Experimental evolution of multicellularity

Multicellularity was one of the most significant innovations in the history of life, but its initial evolution remains poorly understood. Using experimental evolution, we show that key steps in this transition could have occurred quickly. We subjected the unicellular yeast Saccharomyces cerevisiae to an environment in which we expected multicellularity to be adaptive. We observed the rapid evolution of clustering genotypes that display a novel multicellular life history characterized by reproduction via multicellular propagules, a juvenile phase, and determinate growth. The multicellular clusters are uniclonal, minimizing within-cluster genetic conflicts of interest. Simple among-cell division of labor rapidly evolved. Early multicellular strains were composed of physiologically similar cells, but these subsequently evolved higher rates of programmed cell death (apoptosis), an adaptation that increases propagule production. These results show that key aspects of multicellular complexity, a subject of central importance to biology, can readily evolve from unicellular eukaryotes.     

Mechanical feedback as a possible regulator of tissue growth

Regulation of cell growth and proliferation has a fundamental role in animal and plant development and in the progression of cancer. In the context of development, it is important to understand the mechanisms that coordinate growth and patterning of tissues. Imaginal discs, which are larval precursors of fly limbs and organs, have provided much of what we currently know about these processes. Here, we consider the mechanism that is responsible for the observed uniformity of growth in wing imaginal discs, which persists in the presence of gradients in growth inducing morphogens in spite of the stochastic nature of cell division. The phenomenon of “cell competition,” which manifests in apoptosis of slower-growing cells in the vicinity of faster growing tissue, suggests that uniform growth is not a default state but a result of active regulation. How can a patch of tissue compare its growth rate with that of its surroundings? A possible way is furnished by mechanical interactions. To demonstrate this mechanism, we formulate a mathematical model of nonuniform growth in a layer of tissue and examine its mechanical implications. We show that a clone growing faster or slower than the surrounding tissue is subject to mechanical stress, and we propose that dependence of the rate of cell division on local stress could provide an “integral-feedback” mechanism stabilizing uniform growth. The proposed mechanism of growth control is not specific to imaginal disc growth and could be of general relevance. Several experimental tests of the proposed mechanism are suggested.     

Mitochondrial hexokinase II (HKII) and phosphoprotein enriched in astrocytes (PEA15) form a molecular switch governing cellular fate depending on the metabolic state

The metabolic state of a cell is a key determinant in the decision to live and proliferate or to die. Consequently, balanced energy metabolism and the regulation of apoptosis are critical for the development and maintenance of differentiated organisms. Hypoxia occurs physiologically during development or exercise and pathologically in vascular disease, tumorigenesis, and inflammation, interfering with homeostatic metabolism. Here, we show that the hypoxia-inducible factor (HIF)-1-regulated glycolytic enzyme hexokinase II (HKII) acts as a molecular switch that determines cellular fate by regulating both cytoprotection and induction of apoptosis based on the metabolic state. We provide evidence for a direct molecular interactor of HKII and show that, together with phosphoprotein enriched in astrocytes (PEA15), HKII inhibits apoptosis after hypoxia. In contrast, HKII accelerates apoptosis in the absence of PEA15 and under glucose deprivation. HKII both protects cells from death during hypoxia and functions as a sensor of glucose availability during normoxia, inducing apoptosis in response to glucose depletion. Thus, HKII-mediated apoptosis may represent an evolutionarily conserved altruistic mechanism to eliminate cells during metabolic stress to the advantage of a multicellular organism.     

Kinetochore asymmetry defines a single yeast lineage

Asymmetric cell division is of fundamental importance in biology as it allows for the establishment of separate cell lineages during the development of multicellular organisms. Although microbial systems, including the yeast Saccharomyces cerevisiae, are excellent models of asymmetric cell division, this phenotype occurs in all cell divisions; consequently, models of lineage-specific segregation patterns in these systems do not exist. Here, we report the first example of lineage-specific asymmetric division in yeast. We used fluorescent tags to show that components of the yeast kinetochore, the protein complex that anchors chromosomes to the mitotic spindle, divide asymmetrically in a single postmeiotic lineage. This phenotype is not seen in vegetatively dividing haploid or diploid cells. This kinetochore asymmetry suggests a mechanism for the selective segregation of sister centromeres to daughter cells to establish different cell lineages or fates. These results provide a mechanistic link between lineage-defining asymmetry of metazoa with unicellular eukaryotes.     

WNK1 is required for mitosis and abscission

WNK [with no lysine (K)] protein kinases are found in all sequenced multicellular and many unicellular organisms. WNKs influence ion balance. Two WNK family members are associated with a single gene form of hypertension. RNA interference screens have implicated WNKs in survival and growth, and WNK1 is essential for viability of mice. We found that the majority of WNK1 is localized on cytoplasmic puncta in resting cells. During cell division, WNK1 localizes to mitotic spindles. Therefore, we analyzed mitotic phenotypes in WNK1 knockdown cells. A large percentage of WNK1 knockdown cells fail to complete cell division, displaying defects in mitotic spindles and also in abscission and cell survival. One of the best-characterized WNK1 targets is the protein kinase OSR1 (oxidative stress responsive 1). OSR1 regulates ion cotransporters, is activated in response to osmotic stress by WNK family members, and is largely associated with WNK1. In resting cells, the majority of OSR1, like WNK1, is on cytoplasmic puncta. OSR1 is also in nuclei. In contrast to WNK1, however, OSR1 does not concentrate around spindles during mitosis and does not show a WNK1-like localization pattern in mitotic cells. Knockdown of OSR1 has only a modest effect on cell survival and does not lead to spindle defects. We conclude that decreased cell survival associated with loss of WNK1 is attributable to defects in chromosome segregation and abscission and is independent of the effector kinase OSR1.     

Solid stress generated by spheroid growth estimated using a linear poroelasticity model small star, filled

The unchecked growth of a solid tumor produces solid stress, causing deformation of the surrounding tissue. This stress can result in clinical complications, especially in confined environments such as the brain, and may also be responsible for pathophysiological anomalies such as the collapse of blood and lymphatic vessels. High stress levels may also inhibit further cell division within tumors. Unfortunately, little is known about the dynamics of stress accumulation in tumors or its effects on cell biology. We present a mathematical model for tumor growth in a confined, elastic environment such as living tissue. The model, developed from theories of thermal expansion using the current configuration of the material element, allows the stresses within the growing tumor and the surrounding medium to be calculated. The experimental observation that confining environments limit the growth of tumor spheroids to less than the limit imposed by nutrient diffusion is incorporated into the model using a stress dependent rate for tumor growth. The model is validated against experiments for MU89 tumor spheroid growth in Type VII agarose gel. Using the mathematical model and the experimental evidence we show that the tumor cell size is reduced by solid stress inside the tumor spheroid. This leads to the interesting possibility that cell size could be a direct indicator of solid stress level inside the tumors in clinical setting.     

Effect of calorie restriction on the metabolic history of chronologically aging yeast

Aging is a highly complex, multifactorial process. We use the yeast Saccharomyces cerevisiae as a model to study the mechanisms of cellular aging in multicellular eukaryotes. To address the inherent complexity of aging from a systems perspective and to build an integrative model of aging process, we investigated the effect of calorie restriction (CR), a low-calorie dietary regimen, on the metabolic history of chronologically aging yeast. We examined how CR influences the age-related dynamics of changes in the intracellular levels of numerous proteins and metabolites, carbohydrate and lipid metabolism, interorganellar metabolic flow, concentration of reactive oxygen species, mitochondrial morphology, essential oxidation–reduction processes in mitochondria, mitochondrial proteome, cardiolipin in the inner mitochondrial membrane, frequency of mitochondrial DNA mutations, dynamics of mitochondrial nucleoid, susceptibility to mitochondria-controlled apoptosis, and stress resistance. Based on the comparison of the metabolic histories of long-lived CR yeast and short-lived non-CR yeast, we propose that yeast define their long-term viability by designing a diet-specific pattern of metabolism and organelle dynamics prior to reproductive maturation. Thus, our data suggest that longevity in chronologically aging yeast is programmed by the level of metabolic capacity and organelle organization they developed, in a diet-specific fashion, prior to entry into a non-proliferative state.     

Phylogenetic fate mapping

Cell fate maps describe how the sequence of cell division, migration, and apoptosis transform a zygote into an adult. Yet, it is only in Caenorhabditis elegans where microscopic observation of each cell division has allowed for construction of a complete fate map. More complex, and opaque, animals prove less yielding. DNA replication, however, generates somatic mutations. Consequently, multicellular organisms comprise mosaics where most cells acquire unique genomes that are potentially capable of delineating their ancestry. Here we take a phylogenetic approach to passively retrace embryonic relationships by deducing the order in which mutations have arisen during development. We show that polyguanine repeat DNA sequences are particularly useful genetic markers, because they frequently change length during mitosis. To demonstrate feasibility, we phylogenetically reconstruct the lineage of cultured mouse NIH 3T3 cells based on mutations affecting the length of polyguanine markers. We then employ whole genome amplification to genotype polyguanine markers in single cells taken from a mouse and use phylogenetics to infer the developmental relationships of the sampled tissues. The result is consistent with the present understanding of embryogenesis and demonstrates the large scale potential of this method for producing a complete mammalian cell fate at the resolution of a single cell.     

Asymmetric Stem Cell Division and function of the Niche in the <Emphasis Type="Italic">Drosophila</Emphasis> Male Germ Line

The balance between stem cell and differentiating cell populations is critical for the long-term maintenance of tissue renewal for cell types derived from adult stem cell lineages such as blood, skin, intestinal epithelium, and sperm. To keep this balance, stem cells have the potential to divide asymmetrically, producing one daughter cell that maintains stem cell identity and one daughter cell that initiates differentiation. In many adult stem cell systems, the maintenance, proliferation, and number of stem cells appear to be controlled by the microenvironment, or niche. The Drosophila male and female germ line provide excellent model systems in which to study asymmetric stem cell divisions within the stem cell niche. In addition to signals from the niche that specify stem cell self-renewal, the stem cells themselves have elaborate cellular mechanisms to ensure the asymmetric outcome of cell division.     

Multicellularity and the functional interdependence of motility and molecular transport

Benefits, costs, and requirements accompany the transition from motile totipotent unicellular organisms to multicellular organisms having cells specialized into reproductive (germ) and vegetative (sterile soma) functions such as motility. In flagellated colonial organisms such as the volvocalean green algae, organized beating by the somatic cells' flagella yields propulsion important in phototaxis and chemotaxis. It has not been generally appreciated that for the larger colonies flagellar stirring of boundary layers and remote transport are fundamental for maintaining a sufficient rate of metabolite turnover, one not attainable by diffusive transport alone. Here, we describe experiments that quantify the role of advective dynamics in enhancing productivity in germ soma-differentiated colonies. First, experiments with suspended deflagellated colonies of Volvox carteri show that forced advection improves productivity. Second, particle imaging velocimetry of fluid motion around colonies immobilized by micropipette aspiration reveals flow fields with very large characteristic velocities U extending to length scales exceeding the colony radius R. For a typical metabolite diffusion constant D, the associated Peclet number Pe = 2UR/D > 1, indicative of the dominance of advection over diffusion, with striking augmentation at the cell division stage. Near the colony surface, flows generated by flagella can be chaotic, exhibiting mixing due to stretching and folding. These results imply that hydrodynamic transport external to colonies provides a crucial boundary condition, a source for supplying internal diffusional dynamics.     

Tunable interplay between epidermal growth factor and cell–cell contact governs the spatial dynamics of epithelial growth

Contact-inhibition of proliferation constrains epithelial tissue growth, and the loss of contact-inhibition is a hallmark of cancer cells. In most physiological scenarios, cell–cell contact inhibits proliferation in the presence of other growth-promoting cues, such as soluble growth factors (GFs). How cells quantitatively reconcile the opposing effects of cell–cell contact and GFs, such as epidermal growth factor (EGF), remains unclear. Here, using quantitative analysis of single cells within multicellular clusters, we show that contact is not a “master switch” that overrides EGF. Only when EGF recedes below a threshold level, contact inhibits proliferation, causing spatial patterns in cell cycle activity within epithelial cell clusters. Furthermore, we demonstrate that the onset of contact-inhibition and the timing of spatial patterns in proliferation may be reengineered. Using micropatterned surfaces to amplify cell–cell interactions, we induce contact-inhibition at a higher threshold level of EGF. Using a complementary molecular genetics approach to enhance cell–cell interactions by overexpressing E-cadherin also increases the threshold level of EGF at which contact-inhibition is triggered. These results lead us to propose a state diagram in which epithelial cells transition from a contact-uninhibited state to a contact-inhibited state at a critical threshold level of EGF, a property that may be tuned by modulating the extent of cell–cell contacts. This quantitative model of contact-inhibition has direct implications for how tissue size may be determined and deregulated during development and tumor formation, respectively, and provides design principles for engineering epithelial tissue growth in applications such as tissue engineering.     

Depletion of minichromosome maintenance protein 5 in the zebrafish retina causes cell-cycle defect and apoptosis

In multicellular organisms, the control of genome duplication and cell division must be tightly coordinated. Essential roles of the minichromosome maintenance (MCM) proteins for genome duplication have been well established. However, no genetic model has been available to address the function of MCM proteins in the context of vertebrate organogenesis. Here, we present positional cloning of a zebrafish mcm5 mutation and characterization of its retina phenotype. In the retina, mcm5 expression correlates closely with the pattern of cell proliferation. By the third day of development, mcm5 is down-regulated in differentiated cells but is maintained in regions containing retinal stem cells. We demonstrate that a gradual depletion of maternally derived MCM5 protein leads to a prolonged S phase, cell-cycle-exit failure, apoptosis, and reduction in cell number in mcm5(m850) mutant embryos. Interestingly, by the third day of development, increased apoptosis is detectable only in the retina, tectum, and hindbrain but not in other late-proliferating tissues, suggesting that different tissues may employ distinct cellular programs in responding to the depletion of MCM5.     

Survivin在消化系肿瘤的研究进展

恶性肿瘤的形成原因尚不明确,目前认为细胞增殖与细胞凋亡的平衡遭到破坏,导致肿瘤的发生与发展。Survivin是近年发现的凋亡抑制蛋白家族成员,在分化成熟的组织中无表达或微量表达,而在胚胎和多数人类恶性肿瘤组织中高表达;其抑制肿瘤细胞凋亡进而促进细胞生长,调节细胞分裂,对肿瘤的形成和发展起了重要的作用,并有望成为肿瘤治疗的新靶点。本文就Survivin的结构、功能及其在消化系肿瘤的研究进展综述如下。     

Evidence of RedOX Imbalance during Zika Virus Infection Promoting the Formation of Disulfide-Bond-Dependent Oligomers of the Envelope Protein

Flaviviruses replicate in membrane factories associated with the endoplasmic reticulum (ER). Significant levels of flavivirus viral protein accumulation contribute to ER stress. As a consequence, the host cell exhibits an Unfolded Protein Response (UPR), subsequently stimulating appropriate cellular responses such as adaptation, autophagy or apoptosis. The correct redox conditions of this compartment are essential to forming native disulfide bonds in proteins. Zika virus (ZIKV) has the ability to induce persistent ER stress leading to the activation of UPR pathways. In this study, we wondered whether ZIKV affects the redox balance and consequently the oxidative protein folding in the ER. We found that ZIKV replication influences the redox state, leading to the aggregation of the viral envelope protein as amyloid-like structures in the infected cells.     

Evidence of asymmetric cell division and centrosome inheritance in human neuroblastoma cells

Asymmetric cell division (ACD) is believed to be a physiological event that occurs during development and tissue homeostasis in a large variety of organisms. ACD produces two unequal daughter cells, one of which resembles a multipotent stem and/or progenitor cell, whereas the other has potential for differentiation. Although recent studies have shown that the balance between self-renewal and differentiation potentials is precisely controlled and that alterations in the balance may lead to tumorigenesis in Drosophila neuroblasts, it is largely unknown whether human cancer cells directly show ACD in an evolutionarily conserved manner. Here, we show that the conserved polarity/spindle protein NuMA is preferentially localized to one side of the cell cortex during cell division, generating unequal inheritance of fate-altering molecules in human neuroblastoma cell lines. We also show that the cells with a single copy of MYCN showed significantly higher percentages of ACD than those with MYCN amplification. Moreover, suppression of MYCN in MYCN-amplified cells caused ACD, whereas expression of MYCN in MYCN-nonamplified cells enhanced symmetric cell division. Furthermore, we demonstrate that centrosome inheritance follows a definite rule in ACD: The daughter centrosome with younger mother centriole is inherited to the daughter cell with NuMA preferentially localized to the cell cortex, whereas the mother centrosome with the older mother centriole migrates to the other daughter cell. Thus, the mechanisms of cell division of ACD or symmetric cell division and centrosome inheritance are recapitulated in human cancer cells, and these findings may facilitate studies on cancer stem cells.     

Caspase-9 regulates apoptosis/proliferation balance during metamorphic brain remodeling in Xenopus

During anuran metamorphosis, the tadpole brain is transformed producing the sensorial and motor systems required for the frog's predatory lifestyle. Nervous system remodeling simultaneously implicates apoptosis, cell division, and differentiation. The molecular mechanisms underlying this remodeling have yet to be characterized. Starting from the observation that active caspase-9 and the Bcl-X(L) homologue, XR11 are highly expressed in tadpole brain during metamorphosis, we determined their implication in regulating the balance of apoptosis and proliferation in the developing tadpole brain. In situ hybridization showed caspase-9 mRNA to be expressed mainly in the ventricular area, a site of neuroblast proliferation. To test the functional role of caspase-9 in equilibrating neuroblast production and elimination, we overexpressed a dominant-negative caspase-9 protein, DN9, in the tadpole brain using somatic gene transfer and germinal transgenesis. In both cases, abrogating caspase-9 activity significantly decreased brain apoptosis and increased numbers of actively proliferating cells in the ventricular zone. Moreover, overexpression of XR11 with or without DN9 was also effective in decreasing apoptosis and increasing cell division in the tadpole brain. We conclude that XR11 and caspase-9, two key members of the mitochondrial death pathway, are implicated in controlling the proliferative status of neuroblasts in the metamorphosing Xenopus brain. Modification of their expression during the critical period of metamorphosis alters the outcome of metamorphic neurogenesis, resulting in a modified brain phenotype in juvenile Xenopus.     

Nuclear receptors in cell life and death

The balance between cell proliferation and programmed cell death (apoptosis) determines body patterns during animal development and controls compartment sizes, tissue architecture and remodeling. The removal of primordial structures by apoptosis allows the organism to develop sex specifically and to adapt for novel functions at later stages; apoptosis also limits the size of evolving structures. It is a ubiquitous function that is essential for all cells. Although inappropriate regulation or execution of apoptosis leads to disease, such as cancer, there is now evidence for its great therapeutic potential. This would be particularly true if apoptosis could be targeted at defined cell compartments, rather than acting ubiquitously like chemotherapy. Here, we discuss the potential of nuclear receptor ligands, many of which act through their cognate receptors in defined body compartments as modulators of cell life and death, with special emphasis on the molecular pathways by which these receptors affect cell-cycle progression, survival and apoptosis.     

脂肪因子在肝硬化发病机制中的作用

脂肪组织不再只是一个储存过剩能量的惰性组织,同时也是一个活跃的内分泌器官.脂肪组织作为旺盛的内分泌器官已得到广泛认可,其分泌的大量脂肪因子如瘦素、细胞因子等参与维持机体能量稳态和调控糖、脂代谢平衡,对全身器官系统有重要的调节功能.研究发现脂肪因子影响肝脏的炎症、纤维化和细胞死亡.本文就各种脂肪因子与肝硬化的关系作一综述.     

Ectopic hTERT expression extends the life span of human CD4+ helper and regulatory T-cell clones and confers resistance to oxidative stress-induced apoptosis

Human somatic cells have a limited life span in vitro. Upon aging and with each cell division, shortening of telomeres occurs, which eventually will lead to cell cycle arrest. Ectopic hTERT expression has been shown to extend the life span of human T cells by preventing this telomere erosion. In the present study, we have shown that ectopic hTERT expression extends the life span of CD4+ T helper type 1 or 2 and regulatory T-cell clones and affected neither the in vitro cytokine production profile nor their specificity for antigen. In mixed cell cultures, ectopic hTERT-expressing clones were found to expand in greater numbers than untransduced cells of the same replicative age. This ectopic hTERT-induced growth advantage was not due to an enhanced cell division rate or number of divisions following T-cell receptor-mediated activation, as determined in carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling experiments. Moreover, the susceptibility to activation-induced cell death of both cell types was similar. However, cultures of resting hTERT-transduced T cells contained higher frequencies of Bcl-2-expressing cells and lower active caspase-3-expressing cells, compared with wild-type cells. Furthermore, hTERT-transduced cells were more resistant to oxidative stress, which causes preferential DNA damage in telomeres. Taken together, these results show that ectopic hTERT expression not only protects proliferating T cells from replicative senescence but also confers resistance to apoptosis induced by oxidative stress.