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The non-catalytic domains of Drosophila katanin regulate its abundance and microtubule-disassembly activity.

Grode KD, Rogers SL - PLoS ONE (2015)

Bottom Line: First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation.The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60.Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

ABSTRACT
Microtubule severing is a biochemical reaction that generates an internal break in a microtubule and regulation of microtubule severing is critical for cellular processes such as ciliogenesis, morphogenesis, and meiosis and mitosis. Katanin is a conserved heterodimeric ATPase that severs and disassembles microtubules, but the molecular determinants for regulation of microtubule severing by katanin remain poorly defined. Here we show that the non-catalytic domains of Drosophila katanin regulate its abundance and activity in living cells. Our data indicate that the microtubule-interacting and trafficking (MIT) domain and adjacent linker region of the Drosophila katanin catalytic subunit Kat60 cooperate to regulate microtubule severing in two distinct ways. First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation. The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60. Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules. On the basis of our data, we propose that the non-catalytic domains of Drosophila katanin serve as the principal sites of integration of regulatory inputs, thereby controlling its ability to sever and disassemble microtubules.

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Inducible expression of Kat60 results in measurable microtubule disassembly in our single-cell assay.(A and B) Histograms of normalized levels of alpha-tubulin (Left) and fold overexpression levels of Kat60 (Middle) in Drosophila S2 cells stably expressing GFP and copper-inducible Kat60 (A) or FLAG-Kat80 (B) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A and B were also treated with 0 (light gray), 0.01 (medium gray), 0.1 (dark gray), or 1.0 mM CuSO4 (black) for 20 hours and immunostained for alpha-tubulin and Kat60. Normalized levels of alpha-tubulin are expressed as a percentage of the mean levels of alpha-tubulin in cells stably expressing GFP alone that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. Fold overexpression levels of Kat60 are expressed as a fraction of the difference in the mean levels of Kat60 between cells stably expressing GFP alone that were treated with control and both Kat60 and Kat80 UTR dsRNA for 7 days total. Data are pooled from three independent experiments (see S1 Table for summary statistics of the single-cell measurements collected). (Right) Immunoblots of cell lysates prepared from the cells described in A and B. Molecular weights (in Kd) are shown on the left.
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pone.0123912.g003: Inducible expression of Kat60 results in measurable microtubule disassembly in our single-cell assay.(A and B) Histograms of normalized levels of alpha-tubulin (Left) and fold overexpression levels of Kat60 (Middle) in Drosophila S2 cells stably expressing GFP and copper-inducible Kat60 (A) or FLAG-Kat80 (B) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A and B were also treated with 0 (light gray), 0.01 (medium gray), 0.1 (dark gray), or 1.0 mM CuSO4 (black) for 20 hours and immunostained for alpha-tubulin and Kat60. Normalized levels of alpha-tubulin are expressed as a percentage of the mean levels of alpha-tubulin in cells stably expressing GFP alone that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. Fold overexpression levels of Kat60 are expressed as a fraction of the difference in the mean levels of Kat60 between cells stably expressing GFP alone that were treated with control and both Kat60 and Kat80 UTR dsRNA for 7 days total. Data are pooled from three independent experiments (see S1 Table for summary statistics of the single-cell measurements collected). (Right) Immunoblots of cell lysates prepared from the cells described in A and B. Molecular weights (in Kd) are shown on the left.

Mentions: To establish baseline single-cell measurements for the effects of inducible expression of Kat60 and Kat80 on microtubules, we used our assay to measure the loss of microtubules in cells stably expressing GFP and copper-inducible Kat60 or FLAG-Kat80 that were induced with increasing concentrations of CuSO4. Cells induced to express Kat60 had reduced levels of alpha-tubulin compared to control cells and the degree of reduction increased with increasing concentrations of CuSO4 over a 100-fold CuSO4 concentration range (Fig 3A and S1 Table). Appropriately, Kat60 accumulated at levels that increased with increasing concentrations of CuSO4 in these cells. As expected, cells induced to express FLAG-Kat80 had almost identical levels of alpha-tubulin and Kat60 relative to control cells and FLAG-Kat80 accumulated at levels that increased with increasing concentrations of CuSO4 in these cells (Fig 3B and S1 Table). Collectively, these data demonstrate that inducible expression of Kat60, but not Kat80, results in measurable microtubule disassembly in our single-cell assay.


The non-catalytic domains of Drosophila katanin regulate its abundance and microtubule-disassembly activity.

Grode KD, Rogers SL - PLoS ONE (2015)

Inducible expression of Kat60 results in measurable microtubule disassembly in our single-cell assay.(A and B) Histograms of normalized levels of alpha-tubulin (Left) and fold overexpression levels of Kat60 (Middle) in Drosophila S2 cells stably expressing GFP and copper-inducible Kat60 (A) or FLAG-Kat80 (B) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A and B were also treated with 0 (light gray), 0.01 (medium gray), 0.1 (dark gray), or 1.0 mM CuSO4 (black) for 20 hours and immunostained for alpha-tubulin and Kat60. Normalized levels of alpha-tubulin are expressed as a percentage of the mean levels of alpha-tubulin in cells stably expressing GFP alone that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. Fold overexpression levels of Kat60 are expressed as a fraction of the difference in the mean levels of Kat60 between cells stably expressing GFP alone that were treated with control and both Kat60 and Kat80 UTR dsRNA for 7 days total. Data are pooled from three independent experiments (see S1 Table for summary statistics of the single-cell measurements collected). (Right) Immunoblots of cell lysates prepared from the cells described in A and B. Molecular weights (in Kd) are shown on the left.
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pone.0123912.g003: Inducible expression of Kat60 results in measurable microtubule disassembly in our single-cell assay.(A and B) Histograms of normalized levels of alpha-tubulin (Left) and fold overexpression levels of Kat60 (Middle) in Drosophila S2 cells stably expressing GFP and copper-inducible Kat60 (A) or FLAG-Kat80 (B) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A and B were also treated with 0 (light gray), 0.01 (medium gray), 0.1 (dark gray), or 1.0 mM CuSO4 (black) for 20 hours and immunostained for alpha-tubulin and Kat60. Normalized levels of alpha-tubulin are expressed as a percentage of the mean levels of alpha-tubulin in cells stably expressing GFP alone that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. Fold overexpression levels of Kat60 are expressed as a fraction of the difference in the mean levels of Kat60 between cells stably expressing GFP alone that were treated with control and both Kat60 and Kat80 UTR dsRNA for 7 days total. Data are pooled from three independent experiments (see S1 Table for summary statistics of the single-cell measurements collected). (Right) Immunoblots of cell lysates prepared from the cells described in A and B. Molecular weights (in Kd) are shown on the left.
Mentions: To establish baseline single-cell measurements for the effects of inducible expression of Kat60 and Kat80 on microtubules, we used our assay to measure the loss of microtubules in cells stably expressing GFP and copper-inducible Kat60 or FLAG-Kat80 that were induced with increasing concentrations of CuSO4. Cells induced to express Kat60 had reduced levels of alpha-tubulin compared to control cells and the degree of reduction increased with increasing concentrations of CuSO4 over a 100-fold CuSO4 concentration range (Fig 3A and S1 Table). Appropriately, Kat60 accumulated at levels that increased with increasing concentrations of CuSO4 in these cells. As expected, cells induced to express FLAG-Kat80 had almost identical levels of alpha-tubulin and Kat60 relative to control cells and FLAG-Kat80 accumulated at levels that increased with increasing concentrations of CuSO4 in these cells (Fig 3B and S1 Table). Collectively, these data demonstrate that inducible expression of Kat60, but not Kat80, results in measurable microtubule disassembly in our single-cell assay.

Bottom Line: First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation.The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60.Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

ABSTRACT
Microtubule severing is a biochemical reaction that generates an internal break in a microtubule and regulation of microtubule severing is critical for cellular processes such as ciliogenesis, morphogenesis, and meiosis and mitosis. Katanin is a conserved heterodimeric ATPase that severs and disassembles microtubules, but the molecular determinants for regulation of microtubule severing by katanin remain poorly defined. Here we show that the non-catalytic domains of Drosophila katanin regulate its abundance and activity in living cells. Our data indicate that the microtubule-interacting and trafficking (MIT) domain and adjacent linker region of the Drosophila katanin catalytic subunit Kat60 cooperate to regulate microtubule severing in two distinct ways. First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation. The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60. Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules. On the basis of our data, we propose that the non-catalytic domains of Drosophila katanin serve as the principal sites of integration of regulatory inputs, thereby controlling its ability to sever and disassemble microtubules.

Show MeSH