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Reconstitution of a hierarchical +TIP interaction network controlling microtubule end tracking of dynein

Nature Cell Biology volume 16, pages 804–811 (2014)Cite this article

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Abstract

Growing microtubule end regions recruit a variety of proteins collectively termed +TIPs, which confer local functions to the microtubule cytoskeleton1. +TIPs form dynamic interaction networks whose behaviour depends on a number of potentially competitive and hierarchical interaction modes2. The rules that determine which of the various +TIPs are recruited to the limited number of available binding sites at microtubule ends remain poorly understood. Here we examined how the human dynein complex, the main minus-end-directed motor and an important +TIP (refs 2, 3, 4), is targeted to growing microtubule ends in the presence of different +TIP competitors. Using a total internal reflection fluorescence microscopy-based reconstitution assay, we found that a hierarchical recruitment mode targets the large dynactin subunit p150Glued to growing microtubule ends via EB1 and CLIP-170 in the presence of competing SxIP-motif-containing peptides. We further show that the human dynein complex is targeted to growing microtubule ends through an interaction of the tail domain of dynein with p150Glued. Our results highlight how the connectivity and hierarchy within dynamic +TIP networks are orchestrated.

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Figure 1: SxIP-motif-containing peptides disrupt EB1-mediated targeting of p150 to growing microtubule ends.
Figure 2: SxIP motif and CAP-Gly domain binding to EB1 is mutually exclusive.
Figure 3: CLIP-170 restores EB1-dependent end tracking of p150 in the presence of SxIP peptides.
Figure 4: EB1 and p150 target the human dynein complex to growing microtubule ends.

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Acknowledgements

We thank R. M. Buey for cloning of the p150–GCN4 construct, I. Lüke for help with insect cell culture and protein expressions, the Peptide Chemistry facility LRI for peptide synthesis, J. Roostalu for Atto488-labelled tubulin, N. Cade for microscopy support and critical reading of the manuscript, and H. Walden for useful advice on protein purification. C.D. and T.S. acknowledge financial support from the European Research Council (ERC project ID 323042) and the German Research Foundation (DFG SU 175/7-1). M.O.S. is supported by a grant from the Swiss National Science Foundation (310030B_138659).

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Authors and Affiliations

  1. London Research Institute, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK

    Christian Duellberg, Martina Trokter, Rupam Jha & Thomas Surrey

  2. European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

    Christian Duellberg, Martina Trokter & Thomas Surrey

  3. Department of Biology and Chemistry, Laboratory of Biomolecular Research, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland

    Indrani Sen & Michel O. Steinmetz

  4. Present address: Institute of Structural and Molecular Biology, University College London and Birkbeck, Malet Street, London WC1E 7HX, UK.,

    Martina Trokter

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Contributions

C.D. performed experiments, C.D., M.T., R.J. and I.S. prepared reagents, and C.D., M.O.S. and T.S. analysed data, designed research and wrote the manuscript.

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Correspondence to Thomas Surrey.

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Integrated supplementary information

Supplementary Figure 1 EB1 targets the neuronal splice variant of p150 to growing microtubule ends in vitro.

(a) Top: Schematics illustrating the difference between the tissue and neuronal p150 isoforms: insertion of a positively charged stretch of 20 amino acids, encoded by exons 5–7. Note: In the main figures, the addition ‘-T’ is omitted, because only experiments with the tissue form of p150 (except in Fig. 2d) are shown there. Bottom: Schematics illustrating the composition of the p150 constructs used for the experiments shown in this figure: mCherry labelled neuronal and tissue p150 isoforms containing the first 547 and 527 amino acids of p150 (mCherry-p150547-N and mCherry-p150527-T, ending at corresponding amino acid positions), respectively; GFP-labelled neuronal p150 isoform containing the first 218 amino acids (with the CAP-Gly domain and the basic domain) followed by the artificial dimerization domain GCN4 (p150218GCN4-N). This latter construct was made for comparison with previously published work4. (b) Illustration of the conversion of a time series (top) to a kymograph (‘space-time plot’, bottom). Example images and corresponding kymographs of a time series (corresponding to Fig. 1b) are shown for the individual fluorescent channels (middle and right) as well as their merge (left, with mCherry-p150 in green and Cy5-microtubules in red). (c) Left column: Dual (top) and single colour overview images (middle) and representative kymographs (bottom) of 125 nM mCherry-p150547-N (green) tracking growing Cy5-microtubule (red) ends in the presence of 150 nM unlabelled EB1. Remaining panels: Comparison of the microtubule binding strength of the two p150 isoforms in the absence of EB1: dual (top) and single (middle) colour overview images and a single colour time-averaged images (from 60 consecutive frames; bottom) showing 125 nM mCherry-p150547-N (green in merge, left pair of columns) or 125 nM mCherry-p150527-T (green in merge, right pair of columns) binding along Cy5-microtubules (red in merge) in Standard TIRFM buffer or standard TIRFM buffer without KCl, as indicated. (d) An overview dual colour TIRF microscopy image (left) and single and dual colour kymographs (right) of 10 nM GFP-p150218GCN4-N (green in merge) tracking growing ends of Cy5-microtubules (red in merge) in the presence of 150 nM unlabelled EB1. Standard TIRFM buffer was supplemented with additional 25 mM KCl and 85 mM K-acetate, which was necessary to keep this construct soluble and to reduce its overall microtubule binding strength. (e) Three colour experiment showing that mCherry-EB3 recruits Alexa647-p150547-N to growing microtubule ends. Left: overview images of 150 nM mCherry-EB3 (top, blue) and 75 nM Alexa647-p150547-N (bottom, green) on Cy5-microtubules (red) in Standard TIRFM buffer (the same image frames are shown). Right: Single colour and merged triple colour kymographs of a representative microtubule in the same experiment (same colour code in merge). Cy5-tubulin concentration was always 20 μM. Horizontal and vertical scale bars are 3 μm and 20 s, respectively. Taken together, these results show that despite the stronger binding of the neuronal p150 isoform to microtubules, both the neuronal and the tissue isoform can track growing microtubule ends in an EB dependent manner (irrespective of the construct of the neuronal isoform used and irrespective of whether EB1 or EB3 is used).

Supplementary Figure 2 CLIP-170 without a putative SxIP motif competes with SxIP peptides for EB1 binding at growing microtubule ends.

(a) Schematic of a mutated version of GFP-CLIP-170ΔC with a putative SxIP motif between the CAP-Gly domains changed to SxNN (GFP-CLIP-170ΔCSxNN). (b) Representative dual-colour images (top) and kymographs (bottom) of 75 nM GFP-CLIP-170ΔC (green, left) or 75 nM GFP-CLIP-170ΔCSxNN (green, right) in the presence of 150 nM unlabelled EB1 and either no additional SxIP peptide or with 15 μM unlabelled SxIP peptide, as indicated. Cy5-microtubules (red) elongated from immobilised seeds in the presence of 20 μM Cy5-tubulin in Standard TIRFM buffer. Horizontal and vertical scale bars are 3 μm and 20 s, respectively. (c) Quantification of fluorescence intensities of the GFP-CLIP-170ΔC and GFP-CLIP-170ΔCSxNN at microtubule end regions and on the microtubule lattice in the presence of unlabelled EB1 and in the presence or absence of competing unlabelled SxIP peptides. Error bars are standard errors of the mean (s.e.m.) and are derived from at least 250 individual frames per condition from 3 independent experiments. These experiments show that GFP-CLIP-170ΔC and GFP-CLIP-170ΔCSxNN are both recruited to growing microtubule ends by EB1 without a significant difference. GFP-CLIP-170ΔCSxNN is also displaced from EB1 at growing microtubule ends by an excess of SxIP peptides, similar to wild type GFP-CLIP-170ΔC. This demonstrates that the putative SxIP motif of CLIP-170 does not contribute significantly to binding of CLIP-170 to EB1, even in the context of the native flanking CLIP-170 domains. This confirms previous results with a short CLIP-170 sequence peptide32 and demonstrates that the CAP-Gly domains, and not the putative SxIP sequence in CLIP-170 are responsible for the competition between CLIP-170 and SxIP peptides for EB binding.

Supplementary Figure 3 An intact distal zinc knuckle of CLIP-170 is required for efficient interaction with the CAP-Gly domain of p150.

(a) Schematics of mCherry-EB3, of the GFP-labelled tissue p150 isoform containing the first 198 amino acids of p150 followed by the artificial dimerisation domain GCN4 (GFP-p150198GCN4-T), and of a construct comprising the final 40 amino acids of CLIP-170 containing the distal zinc knuckle domain and the C-terminal EEY/F motif (CLIP-C40). A thioredoxin tag on CLIP-C40 was added to improve solubility (solubility tag). (b) Single and triple colour kymographs showing the localisation of 75 nM mCherry-EB3 (red in merge) and 20 nM GFP-p150198GCN4-T (green in merge) on a Cy5-microtubule (blue in merge) in the presence of 660 nM unlabelled CLIP-C40 (C-terminal 40 amino acids of CLIP-170), used here as a sequestering agent, in the absence (left) and presence (right) of 5 mM EGTA. EGTA chelates zinc ions33 and is therefore expected to unfold the zinc knuckle domain of CLIP-170. Standard TIRFM buffer without or with 5 mM EGTA was used as indicated. We observe that the localisation of mCherry-EB3 (used here instead of EB1, because N-terminal fusions with fluorescent proteins tend to impair EB1, but not EB3 activity) to growing microtubule ends is unaffected by EGTA, but that GFP-p150198GCN4-T localises to microtubule ends only in the presence of EGTA. This demonstrates that CLIP-C40 efficiently sequesters GFP-p150218GCN4-T in solution only in the absence of EGTA when the distal zinc knuckle domain of CLIP-170 is properly folded. This supports the conclusion from Figs 1–3 that the CAP-Gly domain of p150 can either interact at microtubule ends with the C-terminal part of EBs or with the C-terminal part of EB-recruited CLIP-170. (c) Left: Representative overview images (top) and kymographs (bottom) showing 150 nM unlabelled EB1 targeting 10 nM p150218GCN4-N (green) to the ends of growing Cy5-microtubules (red) only in the absence (left), but not in the presence (right) of unlabelled 660 nM CLIP-170ΔC. Standard TIRFM buffer was supplemented with additional 25 mM KCl and 85 mM K-acetate, which was necessary to keep this construct soluble and to reduce its overall microtubule binding strength. This experiment provides evidence that the N-terminal part of CLIP-170 competes with p150 for EB1 binding at growing microtubule ends. Cy5-tubulin concentration was always 20 μM. Horizontal and vertical scale bars are 3 μm and 20 s, respectively. Right: The scheme illustrates the competition between the N-terminal part of CLIP-170 and p150 for EB1 binding at growing microtubule ends.

Supplementary Figure 4 CLIP-170-mediated recruitment of p150 does not require a direct interaction between EB1 and p150.

(a) Scheme of a fusion protein consisting of EB1 lacking its 4 most C-terminal amino acids followed by the 40 most C-terminal amino acids of CLIP-170 (EB1ΔEEY-CLIP-C40). This fusion protein contains the EBH domain that interacts with both SxIP peptides and with the CAP-Gly domain of p150, but lacks the terminal EEY sequence of EB1 known to also interact with the p150 CAP-Gly domain (see Table 1); instead of the terminal EEY sequence of EB1 the construct contains the distal zinc knuckle and the EEY/F tail motif of CLIP-170 that interact with the CAP-Gly domain of p150, but not with SxIP motif peptides. A thioredoxin tag was added to improve solubility. (b) Single and dual colour kymographs showing that 150 nM unlabelled EB1 (left column of panels) as well as 150 nM unlabelled EB1ΔEEY-CLIP-C40 (second column) recruits 20 nM GFP-p150198GCN4-T (top kymograph, green in bottom merge) to the ends of growing Cy5-microtubules (middle kymograph, and red in bottom merge). This interaction cannot be strongly suppressed by the additional presence of a large excess of 60 μM unlabelled SxIP peptide (far right), although SxIP peptides are recruited by the EB1ΔEEY-CLIP-C40 fusion protein (third column of panels) as demonstrated by end tracking of 150 nM Fluo-SxIP (top kymograph, green in bottom merge). This means that the interaction between the zinc knuckle domain of the EB1ΔEEY-CLIP-C40 fusion protein and the p150 CAP-Gly domain is sufficient to rescue recruitment of p150 in the presence of competing SxIP motif peptides (see Fig. 3) that occupy the EBH domain of EB1. The EEY/F motif of EB1 that is absent in the EB1ΔEEY-CLIP-C40 fusion is not required for this rescue. Representative two or single-colour kymographs for each condition are depicted. ‘488’ indicates excitation at 488 nm which excites either GFP fused to p150 constructs or fluorescein covalently linked to SxIP peptide. Cy5-tubulin concentration was 20 μM. Experiments were performed in TIRFM buffer without EGTA (Methods). Horizontal and vertical scale bars are 3 μm and 20 s, respectively. (c) Scheme illustrating non-competitive binding of SxIP peptides and p150 to the EB1ΔEEY-CLIP-C40 fusion protein: SxIP peptides bind to the EBH domain and the CAP-Gly domain of p150 binds to the sequence corresponding to the C-terminal part of CLIP-170.

Supplementary Figure 5 Purity of recombinant proteins.

Coomassie-stained SDS-PAGE gels of the purified proteins, as indicated. The double band seen for mCherry constructs is likely due to different maturation forms of mCherry as reported previously. Mass spectrometry analysis supported this by showing that both bands correspond to the expected construct and not to a potential contaminant or truncated protein (data not shown).

Supplementary information

Supplementary Information

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EB1 targets p150 to microtubule ends.

Time-lapse TIRF microscopy movie depicting the localisation of 125 nM mCherry-p150 (green) on dynamic Cy5-labelled microtubules (red) in the presence of 150 nM unlabelled EB1. This video corresponds to Fig. 1b. The time stamp (upper left corner) is in seconds. Scale bar: 5 μm. (AVI 17188 kb)

CLIP-170 restores EB1-dependent end tracking of p150 in the presence of SxIP peptides.

Time-lapse TIRF microscopy movies depicting the localisation of 75 nM mCherry-p150 (green) on dynamic Cy5-labelled microtubules (red) in the presence of 150 nM unlabelled EB1 and 6 μM SxNN control peptide (left), 6 μM SxIP peptide (middle) or 6 μM SxIP peptide with additional 75 nM GFP-CLIP-170 (right). Note: GFP-CLIP-170 is not shown here. This video corresponds to Fig. 3a. The time stamp (upper left corner) is in seconds. Scale bar: 5 μm. (AVI 30791 kb)

EB1 and p150 target the dynein complex to microtubule ends.

Time-lapse TIRF microscopy movies depicting the localisation of 14 nM mGFP-tagged human dynein complex (green) on dynamic Cy5-labelled microtubules (red) in the presence of 200 nM EB1 and 125 nM mCherry-p150 (A; left) or in the presence of 200 nM EB1 but without mCherry-p150 (B; middle). EB1 and mCherry-p150 fail to target a tail-less mGFP-tagged dimer of the dynein motor domain (green) to microtubule ends (C; right). Note: mCherry-p150 is not shown here. This video corresponds to Fig. 4b. The time stamp (upper left corner) is in seconds. Scale bar: 5 μm. (AVI 21020 kb)

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Duellberg, C., Trokter, M., Jha, R. et al. Reconstitution of a hierarchical +TIP interaction network controlling microtubule end tracking of dynein. Nat Cell Biol 16, 804–811 (2014). https://doi.org/10.1038/ncb2999

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