Triap1 upregulation promotes escape from mitotic-slippage-induced G1 arrest.

Drugs targeting microtubules rely on the mitotic checkpoint to arrest cell proliferation. The prolonged mitotic arrest induced by such drugs is followed by a G1 arrest. Here, we follow for several weeks the fate of G1-arrested human cells after treatment with nocodazole. We find that a small fraction of cells escapes from the arrest and resumes proliferation. These escaping cells experience reduced DNA damage and p21 activation. Cells surviving treatment are enriched for anti-apoptotic proteins, including Triap1. Increasing Triap1 levels allows cells to survive the first treatment with reduced DNA damage and lower levels of p21; accordingly, decreasing Triap1 re-sensitizes cells to nocodazole. We show that Triap1 upregulation leads to the retention of cytochrome c in the mitochondria, opposing the partial activation of caspases caused by nocodazole. In summary, our results point to a potential role of Triap1 upregulation in the emergence of resistance to drugs that induce prolonged mitotic arrest.

A non-catalytic herpesviral protein reconfigures ERK-RSK signaling by targeting kinase docking systems in the host.

The Kaposi's sarcoma associated herpesvirus protein ORF45 binds the extracellular signal-regulated kinase (ERK) and the p90 Ribosomal S6 kinase (RSK). ORF45 was shown to be a kinase activator in cells but a kinase inhibitor in vitro, and its effects on the ERK-RSK complex are unknown. Here, we demonstrate that ORF45 binds ERK and RSK using optimized linear binding motifs. The crystal structure of the ORF45-ERK2 complex shows how kinase docking motifs recognize the activated form of ERK. The crystal structure of the ORF45-RSK2 complex reveals an AGC kinase docking system, for which we provide evidence that it is functional in the host. We find that ORF45 manipulates ERK-RSK signaling by favoring the formation of a complex, in which activated kinases are better protected from phosphatases and docking motif-independent RSK substrate phosphorylation is selectively up-regulated. As such, our data suggest that ORF45 interferes with the natural design of kinase docking systems in the host.

Epistasis, aneuploidy, and functional mutations underlie evolution of resistance to induced microtubule depolymerization.

Cells with blocked microtubule polymerization are delayed in mitosis, but eventually manage to proliferate despite substantial chromosome missegregation. While several studies have analyzed the first cell division after microtubule depolymerization, we have asked how cells cope long-term with microtubule impairment. We allowed 24 clonal populations of yeast cells with beta-tubulin mutations preventing proper microtubule polymerization, to evolve for ˜150 generations. At the end of the laboratory evolution experiment, cells had regained the ability to form microtubules and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing identified recurrently mutated genes, in particular for tubulins and kinesins, as well as pervasive duplication of chromosome VIII. Recreating these mutations and chromosome VIII disomy prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Most of the identified mutations did not abolish function, but rather restored microtubule functionality. Analysis of the temporal order of resistance development in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by a single additional adaptive mutation in either tubulins or kinesins. Since tubulins are highly conserved among eukaryotes, our results have implications for understanding resistance to microtubule-targeting drugs widely used in cancer therapy.

PP1 and PP2A Use Opposite Phospho-dependencies to Control Distinct Processes at the Kinetochore.

PP1 and PP2A-B56 are major serine/threonine phosphatase families that achieve specificity by colocalizing with substrates. At the kinetochore, however, both phosphatases localize to an almost identical molecular space and yet they still manage to regulate unique pathways and processes. By switching or modulating the positions of PP1/PP2A-B56 at kinetochores, we show that their unique downstream effects are not due to either the identity of the phosphatase or its precise location. Instead, these phosphatases signal differently because their kinetochore recruitment can be either inhibited (PP1) or enhanced (PP2A) by phosphorylation inputs. Mathematical modeling explains how these inverse phospho-dependencies elicit unique forms of cross-regulation and feedback, which allows otherwise indistinguishable phosphatases to produce distinct network behaviors and control different mitotic processes. Furthermore, our genome-wide analysis suggests that these major phosphatase families may have evolved to respond to phosphorylation inputs in opposite ways because many other PP1 and PP2A-B56-binding motifs are also phospho-regulated.

Cellular response upon proliferation in the presence of an active mitotic checkpoint.

Eukaryotic cells treated with microtubule-targeting agents activate the spindle assembly checkpoint to arrest in mitosis and prevent chromosome mis-segregation. A fraction of mitotically arrested cells overcomes the block and proliferates even under persistent checkpoint-activating conditions. Here, we asked what allows proliferation in such unfavourable conditions. We report that yeast cells are delayed in mitosis at each division, implying that their spindle assembly checkpoint remains responsive. The arrest causes their cell cycle to be elongated and results in a size increase. Growth saturates at mitosis and correlates with the repression of various factors involved in translation. Contrary to unperturbed cells, growth of cells with an active checkpoint requires Cdh1. This peculiar cell cycle correlates with global changes in protein expression whose signatures partly overlap with the environmental stress response. Hence, cells dividing with an active checkpoint develop recognisable specific traits that allow them to successfully complete cell division notwithstanding a constant mitotic checkpoint arrest. These properties distinguish them from unperturbed cells. Our observation may have implications for the identification of new therapeutic windows and targets in tumors.

Implications of alternative routes to APC/C inhibition by the mitotic checkpoint complex.

The mitotic checkpoint (also called spindle assembly checkpoint) is a signaling pathway that ensures faithful chromosome segregation. Mitotic checkpoint proteins inhibit the anaphase-promoting complex (APC/C) and its activator Cdc20 to prevent precocious anaphase. Checkpoint signaling leads to a complex of APC/C, Cdc20, and checkpoint proteins, in which the APC/C is inactive. In principle, this final product of the mitotic checkpoint can be obtained via different pathways, whose relevance still needs to be fully ascertained experimentally. Here, we use mathematical models to compare the implications on checkpoint response of the possible pathways leading to APC/C inhibition. We identify a previously unrecognized funneling effect for Cdc20, which favors Cdc20 incorporation into the inhibitory complex and therefore promotes checkpoint activity. Furthermore, we find that the presence or absence of one specific assembly reaction determines whether the checkpoint remains functional at elevated levels of Cdc20, which can occur in cancer cells. Our results reveal the inhibitory logics behind checkpoint activity, predict checkpoint efficiency in perturbed situations, and could inform molecular strategies to treat malignancies that exhibit Cdc20 overexpression.

Cells Escape an Operational Mitotic Checkpoint through a Stochastic Process.

Improperly attached chromosomes activate the mitotic checkpoint that arrests cell division before anaphase. Cells can maintain an arrest for several hours but eventually will resume proliferation, a process we refer to as adaptation. Whether adapting cells bypass an active block or whether the block has to be removed to resume proliferation is not clear. Likewise, it is not known whether all cells of a genetically homogeneous population are equally capable to adapt. Here, we show that the mitotic checkpoint is operational when yeast cells adapt and that each cell has the same propensity to adapt. Our results are consistent with a model of the mitotic checkpoint where adaptation is driven by random fluctuations of APC/CCdc20, the molecular species inhibited by the checkpoint. Our data provide a quantitative framework for understanding how cells overcome a constant stimulus that halts cell cycle progression.

Missing Value Monitoring Enhances the Robustness in Proteomics Quantitation.

In global proteomic analysis, it is estimated that proteins span from millions to less than 100 copies per cell. The challenge of protein quantitation by classic shotgun proteomic techniques relies on the presence of missing values in peptides belonging to low-abundance proteins that lowers intraruns reproducibility affecting postdata statistical analysis. Here, we present a new analytical workflow MvM (missing value monitoring) able to recover quantitation of missing values generated by shotgun analysis. In particular, we used confident data-dependent acquisition (DDA) quantitation only for proteins measured in all the runs, while we filled the missing values with data-independent acquisition analysis using the library previously generated in DDA. We analyzed cell cycle regulated proteins, as they are low abundance proteins with highly dynamic expression levels. Indeed, we found that cell cycle related proteins are the major components of the missing values-rich proteome. Using the MvM workflow, we doubled the number of robustly quantified cell cycle related proteins, and we reduced the number of missing values achieving robust quantitation for proteins over ∼50 molecules per cell. MvM allows lower quantification variance among replicates for low abundance proteins with respect to DDA analysis, which demonstrates the potential of this novel workflow to measure low abundance, dynamically regulated proteins.

Micromanaging checkpoint proteins.

The kinase Mps1, long known to be the 'boss' in mitotic checkpoint signaling, phosphorylates multiple proteins in the checkpoint signaling cascade.

Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells.

Ubiquitination of the epidermal growth factor receptor (EGFR) that occurs when Cbl and Grb2 bind to three phosphotyrosine residues (pY1045, pY1068 and pY1086) on the receptor displays a sharp threshold effect as a function of EGF concentration. Here we use a simple modelling approach together with experiments to show that the establishment of the threshold requires both the multiplicity of binding sites and cooperative binding of Cbl and Grb2 to the EGFR. While the threshold is remarkably robust, a more sophisticated model predicted that it could be modulated as a function of EGFR levels on the cell surface. We confirmed experimentally that the system has evolved to perform optimally at physiological levels of EGFR. As a consequence, this system displays an intrinsic weakness that causes--at the supraphysiological levels of receptor and/or ligand associated with cancer--uncoupling of the mechanisms leading to signalling through phosphorylation and attenuation through ubiquitination.

Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling.

Regulation of macromolecular interactions by phosphorylation is crucial in signaling networks. In the spindle assembly checkpoint (SAC), which enables errorless chromosome segregation, phosphorylation promotes recruitment of SAC proteins to tensionless kinetochores. The SAC kinase Mps1 phosphorylates multiple Met-Glu-Leu-Thr (MELT) motifs on the kinetochore subunit Spc105/Knl1. The phosphorylated MELT motifs (MELT(P)) then promote recruitment of downstream signaling components. How MELT(P) motifs are recognized is unclear. In this study, we report that Bub3, a 7-bladed ß-propeller, is the MELT(P) reader. It contains an exceptionally well-conserved interface that docks the MELT(P) sequence on the side of the ß-propeller in a previously unknown binding mode. Mutations targeting the Bub3 interface prevent kinetochore recruitment of the SAC kinase Bub1. Crucially, they also cause a checkpoint defect, showing that recognition of phosphorylated targets by Bub3 is required for checkpoint signaling. Our data provide the first detailed mechanistic insight into how phosphorylation promotes recruitment of checkpoint proteins to kinetochores. DOI:http://dx.doi.org/10.7554/eLife.01030.001.

Adaptation to the spindle checkpoint is regulated by the interplay between Cdc28/Clbs and PP2ACdc55.

The spindle checkpoint arrests cells in metaphase until all chromosomes are properly attached to the chromosome segregation machinery. Thereafter, the anaphase promoting complex (APC/C) is activated and chromosome segregation can take place. Cells remain arrested in mitosis for hours in response to checkpoint activation, but not indefinitely. Eventually, they adapt to the checkpoint and proceed along the cell cycle. In yeast, adaptation requires the phosphorylation of APC/C. Here, we show that the protein phosphatase PP2A(Cdc55) dephosphorylates APC/C, thereby counteracting the activity of the mitotic kinase Cdc28. We also observe that the key regulator of Cdc28, the mitotic cyclin Clb2, increases before cells adapt and is then abruptly degraded at adaptation. Adaptation is highly asynchronous and takes place over a range of several hours. Our data suggest the presence of a double negative loop between PP2A(Cdc55) and APC/C(Cdc20) (i.e., a positive feedback loop) that controls APC/C(Cdc20) activity. The circuit could guarantee sustained APC/C(Cdc20) activity after Clb2 starts to be degraded.

Threshold-controlled ubiquitination of the EGFR directs receptor fate.

How the cell converts graded signals into threshold-activated responses is a question of great biological relevance. Here, we uncover a nonlinear modality of epidermal growth factor receptor (EGFR)-activated signal transduction, by demonstrating that the ubiquitination of the EGFR at the PM is threshold controlled. The ubiquitination threshold is mechanistically determined by the cooperative recruitment of the E3 ligase Cbl, in complex with Grb2, to the EGFR. This, in turn, is dependent on the simultaneous presence of two phosphotyrosines, pY1045 and either one of pY1068 or pY1086, on the same EGFR moiety. The dose-response curve of EGFR ubiquitination correlate precisely with the non-clathrin endocytosis (NCE) mode of EGFR internalization. Finally, EGFR-NCE mechanistically depends on EGFR ubiquitination, as the two events can be simultaneously re-engineered on a phosphorylation/ubiquitination-incompetent EGFR backbone. Since NCE controls the degradation of the EGFR, our findings have implications for how the cell responds to increasing levels of EGFR signalling, by varying the balance of receptor signalling and degradation/attenuation.

Role of the Mad2 dimerization interface in the spindle assembly checkpoint independent of kinetochores.

BACKGROUND: The spindle assembly checkpoint (SAC) arrests cells when kinetochores are unattached to spindle microtubules. The signaling pathway is initiated at the kinetochores by one SAC component, Mad2, which catalyzes the initial steps of the cascade via the conformational dimerization of its open and closed conformers. Away from kinetochores, the dimerization surface of Mad2 has been proposed, based on data in vitro, to either interact with SAC activators or inactivators and thus to contribute to SAC activation or silencing. Here, we analyze its role in vivo. RESULTS: To analyze the putative pathway downstream of the kinetochores, we used two complementary approaches: we activated the SAC ectopically and independently from kinetochores, and we separated genetically the kinetochore-dependent and independent pools of Mad2. We found that the dimerization surface is required also downstream of kinetochores to mount a checkpoint response. CONCLUSION: Our results show that away from kinetochores the dimerization surface is required for stabilizing the end-product of the pathway, the mitotic checkpoint complex. Surprisingly, downstream of kinetochores the surface does not mediate Mad2 dimerization. Instead, our results are consistent with a role of Mad3 as the main interactor of Mad2 via the dimerization surface.

Endocytosis and signaling: cell logistics shape the eukaryotic cell plan.

Our understanding of endocytosis has evolved remarkably in little more than a decade. This is the result not only of advances in our knowledge of its molecular and biological workings, but also of a true paradigm shift in our understanding of what really constitutes endocytosis and of its role in homeostasis. Although endocytosis was initially discovered and studied as a relatively simple process to transport molecules across the plasma membrane, it was subsequently found to be inextricably linked with almost all aspects of cellular signaling. This led to the notion that endocytosis is actually the master organizer of cellular signaling, providing the cell with understandable messages that have been resolved in space and time. In essence, endocytosis provides the communications and supply routes (the logistics) of the cell. Although this may seem revolutionary, it is still likely to be only a small part of the entire story. A wealth of new evidence is uncovering the surprisingly pervasive nature of endocytosis in essentially all aspects of cellular regulation. In addition, many newly discovered functions of endocytic proteins are not immediately interpretable within the classical view of endocytosis. A possible framework, to rationalize all this new knowledge, requires us to "upgrade" our vision of endocytosis. By combining the analysis of biochemical, biological, and evolutionary evidence, we propose herein that endocytosis constitutes one of the major enabling conditions that in the history of life permitted the development of a higher level of organization, leading to the actuation of the eukaryotic cell plan.

The Eps8/IRSp53/VASP network differentially controls actin capping and bundling in filopodia formation.

There is a body of literature that describes the geometry and the physics of filopodia using either stochastic models or partial differential equations and elasticity and coarse-grained theory. Comparatively, there is a paucity of models focusing on the regulation of the network of proteins that control the formation of different actin structures. Using a combination of in-vivo and in-vitro experiments together with a system of ordinary differential equations, we focused on a small number of well-characterized, interacting molecules involved in actin-dependent filopodia formation: the actin remodeler Eps8, whose capping and bundling activities are a function of its ligands, Abi-1 and IRSp53, respectively; VASP and Capping Protein (CP), which exert antagonistic functions in controlling filament elongation. The model emphasizes the essential role of complexes that contain the membrane deforming protein IRSp53, in the process of filopodia initiation. This model accurately accounted for all observations, including a seemingly paradoxical result whereby genetic removal of Eps8 reduced filopodia in HeLa, but increased them in hippocampal neurons, and generated quantitative predictions, which were experimentally verified. The model further permitted us to explain how filopodia are generated in different cellular contexts, depending on the dynamic interaction established by Eps8, IRSp53 and VASP with actin filaments, thus revealing an unexpected plasticity of the signaling network that governs the multifunctional activities of its components in the formation of filopodia.

Evidence that Aurora B is implicated in spindle checkpoint signalling independently of error correction.

Fidelity of chromosome segregation is ensured by a tension-dependent error correction system that prevents stabilization of incorrect chromosome-microtubule attachments. Unattached or incorrectly attached chromosomes also activate the spindle assembly checkpoint, thus delaying mitotic exit until all chromosomes are bioriented. The Aurora B kinase is widely recognized as a component of error correction. Conversely, its role in the checkpoint is controversial. Here, we report an analysis of the role of Aurora B in the spindle checkpoint under conditions believed to uncouple the effects of Aurora B inhibition on the checkpoint from those on error correction. Partial inhibition of several checkpoint and kinetochore components, including Mps1 and Ndc80, strongly synergizes with inhibition of Aurora B activity and dramatically affects the ability of cells to arrest in mitosis in the presence of spindle poisons. Thus, Aurora B might contribute to spindle checkpoint signalling independently of error correction. Our results support a model in which Aurora B is at the apex of a signalling pyramid whose sensory apparatus promotes the concomitant activation of error correction and checkpoint signalling pathways.

Computational modelling of mitotic exit in budding yeast: the role of separase and Cdc14 endocycles.

The operating principles of complex regulatory networks are best understood with the help of mathematical modelling rather than by intuitive reasoning. Hereby, we study the dynamics of the mitotic exit (ME) control system in budding yeast by further developing the Queralt's model. A comprehensive systems view of the network regulating ME is provided based on classical experiments in the literature. In this picture, Cdc20-APC is a critical node controlling both cyclin (Clb2 and Clb5) and phosphatase (Cdc14) branches of the regulatory network. On the basis of experimental situations ranging from single to quintuple mutants, the kinetic parameters of the network are estimated. Numerical analysis of the model quantifies the dependence of ME control on the proteolytic and non-proteolytic functions of separase. We show that the requirement of the non-proteolytic function of separase for ME depends on cyclin-dependent kinase activity. The model is also used for the systematic analysis of the recently discovered Cdc14 endocycles. The significance of Cdc14 endocycles in eukaryotic cell cycle control is discussed as well.