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Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape.

McNiven MA, Kim L, Krueger EW, Orth JD, Cao H, Wong TW - J. Cell Biol. (2000)

Bottom Line: Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia.Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle.These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, Minnesota 55905, USA.

ABSTRACT
The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin-SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)DeltaPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.

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Dynamin associates with cortical membrane ruffles in growth factor-stimulated fibroblasts. a–e, Immunofluorescence staining of cultured NIH/3T3 fibroblasts with affinity-purified peptide antibodies made to a conserved region of the dynamin NH2 terminus (MC63) or to the proline rich COOH-terminal domain specific for Dyn 2. In resting cells (a and b), dynamin labeling is most prominent as bright spots on the plasma membrane and near the nucleus with a modest accumulation in the cell cortex (arrows). Upon stimulation with PDGF for 10–15 min (c and e), cells display a motile phenotype and dynamin staining becomes significantly enriched at the ruffled membrane at the leading edge of the cell (arrows). This cortical dynamin staining colocalizes markedly with actin in the leading of motile cells (arrows), as confirmed by double labeling with rhodamine phalloidin (e and e′). Immunoblot analysis of immunoprecipitates from cytosolic extract of NIH/3T3 cells using antibodies to cortactin or dynamin that were probed with mAbs to either dynamin (f) or cortactin (g). The lanes for each blot include: lane 1, starting cytosolic extract or IP with; lane 2, nonimmune serum (NI); lane 3, an anticortactin mAb; lane 4, the polyclonal Pan-dynamin antibody MC63; lane 5, the polyclonal antibody Dyn 2 specific for the COOH terminus of Dyn 2. Antibodies to cortactin immunoprecipitate a 105-kD immunoreactive dynamin band and a 95-kD band believed to be a proteolytic fragment of dynamin (f). Antibodies to dynamin immunoprecipitate the actin-binding protein cortactin from cell homogenates (g). The filter from f was stripped and reprobed with antibodies to cortactin (g). Interestingly, whereas the Pan-dynamin antibodies (MC63) immunoprecipitated cortactin (lane 4), the Dyn 2 tail-specific antibody (Dyn 2) did not (lane 5), suggesting that this antibody blocks dynamin–cortactin binding. Bar, 10 μm.
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Figure 1: Dynamin associates with cortical membrane ruffles in growth factor-stimulated fibroblasts. a–e, Immunofluorescence staining of cultured NIH/3T3 fibroblasts with affinity-purified peptide antibodies made to a conserved region of the dynamin NH2 terminus (MC63) or to the proline rich COOH-terminal domain specific for Dyn 2. In resting cells (a and b), dynamin labeling is most prominent as bright spots on the plasma membrane and near the nucleus with a modest accumulation in the cell cortex (arrows). Upon stimulation with PDGF for 10–15 min (c and e), cells display a motile phenotype and dynamin staining becomes significantly enriched at the ruffled membrane at the leading edge of the cell (arrows). This cortical dynamin staining colocalizes markedly with actin in the leading of motile cells (arrows), as confirmed by double labeling with rhodamine phalloidin (e and e′). Immunoblot analysis of immunoprecipitates from cytosolic extract of NIH/3T3 cells using antibodies to cortactin or dynamin that were probed with mAbs to either dynamin (f) or cortactin (g). The lanes for each blot include: lane 1, starting cytosolic extract or IP with; lane 2, nonimmune serum (NI); lane 3, an anticortactin mAb; lane 4, the polyclonal Pan-dynamin antibody MC63; lane 5, the polyclonal antibody Dyn 2 specific for the COOH terminus of Dyn 2. Antibodies to cortactin immunoprecipitate a 105-kD immunoreactive dynamin band and a 95-kD band believed to be a proteolytic fragment of dynamin (f). Antibodies to dynamin immunoprecipitate the actin-binding protein cortactin from cell homogenates (g). The filter from f was stripped and reprobed with antibodies to cortactin (g). Interestingly, whereas the Pan-dynamin antibodies (MC63) immunoprecipitated cortactin (lane 4), the Dyn 2 tail-specific antibody (Dyn 2) did not (lane 5), suggesting that this antibody blocks dynamin–cortactin binding. Bar, 10 μm.

Mentions: Immunostaining of cultured NIH/3T3 fibroblasts with two well characterized, polyclonal antibodies to either the conserved NH2 terminus (MC63) or the proline-rich tail of Dyn 2 (Henley and McNiven 1996) produced an unexpected localization to the cell periphery (Fig. 1, a and b). Most strikingly, this cortical labeling was increased dramatically in fibroblasts treated with growth factors, such as PDGF, which are known to induce cell migration. In these stimulated cells, dynamin staining was localized to clathrin-coated vesicles at the plasma membrane and Golgi apparatus, as well as actin-containing cortical ruffles of the leading edge (Fig. 1, c–e′). Whereas Golgi labeling was observed routinely, in some cells it was absent due to a more rigorous extraction procedure that optimized staining of the cytoskeleton. To define the components of the actin cytoskeleton with which dynamin associates, we conducted an IP-based screening of fibroblast homogenates with antibodies to dynamin and actin-associated proteins. Specifically, Dyn 2 immunoprecipitates were subjected to SDS-PAGE and immunoblot analysis with antibodies to either vinculin, paxillin, cortactin, fimbrin, talin, or zyxin. Whereas the dynamin antibodies did pull down some paxillin protein, (data not shown) only cortactin appeared to bind Dyn 2 in a consistent way. The actin-binding protein cortactin, which is a target for tyrosine kinases and may link growth signals with the actin cytoskeleton (Wu et al. 1991; Downing and Reynolds 1992; Maa et al. 1992), was a particularly attractive candidate for a dynamin binding partner for several reasons. First, cortactin is known to contain an SH3 domain that may have an affinity for the COOH-terminal PRD of dynamin, and second, cortactin has been shown to localize to the cortical leading ruffles of migrating, PDGF stimulated cells, where it is believed to modulate actin assembly and dynamics (Wu and Parsons 1993). The localization of cortactin is similar to that seen in PDGF-treated cells stained with the dynamin antibodies (Fig. 1, c–e). Thus, reciprocal IP experiments were performed (Fig. 1f and Fig. g) on cultured fibroblast homogenates (NIH/3T3 cells) using cortactin and dynamin antibodies. The cortactin antibody precipitated a 105-kD polypeptide, which was recognized by the MC63 Pan-dynamin antibody. The immunoblot also detected an additional polypeptide band that was suspected to be a proteolytic fragment of dynamin or a minor spliced variant (Sontag et al. 1994). Conversely, cortactin was detected in a Dyn 2 immunoprecipitate with the Pan-dynamin antibody. Interestingly, an isoform-specific antibody made to the COOH terminus of the ubiquitously expressed Dyn 2 failed to immunoprecipitate cortactin (Fig. 1 g, lane 5), although the same antibody detected the coimmunoprecipitated dynamin by Western blotting (data not shown). The fact that the Pan-dynamin antibody (MC63) was competent to pull down the cortactin-dynamin complex, whereas the tail-specific (Dyn 2) antibody did not, suggested that cortactin and dynamin may form a stable complex mediated by the COOH–PRD of Dyn 2. Because both of these proteins are likely to participate in a variety of distinct cellular functions, it is likely that only a portion of the total cytoplasmic pool of these proteins form a complex with each other. Indeed, Western blot analysis was implemented to compare the amounts of coimmunoprecipitated Dyn 2–cortactin complexes with that of total cellular protein levels. We estimate that 20–30% of the total Dyn 2 and cortactin are complexed together (data not shown).


Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape.

McNiven MA, Kim L, Krueger EW, Orth JD, Cao H, Wong TW - J. Cell Biol. (2000)

Dynamin associates with cortical membrane ruffles in growth factor-stimulated fibroblasts. a–e, Immunofluorescence staining of cultured NIH/3T3 fibroblasts with affinity-purified peptide antibodies made to a conserved region of the dynamin NH2 terminus (MC63) or to the proline rich COOH-terminal domain specific for Dyn 2. In resting cells (a and b), dynamin labeling is most prominent as bright spots on the plasma membrane and near the nucleus with a modest accumulation in the cell cortex (arrows). Upon stimulation with PDGF for 10–15 min (c and e), cells display a motile phenotype and dynamin staining becomes significantly enriched at the ruffled membrane at the leading edge of the cell (arrows). This cortical dynamin staining colocalizes markedly with actin in the leading of motile cells (arrows), as confirmed by double labeling with rhodamine phalloidin (e and e′). Immunoblot analysis of immunoprecipitates from cytosolic extract of NIH/3T3 cells using antibodies to cortactin or dynamin that were probed with mAbs to either dynamin (f) or cortactin (g). The lanes for each blot include: lane 1, starting cytosolic extract or IP with; lane 2, nonimmune serum (NI); lane 3, an anticortactin mAb; lane 4, the polyclonal Pan-dynamin antibody MC63; lane 5, the polyclonal antibody Dyn 2 specific for the COOH terminus of Dyn 2. Antibodies to cortactin immunoprecipitate a 105-kD immunoreactive dynamin band and a 95-kD band believed to be a proteolytic fragment of dynamin (f). Antibodies to dynamin immunoprecipitate the actin-binding protein cortactin from cell homogenates (g). The filter from f was stripped and reprobed with antibodies to cortactin (g). Interestingly, whereas the Pan-dynamin antibodies (MC63) immunoprecipitated cortactin (lane 4), the Dyn 2 tail-specific antibody (Dyn 2) did not (lane 5), suggesting that this antibody blocks dynamin–cortactin binding. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 1: Dynamin associates with cortical membrane ruffles in growth factor-stimulated fibroblasts. a–e, Immunofluorescence staining of cultured NIH/3T3 fibroblasts with affinity-purified peptide antibodies made to a conserved region of the dynamin NH2 terminus (MC63) or to the proline rich COOH-terminal domain specific for Dyn 2. In resting cells (a and b), dynamin labeling is most prominent as bright spots on the plasma membrane and near the nucleus with a modest accumulation in the cell cortex (arrows). Upon stimulation with PDGF for 10–15 min (c and e), cells display a motile phenotype and dynamin staining becomes significantly enriched at the ruffled membrane at the leading edge of the cell (arrows). This cortical dynamin staining colocalizes markedly with actin in the leading of motile cells (arrows), as confirmed by double labeling with rhodamine phalloidin (e and e′). Immunoblot analysis of immunoprecipitates from cytosolic extract of NIH/3T3 cells using antibodies to cortactin or dynamin that were probed with mAbs to either dynamin (f) or cortactin (g). The lanes for each blot include: lane 1, starting cytosolic extract or IP with; lane 2, nonimmune serum (NI); lane 3, an anticortactin mAb; lane 4, the polyclonal Pan-dynamin antibody MC63; lane 5, the polyclonal antibody Dyn 2 specific for the COOH terminus of Dyn 2. Antibodies to cortactin immunoprecipitate a 105-kD immunoreactive dynamin band and a 95-kD band believed to be a proteolytic fragment of dynamin (f). Antibodies to dynamin immunoprecipitate the actin-binding protein cortactin from cell homogenates (g). The filter from f was stripped and reprobed with antibodies to cortactin (g). Interestingly, whereas the Pan-dynamin antibodies (MC63) immunoprecipitated cortactin (lane 4), the Dyn 2 tail-specific antibody (Dyn 2) did not (lane 5), suggesting that this antibody blocks dynamin–cortactin binding. Bar, 10 μm.
Mentions: Immunostaining of cultured NIH/3T3 fibroblasts with two well characterized, polyclonal antibodies to either the conserved NH2 terminus (MC63) or the proline-rich tail of Dyn 2 (Henley and McNiven 1996) produced an unexpected localization to the cell periphery (Fig. 1, a and b). Most strikingly, this cortical labeling was increased dramatically in fibroblasts treated with growth factors, such as PDGF, which are known to induce cell migration. In these stimulated cells, dynamin staining was localized to clathrin-coated vesicles at the plasma membrane and Golgi apparatus, as well as actin-containing cortical ruffles of the leading edge (Fig. 1, c–e′). Whereas Golgi labeling was observed routinely, in some cells it was absent due to a more rigorous extraction procedure that optimized staining of the cytoskeleton. To define the components of the actin cytoskeleton with which dynamin associates, we conducted an IP-based screening of fibroblast homogenates with antibodies to dynamin and actin-associated proteins. Specifically, Dyn 2 immunoprecipitates were subjected to SDS-PAGE and immunoblot analysis with antibodies to either vinculin, paxillin, cortactin, fimbrin, talin, or zyxin. Whereas the dynamin antibodies did pull down some paxillin protein, (data not shown) only cortactin appeared to bind Dyn 2 in a consistent way. The actin-binding protein cortactin, which is a target for tyrosine kinases and may link growth signals with the actin cytoskeleton (Wu et al. 1991; Downing and Reynolds 1992; Maa et al. 1992), was a particularly attractive candidate for a dynamin binding partner for several reasons. First, cortactin is known to contain an SH3 domain that may have an affinity for the COOH-terminal PRD of dynamin, and second, cortactin has been shown to localize to the cortical leading ruffles of migrating, PDGF stimulated cells, where it is believed to modulate actin assembly and dynamics (Wu and Parsons 1993). The localization of cortactin is similar to that seen in PDGF-treated cells stained with the dynamin antibodies (Fig. 1, c–e). Thus, reciprocal IP experiments were performed (Fig. 1f and Fig. g) on cultured fibroblast homogenates (NIH/3T3 cells) using cortactin and dynamin antibodies. The cortactin antibody precipitated a 105-kD polypeptide, which was recognized by the MC63 Pan-dynamin antibody. The immunoblot also detected an additional polypeptide band that was suspected to be a proteolytic fragment of dynamin or a minor spliced variant (Sontag et al. 1994). Conversely, cortactin was detected in a Dyn 2 immunoprecipitate with the Pan-dynamin antibody. Interestingly, an isoform-specific antibody made to the COOH terminus of the ubiquitously expressed Dyn 2 failed to immunoprecipitate cortactin (Fig. 1 g, lane 5), although the same antibody detected the coimmunoprecipitated dynamin by Western blotting (data not shown). The fact that the Pan-dynamin antibody (MC63) was competent to pull down the cortactin-dynamin complex, whereas the tail-specific (Dyn 2) antibody did not, suggested that cortactin and dynamin may form a stable complex mediated by the COOH–PRD of Dyn 2. Because both of these proteins are likely to participate in a variety of distinct cellular functions, it is likely that only a portion of the total cytoplasmic pool of these proteins form a complex with each other. Indeed, Western blot analysis was implemented to compare the amounts of coimmunoprecipitated Dyn 2–cortactin complexes with that of total cellular protein levels. We estimate that 20–30% of the total Dyn 2 and cortactin are complexed together (data not shown).

Bottom Line: Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia.Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle.These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, Minnesota 55905, USA.

ABSTRACT
The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin-SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)DeltaPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.

Show MeSH
Related in: MedlinePlus