A Dynamic Gene Regulatory Network Model That Recovers the Cyclic Behavior of Arabidopsis thaliana Cell Cycle

Cell cycle control is fundamental in eukaryotic development. Several modeling efforts have been used to integrate the complex network of interacting molecular components involved in cell cycle dynamics. In this paper, we aimed at recovering the regulatory logic upstream of previously known components of cell cycle control, with the aim of understanding the mechanisms underlying the emergence of the cyclic behavior of such components. We focus on Arabidopsis thaliana, but given that many components of cell cycle regulation are conserved among eukaryotes, when experimental data for this system was not available, we considered experimental results from yeast and animal systems. We are proposing a Boolean gene regulatory network (GRN) that converges into only one robust limit cycle attractor that closely resembles the cyclic behavior of the key cell-cycle molecular components and other regulators considered here. We validate the model by comparing our in silico configurations with data from loss- and gain-of-function mutants, where the endocyclic behavior also was recovered. Additionally, we approximate a continuous model and recovered the temporal periodic expression profiles of the cell-cycle molecular components involved, thus suggesting that the single limit cycle attractor recovered with the Boolean model is not an artifact of its discrete and synchronous nature, but rather an emergent consequence of the inherent characteristics of the regulatory logic proposed here. This dynamical model, hence provides a novel theoretical framework to address cell cycle regulation in plants, and it can also be used to propose novel predictions regarding cell cycle regulation in other eukaryotes.     

The Caulobacter cell cycle: timing,spatial organization and checkpoints

In Caulobacter crescentus, a complex regulatory network integrates temporal and spatial information to control the ordered progression of the cell cycle, and to synchronize cell proliferation with development. Periodicity includes the timed synthesis, activation or destruction of key regulatory proteins, which activate a large number of genes at the appropriate time of the cell cycle. Checkpoints serve to couple cell division and polar development to the replication and segregation state of the chromosome. Asymmetrically positioned regulatory components are involved in the sequential positioning of polar organelles. New work sheds light on the spatial organization of cellular components involved in cell cycle progression and polar differentiation, and starts to define the molecular nature of checkpoints involved in cell cycle control and development.     

WI-38 cell long-term quiescence model system: A valuable tool to study molecular events that regulate growth

A number of cell culture model systems have been used to study the regulation of cell cycle progression at the molecular level. In this paper we describe the WI-38 cell long-term quiescence model system. By modulating the length of time that WI-38 cells are density arrested, it is possible to proportionately alter the length of the prereplicative or G-1 phase which the cell traverses after growth factor stimulation in preparation for entry into DNA synthesis. Through studies aimed at understanding the cause and molecular nature of the prolongation of the prereplicative phase, we have determined that gene expression plays an important role in establishing growth factor “competence” and that once the cell becomes “competent” there is a defined order to the molecular events that follow during the remainder of G-1. More specifically, we have determined that the prolongation represents a delay in the ability of long term quiescent cells to become fully “competent” to respond to growth factors which regulate progression through G-1 into S. This prolongation appears to occur as a result of changes during long term quiescence in the ability of immediate early G-1 specific genes (such as c-myc) to activate the expression of early G-1 specific genes (such as ornithine decarboxylase). While ODC is the first and thus far only growth associated gene identified as a target of c-myc (and the Myc/Max protein complex), it is likely that further studies in this model system will reveal other early G-1 growth regulatory genes. We anticipate that future follow-up studies in this model system will provide additional valuable information abuot the function of growth-regulatory genes in controlling growth factor responsiveness and cell cycle progression.     

Integrated actions of progesterone receptor and cell cycle machinery regulate breast cancer cell proliferation

Multiple laboratories have investigated progesterone receptor (PR) involvement in breast cancer cell cycle progression. There is now a growing body of evidence demonstrating complex interactions between PR and cell cycle regulatory proteins. Here we review the current literature linking PR to cell cycle control and discuss gaps in the current knowledge. A more complete understanding of the relationships between PR and cell cycle regulatory molecules may reveal additional avenues for prevention and treatment of steroid receptor positive breast cancers.     

Cyclin E and Cdk2 control GLD-1, the mitosis/meiosis decision, and germline stem cells in Caenorhabditis elegans

Coordination of the cell cycle with developmental events is crucial for generation of tissues during development and their maintenance in adults. Defects in that coordination can shift the balance of cell fates with devastating clinical effects. Yet our understanding of the molecular mechanisms integrating core cell cycle regulators with developmental regulators remains in its infancy. This work focuses on the interplay between cell cycle and developmental regulators in the Caenorhabditis elegans germline. Key developmental regulators control germline stem cells (GSCs) to self-renew or begin differentiation: FBF RNA-binding proteins promote self-renewal, while GLD RNA regulatory proteins promote meiotic entry. We first discovered that many but not all germ cells switch from the mitotic into the meiotic cell cycle after RNAi depletion of CYE-1 (C. elegans cyclin E) or CDK-2 (C. elegans Cdk2) in wild-type adults. Therefore, CYE-1/CDK-2 influences the mitosis/meiosis balance. We next found that GLD-1 is expressed ectopically in GSCs after CYE-1 or CDK-2 depletion and that GLD-1 removal can rescue cye-1/cdk-2 defects. Therefore, GLD-1 is crucial for the CYE-1/CDK-2 mitosis/meiosis control. Indeed, GLD-1 appears to be a direct substrate of CYE-1/CDK-2: GLD-1 is a phosphoprotein; CYE-1/CDK-2 regulates its phosphorylation in vivo; and human cyclin E/Cdk2 phosphorylates GLD-1 in vitro. Transgenic GLD-1(AAA) harbors alanine substitutions at three consensus CDK phosphorylation sites. GLD-1(AAA) is expressed ectopically in GSCs, and GLD-1(AAA) transgenic germlines have a smaller than normal mitotic zone. Together these findings forge a regulatory link between CYE-1/CDK-2 and GLD-1. Finally, we find that CYE-1/CDK-2 works with FBF-1 to maintain GSCs and prevent their meiotic entry, at least in part, by lowering GLD-1 abundance. Therefore, CYE-1/CDK-2 emerges as a critical regulator of stem cell maintenance. We suggest that cyclin E and Cdk-2 may be used broadly to control developmental regulators.     

Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G(0). PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCalpha alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt-villus axis revealed that PKCalpha activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.     

Structure, function and mechanism of the anaphase promoting complex (APC/C)

The complex molecular events responsible for coordinating chromosome replication and segregation with cell division and growth are collectively known as the cell cycle. Progression through the cell cycle is orchestrated by the interplay between controlled protein synthesis and degradation and protein phosphorylation. Protein degradation is primarily regulated through the ubiquitin proteasome system, mediated by two related E3 protein ubiquitin ligases, the Skp1 cullin F-box (SCF) and the anaphase promoting complex (also known as the cyclosome) (APC/C). The APC/C is a multi-subunit cullin-RING E3 ubiquitin ligase that regulates progression through the mitotic phase of the cell cycle and controls entry into S phase by catalysing the ubiquitylation of cyclins and other cell cycle regulatory proteins. Selection of APC/C targets is controlled through recognition of short destruction motifs, predominantly the D-box and KEN-box. APC/C-mediated coordination of cell cycle progression is achieved through the temporal regulation of APC/C activity and substrate specificity, exerted through a combination of co-activator subunits, reversible phosphorylation and inhibitory proteins and complexes. The aim of this article is to discuss the APC/C from a structural and mechanistic perspective. Although an atomic structure of the APC/C is still lacking, a combination of genetic, biochemical, electron microscopy studies of intact APC/C and crystallographic analysis of individual subunits, together with analogies to evolutionarily related E3 ligases of the RING family, has provided deep insights into the molecular mechanisms of catalysis and substrate recognition, and structural organisation of the APC/C.     

Signaling molecules involved in the transition of growth to development of Dictyostelium discoideum

The social amoeba Dictyostelium discoideum, a powerful paradigm provides clear insights into the regulation of growth and development. In addition to possessing complex individual cellular functions like a unicellular eukaryote, D. discoideum cells face the challenge of multicellular development. D. discoideum undergoes a relatively simple differentiation process mainly by cAMP mediated pathway. Despite this relative simplicity, the regulatory signaling pathways are as complex as those seen in metazoan development. However, the introduction of restriction-enzyme-mediated integration (REMI) technique to produce developmental gene knockouts has provided novel insights into the discovery of signaling molecules and their role in D. discoideum development. Cell cycle phase is an important aspect for differentiation of D. discoideum, as cells must reach a specific stage to enter into developmental phase and specific cell cycle regulators are involved in arresting growth phase genes and inducing the developmental genes. In this review, we present an overview of the signaling molecules involved in the regulation of growth to differentiation transition (GDT), molecular mechanism of early developmental events leading to generation of cAMP signal and components of cAMP relay system that operate in this paradigm.     

Fibro-adipogenic progenitors in skeletal muscle homeostasis,regeneration and diseases

Skeletal muscle possesses a remarkable regenerative capacity that relies on the activity of muscle stem cells, also known as satellite cells. The presence of non-myogenic cells also plays a key role in the coordination of skeletal muscle regeneration. Particularly, fibro-adipogenic progenitors (FAPs) emerged as master regulators of muscle stem cell function and skeletal muscle regeneration. This population of muscle resident mesenchymal stromal cells has been initially characterized based on its bi-potent ability to differentiate into fibroblasts or adipocytes. New technologies such as single-cell RNAseq revealed the cellular heterogeneity of FAPs and their complex regulatory network during muscle regeneration. In acute injury, FAPs rapidly enter the cell cycle and secrete trophic factors that support the myogenic activity of muscle stem cells. Conversely, deregulation of FAP cell activity is associated with the accumulation of fibrofatty tissue in pathological conditions such as muscular dystrophies and ageing. Considering their central role in skeletal muscle pathophysiology, the regulatory mechanisms of FAPs and their cellular and molecular crosstalk with muscle stem cells are highly investigated in the field. In this review, we summarize the current knowledge on FAP cell characteristics, heterogeneity and the cellular crosstalk during skeletal muscle homeostasis and regeneration. We further describe their role in muscular disorders, as well as different therapeutic strategies targeting these cells to restore muscle regeneration.     

Interactions and Tradeoffs Between Cell Recruitment,Proliferation, and Differentiation Affect CNS Regeneration

Regeneration of central nervous system (CNS) lesions requires movement of progenitor cells and production of their differentiated progeny. Although damage to the CNS clearly promotes these two processes, the interplay between these complex events and how it affects a response remains elusive. Here, we use spatial stochastic modeling to show that tradeoffs arise between production and recruitment during regeneration. Proper spatial control of cell cycle timing can mitigate these tradeoffs, maximizing recruitment, improving infiltration into the lesion, and reducing wasteful production outside of it. Feedback regulation of cell lineage dynamics alone however leads to spatial defects in cell recruitment, suggesting a novel, to our knowledge, hypothesis for the aggregation of cells to the periphery of a lesion in multiple sclerosis. Interestingly, stronger chemotaxis does not correct this aggregation and instead, substantial random cell motions near the site of the lesion are required to improve CNS regeneration.     

Crassulacean acid metabolism: plastic, fantastic

The occurrence, activity and plasticity of the CAM pathway is described from an introductory viewpoint, framed by the use of the four "Phases" of CAM as comparative indicators of the interplay between environmental constraints and internal molecular and biochemical regulation. Having described a number of "rules" which seem to govern the CAM cycle and apply uniformly to most species, a number of key regulatory points can then be identified. These include temporal separation of carboxylases, based on the circadian expression of key genes and their control by metabolites. The role of a circadian oscillator and interplay between tonoplast and nuclear control are central to maintaining the CAM cycle. Control of reserve carbohydrates is often neglected, but the importance of daily partitioning (for growth and the subsequent night-time CAM activity) and use at night is shown to drive the CAM cycle. Finally, it is shown that the genotypic and phenotypic plasticity in patterns of CAM expression is mediated partly by environmental conditions and molecular signalling, but also by diffusive constraints in succulent tissues. A transformation system is now required to allow these key areas of control to be elucidated.     

Interactions and Tradeoffs Between Cell Recruitment,Proliferation, and Differentiation Affect CNS Regeneration

Regeneration of central nervous system (CNS) lesions requires movement of progenitor cells and production of their differentiated progeny. Although damage to the CNS clearly promotes these two processes, the interplay between these complex events and how it affects a response remains elusive. Here, we use spatial stochastic modeling to show that tradeoffs arise between production and recruitment during regeneration. Proper spatial control of cell cycle timing can mitigate these tradeoffs, maximizing recruitment, improving infiltration into the lesion, and reducing wasteful production outside of it. Feedback regulation of cell lineage dynamics alone however leads to spatial defects in cell recruitment, suggesting a novel, to our knowledge, hypothesis for the aggregation of cells to the periphery of a lesion in multiple sclerosis. Interestingly, stronger chemotaxis does not correct this aggregation and instead, substantial random cell motions near the site of the lesion are required to improve CNS regeneration.     

PKCeta associates with cyclin E/Cdk2 complex in serum-starved MCF-7 and NIH-3T3 cells

Protein kinase C (PKC) encodes a family of enzymes implicated in cellular differentiation, growth control, and tumor promotion. However, very little is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that PKCeta associates with the cyclin E/Cdk2 complex. This is shown for the ectopically overexpressed PKCeta in NIH-3T3 cells, the inducibly expressed PKCeta in MCF-7 cells (under control of the tetracycline-responsive promoter), and the endogenously expressed PKCeta in mouse mammary epithelial HC11 cells. Subcellular cell fractionation experiments revealed that the complex with cyclin E is formed mostly in the nuclear fractions, although in these cells PKCeta is predominantly expressed in the cytosolic fractions. The complex of PKCeta and cyclin E was studied at various phases of the cell cycle, in serum-starved quiescent cells and in cells stimulated with serum to reenter the cell cycle. Interestingly, the interaction between PKCeta and cyclin E was most prominent in serum-starved cells and was disintegrated when cells entered the cells cycle. Immunofluorescence staining demonstrated that in serum-starved cells PKCeta is concentrated at the perinuclear zone, which is also the site of its colocalization with cyclin E. Colocalization of PKCeta and cyclin E in the perinuclear region was observed in serum-starved cells, and less in proliferating cells. These experiments suggest that the interaction between PKCeta and cyclin E is carefully regulated, and is correlated with the inactivated form of the cyclin E/Cdk2 complex. Thus, our studies support an important link between PKC and cell cycle control.     

Mitosis-specific phosphorylation of amyloid precursor protein at Threonine 668 leads to its altered processing and association with centrosomes

Background

Atypical expression of cell cycle regulatory proteins has been implicated in Alzheimer's disease (AD), but the molecular mechanisms by which they induce neurodegeneration are not well understood. We examined transgenic mice expressing human amyloid precursor protein (APP) and presenilin 1 (PS1) for changes in cell cycle regulatory proteins to determine whether there is a correlation between cell cycle activation and pathology development in AD.

Results

Our studies in the AD transgenic mice show significantly higher levels of cyclin E, cyclin D1, E2F1, and P-cdc2 in the cells in the vicinity of the plaques where maximum levels of Threonine 668 (Thr668)-phosphorylated APP accumulation was observed. This suggests that the cell cycle regulatory proteins might be influencing plaque pathology by affecting APP phosphorylation. Using neuroglioma cells overexpressing APP we demonstrate that phosphorylation of APP at Thr668 is mitosis-specific. Cells undergoing mitosis show altered cellular distribution and localization of P-APP at the centrosomes. Also, Thr668 phosphorylation in mitosis correlates with increased processing of APP to generate Aβ and the C-terminal fragment of APP, which is prevented by pharmacological inhibitors of the G1/S transition.

Conclusions

The data presented here suggests that cell cycle-dependent phosphorylation of APP may affect its normal cellular function. For example, association of P-APP with the centrosome may affect spindle assembly and cell cycle progression, further contributing to the development of pathology in AD. The experiments with G1/S inhibitors suggest that cell cycle inhibition may impede the development of Alzheimer's pathology by suppressing modification of βAPP, and thus may represent a novel approach to AD treatment. Finally, the cell cycle regulated phosphorylation and processing of APP into Aβ and the C-terminal fragment suggest that these proteins may have a normal function during mitosis.     

Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

ABSTRACT: In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT) inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC) inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space.     

Shape-dependent control of cell growth, differentiation, and apoptosis: switching between attractors in cell regulatory networks

Development of characteristic tissue patterns requires that individual cells be switched locally between different phenotypes or "fates;" while one cell may proliferate, its neighbors may differentiate or die. Recent studies have revealed that local switching between these different gene programs is controlled through interplay between soluble growth factors, insoluble extracellular matrix molecules, and mechanical forces which produce cell shape distortion. Although the precise molecular basis remains unknown, shape-dependent control of cell growth and function appears to be mediated by tension-dependent changes in the actin cytoskeleton. However, the question remains: how can a generalized physical stimulus, such as cell distortion, activate the same set of genes and signaling proteins that are triggered by molecules which bind to specific cell surface receptors. In this article, we use computer simulations based on dynamic Boolean networks to show that the different cell fates that a particular cell can exhibit may represent a preprogrammed set of common end programs or "attractors" which self-organize within the cell's regulatory networks. In this type of dynamic network model of information processing, generalized stimuli (e.g., mechanical forces) and specific molecular cues elicit signals which follow different trajectories, but eventually converge onto one of a small set of common end programs (growth, quiescence, differentiation, apoptosis, etc.). In other words, if cells use this type of information processing system, then control of cell function would involve selection of preexisting (latent) behavioral modes of the cell, rather than instruction by specific binding molecules. Importantly, the results of the computer simulation closely mimic experimental data obtained with living endothelial cells. The major implication of this finding is that current methods used for analysis of cell function that rely on characterization of linear signaling pathways or clusters of genes with common activity profiles may overlook the most critical features of cellular information processing which normally determine how signal specificity is established and maintained in living cells.