Rab35 regulates cadherin-mediated adherens junction formation and myoblast fusion

Cadherins are homophilic cell–cell adhesion molecules implicated in many fundamental processes, such as morphogenesis, cell growth, and differentiation. They accumulate at cell–cell contact sites and assemble into large macromolecular complexes named adherens junctions (AJs). Cadherin targeting and function are regulated by various cellular processes, many players of which remain to be uncovered. Here we identify the small GTPase Rab35 as a new regulator of cadherin trafficking and stabilization at cell–cell contacts in C2C12 myoblasts and HeLa cells. We find that Rab35 accumulates at cell–cell contacts in a cadherin-dependent manner. Knockdown of Rab35 or expression of a dominant-negative form of Rab35 impaired N- and M-cadherin recruitment to cell–cell contacts, their stabilization at the plasma membrane, and association with p120 catenin and led to their accumulation in transferrin-, clathrin-, and AP-2–positive intracellular vesicles. We also find that Rab35 function is required for PIP5KIγ accumulation at cell–cell contacts and phosphatidyl inositol 4,5-bisphosphate production, which is involved in cadherin stabilization at contact sites. Finally, we show that Rab35 regulates myoblast fusion, a major cellular process under the control of cadherin-dependent signaling. Taken together, these results reveal that Rab35 regulates cadherin-dependent AJ formation and myoblast fusion.     

Phase-dependent lobopodial contact paralysis

Time-lapse films of lobopodial bleb interaction between neighboring pairs of EDTA dissociated Triturus alpestris blastula cells indicate that contact paralysis inhibition of one bleb's circus propagation around its cell, depends sharply on the position of the second cell's bleb at the moment of contact, and reaches a minimum when the two blebs are directly apposed.     

Cell Surface Concentrations and Concentration Ranges for Testing In Vitro Autocrine Loops and Small Molecules

A common assumption made when performing in vitro cellular assays is that the concentration of substances in the culture system is uniform. However, since the cells that internalize and secrete substances reside at the bottom of the well, it is conceivable that a concentration gradient could arise across the fluid layer. Importantly, the concentration of a substance in the vicinity of a cell, which is the concentration of interest, cannot be measured via existing methods. In this work a simple strategy for estimating the concentration of a chemical species at the surface of a cell is presented. Finally, this result is used to outline a method for determining the appropriate concentration ranges for testing in vitro autocrine loops and small molecules.     

Development of the protonema and bud formation in mosses

In the course of their development the protonemata of Funaria hygrometrica produce two different substances which diffuse into the substrate. In the chloronema a thermo-labile growth-promoting substance is formed. In the caulonema, after about 10 days, a substance is produced which is thermostable and soluble in amyl alcohol, which can be dialysed, and which functions as a growth inhibitor. Both substances also influence bud formation. This is at an optimum only when there is a certain balance between these two substances.
This promotion is fundamentally different from that brought about by treatment with kinetin, because kinetin can function only as an additional factor in promoting bud formation. Very probably it acts as an agent which creates centres of attraction toward which morphogenetic substances are drawn. This assumption is supported by the fact that kinetin cannot be transported and therefore has no 'after-effect'. It probably functions only in the caulonema cell it penetrates. It converts every caulonema cell into a 'reaction cell'.     

Costimulation in Tetrahymena. II. A nonspecific response to heterotypic cell--cell interactions

Costimulation, a developmental interaction requiring cell-to-cell contact, is the second stage in a linear sequence of events leading from vegetative growth to mating pairs in the ciliate Tetrahymena thermophila. This paper uses the kinetics of appearance of genetically marked progeny to measure accurately the duration of normal costimulation and then to examine the role of mating type in costimulation. Although diverse mating types are required for costimulation to occur, the costimulated cell's response is not specific to the mating type of the cell contacting it; costimulation by one mating type appears to prepare a cell for mating with any complementary mating type.     

Commitment to germ tube or bud formation during release from stationary phase in Candida albicans.

Cells of the pathogenic yeast Candida albicans accumulate as unbudded singlets at stationary phase in defined medium at 25 °C. When released into fresh medium at 37 °C and pH 6.5, these cells will synchronously form elongate pseudomycelia, and when released into fresh medium at either 25 °C, pH 6.5, or 37 °C, pH 4.5, they will synchronously form buds. Using pH and temperature shift experiments, we have examined when cells become committed to pseudomycelium formation and bud formation under conditions conducive to each growth form respectively. It is demonstrated that in either case commitment occurs long after release from stationary phase, at approximately the same time the first evagination is visible on the cell's surface. In addition, it is demonstrated that once a released cell has formed a bud, it and its progeny lose the capacity to form pseudomycelia until they re-enter stationary phase; on the other hand, elongating pseudomycelia retain the capacity to form buds. The possible relationships of the commitment events to septation and to the cell cycle are discussed.     

Insecticidal activity of Bacillus laterosporus

Strains of Bacillus laterosporus demonstrated pathogenic activity for second-instar larvae of the mosquito, Culex quinquefasciatus, but failed to demonstrate detectable pathogenicity against larvae of the cabbage looper, Trichoplusia ni. Of 29 strains of the bacterium screened, 16 displayed pathogenicity for mosquito larvae. One of the most pathogenic strains, NRS 590, also demonstrated pathogenic activity for larvae of the mosquito, Aedes aegypti, and for larvae of the black fly, Simulium vittatum. The pathogenicity for Culex larvae was associated with the cell mass rather than with the culture supernatant. A suspension of ultraviolet irradiation-killed cells demonstrated no loss in pathogenic activity, an indication that the pathogenicity is toxin mediated. The toxic substance produced by NRS 590 was found to be resistant to heating at 96°C for 10 min. The toxin was not associated with the heat-resistant, bacterial endospore or with the associated paraspore since a suspension consisting primarily of spores was not toxic to mosquito larvae. Toxic activity in stationary phase cells of NRS 590 was associated with the cell's particulate fraction rather than with the soluble fraction.     

Effector cells which mediate antibody-dependent cell-mediated cytotoxicity. II. Ontogeny of effector activity in murine spleen.

The specificity of T cells for syngeneic target cells is directed to both antigens and products of the major histocompatibility complex (MHC) on the target cell surface. This dual requirement is best accounted for by the altered-self hypothesis, which implies that the MHC products on a cell's surface are able to form complexes with many other proteins on the surface of the same cell. To account for the ability of MHC products to bind so many different cell surface antigens we propose that interactions in general among macromolecules on the surface of a membrane may be dramatically enhanced by a purely physical effect. This effect derives from the confinement of membrane macromolecules to an effective volume which is the product of membrane surface area times d, the distance over which the center of mass of the molecules can move in a vertical direction (perpendicular to the membrane surface). Because d is very small the effective concentrations of surface molecules are extremely high and their interactions are correspondingly enhanced.     

Hydrogen ion effects of the vertebrate photoreceptor. The pK's of ionizable groups affecting cell permeability.

The permeability of the vertebrate photoreceptor was studied as a function of pH and other parameters. The permeability was measured as the rate of decay of a sequence of distal PIII responses from an isolated frog retina (Rana catesbeiana) after reestablishing the transmembrane sodium gradient in the presence of ouabain. The rate of decay was slowed at increasing stimulus light intensities, with increased extracellular calcium concentration (0.1 to 5 mm), and at extracellular pH's above 7.6 and below 6.6 at lower stimulus light intensities. The effects of stimulus intensity and calcium are consistent with previously published data which showed that these parameters reduced the sodium conductance of the vertebrate photoreceptor. The pH data indicate that two externally available ionizable groups with pK's of approximately 7.6 and below 6.6 are affecting the permeability of the photoreceptor cell.     

Transient Responses to Sudden Illumination in Cells of the Eye of Limulus

Responses recorded from visual cells of Limulus (presumably eccentric cells) following abrupt and maintained illumination consist of depolarization with superimposed spikes. Both the depolarization and the frequency of firing are greater at the beginning of the response than later on. Frequency of firing decreases with time also during stimulation with constant currents, but the decay is then less than it is during constant illumination. Early and steady-state responses do not increase in the same proportion following illumination at different intensities. Membrane conductance is higher during the early peak of the response than in steady state. Early and late potential changes appear to tend to the same equilibrium value. The results support the assumptions that: (a) discharge of impulses is the consequence of depolarization of a specialized "pacemaker region" in the axon; (b) depolarization induced by light is the consequence of increase of membrane conductance. The major conductance changes occurring during constant illumination may be due to corresponding changes of the "stimulus" supplied by the photoreceptor or to changes of sensitivity of the eccentric cell's membrane to this stimulus. Some accessory phenomena may be the consequence of regenerative properties of the nerve cell itself.     

On the mutability of the yeast mitochondrial genome.

A mechanism is proposed to explain how a mutation in a single molecule of mitochondrial DNA (mitDNA) can come to affect all the other mitDNA molecules of a yeast cell. It is suggested that an initial mutation may be “amplified” by a process which is, in fact, intended to ensure the identity of the cell's complement of mitDNA molecules. It is postulated that this process involves a small number of “reference” copies of mitDNA to which all other (“derived”) copies are compared and corrected once per cell cycle. Asymmetric gene conversion is proposed as the correction mechanism and the means of “amplifying” mutations. The model is shown to be compatible with current data on spontaneous and induced mitochondrial mutation in Saccharomyces cerevisiae.     

Transfilter mitogenic effect of dorsal root ganglia on cultured regeneration blastemata,in the newt,Notophthalmus viridescens

Cone stage forelimb blastemata from adult newts were separated into proximal and distal regions and cultured along with dorsal root ganglia in both transfilter and cis (same side) configurations, for a period of 96 h in modified Parker's medium (CMRL-1415) containing insulin and l-thyroxin. Mitotic index and differentiation of cartilage were assessed in ganglionated and nonganglionated, proximal and distal explants after 4 days in vitro. The results show that the nerve influence on the regeneration blastema appears to be mediated by a chemical substance capable of transmission through thin filters of low porosity Moreover the neurotrophic substance has mitogenic properties. The ganglia stimulated blastema cell proliferation transfilter, increasing it from a basal level (mitotic index = 0.339), observed in noninnervated explants to almost threefold values (M.I. = 1.124) in corresponding distal innervated explants. In addition, this transfilter mitogenic effect was manifested in the form of a proximodistal gradient with the highest mitotic index close to the neurons, which diminished with distance from the nerve source. When blastema explants were grown in physical contact with ganglionic neurons (cis configuration), they transcended the proliferation phase within the 4 days of culture and differentiation of cartilage whorls resulted. Presumably, a critical mass of blastema cells is achieved earlier in the presence of a higher concentration of neurotrophic factor.     

MAGUKs, scaffolding proteins at cell junctions, are substrates of different proteases during apoptosis

A major feature of apoptotic cell death is gross structural changes, one of which is the loss of cell–cell contacts. The caspases, executioners of apoptosis, were shown to cleave several proteins involved in the formation of cell junctions. The membrane-associated guanylate kinases (MAGUKs), which are typically associated with cell junctions, have a major role in the organization of protein–protein complexes at plasma membranes and are therefore potentially important caspase targets during apoptosis. We report here that MAGUKs are cleaved and/or degraded by executioner caspases, granzyme B and several cysteine cathepsins in vitro. When apoptosis was induced by UV-irradiation and staurosporine in different epithelial cell lines, caspases were found to efficiently cleave MAGUKs in these cell models, as the cleavages could be prevented by a pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethylketone. Using a selective lysosomal disrupting agent -leucyl--leucine methyl ester, which induces apoptosis through the lysosomal pathway, it was further shown that MAGUKs are also cleaved by the cathepsins in HaCaT and CaCo-2 cells. Immunohistological data showed rapid loss of MAGUKs at the sites of cell–cell contacts, preceding actual cell detachment, suggesting that cleavage of MAGUKs is an important step in fast and efficient cell detachment.     

Influence of natural substances of phenolic character and diethyldithiocarbamate on the metabolism of L-tryptophan in cabbage,maize and pea

The effect of phenolic substances isolated earlier from cabbage, maize and pea on L-tryptophan-3-¹⁴C (L-Try-¹⁴C) metabolism in those plants was investigated. For the sake of comparison the effect of diethyldithiocarbamate (DIECA) on cabbage was also observed. A phenolic substance of unknown structure isolated from cabbage was utilised in a 0.5 mg/ml concentration, p-coumaric acid (PCA) from maize in 0.7 mg/ml concentration and quercetinglucosyl-coumarate (QGC) found in pea in 8 mg/ml concentration were used. The chosen concentrations were on the limit of their inhibitory effect on the growth of the respective plant apical segments. The effect of DIECA was investigated at 0.1 mg/ml concentration. Using non-labelled L-Try as substrate (5×10^?3 m) biological tests have shown that QGC and DIECA have a weaker but distinct effect on the increase in auxin level (+17% and +15%, respectively). All phenolic compounds, as well as DIECA increase the intake of L-Try-¹⁴C from the incubation solutions. Phenolic substances decrease the conversion of L-Try-¹⁴C to its metabolites. The changes were studied after a 20 hour period of L-Try-¹⁴C metabolism. On chromatograms changes in the proportion of L-Try-¹⁴C metabolites took place, especially in the areas of substances of complex character from which IAA splits off easily,i.e. in the areas of 3-indolylacetylglucose and 3-indolylacetamide. The radioactivity of these areas is generally decreased (e.g. the decrease of radioactivity in the 3-indolylacetylglucose area under the influence of the phenolic substance ofBrassica plants is ?7,4%, under the influence of PCA in maize it is ?8.9% and under the influence of QGC in pea it is ?17.1%). DIECA also decreased the radioactivity of this zone, by ?10,5%. In cabbage a large part of L-Try-¹⁴C was transformed to glucobrassicin; its biosynthesis increases in the presence of the phenolic substance of cabbage by +3%, in the presence of DIECA by +27%. The results are discussed in a working hypothesis based on the key formation of IAA, accompanied by its oxidation and detoxication.     

Influence of natural substances of phenolic character and diethyldithiocarbamate on the metabolism of L-tryptophan in cabbage,maize and pea

The effect of phenolic substances isolated earlier from cabbage, maize and pea on L-tryptophan-3-¹⁴G (L-Try-¹⁴C) metabolism in those plants was investigated. For the sake of comparison the effect of diethyldithiocarbamate (DIECA) on cabbage was also observed. A phenolic substance of unknown structure isolated from cabbage was utilised in a 0.5 mg/ml concentration, p-coumarie acid (PCA) from maize in 0.7 mg/ml concentration and quercetin-glucosyl-coumarate (QGC) found in pea in 8 mg/ml concentration were used. The chosen concentrations were on the limit of their inhibitory effect on the growth of the respective plant apical segments. The effect of DIECA was investigated at 0.1 mg/ml concentration. Using non-labelled L-Try as substrate (5 × 10^?3 M) biological tests have shown that QGC and DIECA have a weaker but distinct effect on the increase in auxin level (+ 17% and + 15%, respectively). All phenolic compounds, as well as DIECA increase the intake of L-Try-¹⁴C from the incubation solutions. Phenolic substances decrease the conversion of L-Try-¹⁴C to its metabolites. The changes were studied after a 20 hour period of L-Try-¹⁴C metabolism. On chromatograms changes in the proportion of L-Try-¹⁴C metabolites took place, especially in the areas of substances of complex character from which IAA splits off easily,i.e. in the areas of 3-indolylacetylglucose and 3-indolylaeetamide. The radioactivity of these areas is generally decreased (e.g. the decrease of radioactivity in the 3-indolylacetylglucose area under the influence of the phenolic substance ofBrassica plants is ?7,4%, under the influence of PCA in maize it is —8.9% and under the influence of QGC in pea it is ?17.1% DIECA also decreased the radioactivity of this zone, by ?10,5%. In cabbage a large part of L-Try-¹⁴C was transformed to glucobrassicin; its biosynthesis increases in the presence of the phenolic substance of cabbage by +3%, in the presence of DIECA by +27%. The results are discussed in a working hypothesis based on the key formation of IAA, accompanied by its oxidation and detoxication.     

A cytokinetic model for heterogeneous mammalian cell populations. I. Cell growth and cell death

A general model is proposed for describing the growth behavior of mammalian cell populations, which features:(a) a cell cycle time distribution function with properties such that mean and variance increase with increasing population size; (b) maturation age and maturation rate functions which constrain the maturational pathways of individual cells; and (c) a death rate function, where cell death is construed as irreparable damage to a cell's reproductive apparatus. The biological implications of the model are discussed, and methods for relating the model to real cell systems by means of commonly used experimental techniques are described. The model is compared with earlier models.     

Isolement,action biologique et evolution des substances paragoniales contenues dans le spermatophore d'Acanthoscelides obtectus (Coleoptère)

In Acanthoscelides obtectus, some male secretions deposited in the spermatophore during mating reach the blood of the females and stimulate oögenesis. Water extracts from spermatophores injected into a female abdomen stimulate oögenesis but do not influence egg-laying or sexual receptivity. After column chromatograph of spermatophores, aqueous extracts on Sephadex G 25 Coarse, G 25 Superfine, and G 15, an active fraction has been isolated. This injected into the abdomen of virgin females stimulates oögenesis at low concentrations, but it is toxic at higher concentrations. This fraction was examined by paper electrophoresis at low voltage and then chromatographed on G 10 Sephadex. Two peaks were obtained: the first corresponds to the paragonial substance A which stimulates oögenesis at 0,2 10^?3 μg/μl concentration. The second contains the paragonial substance B. At a 0,3 10^?3 ug/μl concentration this substance is toxic. First this toxicity inhibits oögenesis and then causes the death of most females at higher concentrations. The toxic effect appears 2 or 3 days after injection. These two substances are purified on paper chromatography and the biological activities are contained in a zone of R_f 0.25 to 0.45 (paragonial substance A) and in a zone of 0.16–0.30 R_f (paragonial substance B).The paragonial substances disappear from the spermatophore after mating. Aqueous extracts of spermatophores obtained 6 hr after mating do not stimulate oögenesis and do not have any toxic effect. The chemical nature of these both fractions is not yet determined because the quantity of extracts obtained at the end of the purification is very low.The action of both paragonial substances is similar to the action of hormones. The paragonial substances influence unknown receptors at low concentration after a latent period. The origin of the paragonial B substance was not determined, but this substance which inhibits oögenesis at low concentrations could be an antagonist of paragonial A substance.     

Alarm substance from adult zebrafish alters early embryonic development in offspring

Alarm substances elicit behavioural responses in a wide range of animals but effects on early embryonic development are virtually unknown. Here we investigated whether skin injury-induced alarm substances caused physiological responses in embryos produced by two Danio species (Danio rerio and Danio albolineatus). Both species showed more rapid physiological development in the presence of alarm substance, although there were subtle differences between them: D. rerio had advanced muscle contraction and heart function, whereas D. albolineatus had advanced heart function only. Hence, alarm cues from injured or dying fish may be of benefit to their offspring, inducing physiological responses and potentially increasing their inclusive fitness.     

Taste receptor cell-based biosensor for taste specific recognition based on temporal firing

Taste receptor cells are the taste sensation elements expressing sour, salty, sweet, bitter and umami receptors, respectively. There are cell-to-cell communications between different types of cells. Nevertheless, the mechanism of taste sensation and taste information coded by taste receptor cell is not well understood at present and it is a long-standing issue. In order to explore taste sensation and analyze taste-firing responses from another point of view, we present a promising biomimetic taste receptor cell-based biosensor. The temporal firing responses to different tastants are recorded. Meanwhile, we investigate the firing rate and temporal firing of taste receptor cells. The experimental results are consistent with that from patch clamp and molecular biology experiment. Firing rate is dependent on the concentration of stimulus. PCA analysis (principal component analysis) of the temporal firing responses shows that the responses from different types of taste receptor cells can be distinguished. Furthermore, exogenous ATP is applied to mimic the effects of transmitter ATP (adenosine triphosphate) released from type II cells onto type III cells. Both enhanced and inhibitory effects on spontaneous firing are observed. This novel biomimetic hybrid biosensor provides a potential solution to investigate the taste sensation and coding mechanisms in a non-invasive way.     

The Temporal Winner-Take-All Readout

How can the central nervous system make accurate decisions about external stimuli at short times on the basis of the noisy responses of nerve cell populations? It has been suggested that spike time latency is the source of fast decisions. Here, we propose a simple and fast readout mechanism, the temporal Winner-Take-All (tWTA), and undertake a study of its accuracy. The tWTA is studied in the framework of a statistical model for the dynamic response of a nerve cell population to an external stimulus. Each cell is characterized by a preferred stimulus, a unique value of the external stimulus for which it responds fastest. The tWTA estimate for the stimulus is the preferred stimulus of the cell that fired the first spike in the entire population. We then pose the questions: How accurate is the tWTA readout? What are the parameters that govern this accuracy? What are the effects of noise correlations and baseline firing? We find that tWTA sensitivity to the stimulus grows algebraically fast with the number of cells in the population, N, in contrast to the logarithmic slow scaling of the conventional rate-WTA sensitivity with N. Noise correlations in first-spike times of different cells can limit the accuracy of the tWTA readout, even in the limit of large N, similar to the effect that has been observed in population coding theory. We show that baseline firing also has a detrimental effect on tWTA accuracy. We suggest a generalization of the tWTA, the n-tWTA, which estimates the stimulus by the identity of the group of cells firing the first n spikes and show how this simple generalization can overcome the detrimental effect of baseline firing. Thus, the tWTA can provide fast and accurate responses discriminating between a small number of alternatives. High accuracy in estimation of a continuous stimulus can be obtained using the n-tWTA.