An unconventional interaction between Dis1/TOG and Mal3/EB1 in fission yeast promotes the fidelity of chromosome segregation - PubMed (original) (raw)

. 2016 Dec 15;129(24):4592-4606.

doi: 10.1242/jcs.197533. Epub 2016 Nov 21.

Affiliations

An unconventional interaction between Dis1/TOG and Mal3/EB1 in fission yeast promotes the fidelity of chromosome segregation

Yuzy Matsuo et al. J Cell Sci. 2016.

Abstract

Dynamic microtubule plus-ends interact with various intracellular target regions such as the cell cortex and the kinetochore. Two conserved families of microtubule plus-end-tracking proteins, the XMAP215, ch-TOG or CKAP5 family and the end-binding 1 (EB1, also known as MAPRE1) family, play pivotal roles in regulating microtubule dynamics. Here, we study the functional interplay between fission yeast Dis1, a member of the XMAP215/TOG family, and Mal3, an EB1 protein. Using an in vitro microscopy assay, we find that purified Dis1 autonomously tracks growing microtubule ends and is a bona fide microtubule polymerase. Mal3 recruits additional Dis1 to microtubule ends, explaining the synergistic enhancement of microtubule dynamicity by these proteins. A non-canonical binding motif in Dis1 mediates the interaction with Mal3. X-ray crystallography shows that this new motif interacts in an unconventional configuration with the conserved hydrophobic cavity formed within the Mal3 C-terminal region that typically interacts with the canonical SXIP motif. Selectively perturbing the Mal3-Dis1 interaction in living cells demonstrates that it is important for accurate chromosome segregation. Whereas, in some metazoans, the interaction between EB1 and the XMAP215/TOG family members requires an additional binding partner, fission yeast relies on a direct interaction, indicating evolutionary plasticity of this critical interaction module.

Keywords: Crystallography; EB1; Microtubule polymerase; TIRF microscopy; TOG; XMAP215.

© 2016. Published by The Company of Biologists Ltd.

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Conflict of interest statement

The authors declare no competing or financial interests.

Figures

Fig. 1.

Fig. 1.

Fission yeast Dis1 is a MT polymerase. (A) The MT-binding activity of Dis1–eGFP. Dis1–eGFP concentrations were kept constant at 0.2 µM, while the tubulin concentration was varied (from left to right, 0, 1, 2 and 4 µM). Both supernatant (S) and pellet (P) were stained with Coomassie Brilliant Blue. (B) Tubulin-binding activity of Dis1–eGFP. 5 µM Dis1–eGFP (green), 10 µM tubulin dimers (blue) and their mixture (red) were fractionated on gel filtration columns. (C) Schematic of the assay (top) and dual-colour TIRF-M kymographs showing binding events between 10 nM Dis1–eGFP (green in merge) and the ends of growing MTs. (D) Dual-colour TIRF-M kymographs showing Cy5-labelled MTs growing in the absence or presence of Dis1–eGFP (green). (E) Plot of the mean growth rate. The Cy5-labelled tubulin concentration was 8 µM in C–E. Data points, black; error bars are s.e.m., _n_=50. (F) Kymographs showing GMPCPP-stabilised Cy5-labelled MTs. Scale bars: 5 µm (horizontal) and 1 min (vertical) (C,D,F).

Fig. 2.

Fig. 2.

Dis1 directly binds to Mal3. (A) Binding between Dis1 and Mal3. GST or GST–Mal3 bound to glutathione beads was mixed with Dis1–eGFP. (B) Analytical gel filtration chromatography. 5 µM Dis1–HA (green), 20 µM Mal3 (blue) or the mixture of both proteins (red) was analysed. (C) TIRF-M kymographs showing binding events between 20 nM Dis1–eGFP (green) and the ends of growing Cy5-labelled MTs (red) in the absence or presence of unlabelled Mal3. The Cy5-labelled tubulin concentration was 20 µM. Snapshots are shown at the bottom. (D) Schematic of the assay (top) and triple-colour TIRF-M kymographs showing binding events between 10 nM Dis1–eGFP (green in merge) and the ends of growing Cy5-labelled MTs (blue in merge) in the presence of 200 nM Mal3–mCherry (red in merge) (bottom). (E) 10 nM Dis1–eGFP accumulates at the ends of a MT in a similar manner to 200 nM Mal3–mCherry. The Cy5-labelled tubulin concentration was 10 µM in D and E. Scale bars: 5 µm (horizontal) and 1 min (vertical) (C–E). (F) Plot of the mean growth rate. 10 nM Dis1 and/or 20 nM Mal3 were added in the presence of 8 µM tubulin. Data points, black; error bars are s.e.m., _n_=50.

Fig. 3.

Fig. 3.

The C-terminal tail region of Dis1 is the primary binding site for Mal3. (A) A schematic representation and a summary of their binding to Mal3. TOG1, TOG2, TOG homology domains; SK rich, rich in serine and lysine residues; CC, coiled-coil. (B) Binding between various truncated Dis1 proteins and Mal3. GST–Mal3 bound to glutathione beads was mixed with truncated Dis1–eGFP. The vertical line found between the last two lanes on the right was introduced unintentionally during scanning of the original data. (C) Binding between the full-length or tail-less Dis1 protein and Mal3. (D) Peptide array analysis. The arrays cover from 505th to 882nd amino acid residues of Dis1. The arrays were incubated with a solution containing Mal3–HA protein, followed by immunoblotting against an anti-HA antibody. Amino acid sequences corresponding to bound residues (red) are shown on the bottom. (E) Binding between the tail region of Dis1 and Mal3. GST–Mal3 bound to the glutathione beads were pre-incubated with various concentrations of the Dis1 peptide (amino acids 833–852) (from left to right, 0, 0.5, 2.5, 5.0, 50, 100 µM) for 30 min before incubation with 0.5 µM Dis1 C2–eGFP for an additional hour. (F) Alanine-scanning mutagenesis analysis. Each position in the Dis1 peptide was replaced with alanine. Amino acid residues shown in red were sensitive to alterations. (G) Binding between Mal3 and the wild-type (WT) or mutated Dis1 protein. Dis1–eGFP proteins were LAPA (L841A and P844A) or LAPAFA (L841A, P844A and F847A).

Fig. 4.

Fig. 4.

The coiled-coil and EBH domains of Mal3 are necessary and sufficient for binding to Dis1. (A) A schematic representation of various truncated Mal3 proteins and a summary of their binding to Dis1. CH, calponin-homology domain; CC, coiled-coil; EBH, EB homology. (B,C) Binding between various truncated Mal3 proteins and Dis1. Dis1 C1 (amino acids 520–882) tagged with eGFP was mixed with various GST-tagged truncated Mal3 proteins (B, FL, N, C1 or C, C2, C3), which were bound to the glutathione beads. (D) Binding between the dimerised EBH domain of Mal3 protein and Dis1. EBH-domain-containing Mal3 protein (amino acids 174–247) was mixed with wild-type (WT) or mutated Dis1-C2–eGFP (LAPA; L841A, P844A) and pulled down by GFP trap beads.

Fig. 5.

Fig. 5.

Physical binding between Dis1 and Mal3 is required for the synergistic impact on MT dynamics. (A) TIRF-M kymographs showing Dis1–eGFP (10 nM, green in merge) in the presence or absence of 20 nM Mal3. The Cy5-labelled tubulin (red) concentration was 8 µM. Scale bars: 5 µm (horizontal) and 1 min (vertical). (B,C) Plot of the mean growth rate (B) or catastrophe frequency (C). 10 nM Dis1-eGFP [wild-type (WT) or LAPA] and/or 20 nM Mal3 were added in the presence of 8 µM tubulin. Data points, black; error bars are s.e.m. _n_=50.

Fig. 6.

Fig. 6.

Molecular details of the Mal3–Dis1 interaction. (A) Ribbon diagram of the complex between the coiled-coil and EBH domains of Mal3 (residues 174–247 forming a homodimer as depicted in blue and pink) and the interacting Dis1 peptide (residues 833–852 as depicted in green). A second peptide binds on the other side of the dimer, but has been omitted from the figure for the sake of clarity. (B) Details of the key interacting residues. Surface conservation plot of the EBH domain of Mal3 from low (blue) to high (purple) similarity. (C,D) Close-up view of the interaction seen between the Mal3-EBH dimer (residues 174–247 depicted in blue and pink) and the Dis1 peptide (residues 833–852 depicted in green) (C) or the human EB1-EBH dimer (residues 191–260 depicted in yellow and green) and the SXIP motif containing the MACF peptide (residues 5468–5497 depicted in orange) (D). (E) Cartoon showing the comparison of the interaction interfaces between Mal3–Dis1 (left) and EB1–MCAF (right). A,A′ and B,B′ represent the EBH domain derived from two dimerised Mal3 molecules.

Fig. 7.

Fig. 7.

Dis1 interacts with Mal3 in vivo and its interaction is crucial for Dis1 function. (A) Interaction between Dis1 and Mal3. Whole-cell extracts (1.0 mg) were pulled down with GFP trap beads. Immunoblotting was performed with anti-GFP and anti-FLAG antibodies. Input: 15 µg. WT, wild type. (B) Summary of genetic interaction. Solid lines, synthetically lethal; dotted line, viable. (C) Tetrad dissection. Five representative tetrads are shown [the parental ditype (1), tetratypes (2–4) and the non-parental ditype (5)]. Double mutants were inviable (red circles). (D) Spot assay. Indicated strains were serially diluted (tenfold each), spotted on YES plates and incubated for 4 days or at 26°C for 4 days in the presence of 10 µg/ml TBZ (right). (E) Mitotic progression and MT dynamics in dis1-LAPA cells. Mitotic progression of wild-type (left, _n_=38) and dis1-LAPA (right, _n_=31) cells containing a tubulin marker (mCherry–Atb2) was followed in live cells under fluorescence microscopy at room temperature (23°C). Phase I (red), the initial stage of spindle elongation; phase II (yellow), the pre-anaphase stage with constant spindle length; Phase III (blue), anaphase B (Nabeshima et al., 1998). Results are mean±s.d.; _n_>30 cells; *P<0.01; n.s., not significant (two-tailed unpaired Student's _t_-tests). (F) Minichromosome loss assay. Indicated strains carrying the minichromosome Ch16 (Niwa et al., 1989) were grown on rich YE plates (lacking adenine) and incubated at 25°C for 6 days. The percentage of red and/or sectored colonies is shown at the bottom (_n_≥1,000).

Fig. 8.

Fig. 8.

Interplay between Dis1 and Mal3 and comparison to metazoans. (A) In fission yeast, Dis1 (orange) binds directly to Mal3 (blue), thereby enhancing the MT growth rate and the catastrophe frequency in a synergistic manner (left). By contrast, in metazoans including humans and Drosophila, ch-TOG and Msps (orange) interact with EB1 (blue) through SLAIN2 and Sentin (yellow), respectively (right), thereby accelerating MT dynamics. Dimer formation of EB1/Mal3 is not shown for simplicity. (B) Summary of individual parameters representing MT dynamics in the presence of Dis1 (wild-type or LAPA) and/or Mal3. –, not altered compared to control samples (tubulin only); blue arrow, increased by up to twofold; green arrow, increased by ∼threefold; red arrow, increased by more than fourfold.

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