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J Cell Sci. 2018 Sep 27. pii: jcs.219550. doi: 10.1242/jcs.219550. [Epub ahead of print]

Spatial positioning of EB family proteins at microtubule tips involves distinct nucleotide-dependent binding properties.

Author information

1
Centre for Mechanochemical Cell Biology, University of Warwick, Coventry, CV4 7AL, UK.
2
Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, CV4 7AL, UK.
3
Molecular Organisation and Assembly in Cells (MOAC) Doctoral Training Centre, University of Warwick, Coventry, CV4 7AL, UK.
4
Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK.
5
Current address: University of Cambridge Metabolic Research Laboratories, Addenbrooke's Hospital, Cambridge, CB2 0QQ, UK.
6
Centre for Mechanochemical Cell Biology, University of Warwick, Coventry, CV4 7AL, UK anne@mechanochemistry.org.

Abstract

EB proteins track the ends of growing microtubules and regulate microtubule dynamics both directly and by acting as the hub of the tip-tracking network. Mammalian cells express cell type-specific combinations of three EB proteins with different cellular roles. Here we reconstitute EB1, EB2 and EB3 tip tracking in vitro We find that all three EBs show rapid exchange at the microtubule tip and that their signal correlates to the microtubule assembly rate. However, the three signals differ in their maxima and the position from the microtubule tip. Using microtubules built with nucleotide analogues and site-directed mutagenesis, we show that EB2 prefers binding to microtubule lattices containing a 1:1 mixture of different nucleotides and its distinct binding specificity is conferred by amino acid substitutions at the right-hand side interface of the EB microtubule-binding domain with tubulin. Our data are consistent with the model that all three EB paralogs sense the nucleotide state of both β-tubulins flanking their binding site. Their different profile of preferred binding sites contributes to occupying spatially distinct domains at the temporally evolving microtubule tip structure.

KEYWORDS:

End-binding proteins; MAPs; Microtubules; Nucleotide state; Tip tracking; Tubulin

PMID:
30262468
DOI:
10.1242/jcs.219550
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