Limits...
The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body.

Boisvert FM, Kruhlak MJ, Box AK, Hendzel MJ, Bazett-Jones DP - J. Cell Biol. (2001)

Bottom Line: In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100.They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains.Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, University of Calgary, Calgary, Alberta T2N 4N1, Canada.

ABSTRACT
The transcription coactivator and histone acetyltransferase CAMP response element-binding protein (CBP) has been demonstrated to accumulate in promyelocytic leukemia (PML) bodies. We show that this accumulation is cell type specific. In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100. Using fluorescence recovery after photobleaching, we demonstrate that CBP moves rapidly into and out of PML bodies. In contrast, Pml and Sp100 are relatively immobile in the nucleoplasm and within PML nuclear bodies. They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains. Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.

Show MeSH

Related in: MedlinePlus

Determination of the direction of movement of GFP–CBP. A whole PML nuclear body was bleached (B, box) in a 293 cell expressing GFP–CBP. 5 s after bleaching, an image showing the fluorescence recovery was recorded (5 s; C). A region just outside the PML nuclear body was then bleached (E, box), indicating that fluorescence can be drained from the PML nuclear body (E), which is followed by a rapid reequilibration (5 s; F) of the fluorescence. Bleached box in Fig. 4 B is 400 nm in length.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2198823&req=5

Figure 4: Determination of the direction of movement of GFP–CBP. A whole PML nuclear body was bleached (B, box) in a 293 cell expressing GFP–CBP. 5 s after bleaching, an image showing the fluorescence recovery was recorded (5 s; C). A region just outside the PML nuclear body was then bleached (E, box), indicating that fluorescence can be drained from the PML nuclear body (E), which is followed by a rapid reequilibration (5 s; F) of the fluorescence. Bleached box in Fig. 4 B is 400 nm in length.

Mentions: The rate of fluorescence recovery of CBP in PML nuclear bodies is equivalent to that found in the nucleoplasm. To determine whether the direction of CBP movement is only into PML nuclear bodies or is bidirectional into and out of these domains, we performed both FRAP and fluorescence loss in photobleaching (FLIP) experiments. To determine the rate of movement from the nucleoplasm into PML nuclear bodies, we bleached an entire PML nuclear body in a cell expressing GFP–CBP (Fig. 4, A–C). Complete fluorescence recovery of the PML nuclear body was observed after 5 s. This indicates that CBP can move rapidly from the nucleoplasm into PML nuclear bodies. To determine whether CBP can leave the PML nuclear body, we bleached a region just outside the domain to see whether we could drain some fluorescence from it (FLIP) (Fig. 4D and Fig. E). Indeed, we observed a loss of fluorescence from the PML nuclear body followed by a quick reequilibration (5 s) of the fluorescence. The integrated intensity of signal from the PML body shown in Fig. 4 E decreased from 180 to 131 (gray values) after bleaching but rebounded to 160 within 5 s. Our interpretation is that fluorescent molecules that moved from the PML body to the bleached region outside were rapidly replaced (5 s) by fluorescent molecules moving into the PML body from unbleached but nearby regions of the nucleoplasm. Therefore, we conclude that the movement of GFP–CBP between the nucleoplasm and the PML nuclear bodies is bidirectional. The movement cannot be described as freely mobile since it is significantly slower than that seen for a freely diffusing molecule such as GFP (Fig. 3 G). These experiments indicate that CBP in PML nuclear bodies is not an insoluble aggregation of molecules which form by random clustering of diffusing molecules. The bidirectional movement further demonstrates that CBP molecules are not recruited to these domains, stored, and then degraded (Maul 1998).


The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body.

Boisvert FM, Kruhlak MJ, Box AK, Hendzel MJ, Bazett-Jones DP - J. Cell Biol. (2001)

Determination of the direction of movement of GFP–CBP. A whole PML nuclear body was bleached (B, box) in a 293 cell expressing GFP–CBP. 5 s after bleaching, an image showing the fluorescence recovery was recorded (5 s; C). A region just outside the PML nuclear body was then bleached (E, box), indicating that fluorescence can be drained from the PML nuclear body (E), which is followed by a rapid reequilibration (5 s; F) of the fluorescence. Bleached box in Fig. 4 B is 400 nm in length.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198823&req=5

Figure 4: Determination of the direction of movement of GFP–CBP. A whole PML nuclear body was bleached (B, box) in a 293 cell expressing GFP–CBP. 5 s after bleaching, an image showing the fluorescence recovery was recorded (5 s; C). A region just outside the PML nuclear body was then bleached (E, box), indicating that fluorescence can be drained from the PML nuclear body (E), which is followed by a rapid reequilibration (5 s; F) of the fluorescence. Bleached box in Fig. 4 B is 400 nm in length.
Mentions: The rate of fluorescence recovery of CBP in PML nuclear bodies is equivalent to that found in the nucleoplasm. To determine whether the direction of CBP movement is only into PML nuclear bodies or is bidirectional into and out of these domains, we performed both FRAP and fluorescence loss in photobleaching (FLIP) experiments. To determine the rate of movement from the nucleoplasm into PML nuclear bodies, we bleached an entire PML nuclear body in a cell expressing GFP–CBP (Fig. 4, A–C). Complete fluorescence recovery of the PML nuclear body was observed after 5 s. This indicates that CBP can move rapidly from the nucleoplasm into PML nuclear bodies. To determine whether CBP can leave the PML nuclear body, we bleached a region just outside the domain to see whether we could drain some fluorescence from it (FLIP) (Fig. 4D and Fig. E). Indeed, we observed a loss of fluorescence from the PML nuclear body followed by a quick reequilibration (5 s) of the fluorescence. The integrated intensity of signal from the PML body shown in Fig. 4 E decreased from 180 to 131 (gray values) after bleaching but rebounded to 160 within 5 s. Our interpretation is that fluorescent molecules that moved from the PML body to the bleached region outside were rapidly replaced (5 s) by fluorescent molecules moving into the PML body from unbleached but nearby regions of the nucleoplasm. Therefore, we conclude that the movement of GFP–CBP between the nucleoplasm and the PML nuclear bodies is bidirectional. The movement cannot be described as freely mobile since it is significantly slower than that seen for a freely diffusing molecule such as GFP (Fig. 3 G). These experiments indicate that CBP in PML nuclear bodies is not an insoluble aggregation of molecules which form by random clustering of diffusing molecules. The bidirectional movement further demonstrates that CBP molecules are not recruited to these domains, stored, and then degraded (Maul 1998).

Bottom Line: In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100.They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains.Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, University of Calgary, Calgary, Alberta T2N 4N1, Canada.

ABSTRACT
The transcription coactivator and histone acetyltransferase CAMP response element-binding protein (CBP) has been demonstrated to accumulate in promyelocytic leukemia (PML) bodies. We show that this accumulation is cell type specific. In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100. Using fluorescence recovery after photobleaching, we demonstrate that CBP moves rapidly into and out of PML bodies. In contrast, Pml and Sp100 are relatively immobile in the nucleoplasm and within PML nuclear bodies. They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains. Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.

Show MeSH
Related in: MedlinePlus