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.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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).
Integrated supplementary information
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.
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.
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.
About this article
Nature Reviews Molecular Cell Biology (2015)