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GPI-anchored uPAR requires Endo180 for rapid directional sensing during chemotaxis.

Sturge J, Wienke D, East L, Jones GE, Isacke CM - J. Cell Biol. (2003)

Bottom Line: Endo180 expression was demonstrated to enhance uPA-mediated filopodia production and promote rapid activation of Cdc42 and Rac.Expression of a noninternalizing Endo180 mutant revealed that promotion of random cell migration requires receptor endocytosis, whereas the chemotactic response to uPA does not.From these studies, we conclude that Endo180 is a crucial link between uPA-uPAR and setting of the internal cellular compass.

View Article: PubMed Central - PubMed

Affiliation: The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London SW3 6JB, UK.

ABSTRACT
Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in cell guidance and chemotaxis during normal and pathological events. uPAR is GPI-anchored and the mechanism by which it transmits intracellular polarity cues across the plasma membrane during directional sensing has not been elucidated. The constitutively recycling endocytic receptor Endo180 forms a trimolecular complex with uPAR in the presence of uPA, hence its alternate name uPAR-associated protein. Here, we demonstrate that Endo180 is a general promoter of random cell migration and has a more specific function in cell chemotaxis up a uPA gradient. Endo180 expression was demonstrated to enhance uPA-mediated filopodia production and promote rapid activation of Cdc42 and Rac. Expression of a noninternalizing Endo180 mutant revealed that promotion of random cell migration requires receptor endocytosis, whereas the chemotactic response to uPA does not. From these studies, we conclude that Endo180 is a crucial link between uPA-uPAR and setting of the internal cellular compass.

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Expression of Endo180 is sufficient to confer a sense of direction up a concentration gradient of uPA. (A) FACS analysis of Endo180 and uPAR cell surface expression levels in MCF-7 cells transfected with vector alone (green), wild-type Endo180 (red), or Endo180(Ala1468/Ala1469) (blue). Profiles in black represent vector transfected cells incubated with secondary antibody alone. (B) Mean migratory speed of transfected MCF-7. The directional data are summarized in a circular histogram showing the number of cells lying within each 18° interval. The mean direction and its 95% confidence interval are represented as a red arrow and green sector, respectively. (C, D) Chemotactic response of untreated or mAb A5/158-treated transfected MCF-7 cells in a gradient of uPA or EGF. Data shown in B–D includes analysis of >70 cells pooled from at least three experiments. Cell directionality was determined using the Rayleigh test and a horizon distance which included 50% of all cells assayed for each treatment group. (C and D) The directional data are summarized in a circular histogram showing the number of cells lying within each 18 degree interval. The mean direction and its 95% confidence interval are represented as a red arrow and green sector, respectively.
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fig3: Expression of Endo180 is sufficient to confer a sense of direction up a concentration gradient of uPA. (A) FACS analysis of Endo180 and uPAR cell surface expression levels in MCF-7 cells transfected with vector alone (green), wild-type Endo180 (red), or Endo180(Ala1468/Ala1469) (blue). Profiles in black represent vector transfected cells incubated with secondary antibody alone. (B) Mean migratory speed of transfected MCF-7. The directional data are summarized in a circular histogram showing the number of cells lying within each 18° interval. The mean direction and its 95% confidence interval are represented as a red arrow and green sector, respectively. (C, D) Chemotactic response of untreated or mAb A5/158-treated transfected MCF-7 cells in a gradient of uPA or EGF. Data shown in B–D includes analysis of >70 cells pooled from at least three experiments. Cell directionality was determined using the Rayleigh test and a horizon distance which included 50% of all cells assayed for each treatment group. (C and D) The directional data are summarized in a circular histogram showing the number of cells lying within each 18 degree interval. The mean direction and its 95% confidence interval are represented as a red arrow and green sector, respectively.

Mentions: To confirm that these effects of Endo180 were not cell type specific or an artifact of the siRNA system, stable populations of the uPAR and EGF receptor positive/Endo180 negative MCF-7 breast cancer cell line transfected with vector alone or Endo180 were generated (Fig. 3 A). In agreement with the data obtained with MDA-MB-231 cells (Fig. 2 B), Endo180 expression promoted a statistically significant (P < 0.0001) twofold increase in random migration compared with vector-alone transfected cells (Fig. 3 B). Although unstimulated parental or vector-alone transfected MCF-7 cells produced small membrane protrusions, they did not translocate on a Matrigel surface (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200302124/DC1) or a variety of other substrata including fibronectin, collagens I and IV. In contrast, MCF-7 cells expressing Endo180 exhibited increased membrane protrusion formation and translocated randomly on Matrigel (Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200302124/DC1) and the other substrata tested. These observations indicate that Endo180 is both necessary and sufficient to evoke motility in this cell type. Parental or vector-alone transfected MCF-7 cells placed in a gradient of uPA displayed increased but nondirectional migration (Fig. 3, B and C; Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200302124/DC1). The inability of vector-alone cells to detect a chemotactic gradient was consistent at 1, 5, 10, 15, 20, and 25 nM uPA, a concentration range within which the directional migration of MDA-MB-231 cells is maximally stimulated (Sturge et al., 2002). In accordance with the MDA-MB-231 cell data, MCF-7 cells expressing Endo180 were endowed with a sense of direction up a uPA gradient (Fig. 3 C; Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200302124/DC1; P < 0.0001). Furthermore, this directional migration was totally inhibited by the anti-Endo180 mAb A5/158 (Fig. 3 C) and was not associated with altered cell surface levels of uPAR (Fig. 3 A). Conversely, both vector-alone cells and cells expressing Endo180 displayed increased migratory speed (Fig. 3 B) and directionality up an EGF gradient that was unaffected by Endo180 mAbs (Fig. 3 D), further confirming that directional sensing of an EGF gradient is an Endo180-independent process.


GPI-anchored uPAR requires Endo180 for rapid directional sensing during chemotaxis.

Sturge J, Wienke D, East L, Jones GE, Isacke CM - J. Cell Biol. (2003)

Expression of Endo180 is sufficient to confer a sense of direction up a concentration gradient of uPA. (A) FACS analysis of Endo180 and uPAR cell surface expression levels in MCF-7 cells transfected with vector alone (green), wild-type Endo180 (red), or Endo180(Ala1468/Ala1469) (blue). Profiles in black represent vector transfected cells incubated with secondary antibody alone. (B) Mean migratory speed of transfected MCF-7. The directional data are summarized in a circular histogram showing the number of cells lying within each 18° interval. The mean direction and its 95% confidence interval are represented as a red arrow and green sector, respectively. (C, D) Chemotactic response of untreated or mAb A5/158-treated transfected MCF-7 cells in a gradient of uPA or EGF. Data shown in B–D includes analysis of >70 cells pooled from at least three experiments. Cell directionality was determined using the Rayleigh test and a horizon distance which included 50% of all cells assayed for each treatment group. (C and D) The directional data are summarized in a circular histogram showing the number of cells lying within each 18 degree interval. The mean direction and its 95% confidence interval are represented as a red arrow and green sector, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172817&req=5

fig3: Expression of Endo180 is sufficient to confer a sense of direction up a concentration gradient of uPA. (A) FACS analysis of Endo180 and uPAR cell surface expression levels in MCF-7 cells transfected with vector alone (green), wild-type Endo180 (red), or Endo180(Ala1468/Ala1469) (blue). Profiles in black represent vector transfected cells incubated with secondary antibody alone. (B) Mean migratory speed of transfected MCF-7. The directional data are summarized in a circular histogram showing the number of cells lying within each 18° interval. The mean direction and its 95% confidence interval are represented as a red arrow and green sector, respectively. (C, D) Chemotactic response of untreated or mAb A5/158-treated transfected MCF-7 cells in a gradient of uPA or EGF. Data shown in B–D includes analysis of >70 cells pooled from at least three experiments. Cell directionality was determined using the Rayleigh test and a horizon distance which included 50% of all cells assayed for each treatment group. (C and D) The directional data are summarized in a circular histogram showing the number of cells lying within each 18 degree interval. The mean direction and its 95% confidence interval are represented as a red arrow and green sector, respectively.
Mentions: To confirm that these effects of Endo180 were not cell type specific or an artifact of the siRNA system, stable populations of the uPAR and EGF receptor positive/Endo180 negative MCF-7 breast cancer cell line transfected with vector alone or Endo180 were generated (Fig. 3 A). In agreement with the data obtained with MDA-MB-231 cells (Fig. 2 B), Endo180 expression promoted a statistically significant (P < 0.0001) twofold increase in random migration compared with vector-alone transfected cells (Fig. 3 B). Although unstimulated parental or vector-alone transfected MCF-7 cells produced small membrane protrusions, they did not translocate on a Matrigel surface (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200302124/DC1) or a variety of other substrata including fibronectin, collagens I and IV. In contrast, MCF-7 cells expressing Endo180 exhibited increased membrane protrusion formation and translocated randomly on Matrigel (Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200302124/DC1) and the other substrata tested. These observations indicate that Endo180 is both necessary and sufficient to evoke motility in this cell type. Parental or vector-alone transfected MCF-7 cells placed in a gradient of uPA displayed increased but nondirectional migration (Fig. 3, B and C; Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200302124/DC1). The inability of vector-alone cells to detect a chemotactic gradient was consistent at 1, 5, 10, 15, 20, and 25 nM uPA, a concentration range within which the directional migration of MDA-MB-231 cells is maximally stimulated (Sturge et al., 2002). In accordance with the MDA-MB-231 cell data, MCF-7 cells expressing Endo180 were endowed with a sense of direction up a uPA gradient (Fig. 3 C; Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200302124/DC1; P < 0.0001). Furthermore, this directional migration was totally inhibited by the anti-Endo180 mAb A5/158 (Fig. 3 C) and was not associated with altered cell surface levels of uPAR (Fig. 3 A). Conversely, both vector-alone cells and cells expressing Endo180 displayed increased migratory speed (Fig. 3 B) and directionality up an EGF gradient that was unaffected by Endo180 mAbs (Fig. 3 D), further confirming that directional sensing of an EGF gradient is an Endo180-independent process.

Bottom Line: Endo180 expression was demonstrated to enhance uPA-mediated filopodia production and promote rapid activation of Cdc42 and Rac.Expression of a noninternalizing Endo180 mutant revealed that promotion of random cell migration requires receptor endocytosis, whereas the chemotactic response to uPA does not.From these studies, we conclude that Endo180 is a crucial link between uPA-uPAR and setting of the internal cellular compass.

View Article: PubMed Central - PubMed

Affiliation: The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London SW3 6JB, UK.

ABSTRACT
Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in cell guidance and chemotaxis during normal and pathological events. uPAR is GPI-anchored and the mechanism by which it transmits intracellular polarity cues across the plasma membrane during directional sensing has not been elucidated. The constitutively recycling endocytic receptor Endo180 forms a trimolecular complex with uPAR in the presence of uPA, hence its alternate name uPAR-associated protein. Here, we demonstrate that Endo180 is a general promoter of random cell migration and has a more specific function in cell chemotaxis up a uPA gradient. Endo180 expression was demonstrated to enhance uPA-mediated filopodia production and promote rapid activation of Cdc42 and Rac. Expression of a noninternalizing Endo180 mutant revealed that promotion of random cell migration requires receptor endocytosis, whereas the chemotactic response to uPA does not. From these studies, we conclude that Endo180 is a crucial link between uPA-uPAR and setting of the internal cellular compass.

Show MeSH
Related in: MedlinePlus