Diplom, Eberhard Karls Universitat Tubingen (2008)
Doctor of Philosophy, Eberhard Karls Universitat Tubingen (2015)
James Ferrell, Postdoctoral Faculty Sponsor
Eukaryotic cells inherit their genomes in the form of chromosomes, which are formed from the compaction of interphase chromatin by the condensin complex. Condensin is a member of the structural maintenance of chromosomes (SMC) family of ATPases, large ring-shaped protein assemblies that entrap DNA to establish chromosomal interactions. Here, we use the budding yeast Saccharomyces cerevisiae to dissect the role of the condensin ATPase and its relationship with cell-cycle-regulated chromosome binding dynamics. ATP hydrolysis-deficient condensin binds to chromosomes but is defective in chromosome condensation and segregation. By modulating the ATPase, we demonstrate that it controls condensin's dynamic turnover on chromosomes. Mitosis-specific phosphorylation of condensin's Smc4 subunit reduces the turnover rate. However, reducing turnover by itself is insufficient to compact chromosomes. We propose that condensation requires fine-tuned dynamic condensin interactions with more than one DNA. These results enhance our molecular understanding of condensin function during chromosome condensation.
View details for DOI 10.1016/j.celrep.2018.04.082
View details for Web of Science ID 000433052200010
View details for PubMedID 29791843
View details for PubMedCentralID PMC5986713
Cell-cycle phosphorylation is temporally ordered, at least in part, through the sequential expression of different cyclins. Recent studies by Swaffer et al. (2016) and Godfrey et al. (2017) show that intrinsic properties of the substrate proteins contribute as well: good kinase substrates tend to be phosphorylated early, and good phosphatase substrates tend to be phosphorylated late.
View details for DOI 10.1016/j.molcel.2017.01.025
View details for PubMedID 28157499
The separation of chromosomes in anaphase is a precarious step in the cell cycle. The separation is irreversible, and any error can lead to cell death or genetic instability. Chromosome separation is controlled by the protease separase. Here we discuss recent work that has revealed additional layers of separase regulation and has deepened our understanding of how separase activation is coordinated with other events of mitotic exit.
View details for DOI 10.1016/j.tcb.2016.07.008
View details for Web of Science ID 000392352800004
View details for PubMedID 27567180
The splitting of chromosomes in anaphase and their delivery into the daughter cells needs to be accurately executed to maintain genome stability. Chromosome splitting requires the degradation of securin, whereas the distribution of the chromosomes into the daughter cells requires the degradation of cyclin B. We show that cells encounter and tolerate variations in the abundance of securin or cyclin B. This makes the concurrent onset of securin and cyclin B degradation insufficient to guarantee that early anaphase events occur in the correct order. We uncover that the timing of chromosome splitting is not determined by reaching a fixed securin level, but that this level adapts to the securin degradation kinetics. In conjunction with securin and cyclin B competing for degradation during anaphase, this provides robustness to the temporal order of anaphase events. Our work reveals how parallel cell-cycle pathways can be temporally coordinated despite variability in protein concentrations.
View details for DOI 10.1016/j.molcel.2015.09.022
View details for Web of Science ID 000368286000014
View details for PubMedID 26527280
In eukaryotic cells, components of the 5' to 3' mRNA degradation machinery can undergo a rapid phase transition. The resulting cytoplasmic foci are referred to as processing bodies (P-bodies). The molecular details of the self-aggregation process are, however, largely undetermined. Herein, we use a bottom-up approach that combines NMR spectroscopy, isothermal titration calorimetry, X-ray crystallography, and fluorescence microscopy to probe if mRNA degradation factors can undergo phase transitions in?vitro. We show that the Schizosaccharomyces pombe Dcp2 mRNA decapping enzyme, its prime activator Dcp1, and the scaffolding proteins Edc3 and Pdc1 are sufficient to reconstitute a phase-separation process. Intermolecular interactions between the Edc3?LSm domain and at least 10?helical leucine-rich motifs in Dcp2 and Pdc1 build the core of the interaction network. We show that blocking of these interactions interferes with the clustering behavior, both in?vitro and in?vivo.
View details for DOI 10.1002/anie.201402885
View details for Web of Science ID 000338989500046
View details for PubMedID 24862735
View details for PubMedCentralID PMC4320757
Chromosome attachment to the mitotic spindle in early mitosis is guarded by an Aurora B kinase-dependent error correction mechanism [1, 2] and by the spindle assembly checkpoint (SAC), which delays cell-cycle progression in response to errors in chromosome attachment [3, 4]. The abrupt loss of sister chromatid cohesion at anaphase creates a type of chromosome attachment that in early mitosis would be recognized as erroneous, would elicit Aurora B-dependent destabilization of kinetochore-microtubule attachment, and would activate the checkpoint [5, 6]. However, in anaphase, none of these responses occurs, which is vital to ensure progression through anaphase and faithful chromosome segregation. The difference has been attributed to the drop in CDK1/cyclin B activity that accompanies anaphase and causes Aurora B translocation away from centromeres [7-12] and to the inactivation of the checkpoint by the time of anaphase [10, 11, 13, 14]. Here, we show that checkpoint inactivation may not be crucial because checkpoint activation by anaphase chromosomes is too slow to take effect on the timescale during which anaphase is executed. In addition, we observe that checkpoint activation can still occur for a considerable time after the anaphase-promoting complex/cyclosome (APC/C) becomes active, raising the question whether the checkpoint is indeed completely inactivated by the time of anaphase under physiologic conditions.
View details for DOI 10.1016/j.cub.2014.02.005
View details for Web of Science ID 000333233300024
View details for PubMedID 24583014
The spindle assembly checkpoint is a conserved signalling pathway that protects genome integrity. Given its central importance, this checkpoint should withstand stochastic fluctuations and environmental perturbations, but the extent of and mechanisms underlying its robustness remain unknown. We probed spindle assembly checkpoint signalling by modulating checkpoint protein abundance and nutrient conditions in fission yeast. For core checkpoint proteins, a mere 20% reduction can suffice to impair signalling, revealing a surprising fragility. Quantification of protein abundance in single cells showed little variability (noise) of critical proteins, explaining why the checkpoint normally functions reliably. Checkpoint-mediated stoichiometric inhibition of the anaphase activator Cdc20 (Slp1 in Schizosaccharomyces pombe) can account for the tolerance towards small fluctuations in protein abundance and explains our observation that some perturbations lead to non-genetic variation in the checkpoint response. Our work highlights low gene expression noise as an important determinant of reliable checkpoint signalling.
View details for DOI 10.1038/ncb2864
View details for Web of Science ID 000326680700009
View details for PubMedID 24161933
The Dcp1:Dcp2 decapping complex catalyses the removal of the mRNA 5' cap structure. Activator proteins, including Edc3 (enhancer of decapping 3), modulate its activity. Here, we solved the structure of the yeast Edc3 LSm domain in complex with a short helical leucine-rich motif (HLM) from Dcp2. The motif interacts with the monomeric Edc3 LSm domain in an unprecedented manner and recognizes a noncanonical binding surface. Based on the structure, we identified additional HLMs in the disordered C-terminal extension of Dcp2 that can interact with Edc3. Moreover, the LSm domain of the Edc3-related protein Scd6 competes with Edc3 for the interaction with these HLMs. We show that both Edc3 and Scd6 stimulate decapping in vitro, presumably by preventing the Dcp1:Dcp2 complex from adopting an inactive conformation. In addition, we show that the C-terminal HLMs in Dcp2 are necessary for the localization of the Dcp1:Dcp2 decapping complex to P-bodies in vivo. Unexpectedly, in contrast to yeast, in metazoans the HLM is found in Dcp1, suggesting that details underlying the regulation of mRNA decapping changed throughout evolution.
View details for DOI 10.1038/emboj.2011.408
View details for PubMedID 22085934
View details for PubMedCentralID PMC3261563