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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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Kat60 lacking the MIT domain disassembles microtubules at high levels of accumulation in cells.(A-D) 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), Kat60 and FLAG-Kat80 (B), Kat60-ΔMIT (C), or Kat60-AAA (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D 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 S2 Table for summary statistics of the single-cell measurements collected). (Right) Immunoblots of cell lysates prepared from the cells described in A-D. Molecular weights (in Kd) are shown on the left.
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pone.0123912.g004: Kat60 lacking the MIT domain disassembles microtubules at high levels of accumulation in cells.(A-D) 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), Kat60 and FLAG-Kat80 (B), Kat60-ΔMIT (C), or Kat60-AAA (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D 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 S2 Table for summary statistics of the single-cell measurements collected). (Right) Immunoblots of cell lysates prepared from the cells described in A-D. Molecular weights (in Kd) are shown on the left.

Mentions: To assess the contribution of the non-catalytic domains of Drosophila katanin to its microtubule-disassembly activity, we used our assay to measure the loss of microtubules in cells induced to express Kat60 alone or together with FLAG-Kat80, Kat60 lacking the MIT domain (Kat60-ΔMIT), or Kat60 lacking the MIT domain and linker region (Kat60-AAA). Compared to cells induced to express Kat60 alone, cells induced to express Kat60 together with FLAG-Kat80 had reduced levels of alpha-tubulin (Fig 4A and 4B and S2 Table), suggesting that Kat80 affects the microtubule-disassembly activity of Kat60. However, uninduced cells also had reduced levels of alpha-tubulin, thereby complicating an assessment of the contribution of Kat80 to the microtubule-disassembly activity of Kat60. In support of the notion that Kat80 increases the abundance of Kat60, Kat60 in the presence of FLAG-Kat80 accumulated at slightly higher levels than Kat60 alone in cells. Surprisingly, cells induced to express Kat60-ΔMIT either did not have reduced levels of alpha-tubulin or they had reduced levels of alpha-tubulin similar to cells induced to express Kat60 (Fig 4A and 4C and S2 Table), indicating that the MIT domain of Kat60 is, at the very least, dispensable for its microtubule-disassembly activity. Coincidentally, Kat60-ΔMIT either did not accumulate at detectable levels or it accumulated at notably higher levels than Kat60 in cells, suggesting that once expressed, the MIT domain of Kat60 functions to decrease its abundance. Thus, in addition to providing strong evidence that the MIT domain of Kat60 is dispensable for its microtubule-disassembly activity at high levels of accumulation, these results point to the need for alternative strategies to accurately assess the importance of the MIT domain of Kat60 to its activity. Consistent with the hypothesis that the non-catalytic domains of katanin are crucial to its microtubule-disassembly activity, cells induced to express Kat60-AAA did not have reduced levels of alpha-tubulin relative to cells induced to express Kat60 (Fig 4A and 4D and S2 Table). Remarkably, Kat60-AAA accumulated at higher levels than Kat60-ΔMIT in cells, strongly suggesting that the MIT domain and linker region of Kat60 cooperate to decrease its abundance.


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

Grode KD, Rogers SL - PLoS ONE (2015)

Kat60 lacking the MIT domain disassembles microtubules at high levels of accumulation in cells.(A-D) 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), Kat60 and FLAG-Kat80 (B), Kat60-ΔMIT (C), or Kat60-AAA (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D 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 S2 Table for summary statistics of the single-cell measurements collected). (Right) Immunoblots of cell lysates prepared from the cells described in A-D. Molecular weights (in Kd) are shown on the left.
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pone.0123912.g004: Kat60 lacking the MIT domain disassembles microtubules at high levels of accumulation in cells.(A-D) 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), Kat60 and FLAG-Kat80 (B), Kat60-ΔMIT (C), or Kat60-AAA (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D 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 S2 Table for summary statistics of the single-cell measurements collected). (Right) Immunoblots of cell lysates prepared from the cells described in A-D. Molecular weights (in Kd) are shown on the left.
Mentions: To assess the contribution of the non-catalytic domains of Drosophila katanin to its microtubule-disassembly activity, we used our assay to measure the loss of microtubules in cells induced to express Kat60 alone or together with FLAG-Kat80, Kat60 lacking the MIT domain (Kat60-ΔMIT), or Kat60 lacking the MIT domain and linker region (Kat60-AAA). Compared to cells induced to express Kat60 alone, cells induced to express Kat60 together with FLAG-Kat80 had reduced levels of alpha-tubulin (Fig 4A and 4B and S2 Table), suggesting that Kat80 affects the microtubule-disassembly activity of Kat60. However, uninduced cells also had reduced levels of alpha-tubulin, thereby complicating an assessment of the contribution of Kat80 to the microtubule-disassembly activity of Kat60. In support of the notion that Kat80 increases the abundance of Kat60, Kat60 in the presence of FLAG-Kat80 accumulated at slightly higher levels than Kat60 alone in cells. Surprisingly, cells induced to express Kat60-ΔMIT either did not have reduced levels of alpha-tubulin or they had reduced levels of alpha-tubulin similar to cells induced to express Kat60 (Fig 4A and 4C and S2 Table), indicating that the MIT domain of Kat60 is, at the very least, dispensable for its microtubule-disassembly activity. Coincidentally, Kat60-ΔMIT either did not accumulate at detectable levels or it accumulated at notably higher levels than Kat60 in cells, suggesting that once expressed, the MIT domain of Kat60 functions to decrease its abundance. Thus, in addition to providing strong evidence that the MIT domain of Kat60 is dispensable for its microtubule-disassembly activity at high levels of accumulation, these results point to the need for alternative strategies to accurately assess the importance of the MIT domain of Kat60 to its activity. Consistent with the hypothesis that the non-catalytic domains of katanin are crucial to its microtubule-disassembly activity, cells induced to express Kat60-AAA did not have reduced levels of alpha-tubulin relative to cells induced to express Kat60 (Fig 4A and 4D and S2 Table). Remarkably, Kat60-AAA accumulated at higher levels than Kat60-ΔMIT in cells, strongly suggesting that the MIT domain and linker region of Kat60 cooperate to decrease its abundance.

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