Format

Send to

Choose Destination

See 1 citation found by title matching your search:

See comment in PubMed Commons below
Eur J Cell Biol. 2016 Dec;95(12):521-530. doi: 10.1016/j.ejcb.2016.08.004. Epub 2016 Aug 31.

Insights into the process of EB1-dependent tip-tracking of kinesin-14 Ncd. The role of the microtubule.

Author information

  • 1Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093 Warsaw, Poland. Electronic address: e.szczesna@nencki.gov.pl.
  • 2Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093 Warsaw, Poland. Electronic address: a.kasprzak@nencki.gov.pl.

Abstract

End-binding proteins are capable of tracking the plus-ends of growing microtubules (MTs). The motor protein Ncd, a member of the kinesin-14 family, interacts with EB1 protein and becomes a non-autonomous tip-tracker. Here, we attempted to find out whether at least for Ncd, the efficient EB1-mediated tip-tracking involves the interaction of the kinesin with the MT surface. We prepared a series of Ncd tail mutants in which the MT-binding sites were altered or eliminated. Using TIRF microscopy, we characterized their behavior as tip-trackers and measured the dwell times of single molecules of EB1 and Ncd tail or its mutated forms. The mutated forms of Ncd tail exhibited tip-tracking in the presence of EB1 and the effectiveness of this process was proportional to the affinity of the mutant's tail to MT. Even though the interaction of Ncd with EB1 was weak (Kd∼9μM) the half saturating concentration of EB1 for tip-tracking was 7nM. The dwell time of Ncd tail in the presence of EB1 was ∼1s. The dwell time of EB1 alone was shorter (∼0.3s) and increased considerably in the presence of a large excess of Ncd tail. We demonstrated that tip-tracking of kinesin-14 occurs through several concurrent mechanisms: binding of kinesin only to EB1 located at the MT end, interaction of the kinesin molecules with a composite site formed by EB1 and the MT tip, and probably surface diffusion of the tail along MT. The second mechanism seems to play a crucial role in efficient tip-tracking.

KEYWORDS:

+TIPs; EB1; Kinesin-14; Microtubule plus end; Ncd; Tip-tracking

PMID:
27608966
DOI:
10.1016/j.ejcb.2016.08.004
[PubMed - indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Elsevier Science
    Loading ...
    Support Center