NPP-16/Nup50 Function and CDK-1 Inactivation Are Associated with Anoxia-induced Prophase Arrest in Caenorhabditis elegans

Oxygen, an essential nutrient, is sensed by a multiple of cellular pathways that facilitate the responses to and survival of oxygen deprivation. The Caenorhabditis elegans embryo exposed to severe oxygen deprivation (anoxia) enters a state of suspended animation in which cell cycle progression reversibly arrests at specific stages. The mechanisms regulating interphase, prophase, or metaphase arrest in response to anoxia are not completely understood. Characteristics of arrested prophase blastomeres and oocytes are the alignment of condensed chromosomes at the nuclear periphery and an arrest of nuclear envelope breakdown. Notably, anoxia-induced prophase arrest is suppressed in mutant embryos lacking nucleoporin NPP-16/NUP50 function, indicating that this nucleoporin plays an important role in prophase arrest in wild-type embryos. Although the inactive form of cyclin-dependent kinase (CDK-1) is detected in wild-type–arrested prophase blastomeres, the inactive state is not detected in the anoxia exposed npp-16 mutant. Furthermore, we found that CDK-1 localizes near chromosomes in anoxia-exposed embryos. These data support the notion that NPP-16 and CDK-1 function to arrest prophase blastomeres in C. elegans embryos. The anoxia-induced shift of cells from an actively dividing state to an arrested state reveals a previously uncharacterized prophase checkpoint in the C. elegans embryo.     

Dephosphorylation of cell cycle-regulated proteins correlates with anoxia-induced suspended animation in Caenorhabditis elegans

Some metazoans have evolved the capacity to survive severe oxygen deprivation. The nematode, Caenorhabditis elegans, exposed to anoxia (0 kPa, 0% O(2)) enters into a recoverable state of suspended animation during all stages of the life cycle. That is, all microscopically observable movement ceases including cell division, developmental progression, feeding, and motility. To understand suspended animation, we compared oxygen-deprived embryos to nontreated embryos in both wild-type and hif-1 mutants. We found that hif-1 mutants survive anoxia, suggesting that the mechanisms for anoxia survival are different from those required for hypoxia. Examination of wild-type embryos exposed to anoxia show that blastomeres arrest in interphase, prophase, metaphase, and telophase but not anaphase. Analysis of the energetic state of anoxic embryos indicated a reversible depression in the ATP to ADP ratio. Given that a decrease in ATP concentrations likely affects a variety of cellular processes, including signal transduction, we compared the phosphorylation state of several proteins in anoxic embryos and normoxic embryos. We found that the phosphorylation state of histone H3 and cell cycle-regulated proteins recognized by the MPM-2 antibody were not detectable in anoxic embryos. Thus, dephosphorylation of specific proteins correlate with the establishment and/or maintenance of a state of anoxia-induced suspended animation.     

Anoxia-Reoxygenation Regulates Mitochondrial Dynamics through the Hypoxia Response Pathway,SKN-1/Nrf,and Stomatin-Like Protein STL-1/SLP-2

Many aerobic organisms encounter oxygen-deprived environments and thus must have adaptive mechanisms to survive such stress. It is important to understand how mitochondria respond to oxygen deprivation given the critical role they play in using oxygen to generate cellular energy. Here we examine mitochondrial stress response in C. elegans, which adapt to extreme oxygen deprivation (anoxia, less than 0.1% oxygen) by entering into a reversible suspended animation state of locomotory arrest. We show that neuronal mitochondria undergo DRP-1-dependent fission in response to anoxia and undergo refusion upon reoxygenation. The hypoxia response pathway, including EGL-9 and HIF-1, is not required for anoxia-induced fission, but does regulate mitochondrial reconstitution during reoxygenation. Mutants for egl-9 exhibit a rapid refusion of mitochondria and a rapid behavioral recovery from suspended animation during reoxygenation; both phenotypes require HIF-1. Mitochondria are significantly larger in egl-9 mutants after reoxygenation, a phenotype similar to stress-induced mitochondria hyperfusion (SIMH). Anoxia results in mitochondrial oxidative stress, and the oxidative response factor SKN-1/Nrf is required for both rapid mitochondrial refusion and rapid behavioral recovery during reoxygenation. In response to anoxia, SKN-1 promotes the expression of the mitochondrial resident protein Stomatin-like 1 (STL-1), which helps facilitate mitochondrial dynamics following anoxia. Our results suggest the existence of a conserved anoxic stress response involving changes in mitochondrial fission and fusion.     

Characterization of sub-nuclear changes in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> embryos exposed to brief,intermediate and long-term anoxia to analyze anoxia-induced cell cycle arrest

Background  

The soil nematode C. elegans survives oxygen-deprived conditions (anoxia; <.001 kPa O₂) by entering into a state of suspended animation in which cell cycle progression reversibly arrests. The majority of blastomeres of embryos exposed to anoxia arrest at interphase, prophase and metaphase. The spindle checkpoint proteins SAN-1 and MDF-2 are required for embryos to survive 24 hours of anoxia. To further investigate the mechanism of cell-cycle arrest we examined and compared sub-nuclear changes such as chromatin localization pattern, post-translational modification of histone H3, spindle microtubules, and localization of the spindle checkpoint protein SAN-1 with respect to various anoxia exposure time points. To ensure analysis of embryos exposed to anoxia and not post-anoxic recovery we fixed all embryos in an anoxia glove box chamber.     

Coordinating cell polarity and cell cycle progression: what can we learn from flies and worms?

Spatio-temporal coordination of events during cell division is crucial for animal development. In recent years, emerging data have strengthened the notion that tight coupling of cell cycle progression and cell polarity in dividing cells is crucial for asymmetric cell division and ultimately for metazoan development. Although it is acknowledged that such coupling exists, the molecular mechanisms linking the cell cycle and cell polarity machineries are still under investigation. Key cell cycle regulators control cell polarity, and thus influence cell fate determination and/or differentiation, whereas some factors involved in cell polarity regulate cell cycle timing and proliferation potential. The scope of this review is to discuss the data linking cell polarity and cell cycle progression, and the importance of such coupling for asymmetric cell division. Because studies in model organisms such as Caenorhabditis elegans and Drosophila melanogaster have started to reveal the molecular mechanisms of this coordination, we will concentrate on these two systems. We review examples of molecular mechanisms suggesting a coupling between cell polarity and cell cycle progression.     

Minichromosome maintenance protein 5 homologue in Caenorhabditis elegans plays essential role for postembryonic development

Genome duplication is tightly controlled in multicellular organisms to ensure the genome stability. Studies in Saccharomyces cerevisiae and Xenopus show that minichromosome maintenance (MCM) proteins are essential for genome duplication. However, the development role of MCM proteins in multicellular organisms is not well known. MCM5 encodes a member of the MCM2-7 protein family involved in the initiation of DNA replication. The sequences of all Mcm5 homologues from yeast to human are highly conserved and suggest that their functions are also conserved. Here, we isolated the first mutant allele of mcm-5 (fw7) in Caenorhabditis elegans. Homozygous mcm-5 (fw7) mutants from heterozygous parents exhibited variable larval lethality and adult sterility. The postembryonically born neuron number was decreased and also showed aberrant axon morphology. Our study revealed that the losses of neurons in mcm-5 (fw7) mutants were caused by cell cycle defects not by programmed cell death. The examination showed that mcm-5 was widely used for postembryonic development in multiple cells such as seam cells, gonad and intestinal cells. Knockdown of mcm-5 by RNAi caused 98.1% embryonic arrest, suggesting that mcm-5 was also required for embryonic development. After RNAi treatment of the other MCM2-7 family members, we found that they all exhibited similar phenotypes as mcm-5, suggesting that the MCM2-7 family in C. elegans might function associated with cell division as its homologues in S. cerevisiae.     

Suspended animation,diapause and quiescence: Arresting the cell cycle in C. elegans

Developing organisms require nutrients to support cell division vital for growth and development. An adaptation to stress, used by many organisms, is to reversibly enter an arrested state by reducing energy-requiring processes, such as development and cell division. This “wait it out” approach to survive stress until the environment is conductive for growth and development is used by many metazoans. Much is known about the molecular regulation of cell division, metazoan development and responses to environmental stress. However, how these biological processes intersect is less understood. Here, we review studies conducted in Caenorhabditis elegans that investigate how stresses such as oxygen deprivation (hypoxia and anoxia), exogenous chemicals or starvation affect cellular processes in the embryo, larvae or adult germline. Using C. elegans to identify how stress signals biological arrest can help in our understanding of evolutionary pressures as well as human health-related issues.     

Functional Dissection of Caenorhabditis elegans CLK-2/TEL2 Cell Cycle Defects during Embryogenesis and Germline Development

CLK-2/TEL2 is essential for viability from yeasts to vertebrates, but its essential functions remain ill defined. CLK-2/TEL2 was initially implicated in telomere length regulation in budding yeast, but work in Caenorhabditis elegans has uncovered a function in DNA damage response signalling. Subsequently, DNA damage signalling defects associated with CLK-2/TEL2 have been confirmed in yeast and human cells. The CLK-2/TEL2 interaction with the ATM and ATR DNA damage sensor kinases and its requirement for their stability led to the proposal that CLK-2/TEL2 mutants might phenocopy ATM and/or ATR depletion. We use C. elegans to dissect developmental and cell cycle related roles of CLK-2. Temperature sensitive (ts) clk-2 mutants accumulate genomic instability and show a delay of embryonic cell cycle timing. This delay partially depends on the worm p53 homolog CEP-1 and is rescued by co-depletion of the DNA replication checkpoint proteins ATL-1 (C. elegans ATR) and CHK-1. In addition, clk-2 ts mutants show a spindle orientation defect in the eight cell stages that lead to major cell fate transitions. clk-2 deletion worms progress through embryogenesis and larval development by maternal rescue but become sterile and halt germ cell cycle progression. Unlike ATL-1 depleted germ cells, clk-2–null germ cells do not accumulate DNA double-strand breaks. Rather, clk-2 mutant germ cells arrest with duplicated centrosomes but without mitotic spindles in an early prophase like stage. This germ cell cycle arrest does not depend on cep-1, the DNA replication, or the spindle checkpoint. Our analysis shows that CLK-2 depletion does not phenocopy PIKK kinase depletion. Rather, we implicate CLK-2 in multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development.     

Efficient chaperone-mediated tubulin biogenesis is essential for cell division and cell migration in C. elegans

The efficient folding of actin and tubulin in vitro and in Saccharomyces cerevisiae is known to require the molecular chaperones prefoldin and CCT, yet little is known about the functions of these chaperones in multicellular organisms. Whereas none of the six prefoldin genes are essential in yeast, where prefoldin-independent folding of actin and tubulin is sufficient for viability, we demonstrate that reducing prefoldin function by RNAi in Caenorhabditis elegans causes defects in cell division that result in embryonic lethality. Our analyses suggest that these defects result mainly from a decrease in α-tubulin levels and a subsequent reduction in the microtubule growth rate. Prefoldin subunit 1 (pfd-1) mutant animals with maternally contributed PFD-1 develop to the L4 larval stage with gonadogenesis defects that include aberrant distal tip cell migration. Importantly, RNAi knockdown of prefoldin, CCT or tubulin in developing animals phenocopy the pfd-1 cell migration phenotype. Furthermore, reducing CCT function causes more severe phenotypes (compared with prefoldin knockdown) in the embryo and developing gonad, consistent with a broader role for CCT in protein folding. Overall, our results suggest that efficient chaperone-mediated tubulin biogenesis is essential in C. elegans, owing to the critical role of the microtubule cytoskeleton in metazoan development.     

Suspended animation,diapause and quiescence

Developing organisms require nutrients to support cell division vital for growth and development. An adaptation to stress, used by many organisms, is to reversibly enter an arrested state by reducing energy-requiring processes, such as development and cell division. This “wait it out” approach to survive stress until the environment is conductive for growth and development is used by many metazoans. Much is known about the molecular regulation of cell division, metazoan development and responses to environmental stress. However, how these biological processes intersect is less understood. Here, we review studies conducted in Caenorhabditis elegans that investigate how stresses such as oxygen deprivation (hypoxia and anoxia), exogenous chemicals or starvation affect cellular processes in the embryo, larvae or adult germline. Using C. elegans to identify how stress signals biological arrest can help in our understanding of evolutionary pressures as well as human health-related issues.     

An intrinsic cell cycle checkpoint pathway mediated by MEK and ERK in Drosophila

Cell cycle checkpoints are surveillance mechanisms that safeguard genome integrity. While the extrinsic pathways that halt the cell cycle in response to DNA damages have been well documented, the intrinsic pathways that ensure orderly progression of cell cycle events are not well understood. We demonstrate that Drosophila MEK and ERK constitute an essential intrinsic checkpoint pathway that restrains cell cycle progression in the absence of DNA damage and also responds to ionizing radiation to arrest the cell cycle. Embryos lacking MEK exhibit faster and extra division cycles and fail to undergo timely midblastula transition (MBT) or arrest following ionizing radiation. Conversely, constitutively activated MEK causes cell cycle arrest. Further, MEK activation in the early embryo is cell cycle-dependent and Raf independent and increases in response to ionizing radiation or in the absence of Chk1. Thus, MEK/ERK activation is required for multiple checkpoints and is essential for orderly cell cycle progression.     

PAR-4/LKB1 regulates DNA replication during asynchronous division of the early C. elegans embryo

Regulation of cell cycle duration is critical during development, yet the underlying molecular mechanisms are still poorly understood. The two-cell stage Caenorhabditis elegans embryo divides asynchronously and thus provides a powerful context in which to study regulation of cell cycle timing during development. Using genetic analysis and high-resolution imaging, we found that deoxyribonucleic acid (DNA) replication is asymmetrically regulated in the two-cell stage embryo and that the PAR-4 and PAR-1 polarity proteins dampen DNA replication dynamics specifically in the posterior blastomere, independently of regulators previously implicated in the control of cell cycle timing. Our results demonstrate that accurate control of DNA replication is crucial during C. elegans early embryonic development and further provide a novel mechanism by which PAR proteins control cell cycle progression during asynchronous cell division.     

Balanced Production of Ribosome Components Is Required for Proper G1/S Transition in Saccharomyces cerevisiae

Cell cycle regulation is a very accurate process that ensures cell viability and the genomic integrity of daughter cells. A fundamental part of this regulation consists in the arrest of the cycle at particular points to ensure the completion of a previous event, to repair cellular damage, or to avoid progression in potentially risky situations. In this work, we demonstrate that a reduction in nucleotide levels or the depletion of RNA polymerase I or III subunits generates a cell cycle delay at the G₁/S transition in Saccharomyces cerevisiae. This delay is concomitant with an imbalance between ribosomal RNAs and proteins which, among others, provokes an accumulation of free ribosomal protein L5. Consistently with a direct impact of free L5 on the G₁/S transition, rrs1 mutants, which weaken the assembly of L5 and L11 on pre-60S ribosomal particles, enhance both the G₁/S delay and the accumulation of free ribosomal protein L5. We propose the existence of a surveillance mechanism that couples the balanced production of yeast ribosomal components and cell cycle progression through the accumulation of free ribosomal proteins. This regulatory pathway resembles the p53-dependent nucleolar-stress checkpoint response described in human cells, which indicates that this is a general control strategy extended throughout eukaryotes.     

Activation of the Cell Wall Integrity Pathway Promotes Escape from G2 in the Fungus Ustilago maydis

It is widely accepted that MAPK activation in budding and fission yeasts is often associated with negative effects on cell cycle progression, resulting in delay or arrest at a specific stage in the cell cycle, thereby enabling cells to adapt to changing environmental conditions. For instance, activation of the Cell Wall Integrity (CWI) pathway in the budding yeast Saccharomyces cerevisiae signals an increase in CDK inhibitory phosphorylation, which leads cells to remain in the G2 phase. Here we characterized the CWI pathway of Ustilago maydis, a fungus evolutionarily distant from budding and fission yeasts, and show that activation of the CWI pathway forces cells to escape from G2 phase. In spite of these disparate cell cycle responses in S. cerevisiae and U. maydis, the CWI pathway in both organisms appears to respond to the same class cell wall stressors. To understand the basis of such a difference, we studied the mechanism behind the U. maydis response. We found that activation of CWI pathway in U. maydis results in a decrease in CDK inhibitory phosphorylation, which depends on the mitotic phosphatase Cdc25. Moreover, in response to activation of the CWI pathway, Cdc25 accumulates in the nucleus, providing a likely explanation for the increase in the unphosphorylated form of CDK. We also found that the extended N-terminal domain of Cdc25, which is dispensable under normal growth conditions, is required for this G2 escape as well as for resistance to cell wall stressors. We propose that the process of cell cycle adaptation to cell stress evolved differently in these two divergent organisms so that each can move towards a cell cycle phase most appropriate for responding to the environmental signals encountered.     

DAF-18/PTEN inhibits germline zygotic gene activation during primordial germ cell quiescence

Quiescence, an actively-maintained reversible state of cell cycle arrest, is not well understood. PTEN is one of the most frequently lost tumor suppressors in human cancers and regulates quiescence of stem cells and cancer cells. The sole PTEN ortholog in Caenorhabditis elegans is daf-18. In a C. elegans loss-of-function mutant for daf-18, primordial germ cells (PGCs) divide inappropriately in L1 larvae hatched into starvation conditions, in a TOR-dependent manner. Here, we further investigated the role of daf-18 in maintaining PGC quiescence in L1 starvation. We found that maternal or zygotic daf-18 is sufficient to maintain cell cycle quiescence, that daf-18 acts in the germ line and soma, and that daf-18 affects timing of PGC divisions in fed animals. Importantly, our results also implicate daf-18 in repression of germline zygotic gene activation, though not in germline fate specification. However, TOR is less important to germline zygotic gene expression, suggesting that in the absence of food, daf-18/PTEN prevents inappropriate germline zygotic gene activation and cell division by distinct mechanisms.     

The architecture and conservation pattern of whole-cell control circuitry

The control circuitry that directs and paces Caulobacter cell cycle progression involves the entire cell operating as an integrated system. This control circuitry monitors the environment and the internal state of the cell, including the cell topology, as it orchestrates orderly activation of cell cycle subsystems and Caulobacter's asymmetric cell division. The proteins of the Caulobacter cell cycle control system and its internal organization are co-conserved across many alphaproteobacteria species, but there are great differences in the regulatory apparatus' functionality and peripheral connectivity to other cellular subsystems from species to species. This pattern is similar to that observed for the “kernels” of the regulatory networks that regulate development of metazoan body plans. The Caulobacter cell cycle control system has been exquisitely optimized as a total system for robust operation in the face of internal stochastic noise and environmental uncertainty. When sufficient details accumulate, as for Caulobacter cell cycle regulation, the system design has been found to be eminently rational and indeed consistent with good design practices for human-designed asynchronous control systems.     

Regulation of the Cell Cycle by Protein Phosphatase 2A in Saccharomyces cerevisiae

Protein phosphatase 2A (PP2A) has long been implicated in cell cycle regulation in many different organisms. In the yeast Saccharomyces cerevisiae, PP2A controls cell cycle progression mainly through modulation of cyclin-dependent kinase (CDK) at the G₂/M transition. However, CDK does not appear to be a direct target of PP2A. PP2A affects CDK activity through its roles in checkpoint controls. Inactivation of PP2A downregulates CDK by activating the morphogenesis checkpoint and, consequently, delays mitotic entry. Defects in PP2A also compromise the spindle checkpoint and predispose the cell to an error-prone mitotic exit. In addition, PP2A is involved in controlling the G₁/S transition and cytokinesis. These findings suggest that PP2A functions in many stages of the cell cycle and its effect on cell cycle progression is pleiotropic.     

Balance between cell division and differentiation during plant development

The processes which make possible that a cell gives rise to two daughter cells define the cell division cycle. In individual cells, this is strictly controlled both in time and space. In multicellular organisms extra layers of regulation impinge on the balance between cell proliferation and cell differentiation within particular ontogenic programs. In contrast to animals, organogenesis in plants is a post-embryonic process that requires developmentally programmed reversion of sets of cells from different differentiated states to a pluripotent state followed by regulated proliferation and progression through distinct differentiation patterns. This implies a fine coupling of cell division control, cell cycle arrest and reactivation, endoreplication and differentiation. The emerging view is that cell cycle regulators, in addition to controlling cell division, also function as targets for maintaining cell homeostasis during development. The mechanisms and cross talk among different cell cycle regulatory pathways are discussed here in the context of a developing plant.