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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 or the MIT domain and linker region detectably colocalizes with only a few, if any, microtubules in cells.(A-D) TIRF microscopy images of living Drosophila S2 cells stably expressing RFP-alpha-tubulin and copper-inducible GFP-Kat60-K339A (A), GFP-Kat60-K339A and Myc-Kat80 (B), GFP-Kat60-ΔMIT-K339A (C), or GFP-Kat60-AAA-K339A (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 (A), 0.1 (B), 0.1 (C), or 0.01 mM CuSO4 (D) for 20 hours. GFP images are displayed with the same scaling.
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pone.0123912.g007: Kat60 lacking the MIT domain or the MIT domain and linker region detectably colocalizes with only a few, if any, microtubules in cells.(A-D) TIRF microscopy images of living Drosophila S2 cells stably expressing RFP-alpha-tubulin and copper-inducible GFP-Kat60-K339A (A), GFP-Kat60-K339A and Myc-Kat80 (B), GFP-Kat60-ΔMIT-K339A (C), or GFP-Kat60-AAA-K339A (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 (A), 0.1 (B), 0.1 (C), or 0.01 mM CuSO4 (D) for 20 hours. GFP images are displayed with the same scaling.

Mentions: To test the hypothesis that the non-catalytic domains of Drosophila katanin affect its association with microtubules, we first expressed GFP-Kat60-K339A alone or together with Myc-Kat80, GFP-Kat60-ΔMIT-K339A, or GFP-Kat60-AAA-K339A in cells. We then used our live-cell assay to examine the colocalization of each of these proteins with microtubules. GFP-Kat60-K339A colocalized with the majority of microtubules in cells and it exhibited a discontinuous and punctate localization pattern on these microtubules (Fig 7A). Interestingly, GFP-Kat60-K339A in the presence of Myc-Kat80 also colocalized with the majority of microtubules in cells, however it exhibited a continuous localization pattern on these microtubules (Fig 7B), suggesting that Kat80 alters the association of Kat60 with microtubules. In contrast, GFP-Kat60-ΔMIT-K339A and GFP-Kat60-AAA-K339A detectably colocalized with only a few, if any, microtubules in cells (Fig 7C and 7D), providing strong evidence that the MIT domain and linker region of Kat60 are required for its association with microtubules. From these results, we conclude that the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules.


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 or the MIT domain and linker region detectably colocalizes with only a few, if any, microtubules in cells.(A-D) TIRF microscopy images of living Drosophila S2 cells stably expressing RFP-alpha-tubulin and copper-inducible GFP-Kat60-K339A (A), GFP-Kat60-K339A and Myc-Kat80 (B), GFP-Kat60-ΔMIT-K339A (C), or GFP-Kat60-AAA-K339A (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 (A), 0.1 (B), 0.1 (C), or 0.01 mM CuSO4 (D) for 20 hours. GFP images are displayed with the same scaling.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123912.g007: Kat60 lacking the MIT domain or the MIT domain and linker region detectably colocalizes with only a few, if any, microtubules in cells.(A-D) TIRF microscopy images of living Drosophila S2 cells stably expressing RFP-alpha-tubulin and copper-inducible GFP-Kat60-K339A (A), GFP-Kat60-K339A and Myc-Kat80 (B), GFP-Kat60-ΔMIT-K339A (C), or GFP-Kat60-AAA-K339A (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 (A), 0.1 (B), 0.1 (C), or 0.01 mM CuSO4 (D) for 20 hours. GFP images are displayed with the same scaling.
Mentions: To test the hypothesis that the non-catalytic domains of Drosophila katanin affect its association with microtubules, we first expressed GFP-Kat60-K339A alone or together with Myc-Kat80, GFP-Kat60-ΔMIT-K339A, or GFP-Kat60-AAA-K339A in cells. We then used our live-cell assay to examine the colocalization of each of these proteins with microtubules. GFP-Kat60-K339A colocalized with the majority of microtubules in cells and it exhibited a discontinuous and punctate localization pattern on these microtubules (Fig 7A). Interestingly, GFP-Kat60-K339A in the presence of Myc-Kat80 also colocalized with the majority of microtubules in cells, however it exhibited a continuous localization pattern on these microtubules (Fig 7B), suggesting that Kat80 alters the association of Kat60 with microtubules. In contrast, GFP-Kat60-ΔMIT-K339A and GFP-Kat60-AAA-K339A detectably colocalized with only a few, if any, microtubules in cells (Fig 7C and 7D), providing strong evidence that the MIT domain and linker region of Kat60 are required for its association with microtubules. From these results, we conclude that the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules.

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