摘要:

AbstractWe recently demonstrated that elevated concentrations (≥ 20 μM) of the dynein substrate MgATP2−inhibit the spontaneous ATP‐induced sliding disintegration of isolated, Triton‐demembranted Tetrahymena cilia. We have used a turbidimetric assay (ΔA350 nm) and electron microscopy to examine the effect of ATP on sliding disintegration when activated by other divalent cations. Mg2+, Ca2+, and Mn2+are each capable of activating sliding, but only with Mg2+and Mn2+is disintegration inhibited by elevated ATP concentrations (≥ 1 mM). The two major ATPase activities obtained by KCI extraction of Tetrahymena axonemes differ in their cation specificities such that Mg2+and Ca2+activate the 21S dynein ATPase with equal efficiency, whereas the 13S axonemal ATPase activity is reduced by ∼ 50% when CaATP2−replaces MgATP2−as substrate. With 1 mM MgATP2−as substrate, 10−7to 10−2M added CaC12alleviates the ATP‐dependent inhibition of disintegration and likewise represses 13S MgATPase activity. In contrast, free Ca2+has no effect on either the disintegration response or MgATPase activity. In contrast to Triton‐treated cilia, glycerinated cilia, which beat in 1 mM MgATP2−, are inhibited from beating by high CaATP2−concentrations. These substrate specificities suggest that concentration‐dependent, substrate inhibition of sliding disintegration may be a manifestation of a physiological mechanism that is mediated by the 13S axonemal ATPase and that may function to modulate sliding during bend formation. However, the effects of added CaCl2probably do not reflect a physiological mechanism for regulating beat parameters, but rather may result from CaATP2−competing for MgATP2−binding sites on the 13S ATPase, thereby

ISSN:0271-6585    

DOI:10.1002/cm.970020603

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractExtraction of isolated, demembranated flagellar axonemes of Chlamydomonas reinhardii with 0.6 M KCl solubilized 77–92% of the total axonemal Mg++or Ca++‐ATPase activity, which sedimented as 18S and 12S peaks in sucrose density gradients. The ATPases of these two peaks were further purified by hydroxyapatite (HAP) column chromatography. The ATPase activity of the 18S peak eluted from the HAP column as a single peak coinciding with the protein peak. The HAP purified 18S ATPase had a specific activity of ∼2.0 ± 0.5 μmoles Pihydrolyzed min/mg and was associated with four high molecular weight (HMW) polypeptides of ∼ 310,000‐340,000 daltons, two intermediate molecular weight (IMW) polypeptides of 78,000 and 69,000 daltons, and eight low molecular weight (LMW) polypeptides of 7,800–19,600 daltons. When the 12S sucrose gradient peak together with a trailing shoulder were chromatographed on HAP, the ATPase activity was eluted in two peaks designated 12S and 10.5S on the basis of the sedimentation properties of their associated polypeptides. The 12S peak contained a single dynein ATPase having a specific activity of ∼ 0.6 ± 0.3 μmoles Pihydrolyzed min/mg and associated with ∼ 330,000‐, 21,700‐, and 18, 100‐dalton polypeptides. The 10.5S peak contained several high, intermediate, and low molecular weight polypeptides; of these, on HMW polypeptide and one 28,700‐dalton polypeptide correlated well with the ATPase activity. The purified ATPases had no polypeptides in common; each therefore represents a discrete dynein. Based on protein recovered in the purified fractions, 18S dynein represents ∼ 9.2% of the total axonemal protein; 12S dynein represents

ISSN:0271-6585    

DOI:10.1002/cm.970020604

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractThe distal tips of the central pair and A‐microtubules are capped in mammalian and avian tracheal cilia. The capping structures are similar to those found in protozoan cilia and flagella [Dentler, 1981], and consist of a central microtubule cap that links the central microtubules to the membrane or to the ciliary crown and A‐microtubule plugs that insert into the lumen of each of the A‐microtubule plugs is bound to the central microtubule cap by distal filaments. The ends of the central and outer doublet microtubules are tightly bound to the cap in both intact and in demembranated and reactivated tracheal cilia. Analysis of the displacement of the microtubule tips in cilia fixed at various bend angles revealed that the displacements of A‐microtubules are only partially in agreement with those predicted by the sliding filament model [Satir, 1968]. These results are discussed with respect to the regulation of microtubule sliding in capped cilia and the role of the microtubule capping structures in microtubule a

ISSN:0271-6585    

DOI:10.1002/cm.970020605

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractWhen the area of the stigma of Euglena was irradiated with an infrared laser beam at a dose too low to cause permanent loss of motility, a reduction in flagellar motility was observed only when the external medium contained less than 1 mM Mg2+. At these low Mg2+concentrations, the laser caused a decrease in flagellar frequency and a tendency for the flagellar waveform to shift towards that taken during reversed swimming. This suggests that the effect of the laser irrdiation was to deplete the cells of Mg2+. After the laser pulse the reversal response remained sensitive to the wavelength of the illuminating light. In white light (420–700 nm) 60% of the Euglena showed a reversed waveform; in orange light (530–700 nm) this increased to 90%. This shows that the photoreceptor was not destroyed by the laser irradiation.These experiments were performed on cells that had been impaled on a microelectrode. If direct electric current was passed into the laser‐irradiated cells, the current necessary to cause flagellar arrest was 2 to 4 times less than that for cells not laser irradiated.It is concluded that an internal Mg2+store is present in the Euglena, localized in the area of the paraflagellar swelling; and that the laser irradiation eliminates this Mg2+store, but at the power used it does not destroy the ability of the stigmaparaflagella to control the flagellar act

ISSN:0271-6585    

DOI:10.1002/cm.970020606

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractLigand binding to cell surface receptors induces rapid internalization of ligandreceptor complexes by receptor mediated endocytosis. We have examined the intracellular movement of endocytic vesicles, induced by the lectin concanavalin A (Con A), in cultured rat ovarian granulosa cells using fluorescence and electron microscopy. Within 20 minutes of ligand treatment at 37°C, numerous Con A‐containing endocytic vesicles form, which migrate to the cell center by 60 minutes. Double label fluorescence microscopy, using fluorescien‐Con‐A and rhodamine immunofluorescent staining of tubulin or vimentin, indicates that during vesicle migration microtubules and 10‐nm filaments are altered in their organization. By 30 minutes, vesicles are associated with microtubule bundles, which subsequently collapse around the nucleus. Similarly, 10‐nm filaments accumulate around the nucleus in conjunction with the perinuclear aggregation of endocytic vesicles. Electron microscopy of Con A‐horseradish peroxidase‐labeled cells demonstrates that endocytic vesicles fuse to form large receptosome‐like structures during intracellular migration and these structures are associated with cytoplasmic microtubules and 10‐nm filaments. Taxol, a drug that stabilizes microtubules, prevents endocytic vesicle translocation to the Golgi region. Nocodazole, which causes microtubule disassembly, results in the collapse of 10‐nm filaments and the central aggregation of endocytic vesicles. The data indicate that the cytoskeleton participates in the directed intracellular movement of endocytic vesicles; the possible subcellular basis for this m

ISSN:0271-6585    

DOI:10.1002/cm.970020607

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractTwo monoclonal antibodies reactive for α‐tubulin but not for β‐tubulin have been prepared, characterized in terms of their relative binding to tubulins from differnt sources by a solid‐phase binding assay, immunoautoradiography, and indirect immunofluorescence, and utilized to study flagellar motility. Our results demonstrate that α‐tubulins from different species, and even from different tissues of the same species, are nonidentical. Especially interesting was the observation that one of the antibodies, Ab2, immunofluorescently stained microtubules of chick embryo fibroblast cells, but was completely unreactive for microtubules of rat kangaroo (PtK2) fibroblasts; a different antibody, Ab1, stained both cell types. Results of these and additional experiments clearly show that Ab1 and Ab2 recognize discrete and different epitopes on α‐tubulin.Monoclonal antitubulins Ab1 and Ab2 each inhibited the bend amplitude of reactivated sea urchin spermatozoa without affecting beat frequencies or the ability of the outer doublet microtubules to slide past each other in elastase‐digested models. These results, together with those obtained previously using rabbit polyclonal antitubulin antibodies [Asai and Brokaw, 1980], demonstrate that inhibition of bend amplitude is a common property of antitubulin antibodies and is not due to the binding of antibodies to one specific site on the axoneme. Our results suggest that tubulin subunit conformational changes may occur on the outer doublet lattice and may be integrally involved in the mechanism and control of fl

ISSN:0271-6585    

DOI:10.1002/cm.970020608

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

ISSN:0271-6585    

DOI:10.1002/cm.970020602

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY