Reactivation of newt lung cilia: Evidence for a possible temperature‐ and MgATP‐induced activation mechanism

Hard, R; Cypher, Christopher

doi:10.1002/cm.970210303

Cited by 8 publications

(10 citation statements)

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“…

Demembranated axonemes isolated from newt lung ciliated cells show a complex beat frequency response to varying [MgATP] and temperature [Hard and Cypher, 1992, Cell Motil. Cytoskeleton 21:187-1981.

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confidence: 99%

Reactivation of outer‐arm‐depleted lung axonemes: Evidence for functional differences between inner and outer dynein arms in situ

Hard

Blaustein

Scarcello

1992

Cell Motil. Cytoskeleton

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Demembranated axonemes isolated from newt lung ciliated cells show a complex beat frequency response to varying [MgATP] and temperature [Hard and Cypher, 1992, Cell Motil. Cytoskeleton 21:187-198]. The present study was undertaken to ascertain whether the beat frequency of outer-arm-depleted newt lung axonemes is controlled in a manner similar to that of intact axonemes. Populations of demembranated ciliary axonemes were isolated by Triton X-100 extraction of lungs from the newt, Taricha granulosa. Aliquots of the demembranated axonemes were further treated with solutions containing high salt (0.375 M KC1) and 1.25 mM MgATP. This treatment resulted in the selective removal of outer dynein arms and a concomitant decrease in beat frequency to a stable level, 33-35% of control values. The effects of pH, salt concentration, nucleotides, and temperature on the beat frequency of reactivated outer-arm-depleted axonemes were ascertained and compared with those of intact axonemes. Some reactivation properties, such as nucleotide specificity, the effect of pH on beat frequency and the threshold [MgATP] required for reactivation (approximately 5 microM) were similar to those observed for intact axonemes. Other properties, such as the relationship between beat frequency and varying [MgATP] or salt concentration, differed both qualitatively and quantitatively from those of control axonemes, as did their response to temperature over the range, 5 degrees-32 degrees C. The nature of the results obtained with temperature and MgATP suggests that inner and outer dynein arms are not functionally equivalent in situ.

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“…

Demembranated axonemes isolated from newt lung ciliated cells show a complex beat frequency response to varying [MgATP] and temperature [Hard and Cypher, 1992, Cell Motil. Cytoskeleton 21:187-1981.

…”

mentioning

confidence: 99%

Reactivation of outer‐arm‐depleted lung axonemes: Evidence for functional differences between inner and outer dynein arms in situ

Hard

Blaustein

Scarcello

1992

Cell Motil. Cytoskeleton

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“…Beat frequencies of ciliated cells vary considerably according to the method of isolation, culture, etc. Since beat frequency constitutes a “health meter” built into ciliated cells, it is important for subsequent experimentation that maximum frequencies approach 20 Hz or greater, based on data in vertebrate systems 4 . Cilia beating at low, unphysiologic frequencies may be unresponsive to agents that activate signal transduction pathways, thereby giving erroneous results.…”

Section: Discussionmentioning

confidence: 99%

“…Erythrocyte lysis buffer consisted of 0.15 M NH 4 CL, 10 mM KHCO 3 , 0.1 mM Na 2 EDTA in deionized H 2 O and was prepared fresh before each use. Cell viability was determined using trypan blue dye exclusion, and cells were plated at seeding densities of no less than 2 × 10 4 /cm 2 on type I collagen (Cellogen Discs, Costa Mesa, CA) in media consisting of DMEM/F12‐containing antibiotics and 5 g/mL insulin, 1 μM hydrocortisone, 10 ng/mL L‐isoproterenol, 0.5 ng/mL EGF, and 5% FBS and 1 × 10 −8 retinoic acid.…”

Section: Methodsmentioning

confidence: 99%

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Ciliary Activity in Differentiating and Reactivated Human Respiratory Epithelial Cells

et al. 1999

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The authors have demonstrated that ciliary beat frequency of demembranated human respiratory epithelial cells can be modulated by MgATP and can be adjusted to the same level of activity as measured in living cells. This allows them to selectively test whether a drug's effects on mucociliary transport are the result of direct interactions with the ciliary apparatus or are produced through other indirect mechanisms.

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“…Purified, demembranated axoneme models reactivate with exogenous MgATP and beat at frequencies comparable to those recorded for intact cells. Hard and Cypher [1992] have shown that the beat frequency kinetics of these highly coupled axonemes is biphasic in response to MgATP concentration and temperature. Recently, have demonstrated that the outer dynein arms can be selectively removed from isolated, demembranated newt lung ciliary axonemes by high salt (0.375 M) extraction in a manner similar to sea urchin or trout sperm flagellar axonemes.…”

Section: Introductionmentioning

confidence: 99%

Outer arm dynein from newt lung respiratory cilia: Purification and polypeptide composition

Rupp

Hard

1995

Cell Motil. Cytoskeleton

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Dyneins are multimeric ATPases that comprise the inner and outer arms of cilia and flagella. It previously has been shown that salt extraction of newt lung axonemes selectively removes > 95% of the outer arm dynein (OAD), and that the beat frequency of OAD-depleted axonemes cannot be activated as compared to controls [Hard et al., 1992: Cell Motil. Cytoskeleton 21:199-209]. Therefore, expression of the activated state appears to require the presence of outer dynein arms. The present study was undertaken to ascertain basic information on the structure and molecular composition of newt OAD. Populations of demembranated axonemes were extracted with 0.375 M salt. Each lung released approximately 1.4 x 10(7) axonemes during isolation, yielding approximately 120 ng of salt extractable OAD. Electron microscopy of negatively stained samples revealed that newt OAD consisted of two globular heads joined together by a Y-shaped stem, similar to sea urchin and trout sperm OAD. Each head appeared to be roughly spherical in shape, measuring approximately 17 nm in diameter. Electrophoretic analysis of whole axonemes revealed more than six dynein heavy chains when resolved in silver stained 0-8 M urea, 3-5% acrylamide gradients. Extracted OAD, either crude in high salt or purified by alloaffinity, was composed of two heavy chains. UV-induced (366 nm) photolytic cleavage at the V1 site, performed in the presence of Mg2+, vanadate, and ATP, produced four new polypeptides (M(r) 234, 232, 197, and 189 kD). Photolysis was supported by Mg2+ and Ca2+, but did not occur in the presence of Mn2+. The apparent M(r) of the dynein heavy chains was determined to lie between 430-420 kD. Eight discrete polypeptides (putative intermediate chains, IC1-IC8, M(r), 175-56 kD) copurified with the alpha- and beta-heavy chains by microtubule-alloaffinity. Based on its extraction characteristics, polypeptide composition in purified and crude samples, and structure, we conclude that this two-headed particle represents the entire newt respiratory outer arm dynein.

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Reactivation of newt lung cilia: Evidence for a possible temperature‐ and MgATP‐induced activation mechanism

Cited by 8 publications

References 49 publications

Reactivation of outer‐arm‐depleted lung axonemes: Evidence for functional differences between inner and outer dynein arms in situ

Reactivation of outer‐arm‐depleted lung axonemes: Evidence for functional differences between inner and outer dynein arms in situ

Ciliary Activity in Differentiating and Reactivated Human Respiratory Epithelial Cells

Outer arm dynein from newt lung respiratory cilia: Purification and polypeptide composition

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