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A unique insertion in STARD9's motor domain regulates its stability.

Senese S, Cheung K, Lo YC, Gholkar AA, Xia X, Wohlschlegel JA, Torres JZ - Mol. Biol. Cell (2014)

Bottom Line: These phosphorylation events are important for targeting a pool of STARD9-MD for ubiquitination by the SCFβ-TrCP ubiquitin ligase and proteasome-dependent degradation.Of interest, overexpression of nonphosphorylatable/nondegradable STARD9-MD mutants leads to spindle assembly defects.Our results with STARD9-MD imply that in vivo the protein levels of full-length STARD9 could be regulated by Plk1 and SCFβ-TrCP to promote proper mitotic spindle assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095.

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Increased levels of STARD9-MD lead to spindle assembly and cell division defects. (A) STARD9-MD wild-type and serine-to-alanine mutant stable cell lines were induced to express these proteins. Twenty hours postinduction, cells were fixed and stained for DNA, α-tubulin, and STARD9-MD and imaged by fluorescence microscopy. Bar, 5 μm. (B) Quantification of the fluorescence intensity signal from STARD9-MD wild-type and serine-to-alanine mutant proteins within a 3-μm radius surrounding the centrosome/spindle pole. Data represent the average ± SD of three independent experiments, 20 cells counted for each. *p < 0.05, **p < 0.0005. (C) The percentage of cells with spindle defects was quantified. Data represent the average ± SD of three independent experiments, 100 cells counted for each. *p < 0.05, **p < 0.005, ***p < 0.0005. (D) Live-cell time-lapse microscopy of STARD9-MD wild type and S317A mutant. Top, bright-field (BF) snapshots, and bottom, fluorescence (FL), of STARD-MD localization at the indicated time points in minutes. See Supplemental Movies S1–S4.
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Figure 7: Increased levels of STARD9-MD lead to spindle assembly and cell division defects. (A) STARD9-MD wild-type and serine-to-alanine mutant stable cell lines were induced to express these proteins. Twenty hours postinduction, cells were fixed and stained for DNA, α-tubulin, and STARD9-MD and imaged by fluorescence microscopy. Bar, 5 μm. (B) Quantification of the fluorescence intensity signal from STARD9-MD wild-type and serine-to-alanine mutant proteins within a 3-μm radius surrounding the centrosome/spindle pole. Data represent the average ± SD of three independent experiments, 20 cells counted for each. *p < 0.05, **p < 0.0005. (C) The percentage of cells with spindle defects was quantified. Data represent the average ± SD of three independent experiments, 100 cells counted for each. *p < 0.05, **p < 0.005, ***p < 0.0005. (D) Live-cell time-lapse microscopy of STARD9-MD wild type and S317A mutant. Top, bright-field (BF) snapshots, and bottom, fluorescence (FL), of STARD-MD localization at the indicated time points in minutes. See Supplemental Movies S1–S4.

Mentions: To analyze the consequences of accumulating STARD9-MD in mitosis, we used LAP-tagged STARD9-MD wild-type and serine-to-alanine mutant stable cell lines (expressing STARD9-MD from the same single chromosomal loci) to overexpress wild type and nonphosphorylatable/nondegradable STARD9-MD mutants and analyzed their mitotic defects by immunofluorescence microscopy. Consistent with our previous results, overexpressed STARD9-MD localized to the spindle poles and, to a lesser extent, the spindle microtubules (Torres et al., 2011; Figure 7A). However, the nonphosphorylatable/nondegradable S305A, S312A, and S317A mutants accumulated to very high levels at the spindle poles and spindle, consistent with our immunoblot analyses showing their stabilization in mitosis (Figure 7A). As a control, STARD9-MD with the S316A mutation that did not block STARD9-MD degradation remained at similar low levels as wild-type STARD9-MD (Figure 7A). Similarly, measurements of the total fluorescence intensity of LAP-STARD9-MD at the centrosome indicated that STARD9-MD protein levels were markedly increased in the S305A, S312A, and S317A mutants, whereas wild-type STARD9-MD and the degradation-competent S316A mutant remained low (Figure 7B). Also consistent with previous results, overexpression of STARD9-MD led to a modest but reproducible increase in the percentage of cells with spindle defects (PCM fragmentation and multipolar spindles): ∼18% compared with ∼8% control (Torres et al., 2011; Figure 7, A and C). A similar effect was also seen with the degradation-competent S316A mutant (Figure 7, A and C). Of interest, overexpression of the nonphosphorylatable/nondegradable S305A, S312A, and S317A mutants led to significant increase in the percentage of cells with spindle assembly defects, ∼25–30% (Figure 7, A and C). Consistently, live-cell time-lapse microscopy of nonphosphorylatable/nondegradable mutants like S317A indicated that overexpressing these mutants led to an arrest in mitosis, followed by apoptotic cell death (Figure 7D and Supplemental Movies S1–S4). These data indicated that posttranslational modification of STARD9-MD by Plk1 (phosphorylation) and SCFβ-TrCP (ubiquitination) was critical for controlling the levels of STARD9-MD at the centrosome during mitosis and that accumulation of STARD9-MD led to spindle assembly and cell division defects.


A unique insertion in STARD9's motor domain regulates its stability.

Senese S, Cheung K, Lo YC, Gholkar AA, Xia X, Wohlschlegel JA, Torres JZ - Mol. Biol. Cell (2014)

Increased levels of STARD9-MD lead to spindle assembly and cell division defects. (A) STARD9-MD wild-type and serine-to-alanine mutant stable cell lines were induced to express these proteins. Twenty hours postinduction, cells were fixed and stained for DNA, α-tubulin, and STARD9-MD and imaged by fluorescence microscopy. Bar, 5 μm. (B) Quantification of the fluorescence intensity signal from STARD9-MD wild-type and serine-to-alanine mutant proteins within a 3-μm radius surrounding the centrosome/spindle pole. Data represent the average ± SD of three independent experiments, 20 cells counted for each. *p < 0.05, **p < 0.0005. (C) The percentage of cells with spindle defects was quantified. Data represent the average ± SD of three independent experiments, 100 cells counted for each. *p < 0.05, **p < 0.005, ***p < 0.0005. (D) Live-cell time-lapse microscopy of STARD9-MD wild type and S317A mutant. Top, bright-field (BF) snapshots, and bottom, fluorescence (FL), of STARD-MD localization at the indicated time points in minutes. See Supplemental Movies S1–S4.
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Figure 7: Increased levels of STARD9-MD lead to spindle assembly and cell division defects. (A) STARD9-MD wild-type and serine-to-alanine mutant stable cell lines were induced to express these proteins. Twenty hours postinduction, cells were fixed and stained for DNA, α-tubulin, and STARD9-MD and imaged by fluorescence microscopy. Bar, 5 μm. (B) Quantification of the fluorescence intensity signal from STARD9-MD wild-type and serine-to-alanine mutant proteins within a 3-μm radius surrounding the centrosome/spindle pole. Data represent the average ± SD of three independent experiments, 20 cells counted for each. *p < 0.05, **p < 0.0005. (C) The percentage of cells with spindle defects was quantified. Data represent the average ± SD of three independent experiments, 100 cells counted for each. *p < 0.05, **p < 0.005, ***p < 0.0005. (D) Live-cell time-lapse microscopy of STARD9-MD wild type and S317A mutant. Top, bright-field (BF) snapshots, and bottom, fluorescence (FL), of STARD-MD localization at the indicated time points in minutes. See Supplemental Movies S1–S4.
Mentions: To analyze the consequences of accumulating STARD9-MD in mitosis, we used LAP-tagged STARD9-MD wild-type and serine-to-alanine mutant stable cell lines (expressing STARD9-MD from the same single chromosomal loci) to overexpress wild type and nonphosphorylatable/nondegradable STARD9-MD mutants and analyzed their mitotic defects by immunofluorescence microscopy. Consistent with our previous results, overexpressed STARD9-MD localized to the spindle poles and, to a lesser extent, the spindle microtubules (Torres et al., 2011; Figure 7A). However, the nonphosphorylatable/nondegradable S305A, S312A, and S317A mutants accumulated to very high levels at the spindle poles and spindle, consistent with our immunoblot analyses showing their stabilization in mitosis (Figure 7A). As a control, STARD9-MD with the S316A mutation that did not block STARD9-MD degradation remained at similar low levels as wild-type STARD9-MD (Figure 7A). Similarly, measurements of the total fluorescence intensity of LAP-STARD9-MD at the centrosome indicated that STARD9-MD protein levels were markedly increased in the S305A, S312A, and S317A mutants, whereas wild-type STARD9-MD and the degradation-competent S316A mutant remained low (Figure 7B). Also consistent with previous results, overexpression of STARD9-MD led to a modest but reproducible increase in the percentage of cells with spindle defects (PCM fragmentation and multipolar spindles): ∼18% compared with ∼8% control (Torres et al., 2011; Figure 7, A and C). A similar effect was also seen with the degradation-competent S316A mutant (Figure 7, A and C). Of interest, overexpression of the nonphosphorylatable/nondegradable S305A, S312A, and S317A mutants led to significant increase in the percentage of cells with spindle assembly defects, ∼25–30% (Figure 7, A and C). Consistently, live-cell time-lapse microscopy of nonphosphorylatable/nondegradable mutants like S317A indicated that overexpressing these mutants led to an arrest in mitosis, followed by apoptotic cell death (Figure 7D and Supplemental Movies S1–S4). These data indicated that posttranslational modification of STARD9-MD by Plk1 (phosphorylation) and SCFβ-TrCP (ubiquitination) was critical for controlling the levels of STARD9-MD at the centrosome during mitosis and that accumulation of STARD9-MD led to spindle assembly and cell division defects.

Bottom Line: These phosphorylation events are important for targeting a pool of STARD9-MD for ubiquitination by the SCFβ-TrCP ubiquitin ligase and proteasome-dependent degradation.Of interest, overexpression of nonphosphorylatable/nondegradable STARD9-MD mutants leads to spindle assembly defects.Our results with STARD9-MD imply that in vivo the protein levels of full-length STARD9 could be regulated by Plk1 and SCFβ-TrCP to promote proper mitotic spindle assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095.

Show MeSH
Related in: MedlinePlus