Tip-link integrity and mechanical transduction in vertebrate hair cells.

An attractive hypothesis for hair-cell transduction is that fine, filamentous "tip links" pull directly on mechanically sensitive ion channels located at the tips of the stereocilia. We tested the involvement of tip links in the transduction process by treating bundles with a BAPTA-buffered, low-Ca2+ saline (10(-9) M). BAPTA abolished the transduction current in a few hundred milliseconds. BAPTA treatment for a few seconds eliminated the tip links observed by either scanning or transmission electron microscopy. BAPTA also eliminated the voltage-dependent movement and caused a positive bundle displacement of 133 nm, in quantitative agreement with a model for regulation of tension. We conclude that tip links convey tension to the transduction channels of hair cells.     

Sensory transduction and electrical signaling in guard cells

Guard cells are a valuable model system for the study of photoreception, ion transport, and osmoregulation in plant cells. Changes in stomatal apertures occur when sensing mechanisms within the guard cells transduce environmental stimull into the ion fluxes and biosynthesis of organic solutes that regulate turgor. The electrical events mediating sensory transduction in guard cells can be characterized with a variety of electrophysiological recording techniques. Recent experiments applying the patch clamp method to guard cell protoplasts have demonstrated activation of electrogenic pumps by blue and red light as well as the presence of potassium channels in guard cell plasmalemma. Light activation of electrogenic proton pumping and the ensuing gating of voltage-dependent ion channels appear to be components of sensory transduction of the stomatal response to light. Mechanisms underlying stomatal control by environmental signals can be understood by studying electrical events associated with ion transport.     

Sensory adaptation of Dictyostelium discoideum cells to chemotactic signals

Postvegetative Dictyostelium discoideum cells react chemotactically to gradients of cAMP, folic acid, and pterin. In the presence of a constant concentration of 10(-5) M cAMP cells move at random. They still are able to respond to superimposed gradients of cAMP, although the response is less efficient than without the high background level of cAMP. Cells which are accommodated to 10(-5) M cAMP do not react to a gradient of cAMP if the mean cAMP concentration is decreasing with time. This indicates the involvement of adaptation in the detection of chemotactic gradients: cells adapt to the mean concentration of chemoattractant and respond to positive deviations from the mean concentration. Cells adapted to high cAMP concentrations react normally to gradients of folic acid or pterin. Adaptation to one of these compounds does not affect the response to the other attractants. This suggests that cAMP, folic acid, and pterin are detected by different receptors, and that adaptation is localized at a step in the transduction process before the signals from these receptors coincide into one pathway. I discuss the implications of adaptation for chemotaxis and cell aggregation.     

A density-sensing factor regulates signal transduction in Dictyostelium

Dictyostelium discoideum initiates development when cells overgrow their bacterial food source and starve. To coordinate development, the cells monitor the extracellular level of a protein, conditioned medium factor (CMF), secreted by starved cells. When a majority of the cells in a given area have starved, as signaled by CMF secretion, the extracellular level of CMF rises above a threshold value and permits aggregation of the starved cells. The cells aggregate using relayed pulses of cAMP as the chemoattractant. Cells in which CMF accumulation has been blocked by antisense do not aggregate except in the presence of exogenous CMF. We find that these cells are viable but do not chemotax towards cAMP. Videomicroscopy indicates that the inability of CMF antisense cells to chemotax is not due to a gross defect in motility, although both video and scanning electron microscopy indicate that CMF increases the frequency of pseudopod formation. The activations of Ca2+ influx, adenylyl cyclase, and guanylyl cyclase in response to a pulse of cAMP are strongly inhibited in cells lacking CMF, but are rescued by as little as 10 s exposure of cells to CMF. The activation of phospholipase C by cAMP is not affected by CMF. Northern blots indicate normal levels of the cAMP receptor mRNA in CMF antisense cells during development, while cAMP binding assays and Scatchard plots indicate that CMF antisense cells contain normal levels of the cAMP receptor. In Dictyostelium, both adenylyl and guanylyl cyclases are activated via G proteins. We find that the interaction of the cAMP receptor with G proteins in vitro is not measurably affected by CMF, whereas the activation of adenylyl cyclase by G proteins requires cells to have been exposed to CMF. CMF thus appears to regulate aggregation by regulating an early step of cAMP signal transduction.     

Visual transduction in vertebrate photoreceptors

Light activation of guanylate cyclase at different calcium concentrations was studied in the rod outer segments of the toad retina. The enzyme becomes sensitive to calcium ions after a flash of light, showing an enhancement of its activity when Ca²⁺ concentration is lowered from 10⁻⁴ M to 10⁻⁸ M. A possible pathway of guanylate cyclase activation by light was also investigated by means of the antibody 4A to transducin. When added in excess to transducin, the antibody inhibits light activation of phosphodiesterase as well as of cyclase, suggesting a possible coupling of the two enzymes.     

Histochemical comparison of vertebrate mast cells

Synopsis Mast cells from representative species of each of the five vertebrate classes have been studied histochemically. Histamine was found in reptilian, avian and mammalian species. An N-sulfated mucopolysaccharide was present in all species but the carp. A chymotrypsin-like or a trypsin-like esterase or both was found in the turtle and the several mammalian species examined. The mast cell thus appears to have developed the biological armamentarium associated with the mammalian cells in discrete steps during vertebrate evolution.     

Signal transduction pathways controlling multicellular development in Dictyostelium.

The importance of signal transduction pathways in regulating developmental processes in a number of organisms has become evident in recent years. This is exceptionally clear for Dictyostelium, which uses soluble factors to regulate morphogenesis and cellular differentiation. It is now known that many of these processes are controlled by signal transduction pathways mediated by cyclic AMP through cell surface receptors coupled to G proteins, and that others are mediated by the morphogen DIF.     

Sensory transduction in bacterial chemotaxis involves phosphotransfer between Che proteins

The CheA protein of the Salmonella typhimurium chemotaxis system is phosphorylated by ATP. Phospho-CheA transfers its phosphoryl group to a second chemotaxis protein, CheY. Unlike phospho-CheA, phospho-CheY is relatively unstable, rapidly decaying to phosphate and CheY. We propose that phosphorylation of CheY may play a role in its function as a tumble regulator to control motor behavior in response to attractant and repellent stimuli.     

A comparison of potato and vertebrate lactate dehydrogenases.

The incorporation of labelled leucine was measured in protein fractions of muscle in intact control and dystrophic female hamsters and also in cell-free preparations obtained from these animals. The labelling of the soluble sarcoplasmic protein fraction, the microsomal protein fraction and the sarcolemma protein fraction was increased in the dystrophic hindleg muscle. The specific radioactivities of the sarcolemma protein fraction and other fractions were increased markedly relative to that of free leucine in the dystrophic muscle. In cell-free preparations where ribonuclease effects were avoided, the dystrophic muscle exhibited an increased synthesis of peptide bonds.     

Tunable laser resonance Raman spectroscopic investigations of the transduction process in vertebrate rod cells.

Tunable laser resonance Raman spectroscopy has been applied to probe (in vivo) the role of rhodopsin in transducing light energy into the chemical necessary to generate a neural response. These in vivo experiments have suggested that the Schiff base linkage through which retinal is attached to opsin in rhodopsin is protonated. Furthermore, it appears that light eventually stimulates the deprotonation of the Schiff base linkage between the Meta I and Meta II steps in the intermediate sequence which is the result of light interacting with rhodopsin. Our data suggest that this deprotonation of the Schiff base occurs on the same time scale as overall proton release and uptake by the rhodopsin molecule. It is interesting to note that this series of protonations and deprotonations also occurs within the same time scale as the neural response generation in vertebrates and the generation of a proton gradient by bacteriorhodopsin, which is used by the bacterium, Halobacterium halobium, for ATP synthesis. If these data are analyzed within the context of the in vivo resonance Raman experiments (which seem to indicate that proton release is stimulated in the disc membrane during transduction) then there is a strong suggestion that the proton will assume an important role in any working hypothesis of visual transduction. In essence it appears that protons along with ATP and calcium ions must all be essential elements in the transduction process.     

A new model for chemotactic signal transduction in Dictyostelium discoideum

Dictyostelium discoideum amoebae were employed to study the refractoriness and adaptation of the rapid (5sec) accumulation of actin in their Triton-insoluble cytoskeletons following stimulation with specific chemoattractants. Amoebae became refractory within 10sec for this response but no adaptation occurred during this period. Amoebae desensitized for one attractant were not desensitized for another and responses to stimulation with a mixture of attractants were approximately additive. The characteristics of these processes are compared to published studies of adaptation in other chemoattractant-induced responses and a new model for the chemotactic signal transduction pathway is formulated. We conclude that intracellular cGMP accumulation may be on a separate branch of the pathway from the actin response.     

Nuclear plasmids in the simple eukaryotes Saccharomyces cerevisiae and Dictyostelium discoideum

High copy number nuclear plasmids are becoming recognized as common genetic components of simple eukaryotes. Like bacterial plasmids, eukaryotic plasmids ensure their persistence in dividing cells by having a partitioning system and a regulated means of amplifying copy number to correct inherent fluctuations in partitioning. By virtue of their small size and autonomy from the chromosomes, eukaryotic plasmids are useful for studying not only features of eukaryotic replicons but many aspects of gene regulation and DNA organization in nucleated cells.