A microtubule interactome: complexes with roles in cell cycle and mitosis.

PLoS Biology
Julian R HughesJames G Wakefield

Abstract

The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi) screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division.

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Methods Mentioned

BETA
two-hybrid
Y2H
electrophoresis
transfection
Co-immunoprecipitation
immunoprecipitation

Software Mentioned

IPlab
Mascot Daemon
In
Osprey Network Visualisation Tool
Adobe Photoshop
MASCOT
house Perl script
ClustalW
ProteinLynx Global server
Melanie II

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