seen in the merged image (h). (j–l) In nonperforated activated cells, the cytosolic pool of Abp1 largely obscures the weak Abp1 immunostaining (l) at sites of endocytosis [here marked by anti–AP2 immunostaining (j)]. Inserts represent enlargements of the areas boxed in a–l. Bars, 10 μm.
Mentions: Several observations have suggested that dynamins may play an as yet unidentified cytoskeletal role in addition to their endocytic function (for review, see McNiven et al. 2000). At least dynamin2aa was observed at actin-rich sites within cells, which seem independent of sites of endocytosis (Cao et al. 1998; Ochoa et al. 2000). It was thus important to examine whether the dynamin-rich sites detected by the antibody Hudy1, which were observed to be Abp1 immunopositive under conditions also leading to a recruitment of Abp1 to actin-rich lamellipodia, were sites of endocytosis, or whether they reflect a putative cytoskeletal role of dynamin. We therefore stained cells, which had been stimulated and permeabilized as before, with the monoclonal Hudy1 antibody (Fig. 8 a) and a polyclonal antibody against the clathrin coat component Hip1R (Engqvist-Goldstein et al. 1999) (Fig. 8 c). Besides some labeling in and/or near the nucleus, which seemed to be a result of our membrane perforations, both antibodies detected their antigen in cortical spots resistant to the perforation procedure (Fig. 8, a and c). Colocalization experiments demonstrated that the dynamin-rich sites detected by the monoclonal antibody Hudy1 were also positive for Hip1R (Fig. 8 b). We have previously also shown that Hip1R puncta at the cell cortex colocalize quantitatively with the adaptor protein AP2 detected with the monoclonal antibody AP6 (Engqvist-Goldstein et al. 1999). The perforation-resistant, dynamin-rich areas at the cell cortex also contain eps15 (Fig. 8, d–f), another component of clathrin coats (Tebar et al. 1996). Eps15 immunostaining is strongest at puncta at the periphery and decreases towards the center of the cells (Fig. 8 f), whereas dynamin labeling shows the opposite gradient (Fig. 8 d). However Eps15 and dynamin show clear colocalization (Fig. 8 e).

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