Temporal coordination of the metaphase to anaphase transition

The cell cycle is an ordered sequence of events culminating in the formation of two identical daughter cells. Ensuring the order of the events is essential for genomic integrity and cell proliferation. The sudden and synchronous splitting of chromosomes during the metaphase to anaphase transition is one of the visually most dramatic events of the cell cycle. The transition is driven by the activity of the anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase, which initiates the destruction of its two essential targets, cyclin B and securin. Cyclin B degradation inactivates the cyclin-dependent kinase 1 (CDK1) and triggers a multitude of processes during mitotic exit. Degradation of securin releases separase from its inhibition. Active separase subsequently triggers the highly synchronous separation of sister chromatids. The separation is irreversible and therefore needs to be highly accurate and tightly coordinated with mitotic exit. Yet, little is known about the molecular events that determine the timing of the single processes and coordinate the individual processes relative to each other. I have systematically studied the dynamics of the metaphase to anaphase transition in the fission yeast Schizosaccharomyces pombe using live cell imaging assays with high temporal resolution. My analysis shows that the synchronicity of sister chromatid separation directly depends on the degradation kinetic of its upstream regulator securin, which suggests the absence of additional feedback regulation. Stochastic processes dominate the order in which sister chromatids separate, but an intrinsic bias in chromosome segregation exists, which is enhanced by decreased separase activity or securin degradation rates. Sister chromatid separation has to be tightly coordinated with the cyclin B degradation-driven processes of mitotic exit. I find the temporal order of events during the metaphase to anaphase transition to be remarkably robust against changes in securin and cyclin B, even if the overall timing of the respective events is severely altered. Competition of securin and cyclin B for the shared degradation machinery as well as systematic variability in the protein thresholds at which certain events occur contribute to the observed temporal robustness. I further investigated the consequences of potential misregulation between securin and cyclin B degradation-dependent events and show that high CDK1 activity at anaphase results in untimely destabilization of chromosome attachment, activation of the mitotic checkpoint and inhibition of the APC/C. Yet, we find that inhibition of the APC/C occurs with slow kinetics, which might provide an additional buffer against the detrimental consequences of such a loss in coordination

The Apparent Requirement for Protein Synthesis during G2 Phase Is due to Checkpoint Activation

Protein synthesis inhibitors have long been known to prevent G2 phase cells from entering mitosis. Lockhead et al. demonstrate that this G2 arrest is due to the activation of p38 MAPK, not insufficient protein synthesis, arguing that protein synthesis in G2 phase is not absolutely required for mitotic entry

Slow checkpoint activation kinetics as a safety device in anaphase

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

Time To Split Up: Dynamics of Chromosome Separation

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

Bistable, Biphasic Regulation of PP2A-B55 Accounts for the Dynamics of Mitotic Substrate Phosphorylation

The phosphorylation of mitotic proteins is bistable, which contributes to the decisiveness of the transitions into and out of M phase. The bistability in substrate phosphorylation has been attributed to bistability in the activation of the cyclin-dependent kinase Cdk1. However, more recently it has been suggested that bistability also arises from positive feedback in the regulation of the Cdk1-counteracting phosphatase PP2A-B55. Here, we demonstrate biochemically using Xenopus laevis egg extracts that the Cdk1-counteracting phosphatase PP2A-B55 functions as a bistable switch, even when the bistability of Cdk1 activation is suppressed. In addition, Cdk1 regulates PP2A-B55 in a biphasic manner; low concentrations of Cdk1 activate PP2A-B55 and high concentrations inactivate it. As a consequence of this incoherent feedforward regulation, PP2A-B55 activity rises concurrently with Cdk1 activity during interphase and suppresses substrate phosphorylation. PP2A-B55 activity is then sharply downregulated at the onset of mitosis. During mitotic exit, Cdk1 activity initially falls with no obvious change in substrate phosphorylation; dephosphorylation then commences once PP2A-B55 spikes in activity. These findings suggest that changes in Cdk1 activity are permissive for mitotic entry and exit but that the changes in PP2A-B55 activity are the ultimate trigger

Biometric Recognition of African Clawed Frogs

The African clawed frog (Xenopus laevis) is a commonly used model organism for cell biological, developmental, and biomedical research. For health monitoring and experimental quality control purposes, it is desirable to identify individual frogs regularly throughout their life. Current methods for identification are often invasive and associated with significant investment costs. Identification based on images of the biometric pattern on a frog’s back has been implemented in some laboratories, but so far has been performed manually and therefore is time-consuming and limited to small group sizes. This work proposes a novel pipeline for data acquisition, pre-processing, and training of a classification model based on pattern recognition. The pipeline is structured around laboratory frog colonies and smartphone usage. In order to achieve a lightweight system in our evaluation we consider a MobileNet ConvNet pre-trained on ImageNet. Two feature sets are evaluated on a new data set of 1,647 image samples collected from 160 frogs: RGB images, and 3-channel contour maps (i.e. CORF3D). The results indicate that the CORF3D feature set is favoured over RGB. CORF3D achieved the best performance of 99.94% average accuracy, while RGB had the best performance of 98.79%. Analysis of misclassifications shows that bad predictions are often caused by bad lens focus, light reflections, and positional inconsistency in pattern extraction, which can be addressed during data acquisition. The proposed methodology is, therefore, an effective solution for the recognition of Xenopus laevis

Real-Time Monitoring of APC /C-Mediated Substrate Degradation Using Xenopus laevis Egg Extracts

The anaphase promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase, is a key regulator of mitotic progression. Upon activation in mitosis, the APC/C targets its two essential substrates, securin and cyclin B, for proteasomal destruction. Cyclin B is the activator of cyclin-dependent kinase 1 (Cdk1), the major mitotic kinase, and both cyclin B and securin are safeguards of sister chromatid cohesion. Conversely, the degradation of securin and cyclin B promotes sister chromatid separation and mitotic exit. The negative feedback loop between Cdk1 and APC/C—Cdk1 activating the APC/C and the APC/C inactivating Cdk1—constitutes the core of the biochemical cell cycle oscillator. Since its discovery three decades ago, the mechanisms of APC/C regulation have been intensively studied, and several in vitro assays exist to measure the activity of the APC/C in different activation states. However, most of these assays require the purification of numerous recombinant enzymes involved in the ubiquitylation process (e.g., ubiquitin, the E1 and E2 ubiquitin ligases, and the APC/C) and/or the use of radioactive isotopes. In this chapter, we describe an easy-to-implement method to continuously measure APC/C activity in Xenopus laevis egg extracts using APC/C substrates fused to fluorescent proteins and a fluorescence plate reader. Because the egg extract provides all important enzymes and proteins for the reaction, this method can be used largely without the need for recombinant protein purification. It can also easily be adapted to test the activity of APC/C mutants or investigate other mechanisms of APC/C regulation

Cell-Cycle Regulation of Dynamic Chromosome Association of the Condensin Complex

Summary: 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. : The condensin complex is a key determinant of mitotic chromosome formation. Thadani et al. study the dynamic binding of condensin to chromosomes. They reveal how condensin turnover is regulated by its ATPase and by cell-cycle phosphorylation. Chromosome condensation in mitosis requires fine-tuning of this dynamic behavior. Keywords: chromosome condensation, mitosis, cell cycle, phosphorylation, condensin, ABC ATPase, Saccharomyces cerevisia

In Vitro Reconstitution of a Cellular Phase-Transition Process that Involves the mRNA Decapping Machinery

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