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Phosphatase inhibitor 2 promotes acetylation of tubulin in the primary cilium of human retinal epithelial cells.

Wang W, Brautigan DL - BMC Cell Biol. (2008)

Bottom Line: Knockdown of I-2 by siRNA significantly reduced the acetylation of microtubules in cilia, without a net decrease in whole cell tubulin acetylation.There was a reduction in the percentage of I-2 knockdown cells with a primary cilium, but no apparent alteration in the cilium length, suggesting no change in microtubule-based transport processes.Rescue of tubulin acetylation in I-2 knockdown cells by different chemical inhibitors shows that deacetylases and phosphatases are functionally interconnected to regulate microtubules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Cell Signaling, University of Virginia, School of Medicine, Charlottesville, 22908, USA.

ABSTRACT

Background: Primary cilia are flagella-like projections from the centriole of mammalian cells that have a key role in cell signaling. Human diseases are linked to defects in primary cilia. Microtubules make up the axoneme of cilia and are selectively acetylated and this is thought to contribute to the stability of the structure. However, mechanisms to regulate tubulin acetylation in cilia are poorly understood.

Results: Endogenous phosphatase inhibitor-2 (I-2) was found concentrated in cilia of human epithelial cells, and was localized to cilia early in the process of formation, prior to the full acetylation of microtubules. Knockdown of I-2 by siRNA significantly reduced the acetylation of microtubules in cilia, without a net decrease in whole cell tubulin acetylation. There was a reduction in the percentage of I-2 knockdown cells with a primary cilium, but no apparent alteration in the cilium length, suggesting no change in microtubule-based transport processes. Inhibition of either histone deacetylases with trichostatin A, or protein phosphatase-1 with calyculin A in I-2 knockdown cells partially rescued the acetylation of microtubules in cilia and the percentage of cells with a primary cilium.

Conclusion: The regulatory protein I-2 localizes to the primary cilium where it affects both Ser/Thr phosphorylation and is required for full tubulin acetylation. Rescue of tubulin acetylation in I-2 knockdown cells by different chemical inhibitors shows that deacetylases and phosphatases are functionally interconnected to regulate microtubules. As a multifunctional protein, I-2 may link cell cycle progression to structure and stability of the primary cilium.

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Rescue of I-2 knockdown phenotype by chemical inhibition of PP1 or HDAC. (A) ARPE-19 cells were transfected with siRNA as described in Figure 6. Cells were treated with 0.5 μM TSA or 0.5 nM calyculin A at 24 hr after siRNA transfection and 48 hrs later were fixed and immunostained for acetylated tubulin. Percentage of cells possessing a primary cilium with full or reduced levels of tubulin acetylation was counted as described in Figure 6. (B) Wide field images to show the acetylated tubulin staining (red) and DNA staining (blue) in cells treated with control siRNA, I-2 siRNA, I-2 siRNA + 0.5 nM calyculin A, or I-2 siRNA + 0.5 μM TSA. Scale bar represents 20 micron.
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Figure 8: Rescue of I-2 knockdown phenotype by chemical inhibition of PP1 or HDAC. (A) ARPE-19 cells were transfected with siRNA as described in Figure 6. Cells were treated with 0.5 μM TSA or 0.5 nM calyculin A at 24 hr after siRNA transfection and 48 hrs later were fixed and immunostained for acetylated tubulin. Percentage of cells possessing a primary cilium with full or reduced levels of tubulin acetylation was counted as described in Figure 6. (B) Wide field images to show the acetylated tubulin staining (red) and DNA staining (blue) in cells treated with control siRNA, I-2 siRNA, I-2 siRNA + 0.5 nM calyculin A, or I-2 siRNA + 0.5 μM TSA. Scale bar represents 20 micron.

Mentions: One approach was to inhibit multiple HDACs using the relatively non-specific inhibitor trichostatin A (TSA). Treatment with TSA partially restored to 50% the fraction of I-2 knockdown cells that formed a primary cilium, and had more dramatic and significant effect in increasing to > 30% the fraction of cells with full tubulin acetylation in the primary cilium (Fig. 8). Thus, inhibition of HDACs partially compensated for knockdown of I-2.


Phosphatase inhibitor 2 promotes acetylation of tubulin in the primary cilium of human retinal epithelial cells.

Wang W, Brautigan DL - BMC Cell Biol. (2008)

Rescue of I-2 knockdown phenotype by chemical inhibition of PP1 or HDAC. (A) ARPE-19 cells were transfected with siRNA as described in Figure 6. Cells were treated with 0.5 μM TSA or 0.5 nM calyculin A at 24 hr after siRNA transfection and 48 hrs later were fixed and immunostained for acetylated tubulin. Percentage of cells possessing a primary cilium with full or reduced levels of tubulin acetylation was counted as described in Figure 6. (B) Wide field images to show the acetylated tubulin staining (red) and DNA staining (blue) in cells treated with control siRNA, I-2 siRNA, I-2 siRNA + 0.5 nM calyculin A, or I-2 siRNA + 0.5 μM TSA. Scale bar represents 20 micron.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2630314&req=5

Figure 8: Rescue of I-2 knockdown phenotype by chemical inhibition of PP1 or HDAC. (A) ARPE-19 cells were transfected with siRNA as described in Figure 6. Cells were treated with 0.5 μM TSA or 0.5 nM calyculin A at 24 hr after siRNA transfection and 48 hrs later were fixed and immunostained for acetylated tubulin. Percentage of cells possessing a primary cilium with full or reduced levels of tubulin acetylation was counted as described in Figure 6. (B) Wide field images to show the acetylated tubulin staining (red) and DNA staining (blue) in cells treated with control siRNA, I-2 siRNA, I-2 siRNA + 0.5 nM calyculin A, or I-2 siRNA + 0.5 μM TSA. Scale bar represents 20 micron.
Mentions: One approach was to inhibit multiple HDACs using the relatively non-specific inhibitor trichostatin A (TSA). Treatment with TSA partially restored to 50% the fraction of I-2 knockdown cells that formed a primary cilium, and had more dramatic and significant effect in increasing to > 30% the fraction of cells with full tubulin acetylation in the primary cilium (Fig. 8). Thus, inhibition of HDACs partially compensated for knockdown of I-2.

Bottom Line: Knockdown of I-2 by siRNA significantly reduced the acetylation of microtubules in cilia, without a net decrease in whole cell tubulin acetylation.There was a reduction in the percentage of I-2 knockdown cells with a primary cilium, but no apparent alteration in the cilium length, suggesting no change in microtubule-based transport processes.Rescue of tubulin acetylation in I-2 knockdown cells by different chemical inhibitors shows that deacetylases and phosphatases are functionally interconnected to regulate microtubules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Cell Signaling, University of Virginia, School of Medicine, Charlottesville, 22908, USA.

ABSTRACT

Background: Primary cilia are flagella-like projections from the centriole of mammalian cells that have a key role in cell signaling. Human diseases are linked to defects in primary cilia. Microtubules make up the axoneme of cilia and are selectively acetylated and this is thought to contribute to the stability of the structure. However, mechanisms to regulate tubulin acetylation in cilia are poorly understood.

Results: Endogenous phosphatase inhibitor-2 (I-2) was found concentrated in cilia of human epithelial cells, and was localized to cilia early in the process of formation, prior to the full acetylation of microtubules. Knockdown of I-2 by siRNA significantly reduced the acetylation of microtubules in cilia, without a net decrease in whole cell tubulin acetylation. There was a reduction in the percentage of I-2 knockdown cells with a primary cilium, but no apparent alteration in the cilium length, suggesting no change in microtubule-based transport processes. Inhibition of either histone deacetylases with trichostatin A, or protein phosphatase-1 with calyculin A in I-2 knockdown cells partially rescued the acetylation of microtubules in cilia and the percentage of cells with a primary cilium.

Conclusion: The regulatory protein I-2 localizes to the primary cilium where it affects both Ser/Thr phosphorylation and is required for full tubulin acetylation. Rescue of tubulin acetylation in I-2 knockdown cells by different chemical inhibitors shows that deacetylases and phosphatases are functionally interconnected to regulate microtubules. As a multifunctional protein, I-2 may link cell cycle progression to structure and stability of the primary cilium.

Show MeSH
Related in: MedlinePlus