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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 colocalizes with cortactin in the lamellipodia of growth factor-stimulated fibroblasts via the PRD. Immunofluorescence localization of dynamin and cortactin in quiescent, and PDGF-treated NIH/3T3 cells. Labels indicate whether cells were transfected. Resting cells were transfected with a full-length cortactin–GFP construct and double-labeled for dynamin (a–a′′), or untransfected cells were fixed and double-labeled with antibodies to dynamin (MC63) and cortactin (b–b′′). Resting cells (a–a′′)transfected with the Cort–GFP construct displayed a nonpolarized phenotype with cortactin localized around the cell cortex and on actin stress fibers (arrows). The distribution of cortactin in untransfected cells was identical to that of cells transfected with cort-GFP, although there was a reduced localization to stress fibers (b–b′′). Upon stimulation with PDGF, there was a marked recruitment of both Dyn 2 (c) and cortactin (c′) into large cortical ruffles (arrows) situated at the leading edges of the actively migrating cells. An identical localization was observed in PDGF-stimulated NIH/3T3 cells expressing Dyn 2(aa)–GFP (d and e) that were fixed and stained for cortactin (d′ and e′). Similar to untransfected cells stained with antibodies, Dyn 2(aa)–GFP became concentrated in lamellipodia at the leading edge of PDGF stimulated, motile cells (d and e, arrows). Bars, 10 μm.
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Figure 4: Dynamin colocalizes with cortactin in the lamellipodia of growth factor-stimulated fibroblasts via the PRD. Immunofluorescence localization of dynamin and cortactin in quiescent, and PDGF-treated NIH/3T3 cells. Labels indicate whether cells were transfected. Resting cells were transfected with a full-length cortactin–GFP construct and double-labeled for dynamin (a–a′′), or untransfected cells were fixed and double-labeled with antibodies to dynamin (MC63) and cortactin (b–b′′). Resting cells (a–a′′)transfected with the Cort–GFP construct displayed a nonpolarized phenotype with cortactin localized around the cell cortex and on actin stress fibers (arrows). The distribution of cortactin in untransfected cells was identical to that of cells transfected with cort-GFP, although there was a reduced localization to stress fibers (b–b′′). Upon stimulation with PDGF, there was a marked recruitment of both Dyn 2 (c) and cortactin (c′) into large cortical ruffles (arrows) situated at the leading edges of the actively migrating cells. An identical localization was observed in PDGF-stimulated NIH/3T3 cells expressing Dyn 2(aa)–GFP (d and e) that were fixed and stained for cortactin (d′ and e′). Similar to untransfected cells stained with antibodies, Dyn 2(aa)–GFP became concentrated in lamellipodia at the leading edge of PDGF stimulated, motile cells (d and e, arrows). Bars, 10 μm.

Mentions: The observations described above demonstrate that antibodies to dynamin label membrane ruffles and lamellipodia in cultured fibroblasts (Fig. 1) and that Dyn 2 binds directly to cortactin by its proline rich tail domain (Fig. 2 and Fig. 3). To test if Dyn 2 colocalizes with cortactin, double immunofluorescence staining of resting and PDGF-stimulated NIH/3T3 cells was conducted using antibodies to dynamin and cortactin (Fig. 4). In resting fibroblasts, cortactin was localized to a fine cortical rim along the cell periphery and punctate spots on the plasma membrane. Expression of exogenous cortactin, or cortactin–GFP in these cells showed a localization identical to that of untransfected cells with an additional localization to numerous stress fibers (Fig. 4, a–a′′) that also stained positive with rhodamine phalloidin (data not shown). Resting cells displayed only a modest colocalization between dynamin and cortactin in the cortex (Fig. 4, b–b′′). However, upon stimulation with PDGF (Fig. 4, c–c′′), fibroblasts assumed a polarized morphology characteristic of motile cells (Bray and White 1988; Stossel 1993). Most dramatic was the change in the distribution of dynamin staining, which became concentrated in a thin veil at the ruffling edge of the cell. Concomitant with this rearrangement of dynamin was a striking and precise colocalization with cortactin that was shown previously to accumulate in the periphery of PDGF-treated cells (Wu and Parsons 1993).


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 colocalizes with cortactin in the lamellipodia of growth factor-stimulated fibroblasts via the PRD. Immunofluorescence localization of dynamin and cortactin in quiescent, and PDGF-treated NIH/3T3 cells. Labels indicate whether cells were transfected. Resting cells were transfected with a full-length cortactin–GFP construct and double-labeled for dynamin (a–a′′), or untransfected cells were fixed and double-labeled with antibodies to dynamin (MC63) and cortactin (b–b′′). Resting cells (a–a′′)transfected with the Cort–GFP construct displayed a nonpolarized phenotype with cortactin localized around the cell cortex and on actin stress fibers (arrows). The distribution of cortactin in untransfected cells was identical to that of cells transfected with cort-GFP, although there was a reduced localization to stress fibers (b–b′′). Upon stimulation with PDGF, there was a marked recruitment of both Dyn 2 (c) and cortactin (c′) into large cortical ruffles (arrows) situated at the leading edges of the actively migrating cells. An identical localization was observed in PDGF-stimulated NIH/3T3 cells expressing Dyn 2(aa)–GFP (d and e) that were fixed and stained for cortactin (d′ and e′). Similar to untransfected cells stained with antibodies, Dyn 2(aa)–GFP became concentrated in lamellipodia at the leading edge of PDGF stimulated, motile cells (d and e, arrows). Bars, 10 μm.
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Figure 4: Dynamin colocalizes with cortactin in the lamellipodia of growth factor-stimulated fibroblasts via the PRD. Immunofluorescence localization of dynamin and cortactin in quiescent, and PDGF-treated NIH/3T3 cells. Labels indicate whether cells were transfected. Resting cells were transfected with a full-length cortactin–GFP construct and double-labeled for dynamin (a–a′′), or untransfected cells were fixed and double-labeled with antibodies to dynamin (MC63) and cortactin (b–b′′). Resting cells (a–a′′)transfected with the Cort–GFP construct displayed a nonpolarized phenotype with cortactin localized around the cell cortex and on actin stress fibers (arrows). The distribution of cortactin in untransfected cells was identical to that of cells transfected with cort-GFP, although there was a reduced localization to stress fibers (b–b′′). Upon stimulation with PDGF, there was a marked recruitment of both Dyn 2 (c) and cortactin (c′) into large cortical ruffles (arrows) situated at the leading edges of the actively migrating cells. An identical localization was observed in PDGF-stimulated NIH/3T3 cells expressing Dyn 2(aa)–GFP (d and e) that were fixed and stained for cortactin (d′ and e′). Similar to untransfected cells stained with antibodies, Dyn 2(aa)–GFP became concentrated in lamellipodia at the leading edge of PDGF stimulated, motile cells (d and e, arrows). Bars, 10 μm.
Mentions: The observations described above demonstrate that antibodies to dynamin label membrane ruffles and lamellipodia in cultured fibroblasts (Fig. 1) and that Dyn 2 binds directly to cortactin by its proline rich tail domain (Fig. 2 and Fig. 3). To test if Dyn 2 colocalizes with cortactin, double immunofluorescence staining of resting and PDGF-stimulated NIH/3T3 cells was conducted using antibodies to dynamin and cortactin (Fig. 4). In resting fibroblasts, cortactin was localized to a fine cortical rim along the cell periphery and punctate spots on the plasma membrane. Expression of exogenous cortactin, or cortactin–GFP in these cells showed a localization identical to that of untransfected cells with an additional localization to numerous stress fibers (Fig. 4, a–a′′) that also stained positive with rhodamine phalloidin (data not shown). Resting cells displayed only a modest colocalization between dynamin and cortactin in the cortex (Fig. 4, b–b′′). However, upon stimulation with PDGF (Fig. 4, c–c′′), fibroblasts assumed a polarized morphology characteristic of motile cells (Bray and White 1988; Stossel 1993). Most dramatic was the change in the distribution of dynamin staining, which became concentrated in a thin veil at the ruffling edge of the cell. Concomitant with this rearrangement of dynamin was a striking and precise colocalization with cortactin that was shown previously to accumulate in the periphery of PDGF-treated cells (Wu and Parsons 1993).

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