Title page for ETD etd-10272009-084027

Type of Document Master's Thesis
Author Lonergan, Natalie Elaine
URN etd-10272009-084027
Title Characterizing the cargo binding and regulatory function of the tail domain in Ncd motor protein
Degree Master of Science
Department Biology
Advisory Committee
Advisor Name Title
Walker, Richard A. Committee Chair
Sible, Jill C. Committee Member
Wong, Eric A. Committee Member
  • non-claret disjunctional protein
  • nuclear localization signal
  • Kinesin-14 motor proteins
Date of Defense 2009-10-09
Availability unrestricted
Non-claret disjunctional (Ncd) is a kinesin-14 microtubule motor protein involved in the

assembly and stability of meiotic and mitotic spindles in Drosophila oocytes and early embryos,

respectively. Ncd functions by cross-linking microtubules through the tail and motor domains.

It was originally believed that the role of the Ncd tail domain was to only statically bind

microtubules. However, the Ncd tail domain has recently been shown to have properties that

stabilize and bundle microtubules, and contribute to the overall motility of the Ncd protein.

Continued characterization of the Ncd tail domain is essential to understanding the complete role

of Ncd in cell division. This work explored the regulatory function and microtubule binding

properties of the Ncd tail domain.

Ncd activity is regulated during interphase by nuclear sequestration. GFP-Ncd fusion

proteins, containing full length Ncd, individual Ncd domains, or combinations of Ncd domains,

were used to identify the presence of a nuclear localization signal (NLS) in the Ncd polypeptide.

The nuclear localization of only the GFP fusion proteins containing the Ncd tail sequence

indicates that the NLS is contained within the tail domain. Subsequent, experiments performed

with GFP fusion proteins containing segments of the tail domain indicate that essential NLS

amino acid segments may span the length of the tail domain.

Attempts to characterize the microtubule binding properties of the Ncd tail domain, using

bacterially expressed MBP-Ncd tail-stalk, were unsuccessful. MBP-Ncd tail-stalk proteins

aggregated under binding assay conditions, preventing an accurate determination of the

stoichiometric binding relationship between Ncd and the tubulin dimer.

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