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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.


Deacetylation of α-tubulin is required for HDAC6-mediated ciliary disassembly.(A–C) Immunofluorescence images (A), percentage of ciliated cells (B), and ciliary length (C) in RPE1 cells transfected with GFP vector or GFP-α-tubulin wild-type (WT), K40Q, or K40R, serum-starved for 24 hours, and stained with acetylated α-tubulin antibody and DAPI. Scale bar, 5 μm. (D–F) Immunofluorescence images (D), percentage of ciliated cells (E), and ciliary length (F) in RPE1 cells transfected with HA-HDAC6 and GFP vector or GFP-α-tubulin WT, K40Q, or K40R, serum-starved for 24 hours, and stained with HA and acetylated α-tubulin antibodies and DAPI. Scale bar, 5 μm. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. Error bars indicate SEM.
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f6: Deacetylation of α-tubulin is required for HDAC6-mediated ciliary disassembly.(A–C) Immunofluorescence images (A), percentage of ciliated cells (B), and ciliary length (C) in RPE1 cells transfected with GFP vector or GFP-α-tubulin wild-type (WT), K40Q, or K40R, serum-starved for 24 hours, and stained with acetylated α-tubulin antibody and DAPI. Scale bar, 5 μm. (D–F) Immunofluorescence images (D), percentage of ciliated cells (E), and ciliary length (F) in RPE1 cells transfected with HA-HDAC6 and GFP vector or GFP-α-tubulin WT, K40Q, or K40R, serum-starved for 24 hours, and stained with HA and acetylated α-tubulin antibodies and DAPI. Scale bar, 5 μm. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. Error bars indicate SEM.

Mentions: To examine whether the level of α-tubulin acetylation is involved in ciliary homeostasis or is merely a ciliary marker, RPE1 cells were transfected with GFP-α-tubulin wild-type or mutants, including K40Q (mutation of lysine 40 to glutamine to mimic acetylation) and K40R (mutation of lysine 40 to arginine to disrupt acetylation). We found that transfection of RPE1 cells with GFP-α-tubulin wild-type or K40R significantly decreased the percentage of ciliated cells and the length of cilia, compared with transfection with GFP vector (Fig. 6A–C). By contrast, transfection with K40Q slightly decreased the percentage of ciliated cells and did not obviously affect ciliary length (Fig. 6A–C). Cotransfection of β-tubulin with α-tubulin resulted in the same phenotype as transfection of α-tubulin alone (Figure S2), ruling out the possibility that the expression of just α-tubulin causes a dominant effect on ciliary formation.


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)

Deacetylation of α-tubulin is required for HDAC6-mediated ciliary disassembly.(A–C) Immunofluorescence images (A), percentage of ciliated cells (B), and ciliary length (C) in RPE1 cells transfected with GFP vector or GFP-α-tubulin wild-type (WT), K40Q, or K40R, serum-starved for 24 hours, and stained with acetylated α-tubulin antibody and DAPI. Scale bar, 5 μm. (D–F) Immunofluorescence images (D), percentage of ciliated cells (E), and ciliary length (F) in RPE1 cells transfected with HA-HDAC6 and GFP vector or GFP-α-tubulin WT, K40Q, or K40R, serum-starved for 24 hours, and stained with HA and acetylated α-tubulin antibodies and DAPI. Scale bar, 5 μm. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. Error bars indicate SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526867&req=5

f6: Deacetylation of α-tubulin is required for HDAC6-mediated ciliary disassembly.(A–C) Immunofluorescence images (A), percentage of ciliated cells (B), and ciliary length (C) in RPE1 cells transfected with GFP vector or GFP-α-tubulin wild-type (WT), K40Q, or K40R, serum-starved for 24 hours, and stained with acetylated α-tubulin antibody and DAPI. Scale bar, 5 μm. (D–F) Immunofluorescence images (D), percentage of ciliated cells (E), and ciliary length (F) in RPE1 cells transfected with HA-HDAC6 and GFP vector or GFP-α-tubulin WT, K40Q, or K40R, serum-starved for 24 hours, and stained with HA and acetylated α-tubulin antibodies and DAPI. Scale bar, 5 μm. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. Error bars indicate SEM.
Mentions: To examine whether the level of α-tubulin acetylation is involved in ciliary homeostasis or is merely a ciliary marker, RPE1 cells were transfected with GFP-α-tubulin wild-type or mutants, including K40Q (mutation of lysine 40 to glutamine to mimic acetylation) and K40R (mutation of lysine 40 to arginine to disrupt acetylation). We found that transfection of RPE1 cells with GFP-α-tubulin wild-type or K40R significantly decreased the percentage of ciliated cells and the length of cilia, compared with transfection with GFP vector (Fig. 6A–C). By contrast, transfection with K40Q slightly decreased the percentage of ciliated cells and did not obviously affect ciliary length (Fig. 6A–C). Cotransfection of β-tubulin with α-tubulin resulted in the same phenotype as transfection of α-tubulin alone (Figure S2), ruling out the possibility that the expression of just α-tubulin causes a dominant effect on ciliary formation.

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.