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Thyroid hormone receptor alpha1 follows a cooperative CRM1/calreticulin-mediated nuclear export pathway.

Grespin ME, Bonamy GM, Roggero VR, Cameron NG, Adam LE, Atchison AP, Fratto VM, Allison LA - J. Biol. Chem. (2008)

Bottom Line: An important aspect of the shuttling activity of TRalpha is its ability to exit the nucleus through the nuclear pore complex.We show that, in addition to shuttling in heterokaryons, TRalpha shuttles rapidly in an unfused monokaryon system as well.Furthermore, our data show that TRalpha directly interacts with calreticulin, and point to the intriguing possibility that TRalpha follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRalpha from the nucleus to cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of William and Mary, 10675 John Jay Hopkins Drive, Williamsburg, VA 23187, USA.

ABSTRACT
The thyroid hormone receptor alpha1 (TRalpha) exhibits a dual role as an activator or repressor of its target genes in response to thyroid hormone (T(3)). Previously, we have shown that TRalpha, formerly thought to reside solely in the nucleus bound to DNA, actually shuttles rapidly between the nucleus and cytoplasm. An important aspect of the shuttling activity of TRalpha is its ability to exit the nucleus through the nuclear pore complex. TRalpha export is not sensitive to treatment with the CRM1-specific inhibitor leptomycin B (LMB) in heterokaryon assays, suggesting a role for an export receptor other than CRM1. Here, we have used a combined approach of in vivo fluorescence recovery after photobleaching experiments, in vitro permeabilized cell nuclear export assays, and glutathione S-transferase pull-down assays to investigate the export pathway used by TRalpha. We show that, in addition to shuttling in heterokaryons, TRalpha shuttles rapidly in an unfused monokaryon system as well. Furthermore, our data show that TRalpha directly interacts with calreticulin, and point to the intriguing possibility that TRalpha follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRalpha from the nucleus to cytoplasm.

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TRα shuttling is inhibited by treatment with LMB in live monokaryons expressing CRT. A, HeLa cells were transfected with a GFP-TRα expression plasmid and nucleocytoplasmic shuttling was monitored through FRAP (n = 7). White arrowheads indicate photobleached nuclei. Parallel experiments were performed in the presence of LMB (n = 6) to block CRM1-mediated nuclear export and DIC images were taken to delineate cell borders. Fluorescence recovery graphs indicating relative shuttling of GFP-TRα were generated. Black squares indicate relative fluorescence levels within unbleached nuclei and open diamonds represent levels within bleached nuclei. Any apparent change in nucleus morphology is a result of cell movement over the course of the experiment. Error bars, ± 1 S.E. B, as in A using crt+/+ cell line (n = 10, -LMB; n = 6, +LMB). Bar, 10 μm.
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fig1: TRα shuttling is inhibited by treatment with LMB in live monokaryons expressing CRT. A, HeLa cells were transfected with a GFP-TRα expression plasmid and nucleocytoplasmic shuttling was monitored through FRAP (n = 7). White arrowheads indicate photobleached nuclei. Parallel experiments were performed in the presence of LMB (n = 6) to block CRM1-mediated nuclear export and DIC images were taken to delineate cell borders. Fluorescence recovery graphs indicating relative shuttling of GFP-TRα were generated. Black squares indicate relative fluorescence levels within unbleached nuclei and open diamonds represent levels within bleached nuclei. Any apparent change in nucleus morphology is a result of cell movement over the course of the experiment. Error bars, ± 1 S.E. B, as in A using crt+/+ cell line (n = 10, -LMB; n = 6, +LMB). Bar, 10 μm.

Mentions: We first sought to determine whether TRα shuttles under physiological conditions by performing experiments in living cells, independent of heterokaryon formation. To maintain physiological conditions, we used a FRAP assay in multinucleate live cells (monokaryons) to monitor the movement of GFP-TRα from unbleached to bleached nuclei. Unlike in heterokaryons, a monokaryon system does not require cell fusion or other manipulation that would compromise the integrity of the ER and thus, presumably, maintains low levels of cytosolic CRT. Transfected monokaryons were selected and one nucleus within these cells was exposed to intense laser illumination. This exposure resulted in loss of fluorescence within the selected nucleus due to irreversible photobleaching of the GFP fluorophore. The initial bleaching did not, however, result in loss of fluorescence to neighboring nuclei within the same cells (Fig. 1). A series of images was taken for each individual experiment in which fluorescence recovery to bleached nuclei was measured and compared with the concomitant decrease in intensity within unbleached nuclei. Through image analysis and plotting of these fluorescence intensity data, we were able to determine the relative degree of nucleocytoplasmic shuttling within particular cell types and treatments. Specifically, we assayed for TRα shuttling in human HeLa and mouse crt+/+ cell lines, both of which express CRT. We show that, in contrast to the slow nuclear export observed for GR in COS-7 cells (29), TRα in fact shuttles rapidly between nuclei in both HeLa (Fig. 1A) and crt+/+ monokaryons (Fig. 1B). These data are in close agreement with those observed for TRα shuttling kinetics in heterokaryons (1), and suggest that TRα may play an as yet unknown role in cytosolic signaling pathways. After confirming that TRα rapidly shuttles between nuclei in these cells, we sought to determine whether TRα follows a CRM1 or CRT-dependent nuclear export pathway in live, unfused cells.


Thyroid hormone receptor alpha1 follows a cooperative CRM1/calreticulin-mediated nuclear export pathway.

Grespin ME, Bonamy GM, Roggero VR, Cameron NG, Adam LE, Atchison AP, Fratto VM, Allison LA - J. Biol. Chem. (2008)

TRα shuttling is inhibited by treatment with LMB in live monokaryons expressing CRT. A, HeLa cells were transfected with a GFP-TRα expression plasmid and nucleocytoplasmic shuttling was monitored through FRAP (n = 7). White arrowheads indicate photobleached nuclei. Parallel experiments were performed in the presence of LMB (n = 6) to block CRM1-mediated nuclear export and DIC images were taken to delineate cell borders. Fluorescence recovery graphs indicating relative shuttling of GFP-TRα were generated. Black squares indicate relative fluorescence levels within unbleached nuclei and open diamonds represent levels within bleached nuclei. Any apparent change in nucleus morphology is a result of cell movement over the course of the experiment. Error bars, ± 1 S.E. B, as in A using crt+/+ cell line (n = 10, -LMB; n = 6, +LMB). Bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2533095&req=5

fig1: TRα shuttling is inhibited by treatment with LMB in live monokaryons expressing CRT. A, HeLa cells were transfected with a GFP-TRα expression plasmid and nucleocytoplasmic shuttling was monitored through FRAP (n = 7). White arrowheads indicate photobleached nuclei. Parallel experiments were performed in the presence of LMB (n = 6) to block CRM1-mediated nuclear export and DIC images were taken to delineate cell borders. Fluorescence recovery graphs indicating relative shuttling of GFP-TRα were generated. Black squares indicate relative fluorescence levels within unbleached nuclei and open diamonds represent levels within bleached nuclei. Any apparent change in nucleus morphology is a result of cell movement over the course of the experiment. Error bars, ± 1 S.E. B, as in A using crt+/+ cell line (n = 10, -LMB; n = 6, +LMB). Bar, 10 μm.
Mentions: We first sought to determine whether TRα shuttles under physiological conditions by performing experiments in living cells, independent of heterokaryon formation. To maintain physiological conditions, we used a FRAP assay in multinucleate live cells (monokaryons) to monitor the movement of GFP-TRα from unbleached to bleached nuclei. Unlike in heterokaryons, a monokaryon system does not require cell fusion or other manipulation that would compromise the integrity of the ER and thus, presumably, maintains low levels of cytosolic CRT. Transfected monokaryons were selected and one nucleus within these cells was exposed to intense laser illumination. This exposure resulted in loss of fluorescence within the selected nucleus due to irreversible photobleaching of the GFP fluorophore. The initial bleaching did not, however, result in loss of fluorescence to neighboring nuclei within the same cells (Fig. 1). A series of images was taken for each individual experiment in which fluorescence recovery to bleached nuclei was measured and compared with the concomitant decrease in intensity within unbleached nuclei. Through image analysis and plotting of these fluorescence intensity data, we were able to determine the relative degree of nucleocytoplasmic shuttling within particular cell types and treatments. Specifically, we assayed for TRα shuttling in human HeLa and mouse crt+/+ cell lines, both of which express CRT. We show that, in contrast to the slow nuclear export observed for GR in COS-7 cells (29), TRα in fact shuttles rapidly between nuclei in both HeLa (Fig. 1A) and crt+/+ monokaryons (Fig. 1B). These data are in close agreement with those observed for TRα shuttling kinetics in heterokaryons (1), and suggest that TRα may play an as yet unknown role in cytosolic signaling pathways. After confirming that TRα rapidly shuttles between nuclei in these cells, we sought to determine whether TRα follows a CRM1 or CRT-dependent nuclear export pathway in live, unfused cells.

Bottom Line: An important aspect of the shuttling activity of TRalpha is its ability to exit the nucleus through the nuclear pore complex.We show that, in addition to shuttling in heterokaryons, TRalpha shuttles rapidly in an unfused monokaryon system as well.Furthermore, our data show that TRalpha directly interacts with calreticulin, and point to the intriguing possibility that TRalpha follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRalpha from the nucleus to cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of William and Mary, 10675 John Jay Hopkins Drive, Williamsburg, VA 23187, USA.

ABSTRACT
The thyroid hormone receptor alpha1 (TRalpha) exhibits a dual role as an activator or repressor of its target genes in response to thyroid hormone (T(3)). Previously, we have shown that TRalpha, formerly thought to reside solely in the nucleus bound to DNA, actually shuttles rapidly between the nucleus and cytoplasm. An important aspect of the shuttling activity of TRalpha is its ability to exit the nucleus through the nuclear pore complex. TRalpha export is not sensitive to treatment with the CRM1-specific inhibitor leptomycin B (LMB) in heterokaryon assays, suggesting a role for an export receptor other than CRM1. Here, we have used a combined approach of in vivo fluorescence recovery after photobleaching experiments, in vitro permeabilized cell nuclear export assays, and glutathione S-transferase pull-down assays to investigate the export pathway used by TRalpha. We show that, in addition to shuttling in heterokaryons, TRalpha shuttles rapidly in an unfused monokaryon system as well. Furthermore, our data show that TRalpha directly interacts with calreticulin, and point to the intriguing possibility that TRalpha follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRalpha from the nucleus to cytoplasm.

Show MeSH
Related in: MedlinePlus