The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity

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

The actin-microtubule cross-linking activity of Drosophila Short stop is regulated by intramolecular inhibition

Actin and microtubule dynamics must be precisely coordinated during cell migration, mitosis, and morphogenesis—much of this coordination is mediated by proteins that physically bridge the two cytoskeletal networks. We have investigated the regulation of the Drosophila actin-microtubule cross-linker Short stop (Shot), a member of the spectraplakin family. Our data suggest that Shot's cytoskeletal cross-linking activity is regulated by an intramolecular inhibitory mechanism. In its inactive conformation, Shot adopts a “closed” conformation through interactions between its NH2-terminal actin-binding domain and COOH-terminal EF-hand-GAS2 domain. This inactive conformation is targeted to the growing microtubule plus end by EB1. On activation, Shot binds along the microtubule through its COOH-terminal GAS2 domain and binds to actin with its NH2-terminal tandem CH domains. We propose that this mechanism allows Shot to rapidly cross-link dynamic microtubules in response to localized activating signals at the cell cortex

The Spectraplakin Short Stop Is an Actin-Microtubule Cross-Linker That Contributes to Organization of the Microtubule Network

The dynamics of actin and microtubules are coordinated in a variety of cellular and morphogenetic processes; however, little is known about the molecules mediating this cytoskeletal cross-talk. We are studying Short stop (Shot), the sole Drosophila spectraplakin, as a model actin–microtubule cross-linking protein. Spectraplakins are an ancient family of giant cytoskeletal proteins that are essential for a diverse set of cellular functions; yet, we know little about the dynamics of spectraplakins and how they bridge actin filaments and microtubules. In this study we describe the intracellular dynamics of Shot and a structure–function analysis of its role as a cytoskeletal cross-linker. We find that Shot interacts with microtubules using two different mechanisms. In the cell interior, Shot binds growing plus ends through an interaction with EB1. In the cell periphery, Shot associates with the microtubule lattice via its GAS2 domain, and this pool of Shot is actively engaged as a cross-linker via its NH2-terminal actin-binding calponin homology domains. This cross-linking maintains microtubule organization by resisting forces that produce lateral microtubule movements in the cytoplasm. Our results provide the first description of the dynamics of these important proteins and provide key insight about how they function during cytoskeletal cross-talk

Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration

Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. Here we show that the Drosophila Katanin, Dm-Kat60, functions to generate a dynamic cortical-microtubule interface in interphase cells. In S2 cells, Dm-Kat60 concentrates at the interphase cell cortex where it suppresses the polymerization of microtubule plus-ends thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes to the leading edge migratory D17 cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes MTs from their ends. Based on these data, we propose that Dm-Kat60 removes tubulin from microtubule ends or lattice that contact specific cortical sites to preventing stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in MT behaviors involved in cell migration

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

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

Inducible expression of Kat60 results in measurable microtubule disassembly in our single-cell assay.

<p>(A and B) Histograms of normalized levels of alpha-tubulin (Left) and fold overexpression levels of Kat60 (Middle) in <i>Drosophila</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123912#pone.0123912.s005" target="_blank">S1 Table</a> 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.</p

Depletion of Kat80 reduces steady-state Kat60 levels in cells.

<p>(A and B) Immunoblots of <i>Drosophila</i> S2 cell lysates prepared from cells stably expressing GFP alone (A) or GFP and copper-inducible FLAG-Kat80 (B) that were treated with control (lane 1), Kat60 CDS (lane 2), or Kat80 CDS dsRNA (lane 3) for 7 days total. The cells described in B were also treated with 0.1 mM CuSO₄ for 20 hours. Molecular weights (in Kd) are shown on the left.</p

Kat60 lacking the MIT domain does not disassemble microtubules at low levels of accumulation in cells.

<p>(A-D) Immunofluorescence microscopy images of <i>Drosophila</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123912#pone.0123912.s007" target="_blank">S3 Table</a> for summary statistics of the single-cell measurements collected).</p

Kat60 lacking the MIT domain disassembles microtubules at high levels of accumulation in cells.

<p>(A-D) Histograms of normalized levels of alpha-tubulin (Left) and fold overexpression levels of Kat60 (Middle) in <i>Drosophila</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123912#pone.0123912.s006" target="_blank">S2 Table</a> 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.</p

Model for regulation of <i>Drosophila</i> katanin by its non-catalytic domains.

<p>Schematic representation of the proposed contributions of the non-catalytic domains of <i>Drosophila</i> katanin to its proteasome-dependent degradation and microtubule association. Converging solid lines indicate cooperation between the MIT domain and linker region of Kat60 and solid lines with arrowheads indicate enhancement of the proteasome-dependent degradation and microtubule association of Kat60 by its MIT domain and linker region. The solid line with a blunt arrowhead indicates reduction of the proteasome-dependent degradation of Kat60 by Kat80 and the dashed line with an arrowhead indicates alteration of the microtubule association of Kat60 by Kat80. See text for details.</p