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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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Expression of a truncated dynamin (Dyn 2(aa)ΔPRD) induces profound changes in cell shape with a concomitant proliferation of actin stress fibers. Fluorescence micrographs of cultured clone 9 cells expressing either wt Dyn 2(aa)–GFP (a) or Dyn 2(aa)ΔPRD-GFP (b–e). Cells expressing the wt protein (a) display a normal discoidal shape and a punctate distribution of dynamin at both the plasma membrane and Golgi apparatus, identical to that of untransfected cells. In strong contrast, cells expressing the Dyn 2(aa)ΔPRD–GFP became elongated, sprouting long peculiar neurite-like appendages (b–e). Morphological measurements of >200 wt and mutant cells confirmed this shape change showing a six- to sevenfold increase in width versus length (f). To test if changes in actin organization might be responsible for these changes in shape, clone 9 cells expressing the Dyn 2(aa)ΔPRD–GFP (g–i) were fixed and stained for actin using rhodamine phalloidin (g′–i′). Whereas surrounding untransfected cells possessed a cortical ring of filamentous actin with few stress fibers, mutant cells displayed a reduction in cortical actin with a dramatic alteration in the actin cytoskeleton. Specifically, these cells possessed an extensive number of stress fibers that traversed the long axis and contributed to the shape malformation seen in isolated cells that were not grown in a monolayer (b–e). It should be noted that changes in the shape of these mutant cells are minimized in the confluent cultures shown here to provide a comparison of actin organization with the surrounding, untransfected cells (g–i). Shape changes are most prevalent in sparsely plated cells (b–e). Bars, 10 μm.
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Figure 6: Expression of a truncated dynamin (Dyn 2(aa)ΔPRD) induces profound changes in cell shape with a concomitant proliferation of actin stress fibers. Fluorescence micrographs of cultured clone 9 cells expressing either wt Dyn 2(aa)–GFP (a) or Dyn 2(aa)ΔPRD-GFP (b–e). Cells expressing the wt protein (a) display a normal discoidal shape and a punctate distribution of dynamin at both the plasma membrane and Golgi apparatus, identical to that of untransfected cells. In strong contrast, cells expressing the Dyn 2(aa)ΔPRD–GFP became elongated, sprouting long peculiar neurite-like appendages (b–e). Morphological measurements of >200 wt and mutant cells confirmed this shape change showing a six- to sevenfold increase in width versus length (f). To test if changes in actin organization might be responsible for these changes in shape, clone 9 cells expressing the Dyn 2(aa)ΔPRD–GFP (g–i) were fixed and stained for actin using rhodamine phalloidin (g′–i′). Whereas surrounding untransfected cells possessed a cortical ring of filamentous actin with few stress fibers, mutant cells displayed a reduction in cortical actin with a dramatic alteration in the actin cytoskeleton. Specifically, these cells possessed an extensive number of stress fibers that traversed the long axis and contributed to the shape malformation seen in isolated cells that were not grown in a monolayer (b–e). It should be noted that changes in the shape of these mutant cells are minimized in the confluent cultures shown here to provide a comparison of actin organization with the surrounding, untransfected cells (g–i). Shape changes are most prevalent in sparsely plated cells (b–e). Bars, 10 μm.

Mentions: To further define if Dyn 2 and cortactin truly interact in vivo, we performed two additional experiments. We tested if a modest overexpression (three- to fivefold) of one partner would increase the recruitment of the other protein to the ruffle; and second, whether expression of truncated Dyn 2 or cortactin, lacking their putative interaction domains (PRD and SH3, respectively), would alter the cytoplasmic colocalization of these proteins when expressed in cells. The results of these experiments are summarized in Table , and Fig. 5 displays representative images of NIH/3T3 cells expressing various Dyn 2 and cortactin constructs. To test if increased levels of cortactin would recruit additional dynamin to the lamellipodia, cells were transfected with Cort–GFP, allowed to recover for 24–48 h, and were then stained with Dyn 2 antibodies to localize endogenous dynamin. Remarkably, cells expressing Cort–GFP displayed many more ruffles and lamellipodia in which a marked increase in endogenous dynamin protein was apparent. This dramatic increase in dynamin at the ruffle was observed even in the absence of PDGF stimulation (Fig. 5, a and a′). The recruitment of Dyn 2 to the cortex was increased further in cells cotransfected with both cortactin and Dyn 2(aa) expression constructs when stimulated (Fig. 5b and Fig. b′). As shown in Fig. 4, an increased expression of Dyn 2 through the transfection of a Dyn 2(aa)–GFP construct in fibroblasts did not increase cell ruffling or the levels of cortactin sequestered in these structures. These observations suggest that cortactin is first localized to the lamellipodia and subsequently recruits its dynamin binding partner. Additional studies expressing a CortΔSH3 construct in cells supports this prediction. Cells expressing this truncated cortactin continued to display substantial levels of this protein in the cell ruffles that were significantly depleted of endogenous Dyn 2 (Fig. 5c and Fig. d). Thus, altering the cortactin protein in cells had a marked effect on Dyn 2 localization. Finally, expression of a truncated Dyn 2 protein, missing the COOH-terminal 124 amino acids comprising the PRD (Dyn 2(aa)ΔPRD) that mediates cortactin binding (Fig. 2 and Fig. 3) also greatly reduced the levels of dynamin in the leading ruffle (Fig. 5e and Fig. f). Interestingly, although cortactin remained localized to the ruffles of these mutant cells, their polarity and shape appeared markedly altered. Generally, control stimulated fibroblasts displayed an obvious leading edge filled with cortactin and Dyn 2 with only modest levels of these proteins localized to the lateral and posterior cortices. In contrast, mutant cells expressing truncated Dyn 2 or cortactin proteins sequestered little Dyn 2 in the leading ruffle, whereas cortactin appeared distributed in the ruffle and also along most of the cell circumference. Further, these cells also appeared to take on peculiar elongate shapes, with long appendages. Because fibroblasts are generally irregular in shape, we tested whether the Dyn 2(aa)ΔPRD protein, when expressed in a cell line of more uniform size and form, might induce more obvious changes in cell shape. Clone 9 cells, a normal, rat hepatocyte cell line with a consistent discoidal shape were transfected to express Dyn 2(aa)ΔPRD-GFP, and were then allowed to recover for 24–48 h before fixation and viewing with the fluorescence microscope. Fig. 6, a–e, depicts representative pictures of clone 9 cells expressing either wt (Fig. 6 a) or truncated (Fig. 6, b–e) Dyn 2(aa)–GFP. Wt cells show a typical discoid morphology with Dyn 2 localized to clathrin-coated buds both at the plasma membrane and the trans-Golgi network as previously published (Cao et al. 1998). In contrast, mutant cells distributed Dyn 2(aa)ΔPRD-GFP diffusely throughout the cytoplasm. Although these cells appear healthy and survive for extended periods (weeks) in culture, they did possess peculiar, elongated or moon-shaped morphologies. Measurement of 200 cells revealed a consistent six- to sevenfold elongation of mutant cells compared with wt cells (Fig. 6 f). Staining of these malformed cells with rhodamine-phalloidin revealed an extraordinary number of large stress fibers coursing through the cytoplasm along the long axis of the cell. This was in striking contrast to the strong peripheral band of cortical actin observed in wt clone 9 cells (Fig. 6g′–i′). Cells in these images displayed less of a shape change when cultured to confluency to allow comparison of actin staining with surrounding cells. Much greater changes in shape were observed in cells grown more sparsely (Fig. 6, b–e). As discussed below, these changes in actin organization and cell shape in mutant cells are consistent with the concept that the dynamin PRD binds to the SH3 domain of cortactin to regulate actin cytoskeleton dynamics.


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)

Expression of a truncated dynamin (Dyn 2(aa)ΔPRD) induces profound changes in cell shape with a concomitant proliferation of actin stress fibers. Fluorescence micrographs of cultured clone 9 cells expressing either wt Dyn 2(aa)–GFP (a) or Dyn 2(aa)ΔPRD-GFP (b–e). Cells expressing the wt protein (a) display a normal discoidal shape and a punctate distribution of dynamin at both the plasma membrane and Golgi apparatus, identical to that of untransfected cells. In strong contrast, cells expressing the Dyn 2(aa)ΔPRD–GFP became elongated, sprouting long peculiar neurite-like appendages (b–e). Morphological measurements of >200 wt and mutant cells confirmed this shape change showing a six- to sevenfold increase in width versus length (f). To test if changes in actin organization might be responsible for these changes in shape, clone 9 cells expressing the Dyn 2(aa)ΔPRD–GFP (g–i) were fixed and stained for actin using rhodamine phalloidin (g′–i′). Whereas surrounding untransfected cells possessed a cortical ring of filamentous actin with few stress fibers, mutant cells displayed a reduction in cortical actin with a dramatic alteration in the actin cytoskeleton. Specifically, these cells possessed an extensive number of stress fibers that traversed the long axis and contributed to the shape malformation seen in isolated cells that were not grown in a monolayer (b–e). It should be noted that changes in the shape of these mutant cells are minimized in the confluent cultures shown here to provide a comparison of actin organization with the surrounding, untransfected cells (g–i). Shape changes are most prevalent in sparsely plated cells (b–e). Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 6: Expression of a truncated dynamin (Dyn 2(aa)ΔPRD) induces profound changes in cell shape with a concomitant proliferation of actin stress fibers. Fluorescence micrographs of cultured clone 9 cells expressing either wt Dyn 2(aa)–GFP (a) or Dyn 2(aa)ΔPRD-GFP (b–e). Cells expressing the wt protein (a) display a normal discoidal shape and a punctate distribution of dynamin at both the plasma membrane and Golgi apparatus, identical to that of untransfected cells. In strong contrast, cells expressing the Dyn 2(aa)ΔPRD–GFP became elongated, sprouting long peculiar neurite-like appendages (b–e). Morphological measurements of >200 wt and mutant cells confirmed this shape change showing a six- to sevenfold increase in width versus length (f). To test if changes in actin organization might be responsible for these changes in shape, clone 9 cells expressing the Dyn 2(aa)ΔPRD–GFP (g–i) were fixed and stained for actin using rhodamine phalloidin (g′–i′). Whereas surrounding untransfected cells possessed a cortical ring of filamentous actin with few stress fibers, mutant cells displayed a reduction in cortical actin with a dramatic alteration in the actin cytoskeleton. Specifically, these cells possessed an extensive number of stress fibers that traversed the long axis and contributed to the shape malformation seen in isolated cells that were not grown in a monolayer (b–e). It should be noted that changes in the shape of these mutant cells are minimized in the confluent cultures shown here to provide a comparison of actin organization with the surrounding, untransfected cells (g–i). Shape changes are most prevalent in sparsely plated cells (b–e). Bars, 10 μm.
Mentions: To further define if Dyn 2 and cortactin truly interact in vivo, we performed two additional experiments. We tested if a modest overexpression (three- to fivefold) of one partner would increase the recruitment of the other protein to the ruffle; and second, whether expression of truncated Dyn 2 or cortactin, lacking their putative interaction domains (PRD and SH3, respectively), would alter the cytoplasmic colocalization of these proteins when expressed in cells. The results of these experiments are summarized in Table , and Fig. 5 displays representative images of NIH/3T3 cells expressing various Dyn 2 and cortactin constructs. To test if increased levels of cortactin would recruit additional dynamin to the lamellipodia, cells were transfected with Cort–GFP, allowed to recover for 24–48 h, and were then stained with Dyn 2 antibodies to localize endogenous dynamin. Remarkably, cells expressing Cort–GFP displayed many more ruffles and lamellipodia in which a marked increase in endogenous dynamin protein was apparent. This dramatic increase in dynamin at the ruffle was observed even in the absence of PDGF stimulation (Fig. 5, a and a′). The recruitment of Dyn 2 to the cortex was increased further in cells cotransfected with both cortactin and Dyn 2(aa) expression constructs when stimulated (Fig. 5b and Fig. b′). As shown in Fig. 4, an increased expression of Dyn 2 through the transfection of a Dyn 2(aa)–GFP construct in fibroblasts did not increase cell ruffling or the levels of cortactin sequestered in these structures. These observations suggest that cortactin is first localized to the lamellipodia and subsequently recruits its dynamin binding partner. Additional studies expressing a CortΔSH3 construct in cells supports this prediction. Cells expressing this truncated cortactin continued to display substantial levels of this protein in the cell ruffles that were significantly depleted of endogenous Dyn 2 (Fig. 5c and Fig. d). Thus, altering the cortactin protein in cells had a marked effect on Dyn 2 localization. Finally, expression of a truncated Dyn 2 protein, missing the COOH-terminal 124 amino acids comprising the PRD (Dyn 2(aa)ΔPRD) that mediates cortactin binding (Fig. 2 and Fig. 3) also greatly reduced the levels of dynamin in the leading ruffle (Fig. 5e and Fig. f). Interestingly, although cortactin remained localized to the ruffles of these mutant cells, their polarity and shape appeared markedly altered. Generally, control stimulated fibroblasts displayed an obvious leading edge filled with cortactin and Dyn 2 with only modest levels of these proteins localized to the lateral and posterior cortices. In contrast, mutant cells expressing truncated Dyn 2 or cortactin proteins sequestered little Dyn 2 in the leading ruffle, whereas cortactin appeared distributed in the ruffle and also along most of the cell circumference. Further, these cells also appeared to take on peculiar elongate shapes, with long appendages. Because fibroblasts are generally irregular in shape, we tested whether the Dyn 2(aa)ΔPRD protein, when expressed in a cell line of more uniform size and form, might induce more obvious changes in cell shape. Clone 9 cells, a normal, rat hepatocyte cell line with a consistent discoidal shape were transfected to express Dyn 2(aa)ΔPRD-GFP, and were then allowed to recover for 24–48 h before fixation and viewing with the fluorescence microscope. Fig. 6, a–e, depicts representative pictures of clone 9 cells expressing either wt (Fig. 6 a) or truncated (Fig. 6, b–e) Dyn 2(aa)–GFP. Wt cells show a typical discoid morphology with Dyn 2 localized to clathrin-coated buds both at the plasma membrane and the trans-Golgi network as previously published (Cao et al. 1998). In contrast, mutant cells distributed Dyn 2(aa)ΔPRD-GFP diffusely throughout the cytoplasm. Although these cells appear healthy and survive for extended periods (weeks) in culture, they did possess peculiar, elongated or moon-shaped morphologies. Measurement of 200 cells revealed a consistent six- to sevenfold elongation of mutant cells compared with wt cells (Fig. 6 f). Staining of these malformed cells with rhodamine-phalloidin revealed an extraordinary number of large stress fibers coursing through the cytoplasm along the long axis of the cell. This was in striking contrast to the strong peripheral band of cortical actin observed in wt clone 9 cells (Fig. 6g′–i′). Cells in these images displayed less of a shape change when cultured to confluency to allow comparison of actin staining with surrounding cells. Much greater changes in shape were observed in cells grown more sparsely (Fig. 6, b–e). As discussed below, these changes in actin organization and cell shape in mutant cells are consistent with the concept that the dynamin PRD binds to the SH3 domain of cortactin to regulate actin cytoskeleton dynamics.

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