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.