The redoubtable cell

The cell theory—the thesis that all life is made up of one or more cells, the fundamental structural and physiological unit—is one of the most celebrated achievements of modern biological science. And yet from its very inception in the nineteenth century it has faced repeated criticism from some biologists. Why do some continue to criticize the cell theory, and how has it managed nevertheless to keep burying its undertakers? The answers to these questions reveal the complex nature of the cell theory and the cell concept on which it is based. Like other scientific ‘laws’, the assertion that all living things are made of cells purchases its universality at the expense of abstraction. If, however, this law is regarded merely as a widely applicable empirical generalization with notable exceptions, it still remains too important to discard. Debate about whether the cell or the organism standpoint provides the more correct account of anatomical, physiological, and developmental facts illustrates the tension between our attempts to express the truth about reality in conceptual terms conducive to a unified human understanding.     

Microtubules cut loose at the cell cortex

The ability of the microtubule cytoskeleton to rapidly and locally reorganize itself in response to intra- and extracellular signals is essential to its wide range of functions. A site of tightly regulated microtubule dynamics—and the major interface between the microtubule cytoskeleton and the extracellular environment—is the cell cortex, where the selective stabilization and destabilization of microtubule plus-ends is required for normal cell division, morphogenesis and migration. In a recent study, we found that the cortex of Drosophila S2 and D17 cells is coated with the microtubule severing enzyme and plus-end depolymerase, Kat-60, which actively suppresses microtubule growth and stability along the cell edge. We have proposed that cortical Kat-60 functions by uncapping plus-ends, thereby activating another microtubule depolymerase, KLP10A, preloaded onto the end. The localized destruction of microtubule plus-ends at a specific cortical could feed into larger regulatory pathways, such as those in control of the actin cytoskeleton, to influence cell polarization and motility.     

Immunological study of lung development in the mouse embryo. II. First appearance of the great alveolar cell, as shown by immunofluorescence microscopy.

An antiserum that specifically recognizes a lung-specific antigen present in the great alveolar cell in the adult mouse lung was used in immunofluorescence studies to detect the first appearance of this antigen in the embryo. Cellular fluorescence was found to occur in the lung tissue from about Day 14.2 onward and to be due to the presence of the lung-specific or a related antigen. The simultaneous appearance of this antigen (ca. Day 14.2) and the cuboidal type of epithelial cell in which it occurs (ca. Day 14) means that the great alveolar cell—or its precursor—is first detectable around Day 14.2. Since the great alveolar cell—or its precursor—is the first and only type of alveolar epithelial cell to occur in the embryonic lung, it must be the stem cell from which the small alveolar cell derives. The persistent sharp demarcation between the prospective alveolar and bronchial epithelia indicates that the respiratory and the conducting portions of the lung originate from different parts of the tubular system in the prenatal lung.     

Ontogenic evolution of chicken red cell Fc receptor

The ontogenic evolution of chicken red cell Fc receptor was studied in red cells from different age chicken embryos, baby chicken, and adult chicken. The Fc receptor binding capacity for ligands, the number of Fc receptors by red cell, and the association constant between receptor and ligand were analyzed. The Fc receptor is expressed in the red cell surface of 6-day chicken embryo and its binding capacity for ligand—minimal at this moment—is increased in the 8-day chicken embryo red cells. The 12-day chicken embryo erythrocytes binding capacity is similar to the adult chicken red cells. The number of Fc receptors by red cell increase with the age of chicken embryo. After 9 days this number is not modified and it is the same as in adult chicken. Variations of Ko and binding capacity for ligands show a similar evolution in embryogenic development. From these data we suggest that although on Day 9 the number of receptors per cell is the same as in adult chicken, the receptors are not completely exposed at this time and as a consequence, their binding capacity for ligands is lower than in adult chicken erythrocytes.     

The mechanism of guard cell movement

Leaves of higher terrestrial plants have small pores — stomata — responsible for gas exchange. The opening of each stoma results from the osmotic uptake of water by two specialised cells — the guard cells. Because of the involvement in this mechanism of ATPase-proton pumps and active transport of ions across membranes, we have designed an Exploring Guard Cell Movement activity for biology teachers to provide a practical approach to some relevant aspects of bioenergetics and plant physiology.     

Optical biosensors for cell adhesion

Planar optical waveguides offer an ideal substratum for cells on which to reside. The materials from which the waveguides are made—high refractive index transparent dielectrics—correspond to the coatings of medical implants (e.g., the oxides of niobium, tantalum, and titanium) or the high molecular weight polymers used for culture flasks (e.g., polystyrene). The waveguides can furthermore be modified both chemically and morphologically while retaining their full capability for generating an evanescent optical field that has its greatest strength at the interface between the solid substratum and the liquid phase with which it is invariably in contact (i.e., the culture medium bathing the cells), decaying exponentially perpendicular to the interface at a rate controllable by varying the material parameters of the waveguide. Analysis of the perturbation of the evanescent field by the presence of living cells within it enables their size, number density, shape, refractive index (linked to their constitution) and so forth to be determined, the number of parameters depending on the number of waveguide lightmodes analyzed. No labeling of any kind is necessary, and convenient measurement setups are fully compatible with maintaining the cells in their usual environment. If the temporal evolution of the perturbation is analyzed, even more information can be obtained, such as the amount of material (microexudate) secreted by the cell while residing on the surface. Separation of parallel effects simultaneously contributing to the perturbation of the evanescent field can be accomplished by analysis of coupling peak shape when a grating coupler is used to measure the propagation constants of the waveguide lightmodes.     

Isolation and characterization of temperature-sensitive mutants in a Chinese hamster cell line

A replica plating method was used for the isolation of temperature-sensitive (ts) mutants after treatment of Chinese hamster cells with ethyl methanesulfonate (EMS). No significant increase in ts mutants was found after this treatment. The limitations and advantages of the replicating procedure to detect such differences, as well as an alternative method, are discussed.Mutants isolated were classified into two general groups—density-dependent and clear-cut—as measured by survival at low and high cell densities at the restrictive temperature. The density-dependent mutants may be truly “leaky”, losing a metabolite to the medium at an excessive rate at the restrictive temperature. On the other hand, the one clear-cut mutant analyzed extensively dies at a rate determined by its ability to utilize one or more components from the medium. It shows an inverse density relationship in rate of death, as inferred from rates of macromolecular synthesis, as opposed to its growth rate at the permissive temperature.     

Model-based strategy for cell culture seed train layout verified at lab scale

Cell culture seed trains—the generation of a sufficient viable cell number for the inoculation of the production scale bioreactor, starting from incubator scale—are time- and cost-intensive. Accordingly, a seed train offers potential for optimization regarding its layout and the corresponding proceedings. A tool has been developed to determine the optimal points in time for cell passaging from one scale into the next and it has been applied to two different cell lines at lab scale, AGE1.HN_AAT and CHO-K1. For evaluation, experimental seed train realization has been evaluated in comparison to its layout. In case of the AGE1.HN_AAT cell line, the results have also been compared to the formerly manually designed seed train. The tool provides the same seed train layout based on the data of only two batches.     

Induction of unspecific permeabilization of mitochondrial membrane and its role in cell death

Mitochondria participate in various vital cellular processes. Violation of their functions can lead to the development of cardiovascular and neurodegenerative diseases and malignancies. One of the key events responsible for mitochondrial damage—induction of Ca²⁺-dependent mitochondrial permeability transition, due to the opening of a nonspecific pore in the inner mitochondrial membrane. Despite active studies of pore components, its detailed structure has not yet been established. This review analyzes possible constituents and regulators of the pore, the role of the pore in various pathologies, and hypotheses that explain the organization of the pores. Elucidation of these questions can help developing strategies for the treatment of a wide range of pathologies—from Alzheimer and Parkinson’s disease to cancer.     

Isothiocyanate groups of 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS) inhibit cell penetration of octa‐arginine (R8)–fused peptides

Delivering biomolecules, such as antibodies, proteins, and peptides, to the cytosol is an important and challenging aspect of drug development and chemical biology. Polyarginine—a well‐known cell‐penetrating peptide (CPP)—is capable of exploiting its positive charge and guanidium groups to carry a fused cargo into the cytosol. However, the precise mechanism by which this occurs remains ambiguous. In the present study, we established a new method of quantitatively assessing cell penetration. The method involves inducing cell death by using a polyarginine (R8) to deliver a peptide—ie, mitochondrial targeting domain (MTD)—to the cytosol. We found that 4,4′‐diisothiocyanatostilbene‐2,2′‐di‐sulfonate (DIDS)—an anion channel blocker—inhibited the ability of octa‐arginine (R8)–fused MTD to penetrate cells. Other anion channel blockers did not inhibit the penetration of peptides fused with R8. Comparison of DIDS with other structurally similar chemicals revealed that the isothiocyanate group of DIDS may be primarily responsible for the inhibitory effect than its stilbene di‐sulfonate backbone. These results imply that the inhibitory effect of DIDS may not be derived from the interaction between stilbene di‐sulfonate and the anion channels, but from the interaction between the isothiocyanate groups and the cell membrane. Our new MTD method enables the quantitative assessment of cell penetration. Moreover, further studies on the inhibition of CPPs by DIDS may help clarify the mechanism by which penetration occurs and facilitate the design of new penetrative biomolecules.     

The myofibroblast: Paradigm for a mechanically active cell

Tissues lose mechanical integrity when our body is injured. To rapidly restore mechanical stability a multitude of cell types can jump into action by acquiring a reparative phenotype—the myofibroblast. Here, I review the known biomechanics of myofibroblast differentiation and action and speculate on underlying mechanisms. Hallmarks of the myofibroblast are secretion of extracellular matrix, development of adhesion structures with the substrate, and formation of contractile bundles composed of actin and myosin. These cytoskeletal features not only enable the myofibroblast to remodel and contract the extracellular matrix but to adapt its activity to changes in the mechanical microenvironment. Rapid repair comes at the cost of tissue contracture due to the inability of the myofibroblast to regenerate tissue. If contracture and ECM remodeling become progressive and manifests as organ fibrosis, the outcome of myofibroblast activity will have more severe consequences than the initial damage. Whereas the pathological consequences of myofibroblast occurrence are of great interest for physicians, their mechano-responsive features render them attractive for physicists and bioengineers. Their well developed cytoskeleton and responsiveness to a plethora of cytokines fascinate cell biologists and biochemists. Finally, the question of the myofibroblast origin intrigues stem cell biologists and developmental biologists—what else can you ask from a truly interdisciplinary cell?     

Influence of chloroquine treatment on enzymes and phospholipids from rat liver cell fractions

Rats, treated for 12 days with chloroquine show a threefold increase of arylsulfatase activity in the mitochondrial-lysosomal mixed fraction, whereas the succinate: cytochrome c reductase activity is decreased to about 50% in this fraction. Purified lysosomes possess a 35 fold higher arylsulfatase activity, compared with homogenate, whereas neither NADPH: - nor succinate: cytochrome c reductase activity can be detected. In these lysosomes, one third of the phospholipids consists of bis (monoacylglycero) phosphate. The neutral phospholipids — mainly phosphatidylethanolamine — are drastically reduced in these cell organelles during the treatment. Our results indicate that chloroquine is nearly exclusively present in the lysosomal fraction. Furthermore we conclude from our data that bis (monoacylglycero) phosphate — isolated from lysosomal phospholipids — forms complexes with chloroquine.     

Evidence for the occurrence of two forms of β-lipotropin in rat pituitary

A peptide isolated from porcine gut according to its glucagon-like activity in liver (bioactive enteroglucagon) has been characterized immunologically, biologically and chemically: its potency relative to pancreatic glucagon in interacting with an antiglucagon antibody, hepatic glucagon-binding sites and hepatic adenylate cyclase was ～100%, 20% and 10%, respectively. In contrast, it is ～20-times more potent than glucagon in oxyntic glands, justifying the term ‘oxyntomodulin’. Chemically, it consists in the 29 amino acid-peptide glucagon elongated at its C-terminal end by the octapeptide Lys—Arg—Asn—Lys—Asn—Asn—Ile &;—Ala; accordingly, it is called ‘glucagon-37’     

A mechanistic protrusive-based model for 3D cell migration

Cell migration is essential for a variety of biological processes, such as embryogenesis, wound healing, and the immune response. After more than a century of research—mainly on flat surfaces—, there are still many unknowns about cell motility. In particular, regarding how cells migrate within 3D matrices, which more accurately replicate in vivo conditions. We present a novel in silico model of 3D mesenchymal cell migration regulated by the chemical and mechanical profile of the surrounding environment. This in silico model considers cell’s adhesive and nuclear phenotypes, the effects of the steric hindrance of the matrix, and cells ability to degradate the ECM. These factors are crucial when investigating the increasing difficulty that migrating cells find to squeeze their nuclei through dense matrices, which may act as physical barriers. Our results agree with previous in vitro observations where fibroblasts cultured in collagen-based hydrogels did not durotax toward regions with higher collagen concentrations. Instead, they exhibited an adurotactic behavior, following a more random trajectory. Overall, cell’s migratory response in 3D domains depends on its phenotype, and the properties of the surrounding environment, that is, 3D cell motion is strongly dependent on the context.     

Simple and reliable system for transient gene expression for the characteristic signal sequences and the estimation of the localization of target protein in plant cell

The efficiency of transient gene expression in plants credibly demonstrated characteristics of gene functions in numerous studies. Two key strategies of transient expression became favorites among researchers: protoplast transfection and agroinfiltration. Each of them, alongside the advantages, has its own constraints. In this work, an easy, rapid, and reliable system for characterization of the signal sequences and determinations of target protein localization in a plant cell is proposed and tested. This system—called the AgI–PrI—implies production of protoplasts from plant tissues after agroinfiltration. Reliability of the proposed system for transient gene expression has been proved using characterized signal sequences in Nicotiana benthamiana cells. The corresponding protocol is less expensive and depends to a lesser degree on the professional skills in the area of protoplast isolation and transfection; furthermore, it may be applicable to other plant species with either available efficient methods of agroinfiltration and protoplast isolation or with the potential for one of the protocols to be supplemented. Thus, the AgI–PrI technique makes it possible to combine the advantages of two widely used methods for the transient gene expression in plants—agroinfiltration and protoplast isolation and transfection—and concurrently avoids their critical points.     

Apparatus for continuous, large-volume suspended cell cultivation

A 30-liter apparatus for growing cells continuously is described. Samples are taken automatically, thus obviating one of the main causes of contamination. In order to eliminate the continuous introduction of a gas stream, the pH was kept constant by means of the injection of NH₄OH or NaOH. Under these circumstances, and by means of the two methods of agitation described—rotary and “by Vibromixer”—a quantity of cells of approximately 4 × 10¹⁰ is harvested every 48 to 72 hr.