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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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Cultured Drosophila S2 cells overexpressing Kat60 exhibit microtubule-disassembly in a concentration-dependent manner.(A) Schematic of the domain structure of the Drosophila canonical katanin catalytic subunit Kat60 (Top) and the canonical katanin regulatory subunit Kat80 (Bottom). (B and C) Immunofluorescence microscopy images of Drosophila S2 cells overexpressing Kat60 (B) or Myc-Kat80 (C) and immunostained for alpha-tubulin and Kat60 (B) or Myc (C). (B) A cell not overexpressing Kat60 (First) and cells overexpressing Kat60 (Second-Fourth). (C) A cell not overexpressing Myc-Kat80 (First) and cells overexpressing Myc-Kat80 (Second-Fourth). Alpha-tubulin, Kat60, and Myc images in each panel are displayed with the same scaling.
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pone.0123912.g001: Cultured Drosophila S2 cells overexpressing Kat60 exhibit microtubule-disassembly in a concentration-dependent manner.(A) Schematic of the domain structure of the Drosophila canonical katanin catalytic subunit Kat60 (Top) and the canonical katanin regulatory subunit Kat80 (Bottom). (B and C) Immunofluorescence microscopy images of Drosophila S2 cells overexpressing Kat60 (B) or Myc-Kat80 (C) and immunostained for alpha-tubulin and Kat60 (B) or Myc (C). (B) A cell not overexpressing Kat60 (First) and cells overexpressing Kat60 (Second-Fourth). (C) A cell not overexpressing Myc-Kat80 (First) and cells overexpressing Myc-Kat80 (Second-Fourth). Alpha-tubulin, Kat60, and Myc images in each panel are displayed with the same scaling.

Mentions: The Drosophila genome contains single genes that encode the canonical katanin catalytic subunit, Kat60, and the canonical katanin regulatory subunit, Kat80 (Fig 1A). Previous studies have shown that Kat60 by itself can sever and disassemble microtubules in vitro [23,24] and that overexpression of Kat60 is sufficient to promote microtubule disassembly in cultured S2 cells [29] and in larval muscle cells [30]. To date, there have been no functional studies of Kat80. To begin our structure-function analysis of the microtubule-severing activity of Drosophila katanin, we first overexpressed Kat60 in S2 cells and examined the effects on microtubules by immunofluorescence microscopy. Cells overexpressing Kat60 displayed a disorganized array of fragmented microtubules and the overall length of microtubules appeared to decrease with increasing levels of Kat60 (Fig 1B). Many of these cells contained individual microtubules with both ends visible and several of these cells had only short microtubule fragments; cells that showed a complete loss of microtubules were never observed. In addition, cells overexpressing Kat60 had reduced levels of alpha-tubulin compared to control cells and the degree of reduction increased with increasing levels of Kat60. The inverse relationship between alpha-tubulin and Kat60 levels indicates that cells overexpressing Kat60 exhibit microtubule disassembly in a concentration-dependent manner and the disorganized array of short microtubules suggests that this disassembly is due to the microtubule-severing activity of Kat60. The effects of overexpression of Kat60 on microtubules and alpha-tubulin levels in S2 cells are similar to those observed in other animal cell types overexpressing the katanin catalytic subunit [15,31]. To determine if Kat80 possesses microtubule-disassembly activity, we overexpressed Myc-tagged Kat80 (Myc-Kat80) in cells and examined the effects on microtubules by immunofluorescence microscopy. Cells overexpressing Myc-Kat80 did not exhibit microtubule fragmentation or have reduced levels of alpha-tubulin (Fig 1C), consistent with a previous report that the sea urchin katanin regulatory subunit KATNB1 cannot sever and disassemble microtubules in vitro [25]. Taken together, these findings demonstrate that overexpression of Kat60, but not Kat80, is sufficient to promote microtubule disassembly in S2 cells and that the loss of microtubules in these cells occurs as a function of Kat60 levels.


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

Grode KD, Rogers SL - PLoS ONE (2015)

Cultured Drosophila S2 cells overexpressing Kat60 exhibit microtubule-disassembly in a concentration-dependent manner.(A) Schematic of the domain structure of the Drosophila canonical katanin catalytic subunit Kat60 (Top) and the canonical katanin regulatory subunit Kat80 (Bottom). (B and C) Immunofluorescence microscopy images of Drosophila S2 cells overexpressing Kat60 (B) or Myc-Kat80 (C) and immunostained for alpha-tubulin and Kat60 (B) or Myc (C). (B) A cell not overexpressing Kat60 (First) and cells overexpressing Kat60 (Second-Fourth). (C) A cell not overexpressing Myc-Kat80 (First) and cells overexpressing Myc-Kat80 (Second-Fourth). Alpha-tubulin, Kat60, and Myc images in each panel are displayed with the same scaling.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123912.g001: Cultured Drosophila S2 cells overexpressing Kat60 exhibit microtubule-disassembly in a concentration-dependent manner.(A) Schematic of the domain structure of the Drosophila canonical katanin catalytic subunit Kat60 (Top) and the canonical katanin regulatory subunit Kat80 (Bottom). (B and C) Immunofluorescence microscopy images of Drosophila S2 cells overexpressing Kat60 (B) or Myc-Kat80 (C) and immunostained for alpha-tubulin and Kat60 (B) or Myc (C). (B) A cell not overexpressing Kat60 (First) and cells overexpressing Kat60 (Second-Fourth). (C) A cell not overexpressing Myc-Kat80 (First) and cells overexpressing Myc-Kat80 (Second-Fourth). Alpha-tubulin, Kat60, and Myc images in each panel are displayed with the same scaling.
Mentions: The Drosophila genome contains single genes that encode the canonical katanin catalytic subunit, Kat60, and the canonical katanin regulatory subunit, Kat80 (Fig 1A). Previous studies have shown that Kat60 by itself can sever and disassemble microtubules in vitro [23,24] and that overexpression of Kat60 is sufficient to promote microtubule disassembly in cultured S2 cells [29] and in larval muscle cells [30]. To date, there have been no functional studies of Kat80. To begin our structure-function analysis of the microtubule-severing activity of Drosophila katanin, we first overexpressed Kat60 in S2 cells and examined the effects on microtubules by immunofluorescence microscopy. Cells overexpressing Kat60 displayed a disorganized array of fragmented microtubules and the overall length of microtubules appeared to decrease with increasing levels of Kat60 (Fig 1B). Many of these cells contained individual microtubules with both ends visible and several of these cells had only short microtubule fragments; cells that showed a complete loss of microtubules were never observed. In addition, cells overexpressing Kat60 had reduced levels of alpha-tubulin compared to control cells and the degree of reduction increased with increasing levels of Kat60. The inverse relationship between alpha-tubulin and Kat60 levels indicates that cells overexpressing Kat60 exhibit microtubule disassembly in a concentration-dependent manner and the disorganized array of short microtubules suggests that this disassembly is due to the microtubule-severing activity of Kat60. The effects of overexpression of Kat60 on microtubules and alpha-tubulin levels in S2 cells are similar to those observed in other animal cell types overexpressing the katanin catalytic subunit [15,31]. To determine if Kat80 possesses microtubule-disassembly activity, we overexpressed Myc-tagged Kat80 (Myc-Kat80) in cells and examined the effects on microtubules by immunofluorescence microscopy. Cells overexpressing Myc-Kat80 did not exhibit microtubule fragmentation or have reduced levels of alpha-tubulin (Fig 1C), consistent with a previous report that the sea urchin katanin regulatory subunit KATNB1 cannot sever and disassemble microtubules in vitro [25]. Taken together, these findings demonstrate that overexpression of Kat60, but not Kat80, is sufficient to promote microtubule disassembly in S2 cells and that the loss of microtubules in these cells occurs as a function of Kat60 levels.

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