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Murine missing in metastasis (MIM) mediates cell polarity and regulates the motility response to growth factors.

Yu D, Zhan XH, Niu S, Mikhailenko I, Strickland DK, Zhu J, Cao M, Zhan X - PLoS ONE (2011)

Bottom Line: MIM(-/-) cells were also spread less effectively than wild type cells during attachment to dishes and substratum.On the other hand, the level of tyrosine phosphorylation of PDGF receptors was more elevated in MIM depleted cells than wild type cells upon PDGF treatment.Our data suggests that endogenous MIM protein regulates globally the cell architecture and endocytosis that ultimately influence a variety of cellular behaviors, including cell polarity, motility, receptor signaling and membrane ruffling.

View Article: PubMed Central - PubMed

Affiliation: The Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, United of States of America.

ABSTRACT

Background: Missing in metastasis (MIM) is a member of the inverse BAR-domain protein family, and in vitro studies have implied MIM plays a role in deforming membrane curvature into filopodia-like protrusions and cell dynamics. Yet, the physiological role of the endogenous MIM in mammalian cells remains undefined.

Principal findings: We have examined mouse embryonic fibroblasts (MEFs) derived from mice in which the MIM locus was targeted by a gene trapping vector. MIM(-/-) MEFs showed a less polarized architecture characterized by smooth edges and fewer cell protrusions as compared to wild type cells, although the formation of filopodia-like microprotrusions appeared to be normal. Immunofluorescent staining further revealed that MIM(-/-) cells were partially impaired in the assembly of stress fibers and focal adhesions but were enriched with transverse actin filaments at the periphery. Poor assembly of stress fibers was apparently correlated with attenuation of the activity of Rho GTPases and partially relieved upon overexpressing of Myc-RhoA(Q63L), a constitutively activated RhoA mutant. MIM(-/-) cells were also spread less effectively than wild type cells during attachment to dishes and substratum. Upon treatment with PDGF MIM(-/-) cells developed more prominent dorsal ruffles along with increased Rac1 activity. Compared to wild type cells, MIM(-/-) cells had a slower motility in the presence of a low percentage of serum-containing medium but migrated normally upon adding growth factors such as 10% serum, PDGF or EGF. MIM(-/-) cells were also partially impaired in the internalization of transferrin, fluorescent dyes, foreign DNAs and PDGF receptor alpha. On the other hand, the level of tyrosine phosphorylation of PDGF receptors was more elevated in MIM depleted cells than wild type cells upon PDGF treatment.

Conclusions: Our data suggests that endogenous MIM protein regulates globally the cell architecture and endocytosis that ultimately influence a variety of cellular behaviors, including cell polarity, motility, receptor signaling and membrane ruffling.

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Related in: MedlinePlus

PDGF induces prominent dorsal ruffles in MIM−/− MEFs.(A) MIM+/+ (a and b) and MIM−/− (c and d) cells were arrested by incubating for 24 h in 0.2% serum-containing medium and then treated with PDGF for 10 min (b and d) followed by staining with phalloidin. The stained cells were inspected by epifluorescent microscopy. Scale bar: 50 µm. (B) Quantification of dorsal ruffles. Cells were treated with PDGF for the times as indicated. The number of cells showing large ruffling areas was counted. The data shown are the mean ± SEM based on four independent experiments. In each experiment 70 cells were analyzed. The p value was calculated by Anova test, referring to the difference between MIM−/− and MIM+/+ MEFs during the response to PDGF. (C) PDGF treated cells were analyzed for Rac1 activation by pull-down assay followed by Western blot using anti-Rac1 antibody. The Rac1 activation was quantified based on three independent experiments. (D) Quiescent MIM−/− cells grown on fibronectin-coated coverslips were treated with and without Rac1 inhibitor NSC23766 at the concentration of 50 µM for 48 h. The treated cells were then stimulated with PDGF for 10 min, and the Rac1 activation was measured as above. (E) NSC23766 treated cells were also treated with PDGF, and the formation of dorsal ruffles (DR) was quantified based on three independent experiments.
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pone-0020845-g004: PDGF induces prominent dorsal ruffles in MIM−/− MEFs.(A) MIM+/+ (a and b) and MIM−/− (c and d) cells were arrested by incubating for 24 h in 0.2% serum-containing medium and then treated with PDGF for 10 min (b and d) followed by staining with phalloidin. The stained cells were inspected by epifluorescent microscopy. Scale bar: 50 µm. (B) Quantification of dorsal ruffles. Cells were treated with PDGF for the times as indicated. The number of cells showing large ruffling areas was counted. The data shown are the mean ± SEM based on four independent experiments. In each experiment 70 cells were analyzed. The p value was calculated by Anova test, referring to the difference between MIM−/− and MIM+/+ MEFs during the response to PDGF. (C) PDGF treated cells were analyzed for Rac1 activation by pull-down assay followed by Western blot using anti-Rac1 antibody. The Rac1 activation was quantified based on three independent experiments. (D) Quiescent MIM−/− cells grown on fibronectin-coated coverslips were treated with and without Rac1 inhibitor NSC23766 at the concentration of 50 µM for 48 h. The treated cells were then stimulated with PDGF for 10 min, and the Rac1 activation was measured as above. (E) NSC23766 treated cells were also treated with PDGF, and the formation of dorsal ruffles (DR) was quantified based on three independent experiments.

Mentions: The role of MIM in the actin cytoskeleton and morphogenesis was further analyzed when cells were stimulated by PDGF, a growth factor that often induces dorsal ruffles on fibroblasts [24]. Both MIM+/+ and MIM−/− MEFs showed the formation of dorsal ruffles upon PDGF treatment (Fig. 4A). However, those on MIM−/− cells were more prominent than MIM+/+ cells (Fig. 4A, d v b), and the number of MIM−/− cells developing extensive dorsal ruffles was significantly higher than that of MIM+/+ cells during the course of PDGF stimulation (p<0.004) (Fig. 4B). At 5 min more than two-fold difference between MIM−/− and MIM+/+ cells was observed. We also detected higher contents of GTP-Rac1 in MIM−/− cells than those of MIM+/+ cells after 10 and 30 min of PDGF treatment (Fig. 4C) or during early responses to PDGF (Fig. S9). To verify the necessity of Rac activation in membrane ruffling, MIM−/− cells were treated with NSC23766, a Rac selective inhibitor [25]. The drug treatment reduced the PDGF-mediated Rac1 activation in MIM−/− cells by nearly 50% (Fig. 4D), which was apparently correlated with 50% deduction in dorsal ruffling (Fig. 4E), implying a role of Rac activation in the dorsal ruffling mediated by MIM deficiency.


Murine missing in metastasis (MIM) mediates cell polarity and regulates the motility response to growth factors.

Yu D, Zhan XH, Niu S, Mikhailenko I, Strickland DK, Zhu J, Cao M, Zhan X - PLoS ONE (2011)

PDGF induces prominent dorsal ruffles in MIM−/− MEFs.(A) MIM+/+ (a and b) and MIM−/− (c and d) cells were arrested by incubating for 24 h in 0.2% serum-containing medium and then treated with PDGF for 10 min (b and d) followed by staining with phalloidin. The stained cells were inspected by epifluorescent microscopy. Scale bar: 50 µm. (B) Quantification of dorsal ruffles. Cells were treated with PDGF for the times as indicated. The number of cells showing large ruffling areas was counted. The data shown are the mean ± SEM based on four independent experiments. In each experiment 70 cells were analyzed. The p value was calculated by Anova test, referring to the difference between MIM−/− and MIM+/+ MEFs during the response to PDGF. (C) PDGF treated cells were analyzed for Rac1 activation by pull-down assay followed by Western blot using anti-Rac1 antibody. The Rac1 activation was quantified based on three independent experiments. (D) Quiescent MIM−/− cells grown on fibronectin-coated coverslips were treated with and without Rac1 inhibitor NSC23766 at the concentration of 50 µM for 48 h. The treated cells were then stimulated with PDGF for 10 min, and the Rac1 activation was measured as above. (E) NSC23766 treated cells were also treated with PDGF, and the formation of dorsal ruffles (DR) was quantified based on three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111439&req=5

pone-0020845-g004: PDGF induces prominent dorsal ruffles in MIM−/− MEFs.(A) MIM+/+ (a and b) and MIM−/− (c and d) cells were arrested by incubating for 24 h in 0.2% serum-containing medium and then treated with PDGF for 10 min (b and d) followed by staining with phalloidin. The stained cells were inspected by epifluorescent microscopy. Scale bar: 50 µm. (B) Quantification of dorsal ruffles. Cells were treated with PDGF for the times as indicated. The number of cells showing large ruffling areas was counted. The data shown are the mean ± SEM based on four independent experiments. In each experiment 70 cells were analyzed. The p value was calculated by Anova test, referring to the difference between MIM−/− and MIM+/+ MEFs during the response to PDGF. (C) PDGF treated cells were analyzed for Rac1 activation by pull-down assay followed by Western blot using anti-Rac1 antibody. The Rac1 activation was quantified based on three independent experiments. (D) Quiescent MIM−/− cells grown on fibronectin-coated coverslips were treated with and without Rac1 inhibitor NSC23766 at the concentration of 50 µM for 48 h. The treated cells were then stimulated with PDGF for 10 min, and the Rac1 activation was measured as above. (E) NSC23766 treated cells were also treated with PDGF, and the formation of dorsal ruffles (DR) was quantified based on three independent experiments.
Mentions: The role of MIM in the actin cytoskeleton and morphogenesis was further analyzed when cells were stimulated by PDGF, a growth factor that often induces dorsal ruffles on fibroblasts [24]. Both MIM+/+ and MIM−/− MEFs showed the formation of dorsal ruffles upon PDGF treatment (Fig. 4A). However, those on MIM−/− cells were more prominent than MIM+/+ cells (Fig. 4A, d v b), and the number of MIM−/− cells developing extensive dorsal ruffles was significantly higher than that of MIM+/+ cells during the course of PDGF stimulation (p<0.004) (Fig. 4B). At 5 min more than two-fold difference between MIM−/− and MIM+/+ cells was observed. We also detected higher contents of GTP-Rac1 in MIM−/− cells than those of MIM+/+ cells after 10 and 30 min of PDGF treatment (Fig. 4C) or during early responses to PDGF (Fig. S9). To verify the necessity of Rac activation in membrane ruffling, MIM−/− cells were treated with NSC23766, a Rac selective inhibitor [25]. The drug treatment reduced the PDGF-mediated Rac1 activation in MIM−/− cells by nearly 50% (Fig. 4D), which was apparently correlated with 50% deduction in dorsal ruffling (Fig. 4E), implying a role of Rac activation in the dorsal ruffling mediated by MIM deficiency.

Bottom Line: MIM(-/-) cells were also spread less effectively than wild type cells during attachment to dishes and substratum.On the other hand, the level of tyrosine phosphorylation of PDGF receptors was more elevated in MIM depleted cells than wild type cells upon PDGF treatment.Our data suggests that endogenous MIM protein regulates globally the cell architecture and endocytosis that ultimately influence a variety of cellular behaviors, including cell polarity, motility, receptor signaling and membrane ruffling.

View Article: PubMed Central - PubMed

Affiliation: The Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, United of States of America.

ABSTRACT

Background: Missing in metastasis (MIM) is a member of the inverse BAR-domain protein family, and in vitro studies have implied MIM plays a role in deforming membrane curvature into filopodia-like protrusions and cell dynamics. Yet, the physiological role of the endogenous MIM in mammalian cells remains undefined.

Principal findings: We have examined mouse embryonic fibroblasts (MEFs) derived from mice in which the MIM locus was targeted by a gene trapping vector. MIM(-/-) MEFs showed a less polarized architecture characterized by smooth edges and fewer cell protrusions as compared to wild type cells, although the formation of filopodia-like microprotrusions appeared to be normal. Immunofluorescent staining further revealed that MIM(-/-) cells were partially impaired in the assembly of stress fibers and focal adhesions but were enriched with transverse actin filaments at the periphery. Poor assembly of stress fibers was apparently correlated with attenuation of the activity of Rho GTPases and partially relieved upon overexpressing of Myc-RhoA(Q63L), a constitutively activated RhoA mutant. MIM(-/-) cells were also spread less effectively than wild type cells during attachment to dishes and substratum. Upon treatment with PDGF MIM(-/-) cells developed more prominent dorsal ruffles along with increased Rac1 activity. Compared to wild type cells, MIM(-/-) cells had a slower motility in the presence of a low percentage of serum-containing medium but migrated normally upon adding growth factors such as 10% serum, PDGF or EGF. MIM(-/-) cells were also partially impaired in the internalization of transferrin, fluorescent dyes, foreign DNAs and PDGF receptor alpha. On the other hand, the level of tyrosine phosphorylation of PDGF receptors was more elevated in MIM depleted cells than wild type cells upon PDGF treatment.

Conclusions: Our data suggests that endogenous MIM protein regulates globally the cell architecture and endocytosis that ultimately influence a variety of cellular behaviors, including cell polarity, motility, receptor signaling and membrane ruffling.

Show MeSH
Related in: MedlinePlus