Abstract
Plus-end-tracking proteins (+TIPs) are localized at the fast-growing, or plus end, of microtubules, and link microtubule ends to cellular structures. One of the best studied +TIPs is EB1, which forms comet-like structures at the tips of growing microtubules. The molecular mechanisms by which EB1 recognizes and tracks growing microtubule ends are largely unknown. However, one clue is that EB1 can bind directly to a microtubule end in the absence of other proteins. Here we use an in vitro assay for dynamic microtubule growth with two-color total-internal-reflection-fluorescence imaging to investigate binding of mammalian EB1 to both stabilized and dynamic microtubules. We find that under conditions of microtubule growth, EB1 not only tip tracks, as previously shown, but also preferentially recognizes the GMPCPP microtubule lattice as opposed to the GDP lattice. The interaction of EB1 with the GMPCPP microtubule lattice depends on the E-hook of tubulin, as well as the amount of salt in solution. The ability to distinguish different nucleotide states of tubulin in microtubule lattice may contribute to the end-tracking mechanism of EB1.
Highlights
Microtubules are dynamic cytoskeletal polymers that play important roles in intracellular transport, mitosis and cell polarity [1]
Because the lattice of microtubule extensions grown with GTP is known to consist of GDP-tubulin [8], these findings suggest that EB1-GFP can distinguish between the different nucleotide states of tubulin in seeds and extensions
We show that EB1 can distinguish the nucleotide state of tubulin in the microtubule lattice
Summary
Microtubules are dynamic cytoskeletal polymers that play important roles in intracellular transport, mitosis and cell polarity [1]. Defined by their localization at microtubule fast-growing (‘plus’) end, plus-endbinding proteins (+TIPs) regulate a variety of dynamic processes associated with this microtubule end and link microtubule ends to cellular structures such as kinetochores and the cell cortex [2]. EB1 is a highly conserved +TIP, which locates to the ends of polymerizing microtubules in vivo [3]. It directly interacts with many other +TIPs and is potentially central to the assembly of +TIP complexes at microtubule ends [4]. The specifics of this structural feature are not known and several possibilities remain open
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