Noninvasive diagnosis of a single cell with a probe beam

It is important to distinguish a living/dead cell in cell culture, especially in the regenerate medicine field including cell therapy, since those cells are usually in short supply and consequently the ex vivo culture process should be operated strictly. Conventional methods for distinguishing a living from a dead cell usually require labeling with a dye, which spoils the culture of the cell. Here we show a simple noninvasive method for diagnosing a dead or alive cell with a probe beam. If a cell is alive, the active transport of materials across the cell membrane causes a change of concentration gradients, and this change further induces a change of deflection of a probe beam passing through a vicinity of the cell membrane. If a cell is dead, no or little change in deflection of the probe beam is induced because no or little active materials movement across the cell membrane exists. The deflection of the probe beam is monitored, and judgment on whether a cell is dead or alive from the deflection signal agreed with the conventional decision.     

Three-Dimensional Numerical Model of Cell Morphology during Migration in Multi-Signaling Substrates

Cell Migration associated with cell shape changes are of central importance in many biological processes ranging from morphogenesis to metastatic cancer cells. Cell movement is a result of cyclic changes of cell morphology due to effective forces on cell body, leading to periodic fluctuations of the cell length and cell membrane area. It is well-known that the cell can be guided by different effective stimuli such as mechanotaxis, thermotaxis, chemotaxis and/or electrotaxis. Regulation of intracellular mechanics and cell’s physical interaction with its substrate rely on control of cell shape during cell migration. In this notion, it is essential to understand how each natural or external stimulus may affect the cell behavior. Therefore, a three-dimensional (3D) computational model is here developed to analyze a free mode of cell shape changes during migration in a multi-signaling micro-environment. This model is based on previous models that are presented by the same authors to study cell migration with a constant spherical cell shape in a multi-signaling substrates and mechanotaxis effect on cell morphology. Using the finite element discrete methodology, the cell is represented by a group of finite elements. The cell motion is modeled by equilibrium of effective forces on cell body such as traction, protrusion, electrostatic and drag forces, where the cell traction force is a function of the cell internal deformations. To study cell behavior in the presence of different stimuli, the model has been employed in different numerical cases. Our findings, which are qualitatively consistent with well-known related experimental observations, indicate that adding a new stimulus to the cell substrate pushes the cell to migrate more directionally in more elongated form towards the more effective stimuli. For instance, the presence of thermotaxis, chemotaxis and electrotaxis can further move the cell centroid towards the corresponding stimulus, respectively, diminishing the mechanotaxis effect. Besides, the stronger stimulus imposes a greater cell elongation and more cell membrane area. The present model not only provides new insights into cell morphology in a multi-signaling micro-environment but also enables us to investigate in more precise way the cell migration in the presence of different stimuli.     

Rapid assay for cell age response to radiation by electronic volume flow cell sorting

A new technique is described for measuring cell survival as a function of cell cycle position using flow cytometric cell sorting on the basis of electronic volume signals. The sorting of cells into different cell age compartments is demonstrated for three different cell lines commonly used in radiobiological research. Using flow cytometric DNA content analysis and [3H]thymidine autoradiography of the sorted cell populations, we demonstrate that the resolution of the age compartment separation is as good as or better than that reported for other cell synchronizing techniques. The variation in cell survival as a function of position in the cell cycle after a single dose of radiation as measured by volume cell sorting is similar to that determined by other cell synchrony techniques. This new method has several advantages, including: no treatment of the cells is required, thus, this method is noncytotoxic; no cell cycle progression is needed to obtain different cell age compartments; the cell population can be held in complete growth medium at any desired temperature during sorting; and a complete radiation age-response assay can be plated in 2 h. The application of this method to problems in radiobiology and chemotherapy is discussed, along with some of the technical limitations.     

Cell wall analysis

The cell wall is a rigid structure essential for survival of the fungal cell. Because of its absence in mammalian cells, the cell wall is an attractive target for antifungal agents. Thus, for different reasons, it is important to know how the cell wall is synthesized and how different molecules regulate that synthesis. The Schizosaccharomyces pombe cell wall is mainly formed by glucose polysaccharides and some galactomannoproteins. Here, we describe a fast and reliable method to analyze changes in S. pombe cell wall composition by using specific enzymatic degradation and chemical treatment of purified cell walls. This approach provides a powerful means to analyze changes in (1,3)beta-glucan and (1,3)alpha-glucan, two main polysaccharides present in fungal cell walls. Analysis of cell wall polymers will be useful to search for new antifungal drugs that may inhibit cell wall biosynthesis and/or alter cell wall structure.     

Density-dependent tumor cell death and reversible cell cycle arrest: mutually exclusive modes of monocyte-mediated growth control

The population development of five human tumor cell lines is examined under the influence of elutriator-prepared human monocytes in a serum-free hormone- and growth factor-supplemented medium. Analysis was performed by electronic counting and sizing of tumor cell nuclei and flow cytometric detection of cell cycle phases. Tumor cell death is triggered at rather low monocyte:tumor cell ratios (1:2 to 1:4) whereas it is strongly reduced at high monocyte densities. Furthermore, it is shown that confluence of the target cell population is a necessary prerequisite for lysis. The data suggest that in monocyte/tumor cell cocultures the decision on target cell lysis is not made by the effector cell, but rather by the target cell and that the criterion for this decision is the target cell's ability or inability to respond to a monocyte challenge by arresting the cell cycle in G1. Interactions between target cells play an important role in determining the result of this decision process. A common basis is suggested for this kind of density-dependent monocyte-triggered lysis and density-dependent cell death in 3T3 cell cultures as described previously.     

Tracheary Element Differentiation Uses a Novel Mechanism Coordinating Programmed Cell Death and Secondary Cell Wall Synthesis

Tracheary element differentiation requires strict coordination of secondary cell wall synthesis and programmed cell death (PCD) to produce a functional cell corpse. The execution of cell death involves an influx of Ca²⁺ into the cell and is manifested by rapid collapse of the large hydrolytic vacuole and cessation of cytoplasmic streaming. This precise means of effecting cell death is a prerequisite for postmortem developmental events, including autolysis and chromatin degradation. A 40-kD serine protease is secreted during secondary cell wall synthesis, which may be the coordinating factor between secondary cell wall synthesis and PCD. Specific proteolysis of the extracellular matrix is necessary and sufficient to trigger Ca²⁺ influx, vacuole collapse, cell death, and chromatin degradation, suggesting that extracellular proteolysis plays a key regulatory role during PCD. We propose a model in which secondary cell wall synthesis and cell death are coordinated by the concomitant secretion of the 40-kD protease and secondary cell wall precursors. Subsequent cell death is triggered by a critical activity of protease or the arrival of substrate signal precursor corresponding with the completion of a functional secondary cell wall.     

The 'Inka cell' and its associated cells: ultrastructure of the epitracheal glands in the gypsy moth,Lymantria dispar

The epitracheal glands in pharate and young pupae of Lymantria dispar are located at the base of ventrolateral tracheal trunks in the prothoracic and first through eighth abdominal segments. Each gland is composed of four cells the ultrastructure of which is described in this paper. One large cell and one smaller cell have an endocrine function, while a third cell is exocrine. A fourth cell forms a canal running from the exocrine cell into the trachea. The large endocrine cell, but not the smaller endocrine cell has released its secretions in freshly moulted pupae. The exocrine cell is assumed to be involved in the pupal moult events as well. The physiological role of the different cell types is discussed: The large endocrine cell (type I endocrine cell) is supposedly homologous with the 'Inka cell', which produces ecdysis triggering hormone (ETH) and was previously described in Manduca sexta; the functions of the smaller endocrine cell (type II endocrine cell) and the exocrine cell remain unknown.     

Regulation of cell wall biogenesis in Saccharomyces cerevisiae: the cell wall integrity signaling pathway

The yeast cell wall is a strong, but elastic, structure that is essential not only for the maintenance of cell shape and integrity, but also for progression through the cell cycle. During growth and morphogenesis, and in response to environmental challenges, the cell wall is remodeled in a highly regulated and polarized manner, a process that is principally under the control of the cell wall integrity (CWI) signaling pathway. This pathway transmits wall stress signals from the cell surface to the Rho1 GTPase, which mobilizes a physiologic response through a variety of effectors. Activation of CWI signaling regulates the production of various carbohydrate polymers of the cell wall, as well as their polarized delivery to the site of cell wall remodeling. This review article centers on CWI signaling in Saccharomyces cerevisiae through the cell cycle and in response to cell wall stress. The interface of this signaling pathway with other pathways that contribute to the maintenance of cell wall integrity is also discussed.     

Acquisition of embryogenic competency does not require cell division in carrot somatic cell

Totipotency is the ability of a cell to regenerate the entire organism, even after previous differentiation as a specific cell. When totipotency is coupled with active cell division, it was presumed that cell division is essential for this expression. Here, using the stress-induction system of somatic embryos in carrots, we show that cell division is not essential for the expression of totipotency in somatic/embryonic conversion. Morphological and histochemical analyses showed that the cell did not divide during embryo induction. Inhibitors of cell division did not affect the rate of somatic embryo formation. Our results indicate that the newly acquired trait of differentiation appears without cell division, but does not arise with cell division as a newborn cell.     

The cytostat: A new way to study cell physiology in a precisely defined environment

In a cytostat, a continuous culture is monitored and controlled by an automated flow cytometer system, based on the determination of the cell concentration and the single cell property distribution of the growing cell population. The growing culture can be maintained at steady state even at such low cell concentrations that the bioreactor medium composition is negligibly changed by the few cells. Therefore, the cell environment is precisely defined by the feed composition since products of cell growth are not present in significant amounts. Effects on cell growth of nutrients, of toxic compounds such as drugs, or of products made by the cells, if added to the feed medium, can be readily isolated. Using the cytostat, it is shown here that ethanol assumes the triggering function for the increase in cell size in Saccharomyces cerevisiae normally only seen at critical growth rates above critical cell densities. This suggests that ethanol assumes a quorum sensing function on cell growth when a critical cell density is reached.     

东亚飞蝗Locusta migratoria manilensis（Mey．）膝下器中具橛感器的附属细胞和附属结构的超微结构

东亚飞蝗膝下器的具橛感器主要由三类细胞组成．即：感觉细胞、感橛细胞和冠细胞。感觉细胞为具橛感器的主要结构和功能细胞,其超微结构已在其他的文章中描述。感橛细胞是具橛感器的主要支持细胞,从近端到远端依次与神经胶质细胞、感觉细胞的远端树突部分和感觉纤毛部以及顶端细胞外结构——冠、冠细胞直接接触．感橛细胞内最明显的结构为感概,另外,感橛细胞质被高度“空化”。冠细胞紧密包围着感橛细胞和冠,冠细胞中含有大量的纵行微管．并将整个具橛感器连接到体壁上。     

Cell repair through cell fusion in the red algaGriffithsia pacifica

Summary When an intercalary shoot cell of the red algaGriffithsia pacifica is killed, the cell may be replaced through the wound-healing process of cell repair. During cell repair the cells on either side of the dead cell cut off new cells towards the dead cell. The superjacent cell produces a rhizoid; the subjacent cell produces an atypical shoot cell. The two new cells grow towards each other through the lumen of the dead cell. When they meet, they fuse; the resulting cell expands laterally to fill the cavity of the dead cell and is transformed into a typical intercalary shoot cell, morphologically and physiologically indistinguishable from the killed cell it replaces. The entire cell repair process takes 24–30 hours. Three aspects of cell repair suggest that intercellular communication occurs across the dead cell; these are a precocious division of the cell below the dead cell, a reversible change in the morphology and growth of the shoot cell which participates in repair, and a definite attraction between the two cells which fuse. Thus during cell repair we find evidence not only for cellular redifferentiation through cell fusion, but also for extracellular substances which change pathways of morphogenesis.     

Human Speedy: a novel cell cycle regulator that enhances proliferation through activation of Cdk2

The decision for a cell to self-replicate requires passage from G1 to S phase of the cell cycle and initiation of another round of DNA replication. This commitment is a critical one that is tightly regulated by many parallel pathways. Significantly, these pathways converge to result in activation of the cyclin-dependent kinase, cdk2. It is, therefore, important to understand all the mechanisms regulating cdk2 to determine the molecular basis of cell progression. Here we report the identification and characterization of a novel cell cycle gene, designated Speedy (Spy1). Spy1 is 40% homologous to the Xenopus cell cycle gene, X-Spy1. Similar to its Xenopus counterpart, human Speedy is able to induce oocyte maturation, suggesting similar biological characteristics. Spy1 mRNA is expressed in several human tissues and immortalized cell lines and is only expressed during the G1/S phase of the cell cycle. Overexpression of Spy1 protein demonstrates that Spy1 is nuclear and results in enhanced cell proliferation. In addition, flow cytometry profiles of these cells demonstrate a reduction in G1 population. Changes in cell cycle regulation can be attributed to the ability of Spy1 to bind to and prematurely activate cdk2 independent of cyclin binding. We demonstrate that Spy1-enhanced cell proliferation is dependent on cdk2 activation. Furthermore, abrogation of Spy1 expression, through the use of siRNA, demonstrates that Spy1 is an essential component of cell proliferation pathways. Hence, human Speedy is a novel cell cycle protein capable of promoting cell proliferation through the premature activation of cdk2 at the G1/S phase transition.     

Tumor cell "dead or alive": caspase and survivin regulate cell death, cell cycle and cell survival

Cell death and cell cycle progression are two sides of the same coin, and these two different phenomenons are regulated moderately to maintain the cellular homeostasis. Tumor is one of the disease states produced as a result of the disintegrated regulation and is characterized as cells showing an irreversible progression of cell cycle and a resistance to cell death signaling. Several investigations have been performed for the understanding of cell death or cell cycle, and cell death research has remarkably progressed in these 10 years. Caspase is a nomenclature referring to ICE/CED-3 cysteine proteinase family and plays a central role during cell death. Recently, several investigations raised some possible hypotheses that caspase is also involved in cell cycle regulation. In this issue, therefore, we review the molecular basis of cell death and cell cycle regulated by caspase in tumor, especially hepatocellular carcinoma cells.     

Repair on the Go: E. coli Maintains a High Proliferation Rate while Repairing a Chronic DNA Double-Strand Break

DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.     

Numb与神经发育

不对称性细胞分裂是一个母细胞通过一次分裂,产生两个不同命运的子细胞的分裂方式,是单细胞生物向多细胞生物进化的关键一步。根据现有的证据推论,不称性细胞分裂是在器官发育过程中产生细胞多样化的一种基本方式。Numb是第一个被发现决定多细胞生物不对称细胞分裂的信号蛋白。在果蝇中,Numb通过促进Notch泛素化拮抗Notch信号通路,从而决定子细胞的命运,后来的研究表明Numb是细胞内吞调节蛋白,并用通过内吞参与调节神经细胞的粘附,轴突的生长及细胞迁移等过程;并且发现Numb与肿瘤抑制基因p53、泛素化蛋白HDM2形成三聚体抑制p53的泛素化,从而调节肿瘤的恶性程度。本文系统地分析了Numb发现的历史及后来在脊椎动物中的作用和机制,重点介绍了Numb在神经发育过程中的功能。     

Study on multicellular systems using a phase field model

A model of multicellular systems with several types of cells is developed from the phase field model. The model is presented as a set of partial differential equations of the field variables, each of which expresses the shape of one cell. The dynamics of each cell is based on the criteria for minimizing the surface area and retaining a certain volume. The effects of cell adhesion and excluded volume are also taken into account. The proposed model can be used to find the position of the membrane and/or the cortex of each cell without the need to adopt extra variables. This model is suitable for numerical simulations of a system having a large number of cells. The two-dimensional results of cell division, cell adhesion, rearrangement of a cell cluster, chemotaxis, and cell sorting as well as the three-dimensional results of cell clusters on the substrate are presented.     

The relation of temperature and lipid composition to cell adhesion

We have examined as a function of temperature the effect of changes in the composition of the fatty acid chains of membrane phospholipids on the rate of cell to cell adhesion in the neuronal cell line B103. The rate of cell to cell adhesion in this cell line is highly temperature dependent but is not influenced by changes in the fatty acid composition of the plasma membrane generated by growing the cells either in the presence of oleic acid or elaidic acid. In contrast the temperature dependence of the rate of cell to cell adhesion, measured in a monolayer adhesion assay, is highly dependent on the shear force used during the assay. A two-step model of cell to cell adhesion involving multiple adhesion ligands is presented which can be used to explain these observations.     

Cell death by autophagy: facts and apparent artefacts

Autophagy (the process of self-digestion by a cell through the action of enzymes originating within the lysosome of the same cell) is a catabolic process that is generally used by the cell as a mechanism for quality control and survival under nutrient stress conditions. As autophagy is often induced under conditions of stress that could also lead to cell death, there has been a propagation of the idea that autophagy can act as a cell death mechanism. Although there is growing evidence of cell death by autophagy, this type of cell death, often called autophagic cell death, remains poorly defined and somewhat controversial. Merely the presence of autophagic markers in a cell undergoing death does not necessarily equate to autophagic cell death. Nevertheless, studies involving genetic manipulation of autophagy in physiological settings provide evidence for a direct role of autophagy in specific scenarios. This article endeavours to summarise these physiological studies where autophagy has a clear role in mediating the death process and discusses the potential significance of cell death by autophagy.     

New concepts in radiation-induced apoptosis: 'premitotic apoptosis' and 'postmitotic apoptosis'

Formerly, the mechanisms responsible for the killing of cells by ionizing radiation were regarded as being divided into two distinct forms, interphase death and reproductive death. Since they were defined based on the classical radiobiological concepts using a clonogenic cell survival assay, biochemical and molecular biological mechanisms involved in the induction of radiation-induced cell death were not fully understood in relation to the modes of cell death. Recent multidisciplinary approaches to cell death mechanism have revealed that radiation-induced cell death is divided into several distinct pathways by the time course and cell-cycle position, and that apoptotic cell death plays a key role in almost every mode of cell death. This review discusses the mechanisms of radiation-induced apoptosis in relation to cellcycle progression and highlights a new concept of the mode of cell death: 'premitotic apoptosis' and 'postmitotic apoptosis'. The former is a rapid apoptotic cell death associated with a prompt activation of caspase-3, a key enzyme of intracellular signaling of apoptosis. Arapid execution of cell killing in premitotic apoptosis is presumably due to the prompt activation of a set of pre-existed molecules following DNA damages. In contrast, the latter is a delayed apoptotic cell death after cell division, and unlike premitotic apoptosis, it neither requires a rapid activation of caspase-3 nor is inhibited by a specific inhibitor, Ac-DEVD-CHO. A downregulation of anti-apoptotic genes such as MAPK and Bcl-2 may play a key role in this mode of cell death. Characterization of these two types of apoptotic cell death regarding the cell cycle regulation and intrcellular signaling will greatly help to understand the mechanisms of radiation-induced apoptosis.