Inner-arm Dynein Research Articles

Cloning and characterization of the actin-encoding gene of Chlamydomonas reinhardtii

The genomic and complementary DNA sequences were determined for the unique actin-encoding gene in Chlamydomonas reinhardtii (Cr). The deduced amino acid (aa) sequence of this actin was similar to most known actin sequences, with the highest identity (98.1%) being with that of Volvox carteri actin. The Cr actin-encoding gene has one intron in the 5′-untranslated region and eight introns in the coding region. The latter eight introns occur at the same positions as those in the V. carteri actin-encoding gene. The 5′-upstream region contains four short stretches of sequence similar to the so-called ‘tub box’, a characteristic sequence proposed to be responsible for the regulation of synthesis of various axonemal proteins upon deflagellation and during the cell cycle. Southern blot analysis indicated that the Cr genome has only a single actin-encoding gene. An antibody specific for the 11-aa peptide corresponding to the N-terminal sequence of this actin was found to react with a 43-kDa protein associated with flagellar inner-arm dynein. These findings indicate that a single actin functions in both the cytoplasm and flagella of this organism.

Chapter 67 Isolation of Inner- and Outer-Arm Dyneins

Publisher Summary Dyneins are the main constructive body part of the inner and outer arms of eukaryotic flagella and cilia. The composition of axonemal dyneins varies from species to species and within a single axoneme. Within an axoneme, outer-arm dyneins are thought to be homogenous in form, whereas the inner-arm dyneins consist of at least three subforms. Dyneins were first separated from other axonemal components by selective extraction in low-ionic-strength solution containing ethylenediaminetetraacetic acid; however, this procedure causes dynein molecules to dissociate into subunits. It is now most common to use high-ionic-strength (elevated NaCl or KCl) solution to extract dynein from axonemes. Biochemical quantities of dynein can be obtained from five different sources. This chapter describes the procedures for isolation of axonemes and outer-arm dynein from the flagella of the sea urchin Strongylocentrotus purpuratus and for the isolation of inner-arm dynein from the flagella of the green alga Chlamydomonas reinhurdtii . Similar procedures are used to isolate dynein from a number of sources. The chapter discusses isolation of sea urchin sperm axonemes and dynein—collection of sperms, isolation and demembranation of flagella (by osmotic shock and with detergent), and isolation of outer-arm dynein from sea urchin sperm flagella. Because exposure of sea urchin dynein to detergent nonphysiologically activates the dynein ATPase activity, it is advised to demembranate by osmotic shock with high sucrose for investigations into the regulation of axonemes and dynein. There are details on Isolation of Chlamydomonas inner-arm dynein and fractionation of dyneins by sucrose density gradient zonal centrifugation. The existence of numerous outer-arm and inner-arm mutants and the ability to extract both inner and outer arms with high-salt solutions make Chlamydomonas an excellent system for purification of inner-arm dyneins. To isolate inner-arm dyneins from Chlamydomonas , a cell line genetically lacking the entire outer-arm structure is used.

Chapter 68 Separation of Dynein Species by High-Pressure Liquid Chromatography

Publisher Summary High-pressure liquid chromatography (HPLC) on a Mono Q column has been used to separate various axonemal dyneins. This procedure takes only an hour or two. It has the further advantage that it is able to separate multiple inner-arm dyneins, which sucrose gradient centrifugation is unable to separate; however, the sample obtained by Mono Q chromatography may be contaminated by proteins that have similar charges. Therefore, it is recommended that sucrose gradient centrifugation be used in combination with Mono Q chromatography when very pure samples are desired. The procedure described in this chapter is based on the works of Goodenough and associates and has been used to separate seven discrete subspecies of inner-arm dyneins from Chlamydomonas axonemal extract. There is description of the solutions needed and the procedures of dynein extraction and mono Q chromatography. Most studies on isolated dynein have used a purification step with sucrose density gradient centrifugation. Although this method can separate dynein from contaminants having low sedimentation coefficients, it has a significant disadvantage that the centrifugation process takes as long as 5–8 hours. The HPLC method described in the chapter is preferable.

Chapter 22 Nonradioactive Method for ATPase Assays

Publisher Summary There is change in orthophosphate concentration by colorimetry. Nonradioactive ATPase assays follow the change. This happens after quenching an ATP-hydrolyzing reaction at regular time intervals and converting phosphate into a phosphomolybdate complex. A very sensitive method of this kind determines the quantity of phosphomolybdate as a complex with malachite green. The method described in this chapter, developed by Kodama and colleagues, is sensitive enough to measure 0.2–15 μM phosphate, a concentration range that other colorimetric methods cannot measure. The method is simple and reliable; the citric acid added after initiation of the coloring reaction removes excess molybdate and thereby greatly reduces the nonenzymatic hydrolysis of ATP catalyzed by molybdate. Because of its high sensitivity, this assay was used for measuring the ATPase activities of myosin and kinesin on a single microscope coverslip in in vitro motility assays. It has been used to measure the ATPase activities in Chlamydomonas inner-arm dynein fractions in which only small amounts of proteins are available. This chapter discusses the solutions and the procedure involved. There are also suggestions on precautions needed; like, because commercial sodium citrate is often contaminated with trace amounts of phosphate, it has been advised to prepare it from citric acid and NaOH, rather than purchase it. Use of tubes that have been washed with detergent and distilled water often results in a large scatter in the data, so disposable test tubes cannot be used, to be used are test tubes that have been rinsed with 0.5 N HCI and DDW. With higher concentrations of ATP, the time for the coloring reaction needs to be constant.

Immunological detection of actin in the 14S ciliary dynein of Tetrahymena

The association of actin with Tetrahymena ciliary dyneins was examined using a polyclonal antibody against Tetrahymena actin. Western blotting shows that actin is present in the 14S dynein fraction, but not in the 22S dynein fraction, which comprises the outer arm. By anion-exchange chromatography, 14S dynein can be further separated into three major fractions that contain four distinct heavy chains in total. When each fraction was tested by anti-actin immunoblotting, all three fractions contained actin in nearly stoichiometric amounts with the heavy chain. Since Tetrahymena actin differs significantly from acting of other species, the association with inner-arm dynein may be a conserved property of actin.

Isolation of two species of Chlamydomonas reinhardtii flagellar mutants, ida5 and ida6, that lack a newly identified heavy chain of the inner dynein arm.

Two novel Chlamydomonas mutants, ida5 and ida6, that lack subsets of inner-arm dynein have been isolated and mapped to discrete loci on the right arm of linkage group XIV. Of the seven different inner-arm dynein subspecies (a, b, c, d, e, f and g) identified by ion-exchange chromatography, ida5 lacks a, c, d and e, while ida6 lacks e alone; these are the only mutants that have been shown to lack subspecies e. Both strains can swim, albeit more slowly than the wild type. Hence, subspecies e must contribute to flagellar movement although it is unnecessary for the generation of undulating movement.

Translocation and rotation of microtubules caused by multiple species of Chlamydomonas inner-arm dynein

ABSTRACT Dynein was extracted from outer arm-less axonemes of the mutant odal and fractionated by high-pressure liquid chromatography on a MonoQ column into seven distinct subspecies (named a–g). Each subspecies contained one or two heavy chains and several mediumsized and light chains; by vanadate/UV-induced photocleavage and SDS-polyacrylamide gel electrophoresis, eight distinct heavy chains were identified. Analysis of the mutant axonemes indicated that the subspecies f (containing two heavy chains) is missing in the inner-arm mutant idal and the subspecies a, c and d are missing in the mutant ida4. Six subspecies (all but f supported microtubule translocation with the maximal rate ranging from 2 to 12 μm s−1 and the apparent Km for ATP ranging from about 10 to 100 μM. All the subspecies translocated microtubules with the plus end leading, indicating that all the inner-arm dyneins are minus end-directed motors. Five subspecies (all but b and f) displayed microtubule rotation during translocation at rates of up to about 10 Hz. Unexpectedly, the Km values for ATP for translocation and rotation did not always agree; because of this, the pitch of the movement was variable with some subspecies. These observations indicate that axonemes are equipped with several inner-arm subspecies and that torque generation is a feature common to many of them.

Two types of Chlamydomonas flagellar mutants missing different components of inner-arm dynein.

Two types of Chlamydomonas reinhardtii flagellar mutants (idaA and idaB) lacking partial components of the inner-arm dynein were isolated by screening mutations that produce paralyzed phenotypes when present in a mutant missing outer-arm dynein. Of the currently identified three inner-arm subspecies I1, I2, and I3, each containing two heterologous heavy chains (Piperno, G., Z. Ramanis, E. F. Smith, and W. S. Sale. 1990. J. Cell Biol. 110:379-389), idaA and idaB lacked I1 and I2, respectively. The 13 idA isolates comprised three genetically different groups (ida1, ida2, ida3) and the two idaB isolates comprised a single group (ida4). In averaged cross-section electron micrographs, inner dynein arms in wild-type axonemes appeared to have two projections pointing to discrete directions. In ida1-3 and ida4 axonemes, on the other hand, either one of them was missing or greatly diminished. Both projections were weak in the double mutant ida1-3 x ida4. These observations suggest that the inner dynein arms in Chlamydomonas axonemes are aligned not in a single straight row, but in a staggered row or two discrete rows. Both ida1-3 and ida4 swam at reduced speed. Thus, the inner-arm subspecies missing in these mutants are not necessary for flagellar motility. However, the double mutants ida1-3 x ida4 were nonmotile, suggesting that axonemes with significant defects in inner arms cannot function. The inner-arm dynein should be important for the generation of axonemal beating.

Microtubule translocation caused by three subspecies of inner-arm dynein from Chlamydomonas flagella

To help understand the function of inner-arm dynein in nagellar motility, dynein samples from an outer arm-missing mutant of Chlamydomonas (odal) were examined for the ability to translocate microtubules in vitro. High-salt extract ofaxonemes containing inner-arm dynein was separated by ion-exchange chromatography into 7 peak fractions with ATPase activities. Of these, three fractions containing different sets of dynein heavy chains translocated microtubules. The maximal velocities were all between 3 and 5 μm s , which were comparable to the microtubule sliding rate in disintegrating oda axonemes.

High-pressure liquid chromatography fractionation of Chlamydomonas dynein extracts and characterization of inner-arm dynein subunits

A rapid procedure for fractionating salt-stable dynein subunits from high-salt extracts of Chlamydomonas axonemes has been developed using a high-pressure liquid chromatography system with an anion exchange column and gradient salt elution. Five distinct fractions are shown to be highly enriched for five distinct subunits or subunit complexes by SDS/polyacrylamide gel electrophoresis, ATPase activity and electron microscopy. Peaks 1 and 4 contain, respectively, the single-headed γ-subunit and the two-headed α/β-hetero-polymer that form the outer arm in situ and are dissociated by salt exposure; both peaks are absent from the outer arm-less mutant pf-28. Peaks 2, 3 and 5 contain, respectively, two distinct single-headed species and a double-headed species that derive from inner arms; all three peaks are missing from the inner arm-less mutant pf-23. Sucrose-gradient sedimentation analysis confirms these assignments and provides additional information on the intermediate-chain and light-chain composition of the inner-arm species. Electron microscopy of the purified inner-arm species visualized by the quick-freeze deep-etch technique complements a previous analysis of outer-arm species. Each protein is shown to have a unique morphology, and both the inner- and outer-arm proteins clearly belong to a common family whose structural divergence presumably reflects functional specialization.