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 Keywords
- non-claret disjunctional protein
- nuclear localization signal
- Kinesin-14 motor proteins
Date of Defense 2009-10-09 Availability unrestricted AbstractNon-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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