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Regulation of spindle microtubule dynamics in Anaphase

Fachliche Zuordnung Zellbiologie
Förderung Förderung von 2004 bis 2014
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 5434019
 
Erstellungsjahr 2015

Zusammenfassung der Projektergebnisse

Genomic stability relies on proper assembly and function of the mitotic spindle to ensure faithful chromosome segregation. The formation of the mitotic spindle in early mitosis and sister chromatid segregation in late mitosis require global rearrangements of the microtubule cytoskeleton. My laboratory is interested to understand key mechanisms mediating the global switches in microtubule organisation during mitosis. In order to address this problem, we are using cell free extracts of Amphibian eggs, which faithfully reconstitute cell cycle regulation of microtubule behaviour. In this model system, we analysed both the principles underlying spindle assembly as well as the signals required for regulated disassembly of spindle microtubules for chromosome segregation at anaphase onset. Our initially suggested project focused on elucidating (1): which protein phosphatases control anaphase microtubule disassembly, and, (2): which microtubule regulators are targets of regulation by dephosphorylation in anaphase. To analyse the latter aspect, we not only directly tested several well known microtubule regulators in Xenopus cell free extracts but also included an unbiased approach using microtubule sedimentation followed by mass spectrometry to systematically survey the microtubule interactome in metaphase and anaphase. We carried out the following experiments: • We determined the localisation of XKCM1, Op18, XMAP215, EB1 and CLASP by indirect immunofluorescence in Xenopus extract spindles. • We performed microtubule spindown experiments in Xenopus egg extracts followed by immunoblot analysis of XKCM1, Op18, XMAP215, EB1 and CLASP, as well as mass spectrometry analysis of the entire microtubule interactome. • We carried out specific immunodepletions of XKCM1, Op18, XMAP215, EB1 and CLASP. Spindle localisation and microtubule binding were compared in metaphase and in an anaphase-like state as described previously. The relative amount of the microtubule regulators was investigated and compared to the overall microtubule mass. It remained unchanged between metaphase and anaphase for all proteins tested by Immunoblot. Even in systematic mass spectrometry analysis of microtubule spindowns in metaphase and anaphase, we could not detect a significant change in the abundance of a single or a subset of microtubule regulators between metaphase and anaphase. We still tested, using specific immunodepletion experiments, if the reduction of a given protein would destabilise microtubules and mimic anaphase-like behaviour. This was indeed the case for XMAP215, a key regulator of microtubule stability. Upon partial depletion of XMAP215 to ca. 30% residual levels, spindles in metaphase matched respective anaphase samples in terms of overall microtubule density, spindle size and pole-to-pole distribution of microtubules. We therefore analysed the interactome of XMAP215 in metaphase and anaphase using specific immunoprecipitation experiments. Although we identified interesting interaction partners such as EB1, no changes between metaphase and anaphase could be observed. Other proteins described to interact with XMAP215 in a cell cycle dependent manner, such as XKCM1, could not be detected. Moreover, we did not find evidence for a change in posttranslational modification of XMAP215 at the transition from metaphase to anaphase by mass spectrometry. Our goal was also to accompany experiments in cell free extracts with an siRNA-based screen for the identification of phosphatases required for proper anaphase spindle organisation in human tissue culture cells. Although having a promising start in setting up the technology for systematic siRNA-based screens in human cells, the latter project was threatened by a publication of a very similar approach from the laboratory of Daniel Gerlich. Here, our colleagues used their in-depth-expertise in live cell imaging and large scale RNAi screens to systematically investigate the role of human phosphatases in chromosome segregation in anaphase. Their experiments were very much in line with what we had hypothesised from our studies in the Xenopus system: PP2A with its regulatory subunit B55 α triggers key events in anaphase in human cells. Management of time and resources urged us, at this point, to shift the focus of the project, in particular as a parallel approach in our laboratory gained a lot of momentum. We therefore decided to re-focus on the analysis of novel microtubule regulators in spindle formation and function, identified in an alternative approach. In the alternative project, we made use of the systematic identification of potential microtubule regulators, which are generated by de novo synthesis during vertebrate oocyte maturation and entry into meiosis.

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