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Regulation of ubiquitin ligase dynamics by the nucleolus.

Mekhail K, Khacho M, Carrigan A, Hache RR, Gunaratnam L, Lee S - J. Cell Biol. (2005)

Bottom Line: Photobleaching experiments reveal that MDM2 and VHL are highly mobile proteins in settings where their substrates are efficiently degraded.The nucleolar architecture converts MDM2 and VHL to a static state in response to regulatory cues that are associated with substrate stability.Data shown here provide the first evidence that cells have evolved a mechanism to regulate molecular networks by reversibly switching proteins between a mobile and static state.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.

ABSTRACT
Cellular pathways relay information through dynamic protein interactions. We have assessed the kinetic properties of the murine double minute protein (MDM2) and von Hippel-Lindau (VHL) ubiquitin ligases in living cells under physiological conditions that alter the stability of their respective p53 and hypoxia-inducible factor substrates. Photobleaching experiments reveal that MDM2 and VHL are highly mobile proteins in settings where their substrates are efficiently degraded. The nucleolar architecture converts MDM2 and VHL to a static state in response to regulatory cues that are associated with substrate stability. After signal termination, the nucleolus is able to rapidly release these proteins from static detention, thereby restoring their high mobility profiles. A protein surface region of VHL's beta-sheet domain was identified as a discrete [H+]-responsive nucleolar detention signal that targets the VHL/Cullin-2 ubiquitin ligase complex to nucleoli in response to physiological fluctuations in environmental pH. Data shown here provide the first evidence that cells have evolved a mechanism to regulate molecular networks by reversibly switching proteins between a mobile and static state.

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Long-term detention of VHL within the nucleolar space revealed by the inability of VHL to release from nucleoli in a polykaryon fusion assay. (A) MCF7 cells transiently expressing VHL-GFP were fused in a standard PEG fusion assay and incubated in SD media for 30 min (b). Inset shows Hoechst staining of DNA. Cells were replenished with AP media and transferred to hypoxia. VHL-GFP localization was monitored after reaching the pH 6.5 threshold (c–g). Nuclei within a polykaryonic cell were always synchronized in the rates of nucleolar appearance of VHL-GFP. This is not necessarily the case for monokaryonic cells in close proximity under AP conditions (h). (B) Unaltered MCF7 cells were cocultured under standard conditions with either MCF7 (homokaryon assay) or NIH 3T3 (heterokaryon assay) cells transfected to transiently express VHL-GFP. Cells were then transferred to hypoxia in AP media. After nucleolar localization of VHL-GFP, cells were fused and monitored by time-lapse microscopy. Hoechst staining of DNA was used to identify donor and acceptor cells. Arrows indicate the same position in the cell. (C) Unaltered MCF7 cells were cocultured under standard conditions with MCF7 cells transfected to transiently express B23-GFP. Cells were cultured in AP media, fused and monitored as in B. Pseudocolored zooms of area indicated by dashed square are shown.
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fig5: Long-term detention of VHL within the nucleolar space revealed by the inability of VHL to release from nucleoli in a polykaryon fusion assay. (A) MCF7 cells transiently expressing VHL-GFP were fused in a standard PEG fusion assay and incubated in SD media for 30 min (b). Inset shows Hoechst staining of DNA. Cells were replenished with AP media and transferred to hypoxia. VHL-GFP localization was monitored after reaching the pH 6.5 threshold (c–g). Nuclei within a polykaryonic cell were always synchronized in the rates of nucleolar appearance of VHL-GFP. This is not necessarily the case for monokaryonic cells in close proximity under AP conditions (h). (B) Unaltered MCF7 cells were cocultured under standard conditions with either MCF7 (homokaryon assay) or NIH 3T3 (heterokaryon assay) cells transfected to transiently express VHL-GFP. Cells were then transferred to hypoxia in AP media. After nucleolar localization of VHL-GFP, cells were fused and monitored by time-lapse microscopy. Hoechst staining of DNA was used to identify donor and acceptor cells. Arrows indicate the same position in the cell. (C) Unaltered MCF7 cells were cocultured under standard conditions with MCF7 cells transfected to transiently express B23-GFP. Cells were cultured in AP media, fused and monitored as in B. Pseudocolored zooms of area indicated by dashed square are shown.

Mentions: We next studied VHL dynamics using polykaryon fusion assays, which provide an alternative approach to photobleaching in assessing changes in subcellular trafficking of proteins (Walther et al., 2003). Cells expressing VHL-GFP were fused in a standard polyethylene glycol (PEG) fusion assay. VHL remains nuclear-cytoplasmic in polykaryonic cells (Fig. 5 A, a and b; Lee et al., 1999). Transfer to hypoxia resulted in acidification of the media and VHL-GFP displayed its typical two-step localization process to the nucleolus (Fig. 5 A, c–g). It is important to note that nucleolar VHL signal was equally distributed between the nuclei of a polykaryonic cell (Fig. 5 A), indicating that VHL-GFP displays no preference for the nucleoli of one nucleus over another. Next, we cocultured VHL-GFP–expressing and nonexpressing cells under standard conditions, then transferred them to hypoxia in AP media. After the redistribution of VHL-GFP to nucleoli, cells were rapidly fused and replenished with their own acidified AP media. This process yielded a significant number of polykaryonic cells where the fluorescence observed in the cell is only associated with nucleoli of only one or two nuclei, whereas other nuclei displayed no fluorescence (Fig. 5 B). VHL-GFP failed to exhibit any change in localization up to 3 h after fusion. In contrast, under the same conditions B23-GFP (Fig. 5 C) and REV-GFP (unpublished data) redistributed from the nucleoli of a single cell to the nucleo-cytoplasm and nucleoli of the acceptor (nontransfected) cells of polykaryons. In addition to bleaching experiments, results from the fusion assays reveal a role for the nucleolus in regulating the subcellular dynamic profile of the VHL tumor suppressor.


Regulation of ubiquitin ligase dynamics by the nucleolus.

Mekhail K, Khacho M, Carrigan A, Hache RR, Gunaratnam L, Lee S - J. Cell Biol. (2005)

Long-term detention of VHL within the nucleolar space revealed by the inability of VHL to release from nucleoli in a polykaryon fusion assay. (A) MCF7 cells transiently expressing VHL-GFP were fused in a standard PEG fusion assay and incubated in SD media for 30 min (b). Inset shows Hoechst staining of DNA. Cells were replenished with AP media and transferred to hypoxia. VHL-GFP localization was monitored after reaching the pH 6.5 threshold (c–g). Nuclei within a polykaryonic cell were always synchronized in the rates of nucleolar appearance of VHL-GFP. This is not necessarily the case for monokaryonic cells in close proximity under AP conditions (h). (B) Unaltered MCF7 cells were cocultured under standard conditions with either MCF7 (homokaryon assay) or NIH 3T3 (heterokaryon assay) cells transfected to transiently express VHL-GFP. Cells were then transferred to hypoxia in AP media. After nucleolar localization of VHL-GFP, cells were fused and monitored by time-lapse microscopy. Hoechst staining of DNA was used to identify donor and acceptor cells. Arrows indicate the same position in the cell. (C) Unaltered MCF7 cells were cocultured under standard conditions with MCF7 cells transfected to transiently express B23-GFP. Cells were cultured in AP media, fused and monitored as in B. Pseudocolored zooms of area indicated by dashed square are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171338&req=5

fig5: Long-term detention of VHL within the nucleolar space revealed by the inability of VHL to release from nucleoli in a polykaryon fusion assay. (A) MCF7 cells transiently expressing VHL-GFP were fused in a standard PEG fusion assay and incubated in SD media for 30 min (b). Inset shows Hoechst staining of DNA. Cells were replenished with AP media and transferred to hypoxia. VHL-GFP localization was monitored after reaching the pH 6.5 threshold (c–g). Nuclei within a polykaryonic cell were always synchronized in the rates of nucleolar appearance of VHL-GFP. This is not necessarily the case for monokaryonic cells in close proximity under AP conditions (h). (B) Unaltered MCF7 cells were cocultured under standard conditions with either MCF7 (homokaryon assay) or NIH 3T3 (heterokaryon assay) cells transfected to transiently express VHL-GFP. Cells were then transferred to hypoxia in AP media. After nucleolar localization of VHL-GFP, cells were fused and monitored by time-lapse microscopy. Hoechst staining of DNA was used to identify donor and acceptor cells. Arrows indicate the same position in the cell. (C) Unaltered MCF7 cells were cocultured under standard conditions with MCF7 cells transfected to transiently express B23-GFP. Cells were cultured in AP media, fused and monitored as in B. Pseudocolored zooms of area indicated by dashed square are shown.
Mentions: We next studied VHL dynamics using polykaryon fusion assays, which provide an alternative approach to photobleaching in assessing changes in subcellular trafficking of proteins (Walther et al., 2003). Cells expressing VHL-GFP were fused in a standard polyethylene glycol (PEG) fusion assay. VHL remains nuclear-cytoplasmic in polykaryonic cells (Fig. 5 A, a and b; Lee et al., 1999). Transfer to hypoxia resulted in acidification of the media and VHL-GFP displayed its typical two-step localization process to the nucleolus (Fig. 5 A, c–g). It is important to note that nucleolar VHL signal was equally distributed between the nuclei of a polykaryonic cell (Fig. 5 A), indicating that VHL-GFP displays no preference for the nucleoli of one nucleus over another. Next, we cocultured VHL-GFP–expressing and nonexpressing cells under standard conditions, then transferred them to hypoxia in AP media. After the redistribution of VHL-GFP to nucleoli, cells were rapidly fused and replenished with their own acidified AP media. This process yielded a significant number of polykaryonic cells where the fluorescence observed in the cell is only associated with nucleoli of only one or two nuclei, whereas other nuclei displayed no fluorescence (Fig. 5 B). VHL-GFP failed to exhibit any change in localization up to 3 h after fusion. In contrast, under the same conditions B23-GFP (Fig. 5 C) and REV-GFP (unpublished data) redistributed from the nucleoli of a single cell to the nucleo-cytoplasm and nucleoli of the acceptor (nontransfected) cells of polykaryons. In addition to bleaching experiments, results from the fusion assays reveal a role for the nucleolus in regulating the subcellular dynamic profile of the VHL tumor suppressor.

Bottom Line: Photobleaching experiments reveal that MDM2 and VHL are highly mobile proteins in settings where their substrates are efficiently degraded.The nucleolar architecture converts MDM2 and VHL to a static state in response to regulatory cues that are associated with substrate stability.Data shown here provide the first evidence that cells have evolved a mechanism to regulate molecular networks by reversibly switching proteins between a mobile and static state.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.

ABSTRACT
Cellular pathways relay information through dynamic protein interactions. We have assessed the kinetic properties of the murine double minute protein (MDM2) and von Hippel-Lindau (VHL) ubiquitin ligases in living cells under physiological conditions that alter the stability of their respective p53 and hypoxia-inducible factor substrates. Photobleaching experiments reveal that MDM2 and VHL are highly mobile proteins in settings where their substrates are efficiently degraded. The nucleolar architecture converts MDM2 and VHL to a static state in response to regulatory cues that are associated with substrate stability. After signal termination, the nucleolus is able to rapidly release these proteins from static detention, thereby restoring their high mobility profiles. A protein surface region of VHL's beta-sheet domain was identified as a discrete [H+]-responsive nucleolar detention signal that targets the VHL/Cullin-2 ubiquitin ligase complex to nucleoli in response to physiological fluctuations in environmental pH. Data shown here provide the first evidence that cells have evolved a mechanism to regulate molecular networks by reversibly switching proteins between a mobile and static state.

Show MeSH
Related in: MedlinePlus