NONLETHAL DECILIATION OF <i>TETRAHYMENA</i> BY A LOCAL ANESTHETIC AND ITS UTILITY AS A TOOL FOR STUDYING CILIA REGENERATION

Thompson, Guy A.; Baugh, L.C.; Walker, L.F.

doi:10.1083/jcb.61.1.253

Cited by 102 publications

(48 citation statements)

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“…Because Ca 2÷ is necessary and sufficient for deflagellation in detergent-permeabilized cells (Sanders and Salisbury, 1994), agents which induce deflagellation in vivo may act via increases in intracellular [Ca2+]. Deflagellation can be produced in living cells by a variety of stimuli (Minz and Lewin, 1954;Thompson et al, 1974;Lewin et al, 1980;Witman, 1986). We have previously shown that acid flux into the cell triggers deflagellation in vivo (Hartzell et al, 1993) as does external application of the wasp venom peptide, mastoparan (Quarmby et al, 1992).…”

mentioning

confidence: 99%

Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii

Quarmby

Hartzell

1994

The Journal of Cell Biology

108

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Abstract. The molecular machinery of deflagellation can be activated in detergent permeabilized Chlamydomonas reinhardtii by the addition of Ca 2+ (Sanders, M. A., and J. L. Salisbury, 1989. J. Cell Biol. 108:1751-1760). This suggests that stimuli which induce deflagellation in living cells cause an increase in the intracellular concentration of Ca 2+, but this has never been demonstrated. In this paper we report that the wasp venom peptide, mastoparan, and the permeant organic acid, benzoate, activate two different signalling pathways to trigger deflagellation. We have characterized each pathway with respect to: (a) the requirement for extracellular Ca2÷; (b) sensitivity to Ca 2+ channel blockers; and (c) 45Ca influx. We also report that a new mutant strain of C. reinhardtii, adf-1, is specifically defective in the acid-activated signalling pathway. Both signalling pathways appear normal in another mutant, fa-1, that is defective in the machinery of deflagellation (Lewin, R. and C. Burrascano. 1983. Experientia. 39:1397-1398Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751-1760. We conclude that mastoparan induces the release of an intracellular pool of Ca 2+ whereas acid induces an influx of extraceUular Ca 2+ to activate the machinery of deflagellation.D FLAGELLATION is a specific event whereby the flagella are precisely excised from the cell body (Rosenbanm and Carlson, 1969;Satir et al., 1976;Lewin and Lee, 1985;Sanders and Salisbury, 1989; Jarvik and Suhan, 1991). The physical mechanism of flagellar excision appears to involve both a microtubule severing activity (Vale, 1991;Shiina et al., 1992;McNally and Vale, 1993) and a mechanical force generated by centrin (for references see Hartzell et al., 1993). A stellate array of centrin-containing transition zone fibers contract during deflagellation (Sanders and Salisbury, 1989). Chlamydomonas cells permeabilized with the non-ionic detergent, NP-40, deflagellate when Ca 2+ is added in /~M concentrations (Sanders and Salisbury, 1989). Because Ca 2÷ is necessary and sufficient for deflagellation in detergent-permeabilized cells (Sanders and Salisbury, 1994), agents which induce deflagellation in vivo may act via increases in intracellular [Ca2+]. Deflagellation can be produced in living cells by a variety of stimuli (Minz and Lewin, 1954;Thompson et al., 1974;Lewin et al., 1980;Witman, 1986). We have previously shown that acid flux into the cell triggers deflagellation in vivo (Hartzell et al., 1993) as does external application of the wasp venom peptide, mastoparan (Quarmby et al., 1992). We now pose the question: How do these agents generate an intracellular Ca 2÷ signal in vivo?Address all correspondence to L, M, Quarmby, Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 30322.In the only published report to examine the requirement for extracellular Ca 2+ during acid-induced deflagellation, the authors state that a 30-min pretreatment in [Ca 2+] below 0.1 #M inhibited acid-induced deflagellation, but t...

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confidence: 99%

Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii

Quarmby

Hartzell

1994

The Journal of Cell Biology

108

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“…Methods are available for the independent isolation of cilia (24), cytoplasmic microtubules of the oral apparatus (25,26), and the soluble pool of cytoplasmic tubulin (ref. 27 and this report).…”

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confidence: 99%

“…Ciliary axonemes were isolated from Tetrahymena thermophila, clone SB911 (30), as described (24,31).…”

mentioning

confidence: 99%

Multiple forms of tubulin in the cilia and cytoplasm of Tetrahymena thermophila.

Suprenant¹,

Hays²,

LeCluyse³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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Most higher eukaryotic tubulins are separated into a-and J3-tubulin when electrophoresed in NaDodSO4-denaturing gels, while many lower eukaryotic tubulins are poorly resolved under these conditions, which include a stacking gel (pH 6.80) and a separating gel (pH 8.80). By lowering the pH of the separating gel to 8.25, we have found that tubulin isolated from the protozoan Tetrahymena thermophila is resolved by one-dimensional polyacrylamide gel electrophoresis into two a-tubulins and one P-tubulin. Moreover, at least five A-and two .3-tubulin isotypes are identified in Tetrahymena by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis. Three of these a isotypes and one (3 isotype are found specifically in ciliary microtubules, while the other two isotypes are found only in the cytoplasmic tubulin pool that was isolated and induced to self-assemble into microtubules in vitro. Peptide mapping by limited proteolytic digestion indicates that the tubulins are closely related. Possible mechanisms for the generation and selection of these tubulin isotypes are discussed.

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“…In both methods, regeneration of cilia is ensued once the cells are introduced to fresh culture medium. In previous experiments [17] , the motility of T. pyriformis is measured as the percentage of moving cells at each observation during the regeneration process. However, without a measurement confirming that cell velocity returns to pre-deciliation conditions, it cannot be proven that a complete recovery occurs.…”

Section: Introductionmentioning

confidence: 99%

“…Calcium chloride and ethylenediaminetetraacetic acid are utilized for partial deciliation and mechanical shear forces are applied using a syringe fitted with a needle for complete deciliation [13][14][15] . Another method employs a local anesthetic, dibucaine HCl, for complete removal of cilia, eliminating the need for mechanical shear forces [16,17] . In both methods, regeneration of cilia is ensued once the cells are introduced to fresh culture medium.…”

Section: Introductionmentioning

confidence: 99%

Characterization of deciliation-regeneration process of Tetrahymena Pyriformis for cellular robot fabrication

et al. 2011

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Artificial magnetotactic Tetrahymena pyriformis GL (T. pyriformis) cells were created by the internalization of iron oxide nano particles and became controllable with a time-varying external magnetic field. Thus, T. pyriformis can be utilized as a cellular robot to conduct micro-scale tasks such as transportation and manipulation. To complete these tasks, loading inorganic or organic materials onto the cell body is essential, but functionalization of the cell membrane is obstructed by their motile organelles, cilia. Dibucaine HCl, a local anesthetic, removes the cilia from the cell body, and the functional group would be absorbed more efficiently during cilia regeneration. In this paper, we characterize the recovery of artificial magnetotactic T. pyriformis after the deciliation process to optimize a cellular robot fabrication process. After sufficient time to recover, the motility rate and the average velocity of the deciliated cells were six and ten percent lower than that of non-deciliated cells, respectively. We showed that the motile cells after recovery can still be controlled using magnetotaxis, making T. pyriformis a good candidate to be used as a cellular robot.

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NONLETHAL DECILIATION OF TETRAHYMENA BY A LOCAL ANESTHETIC AND ITS UTILITY AS A TOOL FOR STUDYING CILIA REGENERATION

Cited by 102 publications

References 11 publications

Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii

Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii

Multiple forms of tubulin in the cilia and cytoplasm of Tetrahymena thermophila.

Characterization of deciliation-regeneration process of Tetrahymena Pyriformis for cellular robot fabrication

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