Limits...
Polyglutamine expansion accelerates the dynamics of ataxin-1 and does not result in aggregate formation.

Krol HA, Krawczyk PM, Bosch KS, Aten JA, Hol EM, Reits EA - PLoS ONE (2008)

Bottom Line: Instead of being static, insoluble aggregates, nuclear accumulations formed by the polyQ-expanded ataxin-1 showed enhanced intracellular kinetics as compared to wild-type ataxin-1.Interestingly, polyQ expansion did not affect the nuclear-cytoplasmic shuttling of ataxin-1 as proposed before.These results indicate that polyQ expansion does not necessarily lead to aggregate formation, and that the enhanced kinetics may affect the nuclear function of ataxin-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT

Background: Polyglutamine expansion disorders are caused by an expansion of the polyglutamine (polyQ) tract in the disease related protein, leading to severe neurodegeneration. All polyQ disorders are hallmarked by the presence of intracellular aggregates containing the expanded protein in affected neurons. The polyQ disorder SpinoCerebellar Ataxia 1 (SCA1) is caused by a polyQ-expansion in the ataxin-1 protein, which is thought to lead to nuclear aggregates.

Methodology/principal findings: Using advanced live cell fluorescence microscopy and a filter retardation assay we show that nuclear accumulations formed by polyQ-expanded ataxin-1 do not resemble aggregates of other polyQ-expanded proteins. Instead of being static, insoluble aggregates, nuclear accumulations formed by the polyQ-expanded ataxin-1 showed enhanced intracellular kinetics as compared to wild-type ataxin-1. During mitosis, ataxin-1 accumulations redistributed equally among daughter cells, in contrast to polyQ aggregates. Interestingly, polyQ expansion did not affect the nuclear-cytoplasmic shuttling of ataxin-1 as proposed before.

Conclusions/significance: These results indicate that polyQ expansion does not necessarily lead to aggregate formation, and that the enhanced kinetics may affect the nuclear function of ataxin-1. The unexpected findings for a polyQ-expanded protein and their consequences for ongoing SCA1 research are discussed.

Show MeSH

Related in: MedlinePlus

Polyglutamine expansion does not affect ataxin-1 shuttling between the nucleus and the cytoplasm.(A). Cos-7 cells and N2A and differentiated SH-SY5Y neuronal cells contain cytoplasmic accumulations of both Atx1[Q2]GFP and Atx1[Q85]GFP. (B). Representative nucleocytoplasmic shuttling assay in a Cos-7 bikaryon with small and large Atx1[Q2]GFP nuclear accumulations. Shuttling to the bleached nucleus is only observed when small nuclear accumulations are present. (C). Atx1[Q85]GFP can shuttle between nucleus and cytoplasm when bikaryons of Cos-7 cell contain small nuclear accumulations. When large nuclear accumulations are present there is no nucleocytoplasmic shuttling of either Atx1[Q2]GFP or Atx1[Q85]GFP. The bleached region is indicated in white. Sizebar = 1 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2200796&req=5

pone-0001503-g004: Polyglutamine expansion does not affect ataxin-1 shuttling between the nucleus and the cytoplasm.(A). Cos-7 cells and N2A and differentiated SH-SY5Y neuronal cells contain cytoplasmic accumulations of both Atx1[Q2]GFP and Atx1[Q85]GFP. (B). Representative nucleocytoplasmic shuttling assay in a Cos-7 bikaryon with small and large Atx1[Q2]GFP nuclear accumulations. Shuttling to the bleached nucleus is only observed when small nuclear accumulations are present. (C). Atx1[Q85]GFP can shuttle between nucleus and cytoplasm when bikaryons of Cos-7 cell contain small nuclear accumulations. When large nuclear accumulations are present there is no nucleocytoplasmic shuttling of either Atx1[Q2]GFP or Atx1[Q85]GFP. The bleached region is indicated in white. Sizebar = 1 µm.

Mentions: As wild-type ataxin-1 can shuttle between the nucleus and the cytoplasm of a cell, it has been suggested that ataxin-1 is involved in RNA binding and transport to the cellular periphery [7]. This transport may be abolished in SCA1, as polyQ expansion hampers the nuclear export of ataxin-1 [7]. It should however be noted that in this study small wild-type ataxin-1 nuclear accumulations were compared to large polyQ-expanded ataxin-1 nuclear accumulations. As we showed that large accumulations of both polyQ-expanded Atx1[Q85]GFP and wildtype Atx1[Q2]GFP have a lower recovery rate when compared to small accumulations this might have limited the exit of free ataxin-1 from the nucleus. We also observed that Cos-7 cells expressing either Atx1[Q2]GFP or Atx1[Q85]GFP showed cytoplasmic presence of the protein, either diffuse or in body-like structures, independent of the length of the polyQ expansion (Fig 4A). The presence of cytoplasmic polyQ-expanded ataxin-1 is not limited to Cos-7 cells, as cultured neuroblastoma cells such as SH-SY5Y and N2A neuroblastoma cells (Fig 4A) as well as U343MG astrocytoma cells (data not shown) showed cytoplasmic presence and movement of ataxin-1 accumulations. The cytoplasmic presence may reflect the suggested role of ataxin-1 in RNA binding and shuttling to cytoplasmic domains [7]. To test whether polyQ-expansion limits the nuclear-cytoplasmic shuttling of ataxin-1, we performed a nucleocytoplasmic shuttling assay with Atx1[Q2]GFP and Atx1[Q85]GFP transfected in Cos-7 cells. To measure the nuclear shuttling, we performed FRAP analysis on cells containing two nuclei (bikaryons) by photobleaching all fluorescence except for one ataxin-1 nucleus and visualized the exchange and shuttling of GFP-tagged ataxin-1 by imaging the increase of fluorescence in the bleached nucleus. These experiments were performed in the presence of the translation inhibitor cycloheximide to prevent fluorescence recovery due to de novo synthesis of ataxin-1. We distinguished between cells having either only small or large nuclear ataxin-1 accumulations. Cells having only free nuclear ataxin-1 distribution or ataxin-1 present in small nuclear accumulations showed recovery of fluorescence in the bleached nucleus within 30 minutes after bleaching, independent of the length of a polyQ expansion (Fig 4B,C, upper panel). However, the bikaryons with large nuclear ataxin-1 accumulations did not show any recovery within this time span. This observation was independent of the length of the polyQ tract (Fig 4B,C lower panel). Taken together, these data show that the shuttling of ataxin-1 only occurs when the protein is diffusely dispersed in the nucleoplasm or when the protein is present in small nuclear accumulations. More importantly, the ability of ataxin-1 to shuttle from the nucleus is not affected by the length of the polyQ expansion.


Polyglutamine expansion accelerates the dynamics of ataxin-1 and does not result in aggregate formation.

Krol HA, Krawczyk PM, Bosch KS, Aten JA, Hol EM, Reits EA - PLoS ONE (2008)

Polyglutamine expansion does not affect ataxin-1 shuttling between the nucleus and the cytoplasm.(A). Cos-7 cells and N2A and differentiated SH-SY5Y neuronal cells contain cytoplasmic accumulations of both Atx1[Q2]GFP and Atx1[Q85]GFP. (B). Representative nucleocytoplasmic shuttling assay in a Cos-7 bikaryon with small and large Atx1[Q2]GFP nuclear accumulations. Shuttling to the bleached nucleus is only observed when small nuclear accumulations are present. (C). Atx1[Q85]GFP can shuttle between nucleus and cytoplasm when bikaryons of Cos-7 cell contain small nuclear accumulations. When large nuclear accumulations are present there is no nucleocytoplasmic shuttling of either Atx1[Q2]GFP or Atx1[Q85]GFP. The bleached region is indicated in white. Sizebar = 1 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2200796&req=5

pone-0001503-g004: Polyglutamine expansion does not affect ataxin-1 shuttling between the nucleus and the cytoplasm.(A). Cos-7 cells and N2A and differentiated SH-SY5Y neuronal cells contain cytoplasmic accumulations of both Atx1[Q2]GFP and Atx1[Q85]GFP. (B). Representative nucleocytoplasmic shuttling assay in a Cos-7 bikaryon with small and large Atx1[Q2]GFP nuclear accumulations. Shuttling to the bleached nucleus is only observed when small nuclear accumulations are present. (C). Atx1[Q85]GFP can shuttle between nucleus and cytoplasm when bikaryons of Cos-7 cell contain small nuclear accumulations. When large nuclear accumulations are present there is no nucleocytoplasmic shuttling of either Atx1[Q2]GFP or Atx1[Q85]GFP. The bleached region is indicated in white. Sizebar = 1 µm.
Mentions: As wild-type ataxin-1 can shuttle between the nucleus and the cytoplasm of a cell, it has been suggested that ataxin-1 is involved in RNA binding and transport to the cellular periphery [7]. This transport may be abolished in SCA1, as polyQ expansion hampers the nuclear export of ataxin-1 [7]. It should however be noted that in this study small wild-type ataxin-1 nuclear accumulations were compared to large polyQ-expanded ataxin-1 nuclear accumulations. As we showed that large accumulations of both polyQ-expanded Atx1[Q85]GFP and wildtype Atx1[Q2]GFP have a lower recovery rate when compared to small accumulations this might have limited the exit of free ataxin-1 from the nucleus. We also observed that Cos-7 cells expressing either Atx1[Q2]GFP or Atx1[Q85]GFP showed cytoplasmic presence of the protein, either diffuse or in body-like structures, independent of the length of the polyQ expansion (Fig 4A). The presence of cytoplasmic polyQ-expanded ataxin-1 is not limited to Cos-7 cells, as cultured neuroblastoma cells such as SH-SY5Y and N2A neuroblastoma cells (Fig 4A) as well as U343MG astrocytoma cells (data not shown) showed cytoplasmic presence and movement of ataxin-1 accumulations. The cytoplasmic presence may reflect the suggested role of ataxin-1 in RNA binding and shuttling to cytoplasmic domains [7]. To test whether polyQ-expansion limits the nuclear-cytoplasmic shuttling of ataxin-1, we performed a nucleocytoplasmic shuttling assay with Atx1[Q2]GFP and Atx1[Q85]GFP transfected in Cos-7 cells. To measure the nuclear shuttling, we performed FRAP analysis on cells containing two nuclei (bikaryons) by photobleaching all fluorescence except for one ataxin-1 nucleus and visualized the exchange and shuttling of GFP-tagged ataxin-1 by imaging the increase of fluorescence in the bleached nucleus. These experiments were performed in the presence of the translation inhibitor cycloheximide to prevent fluorescence recovery due to de novo synthesis of ataxin-1. We distinguished between cells having either only small or large nuclear ataxin-1 accumulations. Cells having only free nuclear ataxin-1 distribution or ataxin-1 present in small nuclear accumulations showed recovery of fluorescence in the bleached nucleus within 30 minutes after bleaching, independent of the length of a polyQ expansion (Fig 4B,C, upper panel). However, the bikaryons with large nuclear ataxin-1 accumulations did not show any recovery within this time span. This observation was independent of the length of the polyQ tract (Fig 4B,C lower panel). Taken together, these data show that the shuttling of ataxin-1 only occurs when the protein is diffusely dispersed in the nucleoplasm or when the protein is present in small nuclear accumulations. More importantly, the ability of ataxin-1 to shuttle from the nucleus is not affected by the length of the polyQ expansion.

Bottom Line: Instead of being static, insoluble aggregates, nuclear accumulations formed by the polyQ-expanded ataxin-1 showed enhanced intracellular kinetics as compared to wild-type ataxin-1.Interestingly, polyQ expansion did not affect the nuclear-cytoplasmic shuttling of ataxin-1 as proposed before.These results indicate that polyQ expansion does not necessarily lead to aggregate formation, and that the enhanced kinetics may affect the nuclear function of ataxin-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT

Background: Polyglutamine expansion disorders are caused by an expansion of the polyglutamine (polyQ) tract in the disease related protein, leading to severe neurodegeneration. All polyQ disorders are hallmarked by the presence of intracellular aggregates containing the expanded protein in affected neurons. The polyQ disorder SpinoCerebellar Ataxia 1 (SCA1) is caused by a polyQ-expansion in the ataxin-1 protein, which is thought to lead to nuclear aggregates.

Methodology/principal findings: Using advanced live cell fluorescence microscopy and a filter retardation assay we show that nuclear accumulations formed by polyQ-expanded ataxin-1 do not resemble aggregates of other polyQ-expanded proteins. Instead of being static, insoluble aggregates, nuclear accumulations formed by the polyQ-expanded ataxin-1 showed enhanced intracellular kinetics as compared to wild-type ataxin-1. During mitosis, ataxin-1 accumulations redistributed equally among daughter cells, in contrast to polyQ aggregates. Interestingly, polyQ expansion did not affect the nuclear-cytoplasmic shuttling of ataxin-1 as proposed before.

Conclusions/significance: These results indicate that polyQ expansion does not necessarily lead to aggregate formation, and that the enhanced kinetics may affect the nuclear function of ataxin-1. The unexpected findings for a polyQ-expanded protein and their consequences for ongoing SCA1 research are discussed.

Show MeSH
Related in: MedlinePlus