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The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint.

Osmundson EC, Ray D, Moore FE, Gao Q, Thomsen GH, Kiyokawa H - J. Cell Biol. (2008)

Bottom Line: Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis.Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector.These data indicate that Smurf2 is a novel mitotic regulator.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

ABSTRACT
Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 is critical for the metaphase-anaphase transition. APC/C-Cdc20 is required for polyubiquitination and degradation of securin and cyclin B at anaphase onset. The spindle assembly checkpoint delays APC/C-Cdc20 activation until all kinetochores attach to mitotic spindles. In this study, we demonstrate that a HECT (homologous to the E6-AP carboxyl terminus) ubiquitin ligase, Smurf2, is required for the spindle checkpoint. Smurf2 localizes to the centrosome, mitotic midbody, and centromeres. Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis. Smurf2 inactivation prevents nocodazole-treated cells from accumulating cyclin B and securin and prometaphase arrest. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of cyclin B and securin. Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector. Mad2 is mislocalized in Smurf2-depleted cells, suggesting that Smurf2 regulates the localization and stability of Mad2. These data indicate that Smurf2 is a novel mitotic regulator.

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Smurf2 silencing leads to chromosomal misalignment at metaphase and premature onset of anaphase with defective chromosome segregation. HeLa cells stably expressing GFP-H2B were transfected with Smurf2 siRNA #1, and chromosomal movement was monitored 24–25 h after transfection by time-lapse fluorescence microscopy. Quantified data are representative of at least two independent experiments. (a) Defects in chromosomal alignment in Smurf2-depleted cells at metaphase. Metaphase defects in cells transfected with Smurf2 siRNA (shaded bars) or nonspecific dsRNA (siNS, open bars) were categorized into the two indicated groups. (b) Unaligned chromosomes (*) and lack of metaphase plates (#) during mitosis with Smurf2 depletion. Arrowheads denote lagging chromosomes. (c) Chromosomal segregation defects in GFP-H2B HeLa cells transfected with Smurf2 siRNA (shaded bars) or nonspecific dsRNA (open bars) at anaphase. (a and c) Data are means ± SEM (error bars) from three independent experiments. (d) Lagging chromosomes (arrowheads, top row), major segregation defects (arrowheads, middle row), and lack of segregation in GFP-H2B HeLa cells with Smurf2 depletion. (e) Premature anaphase onset in Smurf2-depleted cells. The metaphase–anaphase transition of GFP-H2B HeLa cells was monitored by time-lapse microscopy, and the time from nuclear envelope breakdown (NEBD) until anaphase onset was determined. (f) Representative pictures of the metaphase–anaphase transition. AO, anaphase onset. Bars, 10 μm.
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fig3: Smurf2 silencing leads to chromosomal misalignment at metaphase and premature onset of anaphase with defective chromosome segregation. HeLa cells stably expressing GFP-H2B were transfected with Smurf2 siRNA #1, and chromosomal movement was monitored 24–25 h after transfection by time-lapse fluorescence microscopy. Quantified data are representative of at least two independent experiments. (a) Defects in chromosomal alignment in Smurf2-depleted cells at metaphase. Metaphase defects in cells transfected with Smurf2 siRNA (shaded bars) or nonspecific dsRNA (siNS, open bars) were categorized into the two indicated groups. (b) Unaligned chromosomes (*) and lack of metaphase plates (#) during mitosis with Smurf2 depletion. Arrowheads denote lagging chromosomes. (c) Chromosomal segregation defects in GFP-H2B HeLa cells transfected with Smurf2 siRNA (shaded bars) or nonspecific dsRNA (open bars) at anaphase. (a and c) Data are means ± SEM (error bars) from three independent experiments. (d) Lagging chromosomes (arrowheads, top row), major segregation defects (arrowheads, middle row), and lack of segregation in GFP-H2B HeLa cells with Smurf2 depletion. (e) Premature anaphase onset in Smurf2-depleted cells. The metaphase–anaphase transition of GFP-H2B HeLa cells was monitored by time-lapse microscopy, and the time from nuclear envelope breakdown (NEBD) until anaphase onset was determined. (f) Representative pictures of the metaphase–anaphase transition. AO, anaphase onset. Bars, 10 μm.

Mentions: To explore the mechanism of mitotic failure in Smurf2-depleted cells, we first examined the parameters of chromosomal alignment and segregation because cells defective in the regulation of these events, e.g., depletion of Mad2 or USP44, demonstrate similar mitotic phenotypes (Michel et al., 2004; Stegmeier et al., 2007). We first examined the morphology of mitotic control and Smurf2-depleted HeLa cells during mitosis by staining for the microtubule marker α-tubulin, Smurf2, and chromosomal DNA (Fig. S2 b, available at http://www.jcb.org/cgi/content/full/jcb.200801049/DC1). Of a total of >600 cells examined per group, 7.3% and 8.0% in control and Smurf2-depleted cells, respectively, displayed characteristics of mitosis (i.e., chromatin condensation and formation of mitotic spindles). Although 4.7% of control cells were found to be in metaphase, only 1.9% of Smurf2-depleted cells exhibited typical metaphase morphology with aligned chromosomes. The decrease in the metaphase population suggested that Smurf2 depletion might affect mitotic progression. To further assess the impact of Smurf2 depletion on chromosomal dynamics during mitotic progression, we depleted Smurf2 in HeLa cells stably expressing a GFP–histone H2B fusion protein (GFP-H2B). Cells were then monitored for chromosome movement by time-lapse microscopy, which readily showed that Smurf2-depleted cells initiated anaphase in the presence of misaligned chromosomes. Over 75% of Smurf2-depleted cells exhibited misaligned chromosomes or failed to form a metaphase plate during monitored metaphase–anaphase transition (Fig. 3, a and b; and Video 2). During anaphase, most Smurf2-depleted cells showed defective segregation such as lagging chromosomes or no appreciable chromosome segregation (Fig. 3, c and d). In contrast, <10% of control cells exhibited such defects during mitosis. We also measured the interval between nuclear envelope breakdown and the onset of anaphase, focusing on cells that displayed discernable elements of chromatin segregation. In control cultures, the median interval was ∼55 min, whereas that in Smurf2-depleted cultures was ∼23 min (Fig. 3, e and f; and Video 3). These data indicate that Smurf2 depletion leads to premature anaphase onset together with chromosomal alignment and segregation defects. Thus, Smurf2 seems to play a significant role in the temporal control of the metaphase–anaphase transition, a process normally regulated by the spindle assembly checkpoint.


The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint.

Osmundson EC, Ray D, Moore FE, Gao Q, Thomsen GH, Kiyokawa H - J. Cell Biol. (2008)

Smurf2 silencing leads to chromosomal misalignment at metaphase and premature onset of anaphase with defective chromosome segregation. HeLa cells stably expressing GFP-H2B were transfected with Smurf2 siRNA #1, and chromosomal movement was monitored 24–25 h after transfection by time-lapse fluorescence microscopy. Quantified data are representative of at least two independent experiments. (a) Defects in chromosomal alignment in Smurf2-depleted cells at metaphase. Metaphase defects in cells transfected with Smurf2 siRNA (shaded bars) or nonspecific dsRNA (siNS, open bars) were categorized into the two indicated groups. (b) Unaligned chromosomes (*) and lack of metaphase plates (#) during mitosis with Smurf2 depletion. Arrowheads denote lagging chromosomes. (c) Chromosomal segregation defects in GFP-H2B HeLa cells transfected with Smurf2 siRNA (shaded bars) or nonspecific dsRNA (open bars) at anaphase. (a and c) Data are means ± SEM (error bars) from three independent experiments. (d) Lagging chromosomes (arrowheads, top row), major segregation defects (arrowheads, middle row), and lack of segregation in GFP-H2B HeLa cells with Smurf2 depletion. (e) Premature anaphase onset in Smurf2-depleted cells. The metaphase–anaphase transition of GFP-H2B HeLa cells was monitored by time-lapse microscopy, and the time from nuclear envelope breakdown (NEBD) until anaphase onset was determined. (f) Representative pictures of the metaphase–anaphase transition. AO, anaphase onset. Bars, 10 μm.
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fig3: Smurf2 silencing leads to chromosomal misalignment at metaphase and premature onset of anaphase with defective chromosome segregation. HeLa cells stably expressing GFP-H2B were transfected with Smurf2 siRNA #1, and chromosomal movement was monitored 24–25 h after transfection by time-lapse fluorescence microscopy. Quantified data are representative of at least two independent experiments. (a) Defects in chromosomal alignment in Smurf2-depleted cells at metaphase. Metaphase defects in cells transfected with Smurf2 siRNA (shaded bars) or nonspecific dsRNA (siNS, open bars) were categorized into the two indicated groups. (b) Unaligned chromosomes (*) and lack of metaphase plates (#) during mitosis with Smurf2 depletion. Arrowheads denote lagging chromosomes. (c) Chromosomal segregation defects in GFP-H2B HeLa cells transfected with Smurf2 siRNA (shaded bars) or nonspecific dsRNA (open bars) at anaphase. (a and c) Data are means ± SEM (error bars) from three independent experiments. (d) Lagging chromosomes (arrowheads, top row), major segregation defects (arrowheads, middle row), and lack of segregation in GFP-H2B HeLa cells with Smurf2 depletion. (e) Premature anaphase onset in Smurf2-depleted cells. The metaphase–anaphase transition of GFP-H2B HeLa cells was monitored by time-lapse microscopy, and the time from nuclear envelope breakdown (NEBD) until anaphase onset was determined. (f) Representative pictures of the metaphase–anaphase transition. AO, anaphase onset. Bars, 10 μm.
Mentions: To explore the mechanism of mitotic failure in Smurf2-depleted cells, we first examined the parameters of chromosomal alignment and segregation because cells defective in the regulation of these events, e.g., depletion of Mad2 or USP44, demonstrate similar mitotic phenotypes (Michel et al., 2004; Stegmeier et al., 2007). We first examined the morphology of mitotic control and Smurf2-depleted HeLa cells during mitosis by staining for the microtubule marker α-tubulin, Smurf2, and chromosomal DNA (Fig. S2 b, available at http://www.jcb.org/cgi/content/full/jcb.200801049/DC1). Of a total of >600 cells examined per group, 7.3% and 8.0% in control and Smurf2-depleted cells, respectively, displayed characteristics of mitosis (i.e., chromatin condensation and formation of mitotic spindles). Although 4.7% of control cells were found to be in metaphase, only 1.9% of Smurf2-depleted cells exhibited typical metaphase morphology with aligned chromosomes. The decrease in the metaphase population suggested that Smurf2 depletion might affect mitotic progression. To further assess the impact of Smurf2 depletion on chromosomal dynamics during mitotic progression, we depleted Smurf2 in HeLa cells stably expressing a GFP–histone H2B fusion protein (GFP-H2B). Cells were then monitored for chromosome movement by time-lapse microscopy, which readily showed that Smurf2-depleted cells initiated anaphase in the presence of misaligned chromosomes. Over 75% of Smurf2-depleted cells exhibited misaligned chromosomes or failed to form a metaphase plate during monitored metaphase–anaphase transition (Fig. 3, a and b; and Video 2). During anaphase, most Smurf2-depleted cells showed defective segregation such as lagging chromosomes or no appreciable chromosome segregation (Fig. 3, c and d). In contrast, <10% of control cells exhibited such defects during mitosis. We also measured the interval between nuclear envelope breakdown and the onset of anaphase, focusing on cells that displayed discernable elements of chromatin segregation. In control cultures, the median interval was ∼55 min, whereas that in Smurf2-depleted cultures was ∼23 min (Fig. 3, e and f; and Video 3). These data indicate that Smurf2 depletion leads to premature anaphase onset together with chromosomal alignment and segregation defects. Thus, Smurf2 seems to play a significant role in the temporal control of the metaphase–anaphase transition, a process normally regulated by the spindle assembly checkpoint.

Bottom Line: Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis.Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector.These data indicate that Smurf2 is a novel mitotic regulator.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

ABSTRACT
Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 is critical for the metaphase-anaphase transition. APC/C-Cdc20 is required for polyubiquitination and degradation of securin and cyclin B at anaphase onset. The spindle assembly checkpoint delays APC/C-Cdc20 activation until all kinetochores attach to mitotic spindles. In this study, we demonstrate that a HECT (homologous to the E6-AP carboxyl terminus) ubiquitin ligase, Smurf2, is required for the spindle checkpoint. Smurf2 localizes to the centrosome, mitotic midbody, and centromeres. Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis. Smurf2 inactivation prevents nocodazole-treated cells from accumulating cyclin B and securin and prometaphase arrest. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of cyclin B and securin. Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector. Mad2 is mislocalized in Smurf2-depleted cells, suggesting that Smurf2 regulates the localization and stability of Mad2. These data indicate that Smurf2 is a novel mitotic regulator.

Show MeSH
Related in: MedlinePlus