Limits...
Rho GTPase-independent regulation of mitotic progression by the RhoGEF Net1.

Menon S, Oh W, Carr HS, Frost JA - Mol. Biol. Cell (2013)

Bottom Line: Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation.The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1.These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, TX 77008, USA.

ABSTRACT
Neuroepithelial transforming gene 1 (Net1) is a RhoA-subfamily-specific guanine nucleotide exchange factor that is overexpressed in multiple human cancers and is required for proliferation. Molecular mechanisms underlying its role in cell proliferation are unknown. Here we show that overexpression or knockdown of Net1 causes mitotic defects. Net1 is required for chromosome congression during metaphase and generation of stable kinetochore microtubule attachments. Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation. The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1. We also observe that Net1 expression is required for centrosomal activation of p21-activated kinase and its downstream kinase Aurora A, which are critical regulators of centrosome maturation and spindle assembly. These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

Show MeSH

Related in: MedlinePlus

Overexpression of Net1 isoforms causes nuclear morphology defects. (A) HeLa cells were transfected with HA-Net1, HA-Net1A, or HA-Net1ΔN. Two days later the cells were fixed and stained for HA-epitope proteins (green), α-tubulin (red), and DNA (blue). Shown are representative micrographs. Bar, 10 μm. (B) Functional domains in Net1 proteins. DH, Dbl homology domain; PH, pleckstrin homology domain; orange ovals, nuclear localization signal sequences; purple ovals, C-terminal PDZ domain–binding site; blue bar, Net1-specific sequence (amino acids 1–85); green bar, Net1A-specific sequence (amino acids 1–31); numbers refer to amino acids for mouse Net1 proteins. (C) Quantification of defects in nuclear morphology. Average of three independent experiments. Errors are SEM. Statistical significance compared with control values was determined by Student's t test; *p < 0.05; **p < 0.01; ***p < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3756918&req=5

Figure 1: Overexpression of Net1 isoforms causes nuclear morphology defects. (A) HeLa cells were transfected with HA-Net1, HA-Net1A, or HA-Net1ΔN. Two days later the cells were fixed and stained for HA-epitope proteins (green), α-tubulin (red), and DNA (blue). Shown are representative micrographs. Bar, 10 μm. (B) Functional domains in Net1 proteins. DH, Dbl homology domain; PH, pleckstrin homology domain; orange ovals, nuclear localization signal sequences; purple ovals, C-terminal PDZ domain–binding site; blue bar, Net1-specific sequence (amino acids 1–85); green bar, Net1A-specific sequence (amino acids 1–31); numbers refer to amino acids for mouse Net1 proteins. (C) Quantification of defects in nuclear morphology. Average of three independent experiments. Errors are SEM. Statistical significance compared with control values was determined by Student's t test; *p < 0.05; **p < 0.01; ***p < 0.001.

Mentions: Net1 expression is important for cell proliferation (Dutertre et al., 2010; Leyden et al., 2006), but mechanisms accounting for this effect have not been identified. We reasoned that Net1 might contribute to mitotic progression, since other RhoGEFs have also been shown to regulate mitosis and cytokinesis (Narumiya and Yasuda, 2006). Hemagglutinin (HA)-epitope tagged wild-type Net1, Net1A, or Net1ΔN was expressed in asynchronously growing HeLa cells (Figure 1B). Net1ΔN is a cytoplasmic form of Net1 that constitutively activates RhoA and is transforming in NIH3T3 cells (Alberts and Treisman, 1998; Schmidt and Hall, 2002; Garcia-Mata et al., 2007). Two days later the cells were fixed and stained for Net1 expression, α-tubulin, and DNA. Transfected cells exhibiting abnormal nuclear morphologies were then scored as multinucleated (multiple nuclei of equal size), containing micronuclei (multiple unequally sized nuclei), or containing misshapen nuclei (multilobed, irregularly shaped nuclei). Of importance, the presence of micronuclei or abnormally shaped single nuclei is indicative of errors occurring during mitosis, whereas the presence of multiple, normally shaped nuclei is indicative of errors during cytokinesis (Tatsumoto et al., 1999; Holland and Cleveland, 2012). Representative examples of Net1 isoform–transfected cells and quantification of three independent experiments are shown in Figure 1, A and C. In these experiments, ∼6% of untransfected HeLa cells exhibited abnormal nuclei with morphologies that were evenly distributed among the three phenotypes. On the other hand, cells that overexpressed HA-Net1 exhibited nearly 10 times more abnormal nuclei, with 30% of the cells containing micronuclei,16% containing misshapen nuclei, and 10% multinucleated. In contrast, only 34% of the HA-Net1A–overexpressing cells exhibited abnormal nuclei, with these cells being evenly distributed between micronuclei and multinuclei phenotypes. Cells expressing the N-terminal deletion mutant Net1ΔN also exhibited a high percentage of abnormal nuclei (57%). The majority of these cells, however, showed a multinuclear phenotype (42%), with only a small percentage of cells containing micronuclei (7%) or misshapen single nuclei (8%). Thus these data suggest that Net1 or Net1A overexpression mainly causes errors during mitosis, whereas Net1ΔN expression causes errors during cytokinesis. The effect of Net1ΔN is consistent with the phenotype caused by constitutively active RhoA expression, which also inhibits cytokinesis in HeLa cells (Morin et al., 2009). Intriguingly, the effects of Net1 and Net1A overexpression were qualitatively different, as Net1 overexpression caused the accumulation of cells with micronuclei far more frequently than Net1A.


Rho GTPase-independent regulation of mitotic progression by the RhoGEF Net1.

Menon S, Oh W, Carr HS, Frost JA - Mol. Biol. Cell (2013)

Overexpression of Net1 isoforms causes nuclear morphology defects. (A) HeLa cells were transfected with HA-Net1, HA-Net1A, or HA-Net1ΔN. Two days later the cells were fixed and stained for HA-epitope proteins (green), α-tubulin (red), and DNA (blue). Shown are representative micrographs. Bar, 10 μm. (B) Functional domains in Net1 proteins. DH, Dbl homology domain; PH, pleckstrin homology domain; orange ovals, nuclear localization signal sequences; purple ovals, C-terminal PDZ domain–binding site; blue bar, Net1-specific sequence (amino acids 1–85); green bar, Net1A-specific sequence (amino acids 1–31); numbers refer to amino acids for mouse Net1 proteins. (C) Quantification of defects in nuclear morphology. Average of three independent experiments. Errors are SEM. Statistical significance compared with control values was determined by Student's t test; *p < 0.05; **p < 0.01; ***p < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756918&req=5

Figure 1: Overexpression of Net1 isoforms causes nuclear morphology defects. (A) HeLa cells were transfected with HA-Net1, HA-Net1A, or HA-Net1ΔN. Two days later the cells were fixed and stained for HA-epitope proteins (green), α-tubulin (red), and DNA (blue). Shown are representative micrographs. Bar, 10 μm. (B) Functional domains in Net1 proteins. DH, Dbl homology domain; PH, pleckstrin homology domain; orange ovals, nuclear localization signal sequences; purple ovals, C-terminal PDZ domain–binding site; blue bar, Net1-specific sequence (amino acids 1–85); green bar, Net1A-specific sequence (amino acids 1–31); numbers refer to amino acids for mouse Net1 proteins. (C) Quantification of defects in nuclear morphology. Average of three independent experiments. Errors are SEM. Statistical significance compared with control values was determined by Student's t test; *p < 0.05; **p < 0.01; ***p < 0.001.
Mentions: Net1 expression is important for cell proliferation (Dutertre et al., 2010; Leyden et al., 2006), but mechanisms accounting for this effect have not been identified. We reasoned that Net1 might contribute to mitotic progression, since other RhoGEFs have also been shown to regulate mitosis and cytokinesis (Narumiya and Yasuda, 2006). Hemagglutinin (HA)-epitope tagged wild-type Net1, Net1A, or Net1ΔN was expressed in asynchronously growing HeLa cells (Figure 1B). Net1ΔN is a cytoplasmic form of Net1 that constitutively activates RhoA and is transforming in NIH3T3 cells (Alberts and Treisman, 1998; Schmidt and Hall, 2002; Garcia-Mata et al., 2007). Two days later the cells were fixed and stained for Net1 expression, α-tubulin, and DNA. Transfected cells exhibiting abnormal nuclear morphologies were then scored as multinucleated (multiple nuclei of equal size), containing micronuclei (multiple unequally sized nuclei), or containing misshapen nuclei (multilobed, irregularly shaped nuclei). Of importance, the presence of micronuclei or abnormally shaped single nuclei is indicative of errors occurring during mitosis, whereas the presence of multiple, normally shaped nuclei is indicative of errors during cytokinesis (Tatsumoto et al., 1999; Holland and Cleveland, 2012). Representative examples of Net1 isoform–transfected cells and quantification of three independent experiments are shown in Figure 1, A and C. In these experiments, ∼6% of untransfected HeLa cells exhibited abnormal nuclei with morphologies that were evenly distributed among the three phenotypes. On the other hand, cells that overexpressed HA-Net1 exhibited nearly 10 times more abnormal nuclei, with 30% of the cells containing micronuclei,16% containing misshapen nuclei, and 10% multinucleated. In contrast, only 34% of the HA-Net1A–overexpressing cells exhibited abnormal nuclei, with these cells being evenly distributed between micronuclei and multinuclei phenotypes. Cells expressing the N-terminal deletion mutant Net1ΔN also exhibited a high percentage of abnormal nuclei (57%). The majority of these cells, however, showed a multinuclear phenotype (42%), with only a small percentage of cells containing micronuclei (7%) or misshapen single nuclei (8%). Thus these data suggest that Net1 or Net1A overexpression mainly causes errors during mitosis, whereas Net1ΔN expression causes errors during cytokinesis. The effect of Net1ΔN is consistent with the phenotype caused by constitutively active RhoA expression, which also inhibits cytokinesis in HeLa cells (Morin et al., 2009). Intriguingly, the effects of Net1 and Net1A overexpression were qualitatively different, as Net1 overexpression caused the accumulation of cells with micronuclei far more frequently than Net1A.

Bottom Line: Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation.The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1.These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, TX 77008, USA.

ABSTRACT
Neuroepithelial transforming gene 1 (Net1) is a RhoA-subfamily-specific guanine nucleotide exchange factor that is overexpressed in multiple human cancers and is required for proliferation. Molecular mechanisms underlying its role in cell proliferation are unknown. Here we show that overexpression or knockdown of Net1 causes mitotic defects. Net1 is required for chromosome congression during metaphase and generation of stable kinetochore microtubule attachments. Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation. The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1. We also observe that Net1 expression is required for centrosomal activation of p21-activated kinase and its downstream kinase Aurora A, which are critical regulators of centrosome maturation and spindle assembly. These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

Show MeSH
Related in: MedlinePlus