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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 cortactin 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 Flag vector or Flag-cortactin wild-type (WT), 9KQ, or 9KR, serum-starved for 24 hours, and stained with acetylated α-tubulin and Flag antibodies 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 GFP-HDAC6 and Flag vector or Flag-cortactin WT, 9KQ, or 9KR, serum-starved for 24 hours, and stained with acetylated α-tubulin and Flag antibodies and DAPI. Scale bar, 5 μm. *P < 0.05, ***P < 0.001; ns, not significant. Error bars indicate SEM.
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f7: Deacetylation of cortactin 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 Flag vector or Flag-cortactin wild-type (WT), 9KQ, or 9KR, serum-starved for 24 hours, and stained with acetylated α-tubulin and Flag antibodies 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 GFP-HDAC6 and Flag vector or Flag-cortactin WT, 9KQ, or 9KR, serum-starved for 24 hours, and stained with acetylated α-tubulin and Flag antibodies and DAPI. Scale bar, 5 μm. *P < 0.05, ***P < 0.001; ns, not significant. Error bars indicate SEM.

Mentions: Cortactin is known to interact with filamentous actin (F-actin) and promote actin polymerization323334. In addition, the acetylation of cortactin is known to impede its interaction with F-actin20. However, it is unknown whether the deacetylation of cortactin is involved in the action of HDAC6 to induce ciliary disassembly. To explore this question, RPE1 cells were transfected with Flag-cortactin wild-type or mutants, including 9KQ (mutation of all nine of the repeat-region lysines to glutamines to mimic acetylation) and 9KR (mutation of all nine of the repeat-region lysines to glutamines to disrupt acetylation). 9KQ is known to be unable to interact with F-actin, and 9KR is known to preserve the ability to interact with F-actin20. We found that transfection of RPE1 cells with Flag-cortactin wild-type or 9KR significantly decreased the percentage of ciliated cells and the length of cilia, compared with transfection with Flag vector (Fig. 7A–C). By contrast, transfection with 9KQ slightly decreased ciliary length and did not obviously affect the percentage of ciliated cells (Fig. 7A–C).


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 cortactin 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 Flag vector or Flag-cortactin wild-type (WT), 9KQ, or 9KR, serum-starved for 24 hours, and stained with acetylated α-tubulin and Flag antibodies 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 GFP-HDAC6 and Flag vector or Flag-cortactin WT, 9KQ, or 9KR, serum-starved for 24 hours, and stained with acetylated α-tubulin and Flag antibodies and DAPI. Scale bar, 5 μm. *P < 0.05, ***P < 0.001; ns, not significant. Error bars indicate SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f7: Deacetylation of cortactin 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 Flag vector or Flag-cortactin wild-type (WT), 9KQ, or 9KR, serum-starved for 24 hours, and stained with acetylated α-tubulin and Flag antibodies 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 GFP-HDAC6 and Flag vector or Flag-cortactin WT, 9KQ, or 9KR, serum-starved for 24 hours, and stained with acetylated α-tubulin and Flag antibodies and DAPI. Scale bar, 5 μm. *P < 0.05, ***P < 0.001; ns, not significant. Error bars indicate SEM.
Mentions: Cortactin is known to interact with filamentous actin (F-actin) and promote actin polymerization323334. In addition, the acetylation of cortactin is known to impede its interaction with F-actin20. However, it is unknown whether the deacetylation of cortactin is involved in the action of HDAC6 to induce ciliary disassembly. To explore this question, RPE1 cells were transfected with Flag-cortactin wild-type or mutants, including 9KQ (mutation of all nine of the repeat-region lysines to glutamines to mimic acetylation) and 9KR (mutation of all nine of the repeat-region lysines to glutamines to disrupt acetylation). 9KQ is known to be unable to interact with F-actin, and 9KR is known to preserve the ability to interact with F-actin20. We found that transfection of RPE1 cells with Flag-cortactin wild-type or 9KR significantly decreased the percentage of ciliated cells and the length of cilia, compared with transfection with Flag vector (Fig. 7A–C). By contrast, transfection with 9KQ slightly decreased ciliary length and did not obviously affect the percentage of ciliated cells (Fig. 7A–C).

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.