Abstract
Microtubules dramatically change their dynamics and organization at the entry into mitosis. Although this change is mediated by microtubule-associated proteins (MAPs), how MAPs themselves are regulated is not well understood. Here we used an integrated multi-level approach to establish the framework and biological significance of MAP regulation critical for the interphase/mitosis transition. Firstly, we applied quantitative proteomics to determine global cell cycle changes in the profiles of MAPs in human and Drosophila cells. This uncovered a wide range of cell cycle regulations of MAPs previously unidentified. Secondly, systematic studies of human kinesins highlighted an overlooked aspect of kinesins: most mitotic kinesins suppress their affinity to microtubules or reduce their protein levels in interphase in combination with nuclear localization. Thirdly, in-depth analysis of a novel Drosophila MAP (Mink) revealed that the suppression of the microtubule affinity of this mitotic MAP in combination with nuclear localization is essential for microtubule organization in interphase, and phosphorylation of Mink is needed for kinetochore-microtubule attachment in mitosis. Thus, this first comprehensive analysis of MAP regulation for the interphase/mitosis transition advances our understanding of kinesin biology and reveals the prevalence and importance of multi-layered MAP regulation.
Highlights
Microtubules dramatically change their dynamics and organization at the entry into mitosis
Systematic studies of human kinesins highlighted an overlooked aspect of kinesins: most mitotic kinesins suppress their affinity to microtubules or reduce their protein levels in interphase in combination with nuclear localization
SILAC-based Quantitative Proteomics to Identify Cellcycle-regulated microtubule-associated proteins (MAPs)—Our first goal was to systematically identify MAPs whose microtubule-binding activity or protein level differed in mitosis and interphase in human and Drosophila cells
Summary
Molecular and Protein Techniques—Gateway molecular cloning technology was used to generate entry and expression clones of Mink protein. Restriction digestion and selfligation resulted in the desired plasmid This second method with the inclusion of the human PKI␣ NES coding sequence within the forward primer was used for the generation of NES fusion proteins. Images of live transfected cells were obtained using a microscope (Axiovert, Carl Zeiss, Jena, Germany) attached to a spinning-disc confocal head (Yokogawa, Tokyo, Japan) controlled by Volocity (PerkinElmer Life Sciences) using a laser intensity of 3% and an exposure of 250 ms so expression levels could be assessed. Cells were visualized using a Zeiss Axioplan 2 microscope, and images were captured with a charge-coupled device camera (Hamamatsu, Hamamatsu, Japan) controlled by Openlab 2.2.1 software (PerkinElmer Life Sciences) or using a Plan-Apochromat lens (63ϫ, 1.4 numerical aperture, Zeiss, Jena, Germany) attached to an Axiovert 200 M (Zeiss) with a confocal scan head (LSM5 Exciter, Zeiss). Cells were stained before and after lysis with DAPI (1:50), and images were taken with an Axioplan 2 microscope (Zeiss) attached to a charge-coupled device camera (Hamamatsu) controlled by OpenLab 2.2.1 software (Improvision, Warwick, UK) and were processed with Photoshop (Adobe, San Jose, CA)
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