Microtubule-membrane interactions in cilia. II. Photochemical cross-linking of bridge structures and the identification of a membrane-associated dynein-like ATPase

This is the publisher's version, also available electronically from http://jcb.rupress.org/content/84/2/381.Photochemical cross-linking of both Tetrahymena and Aequipecten ciliary membrane proteins with the lipophilic reagent 4,4'-dithiobisphenylazide links together a high molecular weight dynein-like ATPase, membrane tubulin, and at least two other proteins. Electron microscopy of detergent-extracted cilia reveals that the cross-linked complex remains attached to the outer-doublet microtubules by a microtubule-membrane bridge. Cleavage of the reagent's disulfide bond releases the bridge-membrane complex and the dynein-like membrane-associated ATPase. Electron microscopy was used to ensure that the dynein-like protein did not result from the solubilization of the dynein arms attached to the outer-doublet microtubules. The dynein-like protein has been isolated using sucrose gradients and is similar to axonemal dynein with respect to its sedimentation characteristics nucleotide specificity, and divalent cation requirements. Photochemical cross-linking of ciliary membrane porteins in vivo results initially in the modification of ciliary beat and, eventually, in the cessation of ciliary movement. These results suggest that a dynein-like ATPase comprises the bridge which links the ciliary membrane to the outer-doublet microtubules and that this bridge is involved in the modulation of normal ciliary movement

Ciliary microtubule capping structures contain a mammalian kinetochore antigen

This is the publisher's version, also available electronically from http://jcb.rupress.org/content/110/3/703.Structures that cap the plus ends of microtubules may be involved in the regulation of their assembly and disassembly. Growing and disassembling microtubules in the mitotic apparatus are capped by kinetochores and ciliary and flagellar microtubules are capped by the central microtubule cap and distal filaments. To compare the ciliary caps with kinetochores, isolated Tetrahymena cilia were stained with CREST (Calcinosis/phenomenon esophageal dysmotility, sclerodactyly, telangiectasia) antisera known to stain kinetochores. Immunofluorescence microscopy revealed that a CREST antiserum stained the distal tips of cilia that contained capping structures but did not stain axonemes that lacked capping structures. Both Coomassie blue-stained gels and Western blots probed with CREST antiserum revealed that a 97-kD antigen copurifies with the capping structures. Affinity-purified antibodies to the 97-kD ciliary protein stained the tips of cap-containing Tetrahymena cilia and the kinetochores in HeLa, Chinese hamster ovary, and Indian muntjak cells. These results suggest that at least one polypeptide found in the kinetochore is present in ciliary microtubule capping structures and that there may be a structural and/or functional homology between these structures that cap the plus ends of microtubules

Language support for immigrant children: a study of state schools in the UK and US

In recent decades, immigrants, refugees and asylum seekers have sought a new way of life in large numbers, often leaving their countries of origin behind in search of places that offer a better way of life. The purpose of this study was to investigate how elementary and middle school students in state schools in Reading, England (primarily speakers of Asian languages), and Richmond, Virginia (primarily speakers of Spanish), were supported academically, when most children’s first language was not English. The authors were interested in exploring whether or not there were cultural or structural differences in the way each country helped or hindered these students as they progressed through the school systems. Three UK schools in a district of approximately 100,000 and three US schools in a district of approximately 250,000 were the focus of this exploration from 2000 to 2003. Findings indicated that there were cultural and legislative differences and similarities. Teachers and administrators in both countries attempted to provide services with limited and sometimes diminishing resources. Community support varied based on resources, attitudes toward various ethnic groups, and the coping strategies adopted by these groups in their new environments. Marked differences appeared with regard to the manner in which assessments took place and how the results were made available to the public

FAP206 is a Microtubule-Docking Adapter for Ciliary Radial Spoke 2 and Dynein c

Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo–electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong. Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo. Thus FAP206 is likely part of the front prong and docks RS2 and dynein c to the microtubule

GTP avoidance in Tetrahymena thermophila requires tyrosine kinase activity, intracellular calcium, NOS, and guanylyl cyclase

Guanosine 5'-triphosphate (GTP) is a chemorepellent in Tetrahymena thermophila that has been shown to stimulate cell division as well as ciliary reversal. Previous studies have proposed that GTP avoidance is linked to a receptor-mediated, calcium-based depolarization. However, the intracellular mechanisms involved in GTP avoidance have not been previously documented. In this study, we examine the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila. Using behavioral assays, enzyme immunosorbent assays, Western blotting, and immunofluorescence, we present data that implicate a tyrosine kinase, phospholipase C, intracellular calcium, nitric oxide synthase (NOS) and guanylyl cyclase in GTP signaling. The tyrosine kinase inhibitor genistein eliminates GTP avoidance in Tetrahymena in behavioral assays. Similarly, pharmacological inhibitors of phospholipase C, NOS, and guanylyl cyclase all eliminated Tetrahymena avoidance to GTP. Immunofluorescence data shows evidence of tyrosine kinase activity in the cilia, suggesting that this enzyme activity could be directly involved in ciliary reversal

A protein methylation pathway in Chlamydomonas flagella is active during flagellar resorption

Author Posting. © American Society for Cell Biology, 2008. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 19 (2008): 4319-4327, doi:10.1091/mbc.E08-05-0470.During intraflagellar transport (IFT), the regulation of motor proteins, the loading and unloading of cargo and the turnover of flagellar proteins all occur at the flagellar tip. To begin an analysis of the protein composition of the flagellar tip, we used difference gel electrophoresis to compare long versus short (i.e., regenerating) flagella. The concentration of tip proteins should be higher relative to that of tubulin (which is constant per unit length of the flagellum) in short compared with long flagella. One protein we have identified is the cobalamin-independent form of methionine synthase (MetE). Antibodies to MetE label flagella in a punctate pattern reminiscent of IFT particle staining, and immunoblot analysis reveals that the amount of MetE in flagella is low in full-length flagella, increased in regenerating flagella, and highest in resorbing flagella. Four methylated proteins have been identified in resorbing flagella, using antibodies specific for asymmetrically dimethylated arginine residues. These proteins are found almost exclusively in the axonemal fraction, and the methylated forms of these proteins are essentially absent in full-length and regenerating flagella. Because most cells resorb cilia/flagella before cell division, these data indicate a link between flagellar protein methylation and progression through the cell cycle.This work was supported by National Institutes of Health Grant DK071720 (R.D.S.) and National Science Foundation Grant MCB 0418877 (R.D.S.)

Predicting invasions of North American basses in Japan using native range data and a genetic algorithm

Largemouth bass Micropterus salmoides and smallmouth bass M. dolomieu have been introduced into freshwater habitats in Japan, with potentially serious consequences for native fish populations. In this paper we apply the technique of ecological niche modeling using the genetic algorithm for rule-set prediction (GARP) to predict the potential distributions of these two species in Japan. This algorithm constructs a niche model based on point occurrence records and ecological coverages. The model can be visualized in geographic space, yielding a prediction of potential geographic range. The model can then be tested by determining how well independent point occurrence data are predicted according to the criteria of sensitivity and specificity provided by receiver–operator curve analysis. We ground-truthed GARP’s ability to forecast the geographic occurrence of each species in its native range. The predictions were statistically significant for both species (P , 0.001). We projected the niche models onto the Japanese landscape to visualize the potential geographic ranges of both species in Japan. We tested these predictions using known occurrences from introduced populations of largemouth bass, both in the aggregate and by habitat type. All analyses robustly predicted known Japanese occurrences (P , 0.001). The number of smallmouth bass in Japan was too small for statistical tests, but the 10 known occurrences were predicted by the majority of models

Model-Independent νˉe\bar\nu_{e} Short-Baseline Oscillations from Reactor Spectral Ratios

We consider the ratio of the spectra measured in the DANSS neutrino experiment at 12.7 and 10.7~m from a nuclear reactor. These data give a new model-independent indication in favor of short-baseline $\bar\nu_{e}$ oscillations which reinforce the model-independent indication found in the late 2016 in the NEOS experiment. The combined analysis of the NEOS and DANSS spectral ratios in the framework of 3+1 active-sterile neutrino mixing favor short-baseline $\bar\nu_{e}$ oscillations with a statistical significance of $3.7\sigma$. The two mixing parameters $\sin^{2}2\vartheta_{ee}$ and $\Delta{m}^{2}_{41}$ are constrained at $2\sigma$ in a narrow-$\Delta{m}^{2}_{41}$ island at $\Delta{m}^2_{41} \simeq 1.3 \, \text{eV}^2$, with $\sin^{2}2\vartheta_{ee} = 0.049 \pm 0.023$ ($2\sigma$). We discuss the implications of the model-independent NEOS+DANSS analysis for the reactor and Gallium anomalies. The NEOS+DANSS model-independent determination of short-baseline $\bar\nu_{e}$ oscillations allows us to analyze the reactor rates without assumptions on the values of the main reactor antineutrino fluxes and the data of the Gallium source experiments with free detector efficiencies. The corrections to the reactor neutrino fluxes and the Gallium detector efficiencies are obtained from the fit of the data. In particular, we confirm the indication in favor of the need for a recalculation of the $^{235}\text{U}$ reactor antineutrino flux found in previous studies assuming the absence of neutrino oscillations.Comment: 10 pages; analysis improved by taking into account the uncertainties of the reactor fission fraction