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Deacetylation of α-tubulin and cortactin is required for HDAC6 to trigger ciliary disassembly.

Ran J, Yang Y, Li D, Liu M, Zhou J - Sci Rep (2015)

Bottom Line: Ciliary dysfunction is associated with a variety of diseases known as ciliopathies.Overexpression of HDAC6 decreases the levels of acetylated α-tubulin and cortactin without affecting the expression or localization of known ciliary regulators.These findings provide mechanistic insight into the ciliary role of HDAC6 and underscore the importance of reversible acetylation in regulating ciliary homeostasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China.

ABSTRACT
Cilia play important roles in sensing extracellular signals and directing fluid flow. Ciliary dysfunction is associated with a variety of diseases known as ciliopathies. Histone deacetylase 6 (HDAC6) has recently emerged as a major driver of ciliary disassembly, but little is known about the downstream players. Here we provide the first evidence that HDAC6-mediated deacetylation of α-tubulin and cortactin is critical for its induction of ciliary disassembly. HDAC6 is localized in the cytoplasm and enriched at the centrosome and basal body. Overexpression of HDAC6 decreases the levels of acetylated α-tubulin and cortactin without affecting the expression or localization of known ciliary regulators. We also find that overexpression of α-tubulin or cortactin or their acetylation-deficient mutants enhances the ability of HDAC6 to induce ciliary disassembly. In addition, acetylation-mimicking mutants of α-tubulin and cortactin counteract HDAC6-induced ciliary disassembly. Furthermore, HDAC6 stimulates actin polymerization, and inhibition of actin polymerization abolishes the activity of HDAC6 to trigger ciliary disassembly. These findings provide mechanistic insight into the ciliary role of HDAC6 and underscore the importance of reversible acetylation in regulating ciliary homeostasis.

No MeSH data available.


HDAC6 overexpression does not affect the expression or localization of known ciliary regulators.(A) Western blot analysis of the indicated ciliary regulators, acetylated α-tubulin, α-tubulin, and GFP in RPE1 cells transfected with GFP vector or GFP-HDAC6. (B) Immunofluorescence images of RPE1 cells transfected with GFP vector or GFP-HDAC6, serum-starved for 24 hours, and stained with antibodies against the indicated ciliary regulators and acetylated α-tubulin and DAPI. Scale bar, 5 μm.
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f5: HDAC6 overexpression does not affect the expression or localization of known ciliary regulators.(A) Western blot analysis of the indicated ciliary regulators, acetylated α-tubulin, α-tubulin, and GFP in RPE1 cells transfected with GFP vector or GFP-HDAC6. (B) Immunofluorescence images of RPE1 cells transfected with GFP vector or GFP-HDAC6, serum-starved for 24 hours, and stained with antibodies against the indicated ciliary regulators and acetylated α-tubulin and DAPI. Scale bar, 5 μm.

Mentions: We then sought to identify downstream proteins that mediate the ciliary role of HDAC6. We first analyzed the effect of HDAC6 overexpression on the expression of several key ciliary regulators, including centrosomal protein 110 (CP110), intraflagellar transport protein 88 (IFT88), IFT140, Meckel syndrome 1 (MKS1), ninein, and centrosomal protein 164 (Cep164)2728293031. Western blot analysis revealed that transfection of RPE1 cells with GFP-HDAC6 did not obviously affect the expression of these ciliary regulators, although GFP-HDAC6 significantly decreased α-tubulin acetylation, compared with transfection with GFP vector (Fig. 5A). Immunofluorescence microscopy further showed that overexpression of HDAC6 did not alter the localization of these ciliary regulators, although it clearly resulted in the shortening of ciliary length (Fig. 5B). These results suggest that HDAC6 is likely to induce ciliary disassembly via mechanisms independent of these known ciliary regulators. We also found that siRNA-mediated depletion of HDAC6 expression did not affect the expression or localization of these ciliary regulators (Figure S1).


Deacetylation of α-tubulin and cortactin is required for HDAC6 to trigger ciliary disassembly.

Ran J, Yang Y, Li D, Liu M, Zhou J - Sci Rep (2015)

HDAC6 overexpression does not affect the expression or localization of known ciliary regulators.(A) Western blot analysis of the indicated ciliary regulators, acetylated α-tubulin, α-tubulin, and GFP in RPE1 cells transfected with GFP vector or GFP-HDAC6. (B) Immunofluorescence images of RPE1 cells transfected with GFP vector or GFP-HDAC6, serum-starved for 24 hours, and stained with antibodies against the indicated ciliary regulators and acetylated α-tubulin and DAPI. Scale bar, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526867&req=5

f5: HDAC6 overexpression does not affect the expression or localization of known ciliary regulators.(A) Western blot analysis of the indicated ciliary regulators, acetylated α-tubulin, α-tubulin, and GFP in RPE1 cells transfected with GFP vector or GFP-HDAC6. (B) Immunofluorescence images of RPE1 cells transfected with GFP vector or GFP-HDAC6, serum-starved for 24 hours, and stained with antibodies against the indicated ciliary regulators and acetylated α-tubulin and DAPI. Scale bar, 5 μm.
Mentions: We then sought to identify downstream proteins that mediate the ciliary role of HDAC6. We first analyzed the effect of HDAC6 overexpression on the expression of several key ciliary regulators, including centrosomal protein 110 (CP110), intraflagellar transport protein 88 (IFT88), IFT140, Meckel syndrome 1 (MKS1), ninein, and centrosomal protein 164 (Cep164)2728293031. Western blot analysis revealed that transfection of RPE1 cells with GFP-HDAC6 did not obviously affect the expression of these ciliary regulators, although GFP-HDAC6 significantly decreased α-tubulin acetylation, compared with transfection with GFP vector (Fig. 5A). Immunofluorescence microscopy further showed that overexpression of HDAC6 did not alter the localization of these ciliary regulators, although it clearly resulted in the shortening of ciliary length (Fig. 5B). These results suggest that HDAC6 is likely to induce ciliary disassembly via mechanisms independent of these known ciliary regulators. We also found that siRNA-mediated depletion of HDAC6 expression did not affect the expression or localization of these ciliary regulators (Figure S1).

Bottom Line: Ciliary dysfunction is associated with a variety of diseases known as ciliopathies.Overexpression of HDAC6 decreases the levels of acetylated α-tubulin and cortactin without affecting the expression or localization of known ciliary regulators.These findings provide mechanistic insight into the ciliary role of HDAC6 and underscore the importance of reversible acetylation in regulating ciliary homeostasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China.

ABSTRACT
Cilia play important roles in sensing extracellular signals and directing fluid flow. Ciliary dysfunction is associated with a variety of diseases known as ciliopathies. Histone deacetylase 6 (HDAC6) has recently emerged as a major driver of ciliary disassembly, but little is known about the downstream players. Here we provide the first evidence that HDAC6-mediated deacetylation of α-tubulin and cortactin is critical for its induction of ciliary disassembly. HDAC6 is localized in the cytoplasm and enriched at the centrosome and basal body. Overexpression of HDAC6 decreases the levels of acetylated α-tubulin and cortactin without affecting the expression or localization of known ciliary regulators. We also find that overexpression of α-tubulin or cortactin or their acetylation-deficient mutants enhances the ability of HDAC6 to induce ciliary disassembly. In addition, acetylation-mimicking mutants of α-tubulin and cortactin counteract HDAC6-induced ciliary disassembly. Furthermore, HDAC6 stimulates actin polymerization, and inhibition of actin polymerization abolishes the activity of HDAC6 to trigger ciliary disassembly. These findings provide mechanistic insight into the ciliary role of HDAC6 and underscore the importance of reversible acetylation in regulating ciliary homeostasis.

No MeSH data available.