SUMMMARY How microtubule-associated motor proteins are regulated is not well understood. A potential mechanism for spatial regulation of motor proteins is provided by post-translational modifications of tubulin subunits that form patterns on microtubules. Glutamylation is a conserved tubulin modification [1] that is enriched in axonemes. The enzymes responsible for this PTM, glutamic acid ligases (E-ligases), belong to a family of proteins with a tubulin tyrosine ligase (TTL) homology domain (TTL-like or TTLL proteins) [2]. We show that in cilia of Tetrahymena, TTLL6 E-ligases generate glutamylation mainly on the B-tubule of outer doublet microtubules, the site of force production by ciliary dynein. Deletion of two TTLL6 paralogs caused severe deficiency in ciliary motility associated with abnormal waveform and reduced beat frequency. In isolated axonemes with a normal dynein arm composition, TTLL6 deficiency did not affect the rate of ATP-induced doublet microtubule sliding. Unexpectedly, the same TTLL6 deficiency increased the velocity of microtubule sliding in axonemes that also lack outer dynein arms, in which forces are generated by inner dynein arms. We conclude that tubulin glutamylation on the B-tubule inhibits the net force imposed on sliding doublet microtubules by inner dynein arms.

Motile cilia have nine doublet microtubules, with hundreds of associated proteins that repeat in modules. Each module contains three radial spokes, which differ in their architecture, protein composition, and function. The conserved proteins FAP61 and FAP251 are crucial for the assembly and stable docking of RS3 and cilia motility.

A combination of genetics, biochemistry, and biophysics was used to show that calmodulin is involved in the regulation of an ion channel. Calmodulin restored the Ca2+-dependent K+ current in pantophobiac, a mutant in Paramecium that lacks this current. The restoration of the current occurred within 2 hours after the injection of 1 picogram of wild-type calmodulin into the mutant. The current remained for approximately 30 hours before the mutant phenotype returned. The injection of calmodulin isolated from pantophobiac had no effect. These results imply that calmodulin is required for the function or regulation of the Ca2+-dependent K+ current in Paramecium.

Single Paramecium caudatum were conditioned by pairing ac-generated electric shock (US) with a vibratory stimulus (CS) produced by an auditory speaker. Naive paramecia subjected to shock reliably exhibited a backwards jerk and axial spinning similar to the avoiding reaction described by Jennings in 1904. Such responses did not occur initially to CS alone, but increasingly appeared during the CS period preceding shock pairing (delayed conditioning paradigm). Control subjects given the CS and UCS at the same intervals, but explicitly unpaired, did not show a sustained increase of responses to the CS alone. Short-term memory was demonstrated by subjects first conditioned and then presented CS alone during extinction. These subjects were readily reconditioned. Paramecia trained and stored for 24 h showed reliable memory savings as compared to stored control subjects. Other paramecia were differentially conditioned by training with two CSs. Following the recommendations of Rescorla (1967), a procedure was designed for truly random presentation of the CS and UCS as an additional control for pseudoconditioning. Single paramecia were conditioned with intervals between CSs randomly ranging from 8 to 32 sec. Control subjects received the same number of CSs and UCSs, which were administered independently and randomly during the same total session duration. Thus, CS and UCS were occasionally paired for control subjects. The responses to CS in the conditioned group were anticipatory conditional responses due to the pairing contingency and not wholly due to pseudoconditioning.After a century of sporadic investigation, the question remains whether protozoa are capable of behavioral change that in higher organisms would be described as learning (reviews of the issue include:

Paramecium, a unicellular ciliated protist, alters its motility in response to various stimuli. Externally added GTP transiently induced alternating forward and backward swimming interspersed with whirling at a concentration as low as 0.1 jFM. ATP was 1000-fold less active, whereas CTP and UTP produced essentially no response. The response to the nonhydrolyzable GTP analogs guanosine 5'-[y-thio]triphosphate and guanosine 5'-[13,y-imido]triphosphate was indistinguishable from that to GTP. This behavioral response was correlated with an unusual transient and oscillating membrane depolarization in both wild-type cells and the mutant pawn B, which is defective in the voltage-dependent Ca2+ current required for action potentials. This is a specific effect of external GTP on the excitability of a eukaryotic cell and, to our knowledge, is the first purinergic effect to be discovered in a microorganism.Paramecium tetraurelia normally swims forward except for occasional brief periods of backward swimming or whirling, a randomly directed motion (1). However, many stimuli, thermal, electrical, mechanical, or chemical, can alter the swimming speed and the frequency and duration of backward swimming and whirling events (2-6). These responses are normally transient. Cells return to their prestimulus behavior even in the continued presence of the stimulus, a form of sensory adaptation. The combination of behavioral response and subsequent adaptation can result in attraction to or repulsion from a stimulus (6). These swimming behaviors generally have clear, well-studied, and readily measurable electrophysiological correlates that can aid in unraveling a signal-transduction pathway. For example, increased swimming speed is correlated with membrane hyperpolarization, whereas decreased swimming speed is correlated with membrane depolarization. Strong depolarizations can elicit graded Ca2+-based action potentials, resulting in whirling and backward swimming due to increased intraciliary Ca2+ (3,4).To quantify these swimming behaviors, we have developed a computerized motion analysis assay that measures the percentage of total path time spent whirling and undergoing transitions between forward and backward swimming [defined as percent directional changes (PDCs) (1)]. While using this assay to quantify the behavioral effects of externally added nucleotides, we found that guanine nucleotides specifically and potently altered the swimming behavior of paramecia. In addition, while attempting to corroborate the GTP-induced behavior of the cell with changes in membrane potential, we discovered an electrophysiological response.

Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. Little is known about the assembly and functions of the three individual radial spokes, RS1, RS2, and RS3. In Tetrahymena, a conserved ciliary protein, FAP206, docks RS2 and dynein c to the doublet microtubule.

Chemosensory adaptation is seen in Tetruhymena thermophila following prolonged exposure (ten minutes) to micromolar concentrations of the chemorepellents lysozyme or guanosine triphosphate (GTP). Since these cells initially show repeated backward swimming episodes (avoidance reactions) in these repellents, behavioral adaptation is seen as a decrease in this repellent-induced behavior. The time course of this behavioral adaptation is paralleled by decreases in the extents of surface binding of either 13*P]GTP or [3H]lysozyme in vivo. Scatchard plot analyses of repellent binding in adapted cells suggests the behavioral adaptation is due to a dramatic decrease in the number of surface binding sites, as represented by decreased Bmax values. The estimated K, values for nonadapted cells are 6.6 pM and 8.4 p M for lysozyme and GTP binding, respectively. Behavioral adaptation and decreased surface receptor binding are specific for each repellent. The GTP adapted cells (20 pM for ten minutes) still respond behaviorally to 50 pM lysozyme and bind [3H]lysozyme normally. Lysozyme adapted cells (50 pM for ten minutes) still bind [)*P]GTP and respond behaviorally to GTP. All the behavioral and binding changes seen are also reversible (deadaptation). Neomycin was shown to be a competitive inhibitor of [3H]lysozyme binding and lysozyme-induced avoidance reactions, but it had no effect on either [32P]GTP binding or GTP-induced avoidance reactions. These results are consistent with the hypothesis that there are two separate repellent receptors, one for GTJ? and the other for lysozyme, that are independently downregulated during adaptation to cause specific receptor desensitization and consequent behavioral adaptation.

SUMMARY1. The isolated Ca2+ current from Paramecium caudatum was examined under voltage clamp with long conditioning depolarizations lasting for up to 5 min.2. The isolated transient Ca2+ current inactivates with tens of milliseconds due to Ca2+-dependent Ca2+-channel inactivation (Brehm & Eckert, 1978). When this fast inactivation was blocked by internally delivered EGTA, a much slower inactivation of the Ca2+ current was discovered. This slow inactivation had time constants of tens of seconds, depending on voltage.3. The development of this slow inactivation was further examined by following the Ca2+ transient after 1 s interruptions of the long depolarization. This development is voltage dependent; the rate of inactivation is higher with a larger depolarization.4. After a long depolarization, the Ca2+ current returns in two clearly separable steps. A portion of the current returns rapidly along an exponential time course with time constants of tens to hundreds of milliseconds. The remainder of the current returns slowly with time constants of tens of seconds. A longer conditioning depolarization generates a larger portion that recovers slowly.5. Internally delivered EGTA, sufficient to prevent most of the fast inactivation, did not change the time course or the extent of either the onset or the removal of the slow inactivation. The compound W-7, which inhibits the Ca2+ current itself, does not block the onset of this slow inactivation during depolarization.6. We conclude that the slow inactivation of the Ca2+ channel is a mechanistically different phenomenon from the fast Ca2+-dependent Ca2+-channel inactivation. The possible physiological and behavioural roles of this slow inactivation are discussed.