Reciprocal antagonism of PIN1-APC/CCDH1 governs mitotic protein stability and cell cycle entry.

Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.

Cell Cycle Mapping Using Multiplexed Immunofluorescence.

The development of technologies that allow measurement of the cell cycle at the single-cell level has revealed novel insights into the mechanisms that regulate cell cycle commitment and progression through DNA replication and cell division. These studies have also provided evidence of heterogeneity in cell cycle regulation among individual cells, even within a genetically identical population. Cell cycle mapping combines highly multiplexed imaging with manifold learning to visualize the diversity of "paths" that cells can take through the proliferative cell cycle or into various states of cell cycle arrest. In this chapter, we describe a general protocol of the experimental and computational components of cell cycle mapping. We also provide a comprehensive guide for the design and analysis of experiments, discussing key considerations in detail (e.g., antibody library preparation, analysis strategies, etc.) that may vary depending on the research question being addressed.

Investigating Heterogeneous Cell-Cycle Progression Using Single-Cell Imaging Approaches.

Investigating cell-cycle progression has been challenging due to the complex interconnectivity of regulatory processes and inherent cell-to-cell heterogeneity, which often require synchronization procedures. However, recent advancements in cell-cycle sensors and single-cell imaging techniques have turned this heterogeneity into an advantage for investigating the molecular mechanisms underlying diverse responses. This has led to significant progress in our understanding of cell-cycle regulation. In this paper, we present a comprehensive live single-cell imaging workflow that leverages cutting-edge live-cell sensors. These advanced single-cell imaging procedures provide promising opportunities for elucidating the molecular mechanisms underpinnings of heterogeneous responses in cell-cycle progression.

Pas de deux: the coordinated coupling of erythroid differentiation with the cell cycle.

PURPOSE OF REVIEW: Recent work reveals that cell cycle duration and structure are remodeled in lock-step with distinct stages of erythroid differentiation. These cell cycle features have regulatory roles in differentiation, beyond the generic function of increasing cell number. RECENT FINDINGS: Developmental progression through the early erythroid progenitor stage (known as colony-forming-erythroid, or 'CFU-e') is characterized by gradual shortening of G1 phase of the cycle. This process culminates in a key transcriptional switch to erythroid terminal differentiation (ETD) that is synchronized with, and dependent on, S phase progression. Further, the CFU-e/ETD switch takes place during an unusually short S phase, part of an exceptionally short cell cycle that is characterized by globally fast replication fork speeds. Cell cycle and S phase speed can alter developmental events during erythroid differentiation, through pathways that are targeted by glucocorticoid and erythropoietin signaling during the erythroid stress response. SUMMARY: There is close inter-dependence between cell cycle structure and duration, S phase and replication fork speeds, and erythroid differentiation stage. Further, modulation of cell cycle structure and speed cycle impacts developmental progression and cell fate decisions during erythroid differentiation. These pathways may offer novel mechanistic insights and potential therapeutic targets.

Triaging between post-translational modification of cell cycle regulators and their therapeutics in neurodegenerative diseases.

Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, present challenges in healthcare because of their complicated etiologies and absence of healing remedies. Lately, the emerging role of post-translational modifications (PTMs), in the context of cell cycle regulators, has garnered big interest as a potential avenue for therapeutic intervention. The review explores the problematic panorama of PTMs on cell cycle regulators and their implications in neurodegenerative diseases. We delve into the dynamic phosphorylation, acetylation, ubiquitination, SUMOylation, Glycation, and Neddylation that modulate the key cell cycle regulators, consisting of cyclins, cyclin-dependent kinases (CDKs), and their inhibitors. The dysregulation of these PTMs is related to aberrant cell cycle in neurons, which is one of the factors involved in neurodegenerative pathologies. Moreover, the effect of exogenous activation of CDKs and CDK inhibitors through PTMs on the signaling cascade was studied in postmitotic conditions of NDDs. Furthermore, the therapeutic implications of CDK inhibitors and associated alteration in PTMs were discussed. Lastly, we explored the putative mechanism of PTMs to restore normal neuronal function that might reverse NDDs.

A simple Turing reaction-diffusion model explains how PLK4 breaks symmetry during centriole duplication and assembly.

Centrioles duplicate when a mother centriole gives birth to a daughter that grows from its side. Polo-like-kinase 4 (PLK4), the master regulator of centriole duplication, is recruited symmetrically around the mother centriole, but it then concentrates at a single focus that defines the daughter centriole assembly site. How PLK4 breaks symmetry is unclear. Here, we propose that phosphorylated and unphosphorylated species of PLK4 form the 2 components of a classical Turing reaction-diffusion system. These 2 components bind to/unbind from the surface of the mother centriole at different rates, allowing a slow-diffusing activator species of PLK4 to accumulate at a single site on the mother, while a fast-diffusing inhibitor species of PLK4 suppresses activator accumulation around the rest of the centriole. This "short-range activation/long-range inhibition," inherent to Turing systems, can drive PLK4 symmetry breaking on a either a continuous or compartmentalised Plk4-binding surface, with PLK4 overexpression producing multiple PLK4 foci and PLK4 kinase inhibition leading to a lack of symmetry-breaking and PLK4 accumulation-as observed experimentally.

A PTP1B-Cdk3 Signaling Axis Promotes Cell Cycle Progression of Human Glioblastoma Cells through an Rb-E2F Dependent Pathway.

PTP1B plays a key role in developing different types of cancer. However, the molecular mechanism underlying this effect is unclear. To identify molecular targets of PTP1B that mediate its role in tumorigenesis, we undertook a SILAC-based phosphoproteomic approach, which allowed us to identify Cdk3 as a novel PTP1B substrate. Substrate trapping experiments and docking studies revealed stable interactions between the PTP1B catalytic domain and Cdk3. In addition, we observed that PTP1B dephosphorylates Cdk3 at tyrosine residue 15 in vitro and interacts with it in human glioblastoma cells. Next, we found that pharmacological inhibition of PTP1B or its depletion with siRNA leads to cell cycle arrest with diminished activity of Cdk3, hypophosphorylation of Rb, and the downregulation of E2F target genes Cdk1, Cyclin A, and Cyclin E1. Finally, we observed that the expression of a constitutively active Cdk3 mutant bypasses the requirement of PTP1B for cell cycle progression and expression of E2F target genes. These data delineate a novel signaling pathway from PTP1B to Cdk3 required for efficient cell cycle progression in an Rb-E2F dependent manner in human GB cells.

Discovery of small molecule degraders for modulating cell cycle.

The cell cycle is a complex process that involves DNA replication, protein expression, and cell division. Dysregulation of the cell cycle is associated with various diseases. Cyclin-dependent kinases (CDKs) and their corresponding cyclins are major proteins that regulate the cell cycle. In contrast to inhibition, a new approach called proteolysis-targeting chimeras (PROTACs) and molecular glues can eliminate both enzymatic and scaffold functions of CDKs and cyclins, achieving targeted degradation. The field of PROTACs and molecular glues has developed rapidly in recent years. In this article, we aim to summarize the latest developments of CDKs and cyclin protein degraders. The selectivity, application, validation and the current state of each CDK degrader will be overviewed. Additionally, possible methods are discussed for the development of degraders for CDK members that still lack them. Overall, this article provides a comprehensive summary of the latest advancements in CDK and cyclin protein degraders, which will be helpful for researchers working on this topic.

Modelling how plant cell-cycle progression leads to cell size regulation.

Populations of cells typically maintain a consistent size, despite cell division rarely being precisely symmetrical. Therefore, cells must possess a mechanism of "size control", whereby the cell volume at birth affects cell-cycle progression. While size control mechanisms have been elucidated in a number of other organisms, it is not yet clear how this mechanism functions in plants. Here, we present a mathematical model of the key interactions in the plant cell cycle. Model simulations reveal that the network of interactions exhibits limit-cycle solutions, with biological switches underpinning both the G1/S and G2/M cell-cycle transitions. Embedding this network model within growing cells, we test hypotheses as to how cell-cycle progression can depend on cell size. We investigate two different mechanisms at both the G1/S and G2/M transitions: (i) differential expression of cell-cycle activator and inhibitor proteins (with synthesis of inhibitor proteins being independent of cell size), and (ii) equal inheritance of inhibitor proteins after cell division. The model demonstrates that both these mechanisms can lead to larger daughter cells progressing through the cell cycle more rapidly, and can thus contribute to cell-size control. To test how these features enable size homeostasis over multiple generations, we then simulated these mechanisms in a cell-population model with multiple rounds of cell division. These simulations suggested that integration of size-control mechanisms at both G1/S and G2/M provides long-term cell-size homeostasis. We concluded that while both size independence and equal inheritance of inhibitor proteins can reduce variations in cell size across individual cell-cycle phases, combining size-control mechanisms at both G1/S and G2/M is essential to maintain size homeostasis over multiple generations. Thus, our study reveals how features of the cell-cycle network enable cell-cycle progression to depend on cell size, and provides a mechanistic understanding of how plant cell populations maintain consistent size over generations.

PLK4 self-phosphorylation drives the selection of a single site for procentriole assembly.

Polo-like kinase 4 (PLK4) is a key regulator of centriole biogenesis, but how PLK4 selects a single site for procentriole assembly remains unclear. Using ultrastructure expansion microscopy, we show that PLK4 localizes to discrete sites along the wall of parent centrioles. While there is variation in the number of sites PLK4 occupies on the parent centriole, most PLK4 localize at a dominant site that directs procentriole assembly. Inhibition of PLK4 activity leads to stable binding of PLK4 to the centriole and increases occupancy to a maximum of nine sites. We show that self-phosphorylation of an unstructured linker promotes the release of active PLK4 from the centriole to drive the selection of a single site for procentriole assembly. Preventing linker phosphorylation blocks PLK4 turnover, leading to supernumerary sites of PLK4 localization and centriole amplification. Therefore, self-phosphorylation is a major driver of the spatial patterning of PLK4 at the centriole and plays a critical role in selecting a single centriole duplication site.

Autophagy modulates the stability of Wee1 and cell cycle G2/M transition.

The mammalian cell cycle is divided into four sequential phases, namely G1 (Gap 1), S (synthesis), G2 (Gap 2), and M (mitosis). Wee1, whose turnover is tightly and finely regulated, is a well-known kinase serving as a gatekeeper for the G2/M transition. However, the mechanism underlying the turnover of Wee1 is not fully understood. Autophagy, a highly conserved cellular process, maintains cellular homeostasis by eliminating intracellular aggregations, damaged organelles, and individual proteins. In the present study, we found autophagy deficiency in mouse liver caused G2/M arrest in two mouse models, namely Fip200 and Atg7 liver-specific knockout mice. To uncover the link between autophagy deficiency and G2/M transition, we combined transcriptomic and proteomic analysis for liver samples from control and Atg7 liver-specific knockout mice. The data suggest that the inhibition of autophagy increases the protein level of Wee1 without any alteration of its mRNA abundance. Serum starvation, an autophagy stimulus, downregulates the protein level of Wee1 in vitro. In addition, the half-life of Wee1 is extended by the addition of chloroquine, an autophagy inhibitor. LC3, a central autophagic protein functioning in autophagy substrate selection and autophagosome biogenesis, interacts with Wee1 as assessed by co-immunoprecipitation assay. Furthermore, overexpression of Wee1 leads to G2/M arrest both in vitro and in vivo. Collectively, our data indicate that autophagy could degrade Wee1-a gatekeeper of the G2/M transition, whereas the inhibition of autophagy leads to the accumulation of Wee1 and causes G2/M arrest in mouse liver.

Rapid adaptation to CDK2 inhibition exposes intrinsic cell-cycle plasticity.

CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, we use several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, this is not true of acute inhibition of CDK2. Upon CDK2 inhibition, cells exhibit a rapid loss of substrate phosphorylation that rebounds within several hours. CDK4/6 activity backstops inhibition of CDK2 and sustains the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and cyclin A2 expression, enabling re-activation of CDK2 in the presence of drug. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 may be required to suppress adaptation to CDK2 inhibitors currently under clinical assessment.

Regulation of Cardiomyocyte Division During Cardiac Regeneration.

PURPOSE OF REVIEW: This review explores efforts made over the previous three decades to determine mechanisms of cardiomyocyte cell division. Many investigators have explored cell therapy strategies in animal models and clinical trials over the past 2 decades with marginal results thus far in clinical testing. Hence, there is a greater focus now on strategies to induce cardiomyocyte proliferation. RECENT FINDINGS: Reports to induce reactivation of the cardiomyocyte cell cycle predated the focus on cell therapy, and we summarize the literature on this topic, which began with the very first transgenic mouse studies in cardiovascular science. These earlier studies form the foundation for the use of cell cycle manipulation in cardiac repair and should inform current and future investigations with respect to rigor of assessment in the degree of cardiomyocyte cell division and gold standard measures of cardiac functional improvement.

Embryonic Exposure to UV-328 Impairs the Cell Cycle in Zebrafish (Danio rerio) by Inhibiting the p38 MAPK/p53/Gadd45a Signaling Pathway.

The benzotriazole UV stabilizer UV-328 is well known for its potent antioxidative properties; however, there are concerns about how it may affect signaling nodes and lead to negative consequences. This study identified the key signaling cascades involved in oxidative stress in zebrafish (Danio rerio) larvae and evaluated the cell cycle arrests and associated developmental alternations. Exposure to UV-328 at 0.25, 0.50, 1.00, 2.00, and 4.00 µg/L downregulated gene expression associated with oxidative stress (cat, gpx, gst, and sod) and apoptosis (caspase-3, caspase-6, caspase-8, and caspase-9) at 3 days postfertilization (dpf). The transcriptome aberration in zebrafish with disrupted p38 mitogen-activated protein kinase (MAPK) cascades was validated based on decreased mRNA expressions of p38 MAPK (0.36-fold), p53 (0.33-fold), and growth arrest and DNA damage-inducible protein 45 α (Gadd45a) (0.52-fold) after a 3- and 14-day exposure alongside a correspondingly decreased protein expression. The percentage of cells in the Gap 1 (G1) phase increased from 69.60% to a maximum of 77.07% (p < 0.05) in the 3 dpf embryos. UV-328 inhibited the p38 MAPK/p53/Gadd45a regulatory circuit but promoted G1 phase cell cycle arrest, abnormally accelerating the embryo hatching and heart rate. This study provided mechanistic insights that enrich the risk profiles of UV-328.

Epithelial tissue confinement inhibits cell growth and leads to volume-reducing divisions.

Cell proliferation is a central process in tissue development, homeostasis, and disease, yet how proliferation is regulated in the tissue context remains poorly understood. Here, we introduce a quantitative framework to elucidate how tissue growth dynamics regulate cell proliferation. Using MDCK epithelial monolayers, we show that a limiting rate of tissue expansion creates confinement that suppresses cell growth; however, this confinement does not directly affect the cell cycle. This leads to uncoupling between rates of cell growth and division in epithelia and, thereby, reduces cell volume. Division becomes arrested at a minimal cell volume, which is consistent across diverse epithelia in vivo. Here, the nucleus approaches the minimum volume capable of packaging the genome. Loss of cyclin D1-dependent cell-volume regulation results in an abnormally high nuclear-to-cytoplasmic volume ratio and DNA damage. Overall, we demonstrate how epithelial proliferation is regulated by the interplay between tissue confinement and cell-volume regulation.

Impact of variability in cell cycle periodicity on cell population dynamics.

The cell cycle consists of a series of orchestrated events controlled by molecular sensing and feedback networks that ultimately drive the duplication of total DNA and the subsequent division of a single parent cell into two daughter cells. The ability to block the cell cycle and synchronize cells within the same phase has helped understand factors that control cell cycle progression and the properties of each individual phase. Intriguingly, when cells are released from a synchronized state, they do not maintain synchronized cell division and rapidly become asynchronous. The rate and factors that control cellular desynchronization remain largely unknown. In this study, using a combination of experiments and simulations, we investigate the desynchronization properties in cervical cancer cells (HeLa) starting from the G1/S boundary following double-thymidine block. Propidium iodide (PI) DNA staining was used to perform flow cytometry cell cycle analysis at regular 8 hour intervals, and a custom auto-similarity function to assess the desynchronization and quantify the convergence to an asynchronous state. In parallel, we developed a single-cell phenomenological model the returns the DNA amount across the cell cycle stages and fitted the parameters using experimental data. Simulations of population of cells reveal that the cell cycle desynchronization rate is primarily sensitive to the variability of cell cycle duration within a population. To validate the model prediction, we introduced lipopolysaccharide (LPS) to increase cell cycle noise. Indeed, we observed an increase in cell cycle variability under LPS stimulation in HeLa cells, accompanied with an enhanced rate of cell cycle desynchronization. Our results show that the desynchronization rate of artificially synchronized in-phase cell populations can be used a proxy of the degree of variance in cell cycle periodicity, an underexplored axis in cell cycle research.

CDK actively contributes to establishment of the stationary phase state in fission yeast.

Upon exhaustion of essential environmental nutrients, unicellular organisms cease cell division and enter stationary phase, a metabolically repressed state essential for cell survival in stressful environments. In the fission yeast Schizosaccharomyces pombe, cell size is reduced by cell division before entry into stationary phase; thus cyclin-dependent kinase (CDK) must actively contribute to stationary phase establishment. However, the contribution of CDK to stationary phase remains largely uncharacterized. Here, we examine the role of the sole S. pombe CDK, Cdc2, in the establishment of stationary phase. We show that in stationary phase, nuclear and chromosomal volumes and the nucleus-to-cell volume ratio are reduced, and sister chromatid separation and chromosome fluctuation are repressed. Furthermore, Cdc2 accumulates in the nucleolus. Most of these changes are induced by glucose depletion. Reduction in Cdc2 activity before and upon stationary phase entry alleviates the changes and shortens the survival time of stationary phase cells, whereas Cdc2 inhibition represses nucleolar Cdc2 accumulation and glucose depletion-induced nuclear volume reduction. These results demonstrate that CDK actively regulates stationary phase, both before and upon stationary phase entry.

[Advances of the regulatory mechanism of cyclin, cyclin- dependent kinases and related kinase inhibitors in cell cycle progression].

Cell cycle plays a crucial role in cell development. Cell cycle progression is mainly regulated by cyclin dependent kinase (CDK), cyclin and endogenous CDK inhibitor (CKI). Among these, CDK is the main cell cycle regulator, binding to cyclin to form the cyclin-CDK complex, which phosphorylates hundreds of substrates and regulates interphase and mitotic progression. Abnormal activity of various cell cycle proteins can cause uncontrolled proliferation of cancer cells, which leads to cancer development. Therefore, understanding the changes in CDK activity, cyclin-CDK assembly and the role of CDK inhibitors will help to understand the underlying regulatory processes in cell cycle progression, as well as provide a basis for the treatment of cancer and disease and the development of CDK inhibitor-based therapeutic agents. This review focuses on the key events of CDK activation or inactivation, and summarizes the regulatory processes of cyclin-CDK at specific times and locations, as well as the progress of research on relevant CDK inhibitor therapeutics in cancer and disease. The review concludes with a brief description of the current challenges of the cell cycle process, with the aim to provide scientific references and new ideas for further research on cell cycle process.

An autonomous mathematical model for the mammalian cell cycle.

A mathematical model for the mammalian cell cycle is developed as a system of 13 coupled nonlinear ordinary differential equations. The variables and interactions included in the model are based on detailed consideration of available experimental data. A novel feature of the model is inclusion of cycle tasks such as origin licensing and initiation, nuclear envelope breakdown and kinetochore attachment, and their interactions with controllers (molecular complexes involved in cycle control). Other key features are that the model is autonomous, except for a dependence on external growth factors; the variables are continuous in time, without instantaneous resets at phase boundaries; mechanisms to prevent rereplication are included; and cycle progression is independent of cell size. Eight variables represent cell cycle controllers: the Cyclin D1-Cdk4/6 complex, APCCdh1, SCFßTrCP, Cdc25A, MPF, NuMA, the securin-separase complex, and separase. Five variables represent task completion, with four for the status of origins and one for kinetochore attachment. The model predicts distinct behaviors corresponding to the main phases of the cell cycle, showing that the principal features of the mammalian cell cycle, including restriction point behavior, can be accounted for in a quantitative mechanistic way based on known interactions among cycle controllers and their coupling to tasks. The model is robust to parameter changes, in that cycling is maintained over at least a five-fold range of each parameter when varied individually. The model is suitable for exploring how extracellular factors affect cell cycle progression, including responses to metabolic conditions and to anti-cancer therapies.