Judith Van Houten

Two mechanisms of chemotaxis inParamecium

SummaryParamecia show chemotaxis, that is, they accumulate in or disperse from the vicinity of chemicals. This study examines both the avoiding reactions (abrupt random changes of swimming direction) and velocities of normal and mutant paramecia in attractants and repellents and shows that the animals accumulate or disperse either by changing the frequency of avoiding reactions or by changing swimming velocity. Mutations or conditions that eliminate avoiding reactions abolish the chemotaxis response to chemicals that cause accumulation or dispersal by modulation of frequency of avoiding reactions but not the response to chemicals that cause chemotaxis by modulation of velocity.The current knowledge of the bioelectric control of the swimming behavior inParamecium and observations of mutants defective in bioelectric control and in chemotaxis are used to develop a hypothesis for membrane potential control of chemotaxis: attractants that require the avoiding reaction slightly hyperpolarize the membrane; repellents that require the avoiding reaction slightly depolarize the membrane; repellents that cause chemitaxis by modulation of velocity strongly hyperpolarize the membrane.

Paramecium genome survey : a pilot project

A consortium of laboratories undertook a pilot sequencing project to gain insight into the genome of Paramecium. Plasmid-end sequencing of DNA fragments from the somatic nucleus together with similarity searches identified 722 potential protein-coding genes. High gene density and uniform small intron size make random sequencing of somatic chromosomes a cost-effective strategy for gene discovery in this organism.

Inbreeding of Wistar–Kyoto rat strain with hyperactivity but without hypertension

A genetic inbreeding program using Wistar-Kyoto rat strains as progenitors was used to combine the hyperactivity trait of the spontaneously hypertensive rat (SHR) with the normotensive trait of the WKY genetic control strain. From an SHR X WKY cross we produced a gene-assorting F2 population from which selected brother-sister matings were carried out through seven successive inbred populations. This program produced a new strain of hyperactive rats with normotensive mean systolic blood pressure levels, and we have designated the new strain as the Wistar-Kyoto hyperactive (WK/HA) rat. Another behavioral characteristic of the SHR rat, poor habituation in a nonreinforcing novel environment, did not appear as a characteristic trait of the new strain of WK/HA rats, suggesting a separate underlying genetic basis for the two traits that had been apparently fortuitously fixed in the SHR genotype as a result of intensive inbreeding of that strain. The new WK/HA strain, together with the WKY control strain, is considered as more suitable for subjects in studying hyperactivity in rats than the original SHR strain with its concomitant hypertension and poor habituation traits.

Chemosensory transduction in paramecium

Summary Paramecium tetraurelia is attracted and repelled by a variety of chemical stimuli. The attractants probably signify the presence of nutrients. For attractants, there are at least three signal transduction pathways that all lead to a hyperpolarization of the cell that results in relatively fast and smooth swimming. The three pathways differ in stimuli, receptor mechanisms and second messengers. Receptors for one and possibly two of the pathways couple surface events to the activation of a plasma membrane calcium pump. One pathway is not receptor-mediated, but affects intracellular pH by diffusion of the stimulus across the membrane. A variety of techniques are described in an effort to probe the steps of each of these pathways, which remain only partially defined.

Two quantitative assays for chemotaxis inParamecium

SummaryWe present two new methods to quantify the attraction and repulsion ofParamecium aurelia by chemicals. These are a T-maze assay and a countercurrent separation assay.In the T-maze assay, cells were placed in one arm of a three-way stopcock. The cells were then allowed to enter the other two arms, one of which contained an attractant or repellent. The ratio of cells collected from these two arms was an index of chemotaxis. The reproducibility of this method was demonstrated, using potassium acetate to study attraction (positive chemotaxis), and quinine-HCl to study repulsion (negative chemotaxis). This method is rapid and convenient.The countercurrent separation assay was adapted from the method of Dusenbery (1973), originally used to study behavior in nematodes. Animals were injected into the center of a slanted tube through which two solutions of different densities were pumped in opposite directions. One solution contained an attractant or repellent. Animals emerging from the tube in each of the two solutions were counted. These counts were used to measure the extent of chemotaxis. This method is useful for studying attraction in populations of over 104 paramecia and for isolating mutants defective in chemotaxis.

CHARACTERIZATION OF A PUTATIVE CA2+-TRANSPORTING CA2+-ATPASE IN THE PELLICLES OF PARAMECIUM TETRAURELIA

In Paramecium, no Ca2(+)-ATPases with the properties of Ca2+ pumps have been identified. Here we report a pellicle associated Ca2(+)-ATPase activity and a corresponding phosphoprotein intermediate characteristic of a pump. The Ca2(+)-ATPase activity requires 3 mM Mg for optimal Ca2+ stimulation (KCa = 90 nM) and is specific for ATP as substrate (Km = 75 microM). Vanadate and calmidazolium inhibit Ca2(+)-stimulated activity with an EC50 of about 2 microM and 0.5 microM, respectively. Likewise, 10 microM trifluoperazine inhibits 80% of Ca2(+)-ATPase activity, but bovine calmodulin fails to stimulate. The Ca2(+)-ATPase is not inhibited by sodium azide (10 mM), oligomycin (10 micrograms/ml) or ouabain (0.2 mM). Incubation of pellicles with [gamma-32P]ATP specifically labels a 133 kDa protein in a Ca2(+)-dependent, hydroxylamine-sensitive manner, and the level of phosphorylation is increased by 100 microM La3+. Phosphorylation of an endoplasmic reticulum-enriched fraction labels a Ca2(+)-dependent protein different from the pellicle protein, being lower in molecular mass and unaffected by La3+. Ca2+ uptake by the alveolar sacs, integral components of the pellicle membrane complex, is poorly coupled to Ca2(+)-stimulated ATP hydrolysis (Ca2+ transported/ATP hydrolysed less than 0.2) and is much less sensitive to vanadate inhibition (EC50 approx. 20 microM) compared to the total Ca2(+)-ATPase activity. Therefore, the majority of the Ca2(+)-ATPase activity is likely to be plasma membrane associated.

Chemosensory transduction in eukaryotic microorganisms: trends for neuroscience?

Abstract It might appear curious to read about yeast, slime molds and protozoa in a journal dedicated to neuroscience. However, despite their distinct lack of synapses, eukaryotic microorganisms hold a wealth of information relevant to the signal-transduction pathways that underly activity in neuronal receptor cells, particularly those subserving the chemical senses. Microorganisms are sensitive to chemical stimuli from their environment and thus have similarities to receptor neurons of the olfactory system and the taste bud. Here, we introduce receptors, second messengers and effectors responsible for chemosensory signal transduction in yeast mating, sea-urchin spermatozoan chemolaxis, slime-mold aggregation and development, and ciliate chemoresponses.

Cloning and Molecular Analysis of the Plasma Membrane Ca2+ ‐ATPase Gene in Paramecium tetraurelia

ABSTRACT. We have determined the DNA sequence of the gene encoding the protein of the plasma membrane Ca2+‐ATPase in Paramecium tetraurelia. The predicted amino acid sequence of the plasma membrane Ca2+‐ATPase shows homology to conserved regions of known plasma membrane Ca2+‐ATPases and contains the known binding sites for ATP (FITC), acylphosphate formation, and calmodulin, as well as the “hinge” region: all characteristics common to plasma membrane Ca2+‐ATPases. The deduced molecular weight for this sequence is 131 kDa. The elucidation of this gene will assist in the studies of the mechanisms by which this excitable cell removes calcium entering through voltage gated calcium channels and the pump functions in chemosensory signal transduction.

A Mutant of Paramecium Defective in Chemotaxis

In an effort to study the sensory-motor pathway of chemotaxis in Paramecium tetraurelia, I have generated mutants defective in their responses to chemicals. One mutant in particular, d4-530, is repelled by sodium acetate, which attracts normal paramecia by klinokinesis. The mutant is repelled by the mechanism of orthokinesis. To my knowledge, this is the first report of orthokinesis in chemotaxis of paramecia.

Computer simulation of Paramecium chemokinesis behavior

Abstract A computer program was designed to simulate the distribution of paramecia in a T-maze assay for chemo-accumulation and dispersal. Simulated values of chemokinesis are compared to experimental values for normal and mutant paramecia. The roles of components of swimming behavior (turning frequency and swimming speed), adaptation, and reaction at the border of solutions are examined.