Todd M. Hennessey

Tubulin glutamylation regulates ciliary motility by altering inner dynein arm activity.

How microtubule-associated motor proteins are regulated is not well understood. A potential mechanism for spatial regulation of motor proteins is provided by posttranslational 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 posttranslational modification, 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.

Chemorepellents in Paramecium and Tetrahymena

Although Paramecium has been widely used as a model sensory cell to study the cellular responses to thermal, mechanical and chemoattractant stimuli, little is known about their responses to chemorepellents. We have used a convenient capillary tube repellent bioassay to describe 4 different compounds that are chemorepellents for Paramecium and compared their response with those of Tetrahymena. The classical Paramecium t‐maze chemokinesis test was also used to verify that this is a reliable chemorepellent assay. The first two compounds, GTP and the oxidant NBT, are known to be depolarizing chemorepellents in Paramecium but this is the first report of them as repellents in Tetrahymena. The second two compounds, the secretagogue alcian blue and the dye cibacron blue, have not previously been described as chemorepellents in either of these ciliates. Two other compounds, the secretagogue AED and the oxidant cytochrome c, were found to be repellents to Paramecium but not to Tetrahymena. The repellent nature of each of these compounds is not related to toxicity because cells are completely viable in all of them. More importantly, all of these repellents are effective at micromolar to nanomolar concentrations, providing an opportunity to use them as excitatory ligands in future works concerning their membrane receptors and possible receptor operated ion channels.

Targeted gene disruption of dynein heavy chain 7 of Tetrahymena thermophila results in altered ciliary waveform and reduced swim speed.

Tetrahymena thermophila swims by the coordinated beating of hundreds of cilia that cover its body. It has been proposed that the outer arm dyneins of the ciliary axoneme control beat frequency, whereas the inner arm dyneins control waveform. To test the role of one of these inner arms, dynein heavy chain 7 protein (Dyh7p), a knockout mutant was generated by targeted biolistic transformation of the vegetative macronucleus. Disruption of DYH7, the gene which encodes Dyh7p, was confirmed by PCR examination of both genomic and cDNA templates. Both intact and detergent extracted, reactivated cell model preparations of these mutants, which we call DYH7neo3, displayed swim speeds that were almost half that of wild-type cells. Although the DYH7neo3 mutants were slower than wild type, they were able to modulate their swim speed and show ciliary reversal in response to depolarizing stimuli. High-speed video microscopy of intact, free-swimming DYH7neo3 mutants revealed an irregular pattern of ciliary beat and waveform. The mutant cilia appeared to be engaging in less coordinated, swiveling movements in which the typical shape, periodicity and coordination seen in wild-type cilia were absent or disturbed. We propose that the axonemal inner arm dynein heavy chain 7 proteins contribute to the formation of normal ciliary waveform, which in turn governs the forward swimming velocity of these cells.

Chemosensory Adaptation to Lysozyme and GTP Involves Independently Regulated Receptors in Tetrahymena thermophila

ABSTRACT. Chemosensory adaptation is seen in Tetrahymena 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 [32P]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 KD values for nonadapted cells are 6.6 μM and 8.4 μM for lysozyme and GTP binding, respectively. Behavioral adaptation and decreased surface receptor binding are specific for each repellent. The GTP adapted cells (20 μM for ten minutes) still respond behaviorally to 50 μM lysozyme and bind [3H]lysozyme normally. Lysozyme adapted cells (50 μM for ten minutes) still bind [32P]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 or avoidance reactions. These results are consistent with the hypothesis that there are two separate repellent receptors, one for GTP and the other for lysozyme, that are independently downregulated during adaptation to cause specific receptor desensitization and consequent behavioral adaptation.

Neomycin inhibits the calcium current of Paramecium

The duration of high-K+ stimulated backward swimming is a commonly used bioassay for estimating the amplitude of the inward calcium current of Paramecium. Electrophysiological analysis confirmed that concentrations of neomycin which decreased the duration of stimulated backward swimming also reduced the isolated inward calcium current. Other polycations were also effective in this bioassay and their effectiveness was correlated with the number of their positive charges. Paramecium is therefore a convenient model system for studying the effects of compounds such as neomycin on calcium currents as well as their mechanisms of action.

Chemosensory adaptation in paramecium involves changes in both repellent binding and the consequent receptor potentials

Abstract Two different chemorepellents, GTP and lysozyme, both produce transient depolarizing somatic receptor potentials and backward swimming (at micromolar concentrations) in Parameium. Behavioral adaptation occurs after 10 min in either repellent and the cells regain normal forward swimming. This is very specific for each repellent. Cells that have behaviorally adapted to 0.1 μM lysozyme (for 10 min) show forward swimming in lysozyme, a decreased amplitude of lysozyme-induced depolarizations and a 10-fold decrease in the estimated number of surface binding sites for 3H-lysozyme (by Scatchard analysis). All of these changes are reversible after 10 min in the absence of lysozyme. The lysozyme-adapted cells have normal responses to other depolarizing stimuli such as 8 mM Ba++, 40 mM K+, 10 mM Na+ and 10.0 μM GTP and to the hyperpolarizing chemoattractant, 5 mM acetate. Their surface binding of 32P-GTP was also unaffected. GTP-adapted cells show dramatic decreases in 32P-GTP surface binding sites and in the frequency of GTP-induced depolarizations but no changes in 3H-lysozyme binding or lysozyme-induced backward swimming. These results suggest that lysozyme and GTP have separate high affinity surface receptors on the somatic membrane that are specifically down-regulated during chemosensory adaptation.

Electrophysiology of Tetrahymena.

Publisher Summary The focus in an electrophysiological analysis is the physical health of the cell. This is represented by a reliable, steady, and deep (hyperpolarized) resting membrane potential. A high membrane resistance is also necessary for an electrophysiological analysis. For any condition, many cell membrane potentials should be measured (at least three to ten cells) to establish the baseline, control level. Normal ciliary beating should also be observed. When a cell is damaged, it is often easy to see irregular or slowed ciliary beating, indicating either an uncoordinated or depolarized state. If a resting membrane potential has been established, an irreversible depolarizing drift may also indicate cell damage. A decreased membrane resistance is also an indication of cell damage. A good test of cell condition is to induce an action potential by current injection through the membrane potential recording electrode. This is easily done by using a 1-nA electrode resistance calibration pulse that is commonly a feature of modern recording devices. If the cell shows a visible jerk backward, it ensures that the electrode is inside the cell and that the cell is healthy enough to show a strong response.

Ion Currents of Paramecium : Effects of Mutations and Drugs

Paramecium is a eukaryotic, single-celled organism which is used as a simple model system for studying the regulatory mechanisms governing excitable and sensory cell functions. In terms of ion channel functions, some strategies may have been so efficient in the primative protozoans that they may remain as universal mechanisms in most sensory and excitable cells while other specialized functions may have been refined and further evolved in higher organisms, leading to diversity in the structure, function, and regulation of ion channels.

The CSC proteins FAP61 and FAP251 build the basal substructures of radial spoke 3 in cilia

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 knockout mutation of a constitutive GPCR in Tetrahymena decreases both G-protein activity and chemoattraction.

Although G-protein coupled receptors (GPCRs) are a common element in many chemosensory transduction pathways in eukaryotic cells, no GPCR or regulated G-protein activity has yet been shown in any ciliate. To study the possible role for a GPCR in the chemoresponses of the ciliate Tetrahymena, we have generated a number of macronuclear gene knockouts of putative GPCRs found in the Tetrahymena Genome database. One of these knockout mutants, called G6, is a complete knockout of a gene that we call GPCR6 (TTHERM_00925490). Based on sequence comparisons, the Gpcr6p protein belongs to the Rhodopsin Family of GPCRs. Notably, Gpcr6p shares highest amino acid sequence homologies to GPCRs from Paramecium and several plants. One of the phenotypes of the G6 mutant is a decreased responsiveness to the depolarizing ions Ba2+ and K+, suggesting a decrease in basal excitability (decrease in Ca2+ channel activity). The other major phenotype of G6 is a loss of chemoattraction to lysophosphatidic acid (LPA) and proteose peptone (PP), two known chemoattractants in Tetrahymena. Using microsomal [35S]GTPγS binding assays, we found that wild-type (CU427) have a prominent basal G-protein activity. This activity is decreased to the same level by pertussis toxin (a G-protein inhibitor), addition of chemoattractants, or the G6 mutant. Since the basal G-protein activity is decreased by the GPCR6 knockout, it is likely that this gene codes for a constitutively active GPCR in Tetrahymena. We propose that chemoattractants like LPA and PP cause attraction in Tetrahymena by decreasing the basal G-protein stimulating activity of Gpcr6p. This leads to decreased excitability in wild-type and longer runs of smooth forward swimming (less interrupted by direction changes) towards the attractant. Therefore, these attractants may work as inverse agonists through the constitutively active Gpcr6p coupled to a pertussis-sensitive G-protein.