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Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells.

Cheresh DA, Leng J, Klemke RL - J. Cell Biol. (1999)

Bottom Line: Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell.Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII.Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.

View Article: PubMed Central - PubMed

Affiliation: Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.

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CAS/Crk association, but not ERK activation, is required for Rac-dependent membrane ruffling. (A) Serum-starved COS-7 cells in the presence or absence of insulin (10 μg/ml for 15 min) were stained with rhodamine-conjugated phalloidin, then analyzed by confocal imaging for F-actin (red) containing membrane ruffles after being transfected with either the empty vector (control) or the vector encoding dominant negative CAS (CAS-SD) along with a reporter vector encoding green fluorescent protein (GFP) to identify transfected cells. In some cases, control cells were pretreated for 2 h with 50 μM of PD98059 to inhibit ERK activity before being exposed to insulin as described above. Photomicrographs were taken with a Bio-Rad Labs 1024 laser and a Zeiss Axiovert microscope (400×). Arrowheads indicate cells with prominent F-actin membrane ruffles. (B) COS-7 cells treated as described above were scored for membrane ruffles as described in Materials and Methods. Results are expressed as the percentage of total transfected cells (i.e., green cells) that displayed prominent F-actin membrane ruffles and are the mean ± SEM of three separate experiments. (C) COS cells were transfected with expression vectors encoding CAS, Crk, and dominant negative RacN17, along with a reporter vector encoding GFP, then examined for actin membrane ruffles as describe above. Photomicrographs of CAS/Crk cells (400×) and RacN17 expressing cells (600×) were taken with a Bio-Rad Labs 1024 laser and a Zeiss Axiovert microscope. Arrowheads indicate cells with prominent F-actin membrane ruffles. (D) COS-7 cells transfected with either wild-type DOCK180, gst-tagged CAS and myc-tagged Crk, or CAS and Crk, together with DOCK180 and/or myc-tagged RacN17 were examined for cell migration as described above. An aliquot of cells transfected with CAS/Crk and DOCK180 (lane 2) or cells mock-transfected with the empty vectors (lane 1) as described for the migration experiment above were lysed in detergent and immunoblotted with antibodies to the phosphorylated/activated form of ERK1/ERK2 as described above (top left). Note that in these experiments cells were transfected with CAS/Crk vectors at DNA levels that give half-maximal migration. Each bar represents the mean ± SEM of at least three independent experiments. (E) An aliquot of cells treated as for the migration experiment above were lysed in detergent then immunoblotted with antibodies to CAS, Crk, DOCK180, or myc to detect myc-tagged RacN17. Lane 1, control cells transfected with the empty vectors. Lane 2, cells transfected with the vector containing the cDNA as indicated. Note that the gstCAS and mycCrk proteins show reduced mobility compared with endogenous wild-type forms of these proteins as the result of the molecular tag. Bars, 10 μm.
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Figure 4: CAS/Crk association, but not ERK activation, is required for Rac-dependent membrane ruffling. (A) Serum-starved COS-7 cells in the presence or absence of insulin (10 μg/ml for 15 min) were stained with rhodamine-conjugated phalloidin, then analyzed by confocal imaging for F-actin (red) containing membrane ruffles after being transfected with either the empty vector (control) or the vector encoding dominant negative CAS (CAS-SD) along with a reporter vector encoding green fluorescent protein (GFP) to identify transfected cells. In some cases, control cells were pretreated for 2 h with 50 μM of PD98059 to inhibit ERK activity before being exposed to insulin as described above. Photomicrographs were taken with a Bio-Rad Labs 1024 laser and a Zeiss Axiovert microscope (400×). Arrowheads indicate cells with prominent F-actin membrane ruffles. (B) COS-7 cells treated as described above were scored for membrane ruffles as described in Materials and Methods. Results are expressed as the percentage of total transfected cells (i.e., green cells) that displayed prominent F-actin membrane ruffles and are the mean ± SEM of three separate experiments. (C) COS cells were transfected with expression vectors encoding CAS, Crk, and dominant negative RacN17, along with a reporter vector encoding GFP, then examined for actin membrane ruffles as describe above. Photomicrographs of CAS/Crk cells (400×) and RacN17 expressing cells (600×) were taken with a Bio-Rad Labs 1024 laser and a Zeiss Axiovert microscope. Arrowheads indicate cells with prominent F-actin membrane ruffles. (D) COS-7 cells transfected with either wild-type DOCK180, gst-tagged CAS and myc-tagged Crk, or CAS and Crk, together with DOCK180 and/or myc-tagged RacN17 were examined for cell migration as described above. An aliquot of cells transfected with CAS/Crk and DOCK180 (lane 2) or cells mock-transfected with the empty vectors (lane 1) as described for the migration experiment above were lysed in detergent and immunoblotted with antibodies to the phosphorylated/activated form of ERK1/ERK2 as described above (top left). Note that in these experiments cells were transfected with CAS/Crk vectors at DNA levels that give half-maximal migration. Each bar represents the mean ± SEM of at least three independent experiments. (E) An aliquot of cells treated as for the migration experiment above were lysed in detergent then immunoblotted with antibodies to CAS, Crk, DOCK180, or myc to detect myc-tagged RacN17. Lane 1, control cells transfected with the empty vectors. Lane 2, cells transfected with the vector containing the cDNA as indicated. Note that the gstCAS and mycCrk proteins show reduced mobility compared with endogenous wild-type forms of these proteins as the result of the molecular tag. Bars, 10 μm.

Mentions: Activation of cell migration is characterized by the assembly of actin into membrane ruffles as well as cell contraction (Lauffenburger and Horwitz 1996; Mitchison and Cramer 1996). To explain how the coupling of CAS/Crk and activation of ERK might influence the migration machinery, cells expressing CAS-SD were stimulated with insulin and then examined for actin-containing membrane ruffles. Exposure of mock-transfected control cells to insulin induced prominent membrane ruffles rich in F-actin (Fig. 4 A). Approximately 18% of the control cell population showed membrane ruffling before stimulation with insulin and this was increased to 80% after cells were exposed to this cytokine (Fig. 4 B). Importantly, expression of CAS-SD in these cells completely blocked the insulin-induced membrane ruffling response (Fig. 4A and Fig. B). As expected, cells within the field of view not transfected with CAS-SD showed prominent membrane ruffles (Fig. 4 A). Expression of Crk-SH2 in cells also blocked insulin-induced membrane ruffles (data not shown). Recently, it was reported that CAS/Crk coupling facilitates Rac activity which can promote membrane ruffling (Ridely et al. 1992; Kiyokawa et al. 1998a). Therefore, we determined whether CAS/Crk-induced membrane ruffles required Rac activity. Cells were transfected with CAS and Crk along with a dominant negative form of Rac (RacN17) and then examined for F-actin containing membrane ruffles. Expression of RacN17 in these cells blocked CAS/Crk-induced ruffles (Fig. 4 C). RacN17 also blocked cell migration without impacting ERK activity (data not shown). Thus, CAS/Crk coupling promotes membrane ruffles that depend on Rac, but not on ERK activity.


Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells.

Cheresh DA, Leng J, Klemke RL - J. Cell Biol. (1999)

CAS/Crk association, but not ERK activation, is required for Rac-dependent membrane ruffling. (A) Serum-starved COS-7 cells in the presence or absence of insulin (10 μg/ml for 15 min) were stained with rhodamine-conjugated phalloidin, then analyzed by confocal imaging for F-actin (red) containing membrane ruffles after being transfected with either the empty vector (control) or the vector encoding dominant negative CAS (CAS-SD) along with a reporter vector encoding green fluorescent protein (GFP) to identify transfected cells. In some cases, control cells were pretreated for 2 h with 50 μM of PD98059 to inhibit ERK activity before being exposed to insulin as described above. Photomicrographs were taken with a Bio-Rad Labs 1024 laser and a Zeiss Axiovert microscope (400×). Arrowheads indicate cells with prominent F-actin membrane ruffles. (B) COS-7 cells treated as described above were scored for membrane ruffles as described in Materials and Methods. Results are expressed as the percentage of total transfected cells (i.e., green cells) that displayed prominent F-actin membrane ruffles and are the mean ± SEM of three separate experiments. (C) COS cells were transfected with expression vectors encoding CAS, Crk, and dominant negative RacN17, along with a reporter vector encoding GFP, then examined for actin membrane ruffles as describe above. Photomicrographs of CAS/Crk cells (400×) and RacN17 expressing cells (600×) were taken with a Bio-Rad Labs 1024 laser and a Zeiss Axiovert microscope. Arrowheads indicate cells with prominent F-actin membrane ruffles. (D) COS-7 cells transfected with either wild-type DOCK180, gst-tagged CAS and myc-tagged Crk, or CAS and Crk, together with DOCK180 and/or myc-tagged RacN17 were examined for cell migration as described above. An aliquot of cells transfected with CAS/Crk and DOCK180 (lane 2) or cells mock-transfected with the empty vectors (lane 1) as described for the migration experiment above were lysed in detergent and immunoblotted with antibodies to the phosphorylated/activated form of ERK1/ERK2 as described above (top left). Note that in these experiments cells were transfected with CAS/Crk vectors at DNA levels that give half-maximal migration. Each bar represents the mean ± SEM of at least three independent experiments. (E) An aliquot of cells treated as for the migration experiment above were lysed in detergent then immunoblotted with antibodies to CAS, Crk, DOCK180, or myc to detect myc-tagged RacN17. Lane 1, control cells transfected with the empty vectors. Lane 2, cells transfected with the vector containing the cDNA as indicated. Note that the gstCAS and mycCrk proteins show reduced mobility compared with endogenous wild-type forms of these proteins as the result of the molecular tag. Bars, 10 μm.
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Figure 4: CAS/Crk association, but not ERK activation, is required for Rac-dependent membrane ruffling. (A) Serum-starved COS-7 cells in the presence or absence of insulin (10 μg/ml for 15 min) were stained with rhodamine-conjugated phalloidin, then analyzed by confocal imaging for F-actin (red) containing membrane ruffles after being transfected with either the empty vector (control) or the vector encoding dominant negative CAS (CAS-SD) along with a reporter vector encoding green fluorescent protein (GFP) to identify transfected cells. In some cases, control cells were pretreated for 2 h with 50 μM of PD98059 to inhibit ERK activity before being exposed to insulin as described above. Photomicrographs were taken with a Bio-Rad Labs 1024 laser and a Zeiss Axiovert microscope (400×). Arrowheads indicate cells with prominent F-actin membrane ruffles. (B) COS-7 cells treated as described above were scored for membrane ruffles as described in Materials and Methods. Results are expressed as the percentage of total transfected cells (i.e., green cells) that displayed prominent F-actin membrane ruffles and are the mean ± SEM of three separate experiments. (C) COS cells were transfected with expression vectors encoding CAS, Crk, and dominant negative RacN17, along with a reporter vector encoding GFP, then examined for actin membrane ruffles as describe above. Photomicrographs of CAS/Crk cells (400×) and RacN17 expressing cells (600×) were taken with a Bio-Rad Labs 1024 laser and a Zeiss Axiovert microscope. Arrowheads indicate cells with prominent F-actin membrane ruffles. (D) COS-7 cells transfected with either wild-type DOCK180, gst-tagged CAS and myc-tagged Crk, or CAS and Crk, together with DOCK180 and/or myc-tagged RacN17 were examined for cell migration as described above. An aliquot of cells transfected with CAS/Crk and DOCK180 (lane 2) or cells mock-transfected with the empty vectors (lane 1) as described for the migration experiment above were lysed in detergent and immunoblotted with antibodies to the phosphorylated/activated form of ERK1/ERK2 as described above (top left). Note that in these experiments cells were transfected with CAS/Crk vectors at DNA levels that give half-maximal migration. Each bar represents the mean ± SEM of at least three independent experiments. (E) An aliquot of cells treated as for the migration experiment above were lysed in detergent then immunoblotted with antibodies to CAS, Crk, DOCK180, or myc to detect myc-tagged RacN17. Lane 1, control cells transfected with the empty vectors. Lane 2, cells transfected with the vector containing the cDNA as indicated. Note that the gstCAS and mycCrk proteins show reduced mobility compared with endogenous wild-type forms of these proteins as the result of the molecular tag. Bars, 10 μm.
Mentions: Activation of cell migration is characterized by the assembly of actin into membrane ruffles as well as cell contraction (Lauffenburger and Horwitz 1996; Mitchison and Cramer 1996). To explain how the coupling of CAS/Crk and activation of ERK might influence the migration machinery, cells expressing CAS-SD were stimulated with insulin and then examined for actin-containing membrane ruffles. Exposure of mock-transfected control cells to insulin induced prominent membrane ruffles rich in F-actin (Fig. 4 A). Approximately 18% of the control cell population showed membrane ruffling before stimulation with insulin and this was increased to 80% after cells were exposed to this cytokine (Fig. 4 B). Importantly, expression of CAS-SD in these cells completely blocked the insulin-induced membrane ruffling response (Fig. 4A and Fig. B). As expected, cells within the field of view not transfected with CAS-SD showed prominent membrane ruffles (Fig. 4 A). Expression of Crk-SH2 in cells also blocked insulin-induced membrane ruffles (data not shown). Recently, it was reported that CAS/Crk coupling facilitates Rac activity which can promote membrane ruffling (Ridely et al. 1992; Kiyokawa et al. 1998a). Therefore, we determined whether CAS/Crk-induced membrane ruffles required Rac activity. Cells were transfected with CAS and Crk along with a dominant negative form of Rac (RacN17) and then examined for F-actin containing membrane ruffles. Expression of RacN17 in these cells blocked CAS/Crk-induced ruffles (Fig. 4 C). RacN17 also blocked cell migration without impacting ERK activity (data not shown). Thus, CAS/Crk coupling promotes membrane ruffles that depend on Rac, but not on ERK activity.

Bottom Line: Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell.Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII.Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.

View Article: PubMed Central - PubMed

Affiliation: Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.

Show MeSH
Related in: MedlinePlus