Limits...
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.

Show MeSH
Kat60 lacking the MIT domain does not disassemble microtubules at low levels of accumulation in cells.(A-D) Immunofluorescence microscopy images of 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 0–1.0 (A), 0–1.0 (B), 0–0.01 (C), or 0–0.01 mM CuSO4 (D) for 20 hours and immunostained for alpha-tubulin and Kat60. Alpha-tubulin and Kat60 images in each panel are displayed with the same scaling. (E) Line graphs of normalized levels of alpha-tubulin as a function of fold overexpression levels of Kat60 for the cells described in A-D. Normalized levels of alpha-tubulin are expressed as a percentage of the mean levels of alpha-tubulin in cells with fold overexpression levels of Kat60 below 0. 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 represent mean values ± standard deviation from cells with fold overexpression levels of Kat60 between 0 and 40, pooled from six independent experiments (see S3 Table for summary statistics of the single-cell measurements collected).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4401518&req=5

pone.0123912.g006: Kat60 lacking the MIT domain does not disassemble microtubules at low levels of accumulation in cells.(A-D) Immunofluorescence microscopy images of 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 0–1.0 (A), 0–1.0 (B), 0–0.01 (C), or 0–0.01 mM CuSO4 (D) for 20 hours and immunostained for alpha-tubulin and Kat60. Alpha-tubulin and Kat60 images in each panel are displayed with the same scaling. (E) Line graphs of normalized levels of alpha-tubulin as a function of fold overexpression levels of Kat60 for the cells described in A-D. Normalized levels of alpha-tubulin are expressed as a percentage of the mean levels of alpha-tubulin in cells with fold overexpression levels of Kat60 below 0. 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 represent mean values ± standard deviation from cells with fold overexpression levels of Kat60 between 0 and 40, pooled from six independent experiments (see S3 Table for summary statistics of the single-cell measurements collected).

Mentions: To circumvent the limitations highlighted above and reassess the contribution of the non-catalytic domains of Drosophila katanin to its microtubule-disassembly activity, we first expressed Kat60 alone or together with FLAG-Kat80, Kat60-ΔMIT, or Kat60-AAA over a broad range of levels in cells. We then used immunofluorescence microscopy and our single-cell assay to carefully examine the effects on microtubules as a function of the levels of these proteins. Cells expressing Kat60 together with FLAG-Kat80 exhibited microtubule disassembly in a concentration-dependent manner analogous to cells expressing Kat60 alone (Fig 6A and 6B). Likewise, these cells had reduced levels of alpha-tubulin similar to cells expressing full-length Kat60 alone (Fig 6E and S3 Table), suggesting that Kat80 does not affect the ability of Kat60 to disassemble microtubules. By contrast, cells expressing Kat60-ΔMIT at low levels did not display noticeably disassembled microtubules, although cells expressing Kat60-ΔMIT at high levels showed partially disassembled microtubules similar to cells expressing Kat60 at low levels (Fig 6A and 6C). Accordingly, the former cells did not have notably reduced levels of alpha-tubulin, whereas the latter cells had equivalent levels of alpha-tubulin compared to cells expressing Kat60 at low levels (Fig 6E and S3 Table). Thus, in addition to providing strong evidence that the MIT domain of Kat60 is important for its ability to disassemble microtubules at low levels of accumulation, these data further underscore the unique concentration-dependent contribution of the MIT domain of Kat60 to its activity. As predicted from our previous observations, cells expressing Kat60-AAA did not exhibit microtubule disassembly or have reduced levels of alpha-tubulin like cells expressing full-length Kat60 (Fig 6A, 6D, and 6E and S3 Table), strongly suggesting that the MIT domain and linker region of Kat60 cooperate to augment its microtubule-disassembly activity.


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 does not disassemble microtubules at low levels of accumulation in cells.(A-D) Immunofluorescence microscopy images of 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 0–1.0 (A), 0–1.0 (B), 0–0.01 (C), or 0–0.01 mM CuSO4 (D) for 20 hours and immunostained for alpha-tubulin and Kat60. Alpha-tubulin and Kat60 images in each panel are displayed with the same scaling. (E) Line graphs of normalized levels of alpha-tubulin as a function of fold overexpression levels of Kat60 for the cells described in A-D. Normalized levels of alpha-tubulin are expressed as a percentage of the mean levels of alpha-tubulin in cells with fold overexpression levels of Kat60 below 0. 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 represent mean values ± standard deviation from cells with fold overexpression levels of Kat60 between 0 and 40, pooled from six independent experiments (see S3 Table for summary statistics of the single-cell measurements collected).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401518&req=5

pone.0123912.g006: Kat60 lacking the MIT domain does not disassemble microtubules at low levels of accumulation in cells.(A-D) Immunofluorescence microscopy images of 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 0–1.0 (A), 0–1.0 (B), 0–0.01 (C), or 0–0.01 mM CuSO4 (D) for 20 hours and immunostained for alpha-tubulin and Kat60. Alpha-tubulin and Kat60 images in each panel are displayed with the same scaling. (E) Line graphs of normalized levels of alpha-tubulin as a function of fold overexpression levels of Kat60 for the cells described in A-D. Normalized levels of alpha-tubulin are expressed as a percentage of the mean levels of alpha-tubulin in cells with fold overexpression levels of Kat60 below 0. 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 represent mean values ± standard deviation from cells with fold overexpression levels of Kat60 between 0 and 40, pooled from six independent experiments (see S3 Table for summary statistics of the single-cell measurements collected).
Mentions: To circumvent the limitations highlighted above and reassess the contribution of the non-catalytic domains of Drosophila katanin to its microtubule-disassembly activity, we first expressed Kat60 alone or together with FLAG-Kat80, Kat60-ΔMIT, or Kat60-AAA over a broad range of levels in cells. We then used immunofluorescence microscopy and our single-cell assay to carefully examine the effects on microtubules as a function of the levels of these proteins. Cells expressing Kat60 together with FLAG-Kat80 exhibited microtubule disassembly in a concentration-dependent manner analogous to cells expressing Kat60 alone (Fig 6A and 6B). Likewise, these cells had reduced levels of alpha-tubulin similar to cells expressing full-length Kat60 alone (Fig 6E and S3 Table), suggesting that Kat80 does not affect the ability of Kat60 to disassemble microtubules. By contrast, cells expressing Kat60-ΔMIT at low levels did not display noticeably disassembled microtubules, although cells expressing Kat60-ΔMIT at high levels showed partially disassembled microtubules similar to cells expressing Kat60 at low levels (Fig 6A and 6C). Accordingly, the former cells did not have notably reduced levels of alpha-tubulin, whereas the latter cells had equivalent levels of alpha-tubulin compared to cells expressing Kat60 at low levels (Fig 6E and S3 Table). Thus, in addition to providing strong evidence that the MIT domain of Kat60 is important for its ability to disassemble microtubules at low levels of accumulation, these data further underscore the unique concentration-dependent contribution of the MIT domain of Kat60 to its activity. As predicted from our previous observations, cells expressing Kat60-AAA did not exhibit microtubule disassembly or have reduced levels of alpha-tubulin like cells expressing full-length Kat60 (Fig 6A, 6D, and 6E and S3 Table), strongly suggesting that the MIT domain and linker region of Kat60 cooperate to augment its microtubule-disassembly activity.

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