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.

Spontaneously slow-cycling subpopulations of human cells originate from activation of stress-response pathways.

Slow-cycling subpopulations exist in bacteria, yeast, and mammalian systems. In the case of cancer, slow-cycling subpopulations have been proposed to give rise to drug resistance. However, the origin of slow-cycling human cells is poorly studied, in large part due to lack of markers to identify these rare cells. Slow-cycling cells pass through a noncycling period marked by low CDK2 activity and high p21 levels. Here, we use this knowledge to isolate these naturally slow-cycling cells from a heterogeneous population and perform RNA sequencing to delineate the transcriptome underlying the slow-cycling state. We show that cellular stress responses-the p53 transcriptional response and the integrated stress response (ISR)-are the most salient causes of spontaneous entry into the slow-cycling state. Finally, we show that cells' ability to enter the slow-cycling state enhances their survival in stressful conditions. Thus, the slow-cycling state is hardwired to stress responses to promote cellular survival in unpredictable environments.

A map of protein dynamics during cell-cycle progression and cell-cycle exit.

The cell-cycle field has identified the core regulators that drive the cell cycle, but we do not have a clear map of the dynamics of these regulators during cell-cycle progression versus cell-cycle exit. Here we use single-cell time-lapse microscopy of Cyclin-Dependent Kinase 2 (CDK2) activity followed by endpoint immunofluorescence and computational cell synchronization to determine the temporal dynamics of key cell-cycle proteins in asynchronously cycling human cells. We identify several unexpected patterns for core cell-cycle proteins in actively proliferating (CDK2-increasing) versus spontaneously quiescent (CDK2-low) cells, including Cyclin D1, the levels of which we find to be higher in spontaneously quiescent versus proliferating cells. We also identify proteins with concentrations that steadily increase or decrease the longer cells are in quiescence, suggesting the existence of a continuum of quiescence depths. Our single-cell measurements thus provide a rich resource for the field by characterizing protein dynamics during proliferation versus quiescence.

Using in vivo biotinylated ubiquitin to describe a mitotic exit ubiquitome from human cells.

Mitotic division requires highly regulated morphological and biochemical changes to the cell. Upon commitment to exit mitosis, cells begin to remove mitotic regulators in a temporally and spatially controlled manner to bring about the changes that reestablish interphase. Ubiquitin-dependent pathways target these regulators to generate polyubiquitin-tagged substrates for degradation by the 26S proteasome. However, the lack of cell-based assays to investigate in vivo ubiquitination limits our knowledge of the identity of substrates of ubiquitin-mediated regulation in mitosis. Here we report an in vivo ubiquitin tagging system used in human cells that allows efficient purification of ubiquitin conjugates from synchronized cell populations. Coupling purification with mass spectrometry, we have identified a series of mitotic regulators targeted for polyubiquitination in mitotic exit. We show that some are new substrates of the anaphase-promoting complex/cyclosome and validate KIFC1 and RacGAP1/Cyk4 as two such targets involved respectively in timely mitotic spindle disassembly and cell spreading. We conclude that in vivo biotin tagging of ubiquitin can provide valuable information about the role of ubiquitin-mediated regulation in processes required for rebuilding interphase cells.

Spatiotemporal organization of Aurora-B by APC/CCdh1 after mitosis coordinates cell spreading through FHOD1.

Spatiotemporal regulation of mitotic kinase activity underlies the extensive rearrangement of cellular components required for cell division. One highly dynamic mitotic kinase is Aurora-B (AurB), which has multiple roles defined by the changing localisation of the chromosome passenger complex (CPC) as cells progress through mitosis, including regulation of cytokinesis and abscission. Like other mitotic kinases, AurB is a target of the anaphase-promoting complex (APC/C) ubiquitin ligase during mitotic exit, but it is not known if APC/C-mediated destruction plays any specific role in controlling AurB activity. We have examined the contribution of the Cdh1 coactivator-associated APC/C(Cdh1) to the organization of AurB activity as cells exit mitosis and re-enter interphase. We report that APC/C(Cdh1)-dependent proteolysis restricts a cell-cortex-associated pool of active AurB in space and time. In early G1 phase this pool of AurB is found at protrusions associated with cell spreading. AurB retention at the cortex depends on a formin, FHOD1, critically required to organize the cytoskeleton after division. We identify AurB phosphorylation sites in FHOD1 and show that phosphomutant FHOD1 is impaired in post-mitotic assembly of oriented actin cables. We propose that Cdh1 contributes to spatiotemporal organization of AurB activity and that organization of FHOD1 activity by AurB contributes to daughter cell spreading after mitosis.

Substrate targeting by the ubiquitin-proteasome system in mitosis.

Both cell cycle progression and the ubiquitin-proteasome system (UPS) that drives it are precisely regulated. Enzymatically, many ubiquitylation and degradation reactions have been characterized in in vitro systems, providing insights into the fundamental mechanisms of the UPS. Biologically, a range of degradation events depending on a ubiquitin ligase called the Anaphase-Promoting Complex (APC/C), have been shown to control mitotic progression through removal of key substrates with extreme temporal precision. However we are only just beginning to understand how the different enzymatic activities of the UPS act collectively - and in cooperation with other cellular factors - for accurate temporal and spatial control of mitotic substrate levels in vivo.

Self-renewing human naïve pluripotent stem cells dedifferentiate in 3D culture and form blastoids spontaneously.

Human naïve pluripotent stem cells (hnPSCs) can generate integrated models of blastocysts termed blastoids upon switch to inductive medium. However, the underlying mechanisms remain obscure. Here we report that self-renewing hnPSCs spontaneously and efficiently give rise to blastoids upon three dimensional (3D) suspension culture. The spontaneous blastoids mimic early stage human blastocysts in terms of structure, size, and transcriptome characteristics and are capable of progressing to post-implantation stages. This property is conferred by the glycogen synthase kinase-3 (GSK3) signalling inhibitor IM-12 present in 5iLAF self-renewing medium. IM-12 upregulates oxidative phosphorylation-associated genes that underly the capacity of hnPSCs to generate blastoids spontaneously. Starting from day one of self-organization, hnPSCs at the boundary of all 3D aggregates dedifferentiate into E5 embryo-like intermediates. Intermediates co-express SOX2/OCT4 and GATA6 and by day 3 specify trophoblast fate, which coincides with cavity and blastoid formation. In summary, spontaneous blastoid formation results from 3D culture triggering dedifferentiation of hnPSCs into earlier embryo-like intermediates which are then competent to segregate blastocyst fates.

Revisiting degron motifs in human AURKA required for its targeting by APC/CFZR1.

Mitotic kinase Aurora A (AURKA) diverges from other kinases in its multiple active conformations that may explain its interphase roles and the limited efficacy of drugs targeting the kinase pocket. Regulation of AURKA activity by the cell is critically dependent on destruction mediated by the anaphase-promoting complex (APC/CFZR1) during mitotic exit and G1 phase and requires an atypical N-terminal degron in AURKA called the "A-box" in addition to a reported canonical D-box degron in the C-terminus. Here, we find that the reported C-terminal D-box of AURKA does not act as a degron and instead mediates essential structural features of the protein. In living cells, the N-terminal intrinsically disordered region of AURKA containing the A-box is sufficient to confer FZR1-dependent mitotic degradation. Both in silico and in cellulo assays predict the QRVL short linear interacting motif of the A-box to be a phospho-regulated D-box. We propose that degradation of full-length AURKA also depends on an intact C-terminal domain because of critical conformational parameters permissive for both activity and mitotic degradation of AURKA.

In vitro generation of mouse morula-like cells.

Generating cells with the molecular and functional properties of embryo cells and with full developmental potential is an aim with fundamental biological significance. Here we report the in vitro generation of mouse transient morula-like cells (MLCs) via the manipulation of signaling pathways. MLCs are molecularly distinct from embryonic stem cells (ESCs) and cluster instead with embryo 8- to 16-cell stage cells. A single MLC can generate a blastoid, and the efficiency increases to 80% when 8-10 MLCs are used. MLCs make embryoids directly, efficiently, and within 4 days. Transcriptomic analysis shows that day 4-5 MLC-derived embryoids contain the cell types found in natural embryos at early gastrulation. Furthermore, MLCs introduced into morulae segregate into epiblast (EPI), primitive endoderm (PrE), and trophectoderm (TE) fates in blastocyst chimeras and have a molecular signature indistinguishable from that of host embryo cells. These findings represent the generation of cells that are molecularly and functionally similar to the precursors of the first three cell lineages of the embryo.

Single-cell RNA sequencing reveals the developmental program underlying proximal-distal patterning of the human lung at the embryonic stage.

The lung is the primary respiratory organ in human, in which the proximal airway and the distal alveoli are responsible for air conduction and gas exchange, respectively. However, the regulation of proximal-distal patterning at the embryonic stage of human lung development is largely unknown. Here we investigated the early lung development of human embryos at weeks 4-8 post fertilization (Carnegie stages 12-21) using single-cell RNA sequencing, and obtained a transcriptomic atlas of 169,686 cells. We observed discernible gene expression patterns of proximal and distal epithelia at week 4, upon the initiation of lung organogenesis. Moreover, we identified novel transcriptional regulators of the patterning of proximal (e.g., THRB and EGR3) and distal (e.g., ETV1 and SOX6) epithelia. Further dissection revealed various stromal cell populations, including an early-embryonic BDNF+ population, providing a proximal-distal patterning niche with spatial specificity. In addition, we elucidated the cell fate bifurcation and maturation of airway and vascular smooth muscle progenitor cells at the early stage of lung development. Together, our study expands the scope of human lung developmental biology at early embryonic stages. The discovery of intrinsic transcriptional regulators and novel niche providers deepens the understanding of epithelial proximal-distal patterning in human lung development, opening up new avenues for regenerative medicine.

Measuring the size and growth of single cells.

The size and growth of a cell can be described by three related physical parameters: volume, density and mass. All the three are coupled to numerous biochemical reactions and biophysical properties of a cell. It is therefore not surprising that cell size and growth pattern are tightly regulated across all kingdoms of life. Indeed, deregulation of cell size and growth has been found to be associated with diseases. Yet, how cells regulate their size and how cell size connects to cell function remain poorly understood, partly due to the difficulties to precisely measure the size and growth of single cells. In this review, we summarize methods of measuring cell volume, density, and mass, and discuss how the new technologies may advance our understanding of cell size control.

Induction of apoptosis by Polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma L929 cells.

BACKGROUND: The Galanthus nivalis agglutinin (GNA)-related lectins have been reported to bear antiproliferative and apoptosis-inducing activities in cancer cells; however, the precise mechanisms by which GNA-related lectins induce cell death are still only rudimentarily understood. METHODS: In the present study, Polygonatum odoratum lectin (designated POL), a mannose-binding specific GNA-related lectin, possessed a remarkable antiproliferative activity toward murine fibrosarcoma L929 cells. And, this lectin induced L929 cell apoptosis in a caspase-dependent manner. In addition, POL treatment increased the levels of FasL and Fas-Associated protein with Death Domain (FADD) proteins and resulted in caspase-8 activation. Also, POL treatment caused mitochondrial transmembrane potential collapse and cytochrome c release, leading to activations of caspase-9 and caspase-3. Moreover, POL treatment enhanced tumor necrosis factor alpha (TNFalpha)-induced L929 cell apoptosis. RESULTS: Our data demonstrate for the first time that this lectin induces apoptosis through both death-receptor and mitochondrial pathways, as well as amplifies TNFalpha-induced L929 cell apoptosis. GENERAL SIGNIFICANCE: These inspiring findings would provide new molecular basis for further understanding cell death mechanisms of the Galanthus nivalis agglutinin (GNA)-related lectins in future cancer investigations.

Temporal integration of mitogen history in mother cells controls proliferation of daughter cells.

Multicellular organisms use mitogens to regulate cell proliferation, but how fluctuating mitogenic signals are converted into proliferation-quiescence decisions is poorly understood. In this work, we combined live-cell imaging with temporally controlled perturbations to determine the time scale and mechanisms underlying this system in human cells. Contrary to the textbook model that cells sense mitogen availability only in the G1 cell cycle phase, we find that mitogenic signaling is temporally integrated throughout the entire mother cell cycle and that even a 1-hour lapse in mitogen signaling can influence cell proliferation more than 12 hours later. Protein translation rates serve as the integrator that proportionally converts mitogen history into corresponding levels of cyclin D in the G2 phase of the mother cell, which controls the proliferation-quiescence decision in daughter cells and thereby couples protein production with cell proliferation.

Visualizing the metazoan proliferation-quiescence decision in vivo.

Cell proliferation and quiescence are intimately coordinated during metazoan development. Here, we adapt a cyclin-dependent kinase (CDK) sensor to uncouple these key events of the cell cycle in Caenorhabditis elegans and zebrafish through live-cell imaging. The CDK sensor consists of a fluorescently tagged CDK substrate that steadily translocates from the nucleus to the cytoplasm in response to increasing CDK activity and consequent sensor phosphorylation. We show that the CDK sensor can distinguish cycling cells in G1 from quiescent cells in G0, revealing a possible commitment point and a cryptic stochasticity in an otherwise invariant C. elegans cell lineage. Finally, we derive a predictive model of future proliferation behavior in C. elegans based on a snapshot of CDK activity in newly born cells. Thus, we introduce a live-cell imaging tool to facilitate in vivo studies of cell-cycle control in a wide-range of developmental contexts.

All living things are made up of cells that form the different tissues, organs and structures of an organism. The human body, for example, is thought to consist of some 37 trillion cells and harbor over 200 cell types. To maintain a working organism, cells divide to create new cells and replace the ones that have died. Cell division is a tightly controlled process consisting of several steps, and cells continuously face a Shakespearean dilemma of deciding whether to continue dividing (also known as cell proliferation) or to halt the process (known as quiescence). This difficult balancing act is critical during all stages of life, from embryonic development to tissue growth in an adult. Problems in the underlying pathways can result in diseases such as cancer. Cell division is driven by proteins called CDKs, which help cells to complete their cell cycle in the correct sequence. To gain more insight into this complex process, scientists have developed tools for monitoring CDKs. One such tool is a fluorescent biosensor, a molecule that can be inserted into cells that glows and moves in response to CDK activity. The biosensor can be studied and measured in each cell using a microscope. Adikes, Kohrman, Martinez et al. adapted and optimized an existing CDK biosensor to help study cell division and the switch between proliferation and quiescence in two common research organisms, the nematode Caenorhabditis elegans and the zebrafish. Analysis of this biosensor showed that CDK activity at the end of cell division is higher if the cells will divide again but is low if the cells are going to become quiescent. This could suggest that the decision of a cell between proliferation and quiescence may happen earlier than expected. The optimized biosensor is sensitive enough to detect these differences and can even measure variations that influence proliferation in a region on C. elegans that was once thought to be unchanging. The development of this biosensor provides a useful research tool that could be used in other living organisms. Many research questions relate to cell division and so the applications of this tool are wide ranging.

Antiproliferative activity and apoptosis-inducing mechanism of Concanavalin A on human melanoma A375 cells.

The objective of this study was to investigate the antiproliferative activity and apoptosis-inducing mechanism of Concanavalin A (ConA) on human melanoma A375 cells. We firstly simulated the three-dimensional structure of ConA. Subsequently, we found that ConA possessed remarkable antiproliferative effect on A375 cells. Further experimental data indicated that there was a link between its hemagglutinating activity, mannose-binding activity and antiproliferative activity. In addition, we showed that ConA induced A375 cell apoptosis in a caspase-dependent manner. Then, we demonstrated that the treatment of ConA caused mitochondrial transmembrane potential (MMP) collapse, cytochrome c release and caspase activation. In conclusion, we report for the first time that there may be a close correlation between carbohydrate-binding activity of ConA and its antiproliferative activity. Also, we demonstrate firstly that ConA induces A375 cell death in a caspase-dependent manner as well as through a mitochondrial apoptotic pathway.

Ki67 is a Graded Rather than a Binary Marker of Proliferation versus Quiescence.

Ki67 staining is widely used as a proliferation indicator in the clinic, despite poor understanding of this protein's function or dynamics. Here, we track Ki67 levels under endogenous control in single cells over time and find that Ki67 accumulation occurs only during S, G2, and M phases. Ki67 is degraded continuously in G1 and G0 phases, regardless of the cause of entry into G0/quiescence. Consequently, the level of Ki67 during G0 and G1 in individual cells is highly heterogeneous and depends on how long an individual cell has spent in G0. Thus, Ki67 is a graded rather than a binary marker both for cell-cycle progression and time since entry into quiescence.

UBE2O remodels the proteome during terminal erythroid differentiation.

During terminal differentiation, the global protein complement is remodeled, as epitomized by erythrocytes, whose cytosol is ~98% globin. The erythroid proteome undergoes a rapid transition at the reticulocyte stage; however, the mechanisms driving programmed elimination of preexisting cytosolic proteins are unclear. We found that a mutation in the murine Ube2o gene, which encodes a ubiquitin-conjugating enzyme induced during erythropoiesis, results in anemia. Proteomic analysis suggested that UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome. In particular, ribosome elimination, a hallmark of reticulocyte differentiation, was defective in Ube2o-/- mutants. UBE2O recognized ribosomal proteins and other substrates directly, targeting them to proteasomes for degradation. Thus, in reticulocytes, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome.

Isolation of Ubiquitinated Proteins to High Purity from In Vivo Samples.

Ubiquitination pathways are widely used within eukaryotic cells. The complexity of ubiquitin signaling gives rise to a number of problems in the study of specific pathways. One problem is that not all processes regulated by ubiquitin are shared among the different cells of an organism (e.g., neurotransmitter release is only carried out in neuronal cells). Moreover, these processes are often highly temporally dynamic. It is essential therefore to use the right system for each biological question, so that we can characterize pathways specifically in the tissue or cells of interest. However, low stoichiometry, and the unstable nature of many ubiquitin conjugates, presents a technical barrier to studying this modification in vivo. Here, we describe two approaches to isolate ubiquitinated proteins to high purity. The first one favors isolation of the whole mixture of ubiquitinated material from a given tissue or cell type, generating a survey of the ubiquitome landscape for a specific condition. The second one favors the isolation of just one specific protein, in order to facilitate the characterization of its ubiquitinated fraction. In both cases, highly stringent denaturing buffers are used to minimize the presence of contaminating material in the sample.

Conserved molecular interactions in centriole-to-centrosome conversion.

Centrioles are required to assemble centrosomes for cell division and cilia for motility and signalling. New centrioles assemble perpendicularly to pre-existing ones in G1-S and elongate throughout S and G2. Fully elongated daughter centrioles are converted into centrosomes during mitosis to be able to duplicate and organize pericentriolar material in the next cell cycle. Here we show that centriole-to-centrosome conversion requires sequential loading of Cep135, Ana1 (Cep295) and Asterless (Cep152) onto daughter centrioles during mitotic progression in both Drosophila melanogaster and human. This generates a molecular network spanning from the inner- to outermost parts of the centriole. Ana1 forms a molecular strut within the network, and its essential role can be substituted by an engineered fragment providing an alternative linkage between Asterless and Cep135. This conserved architectural framework is essential for loading Asterless or Cep152, the partner of the master regulator of centriole duplication, Plk4. Our study thus uncovers the molecular basis for centriole-to-centrosome conversion that renders daughter centrioles competent for motherhood.

Ubiquitin-Mediated Degradation of Aurora Kinases.

The Aurora kinases are essential regulators of mitosis in eukaryotes. In somatic cell divisions of higher eukaryotes, the paralogs Aurora kinase A (AurA) and Aurora kinase B (AurB) play non-overlapping roles that depend on their distinct spatiotemporal activities. These mitotic roles of Aurora kinases depend on their interactions with different partners that direct them to different mitotic destinations and different substrates: AurB is a component of the chromosome passenger complex that orchestrates the tasks of chromosome segregation and cytokinesis, while AurA has many known binding partners and mitotic roles, including a well-characterized interaction with TPX2 that mediates its role in mitotic spindle assembly. Beyond the spatial control conferred by different binding partners, Aurora kinases are subject to temporal control of their activation and inactivation. Ubiquitin-mediated proteolysis is a critical route to irreversible inactivation of these kinases, which must occur for ordered transition from mitosis back to interphase. Both AurA and AurB undergo targeted proteolysis after anaphase onset as substrates of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase, even while they continue to regulate steps during mitotic exit. Temporal control of Aurora kinase destruction ensures that AurB remains active at the midbody during cytokinesis long after AurA activity has been largely eliminated from the cell. Differential destruction of Aurora kinases is achieved despite the fact that they are targeted at the same time and by the same ubiquitin ligase, making these substrates an interesting case study for investigating molecular determinants of ubiquitin-mediated proteolysis in higher eukaryotes. The prevalence of Aurora overexpression in cancers and their potential as therapeutic targets add importance to the task of understanding the molecular determinants of Aurora kinase stability. Here, we review what is known about ubiquitin-mediated targeting of these critical mitotic regulators and discuss the different factors that contribute to proteolytic control of Aurora kinase activity in the cell.