In Vitro Reconstitution of Dynein Force Exertion in a Bulk Viscous Medium.

The forces generated by microtubules (MTs) and their associated motors orchestrate essential cellular processes ranging from vesicular trafficking to centrosome positioning [1, 2]. To date, most studies have focused on MT force exertion by motors anchored to a static surface, such as the cell cortex in vivo or glass surfaces in vitro [2-4]. However, motors also transport large cargos and endomembrane networks, whose hydrodynamic interactions with the viscous cytoplasm should generate sizable forces in bulk. Such forces may contribute to MT aster centration, organization, and orientation [5-14] but have yet to be evidenced and studied in a minimal reconstituted system. By developing a bulk motility assay, based on stabilized MTs and dynein-coated beads freely floating in a viscous medium away from any surface, we demonstrate that the motion of a cargo exerts a pulling force on the MT and propels it in opposite direction. Quantification of resulting MT movements for different motors, motor velocities, over a range of cargo sizes and medium viscosities shows that the efficiency of this mechanism is primarily determined by cargo size and MT length. Forces exerted by cargos are additive, allowing us to recapitulate tug-of-war situations or bi-dimensional motions of minimal asters. These data also reveal unappreciated effects of the nature of viscous crowders and hydrodynamic interactions between cargos and MTs, likely relevant to understand this mode of force exertion in living cells. This study reinforces the notion that endomembrane transport can exert significant forces on MTs.

Purification and crystal structure of human ODA16: Implications for ciliary import of outer dynein arms by the intraflagellar transport machinery.

Motile cilia protrude from cell surfaces and are necessary to create movement of cells and fluids in the body. At the molecular level, cilia contain several dynein molecular motor complexes including outer dynein arms (ODAs) that are attached periodically to the ciliary axoneme, where they hydrolyse ATP to create the force required for bending and motility of the cilium. ODAs are preassembled in the cytoplasm and subsequently trafficked into the cilium by the intraflagellar transport (IFT) system. In the case of the green alga Chlamydomonas reinhardtii, the adaptor protein ODA16 binds to ODAs and directly to the IFT complex component IFT46 to facilitate the ciliary import of ODAs. Here, we purified recombinant human IFT46 and ODA16, determined the high-resolution crystal structure of the ODA16 protein, and carried out direct interaction studies of IFT46 and ODA16. The human ODA16 C-terminal 320 residues adopt the fold of an eight-bladed ß-propeller with high overall structural similarity to the Chlamydomonas ODA16. However, the small 80 residue N-terminal domain, which in Chlamydomonas ODA16 is located on top of the ß-propeller and is required to form the binding cleft for IFT46, has no visible electron density in case of the human ODA16 structure. Furthermore, size exclusion chromatography and pull-down experiments failed to detect a direct interaction between human ODA16 and IFT46. These data suggest that additional factors may be required for the ciliary import of ODAs in human cells with motile cilia.

Development of an expression system in Tetrahymena inner arm dyneins and motile properties of the single-headed subspecies (Dyh8p and Dyh12p).

Diverse inner arm dyneins cooperate with outer arm dyneins to produce ciliary beating. This study demonstrates an expression system for inner arm dyneins in Tetrahymena. The motor domain of inner arm dynein (Dyh8p or Dyh12p) was fused with the tail of outer arm dynein (Dyh3p) and expressed in viable DYH3-knockout (vKO-DYH3) cells. The chimeric dyneins were observed in the oral apparatus and cilia on the cell bodies, and did not change the swimming speed of vKO-DYH3 cells. In a gliding assay, the motor domains of Dyh8p and Dyh12p moved toward the minus ends of microtubules at 0.8 and 0.3 µm/s, respectively. The gliding velocities of Dyh8p and Dyh12p were decreased in 5 mM ATP but not increased in 0.1 or 0.5 mM ADP. This expression system will be useful for molecular studies on diverse inner arm dyneins.

Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots.

We develop magnetic cytoskeleton affinity (MiCA) purification, which allows for rapid isolation of molecular motors conjugated to large multivalent quantum dots, in miniscule quantities, which is especially useful for single-molecule applications. When purifying labeled molecular motors, an excess of fluorophores or labels is usually used. However, large labels tend to sediment during the centrifugation step of microtubule affinity purification, a traditionally powerful technique for motor purification. This is solved with MiCA, and purification time is cut from 2 h to 20 min, a significant time-savings when it needs to be done daily. For kinesin, MiCA works with as little as 0.6 µg protein, with yield of ∼27%, compared to 41% with traditional purification. We show the utility of MiCA purification in a force-gliding assay with kinesin, allowing, for the first time, simultaneous determination of whether the force from each motor in a multiple-motor system drives or hinders microtubule movement. Furthermore, we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD (DDB-QD), ordinarily a difficult protein-complex to purify.

Quantitative Proteomic Analysis of Human Airway Cilia Identifies Previously Uncharacterized Proteins of High Abundance.

Cilia are essential to many diverse cellular processes. Although many major axonemal components have been identified and studied, how they interact to form a functional axoneme is not completely understood. To further our understanding of the protein composition of human airway cilia, we performed a semiquantitative analysis of ciliary axonemes using label-free LC/MSE, which identified over 400 proteins with high confidence. Tubulins were the most abundant proteins identified, with evidence of 20 different isoforms obtained. Twelve different isoforms of axonemal dynein heavy chain were also identified. Absolute quantification of the nontubulin components demonstrated a greater than 75-fold range of protein abundance (RSPH9;1850 fmol vs CCDC103;24 fmol), adding another level of complexity to axonemal structure. Of the identified proteins, ∼70% are known axonemal proteins. In addition, many previously uncharacterized proteins were identified. Unexpectedly, several of these, including ERICH3, C1orf87, and CCDC181, were present at high relative abundance in the cilia. RT-PCR analysis and immunoblotting confirmed cilia-specific expression for eight uncharacterized proteins, and fluorescence microscopy demonstrated unique axonemal localizations. These studies have provided the first quantitative analysis of the ciliary proteome and have identified and characterized several previously unknown proteins as major constituents of human airway cilia.

Studying the Mechanochemistry of Processive Cytoskeletal Motors With an Optical Trap.

Cytoskeletal motors utilize the energy stored in ATP to generate linear motion along rigid filaments. Because their enzymatic cycles are tightly coupled to the production of force and forward movement, the optical-trapping technique is uniquely suited for studying their mechanochemical cycle. Here, we discuss the practical aspects of optical trapping in connection with single-motor assays and describe three distinct experimental modes (fixed-trap, force feedback, and square wave) that are typically used to investigate the enzymatic and biophysical properties of cytoskeletal motors. The principal outstanding questions in the field involve motor regulation by cargo adaptor proteins and cargo transport by teams of motors, ensuring that the optical trap's ability to apply precise forces and measure nanometer-scale displacements will remain crucial to the study of intracellular motility in the foreseeable future.

Optical Tweezers-Based Measurements of Forces and Dynamics at Microtubule Ends.

Microtubules are dynamic cytoskeletal polymers that polymerize and depolymerize while interacting with different proteins and structures within the cell. The highly regulated dynamic properties as well as the pushing and pulling forces generated by dynamic microtubule ends play important roles in processes such as in cell division. For instance, microtubule end-binding proteins are known to affect dramatically the dynamic properties of microtubules, and cortical dyneins are known to mediate pulling forces on microtubule ends. We discuss in this chapter our efforts to reconstitute these systems in vitro and mimic their interactions with structures within the cell using micro-fabricated barriers. Using an optical tweezers setup, we investigate the dynamics and forces of microtubules growing against functionalized barriers in the absence and presence of end-binding proteins and barrier-attached motor proteins. This setup allows high-speed as well as nanometer and piconewton resolution measurements on dynamic microtubules.

Interferometric Scattering Microscopy for the Study of Molecular Motors.

Our understanding of molecular motor function has been greatly improved by the development of imaging modalities, which enable real-time observation of their motion at the single-molecule level. Here, we describe the use of a new method, interferometric scattering microscopy, for the investigation of motor protein dynamics by attaching and tracking the motion of metallic nanoparticle labels as small as 20nm diameter. Using myosin-5, kinesin-1, and dynein as examples, we describe the basic assays, labeling strategies, and principles of data analysis. Our approach is relevant not only for motor protein dynamics but also provides a general tool for single-particle tracking with high spatiotemporal precision, which overcomes the limitations of single-molecule fluorescence methods.

Comparison of interscalene brachial plexus block and intra-articular local anesthetic administration on postoperative pain management in arthroscopic shoulder surgery / Comparação de bloqueio do plexo braquial por via interescalênica e administração de anestésico local intra-articular no manejo da dor no pós-operatório de cirurgia artroscópica do ombro / Comparación de bloqueo del plexo braquial por vía interescalénica y administración de anestésico local intraarticular en el manejo del dolor en el postoperatorio de cirugía artroscópica del hombro

BACKGROUND AND OBJECTIVES: In this study, the aim was to compare postoperative analgesia effects of the administration of ultrasound-guided interscalene brachial plexus block and intra-articular bupivacaine carried out with bupivacaine. METHODS: In the first group of patients 20 mL 0.25% bupivacaine and ultrasound-guided interscalene brachial plexus block (ISPB) were applied, while 20 mL 0.25% bupivacaine was given via intra-articular (IA) administration to the second group patients after surgery. Patients in the third group were considered the control group and no block was performed. Patient-controlled analgesia (PCA) with morphine was used in all three groups for postoperative analgesia. RESULTS: In the ISPB group, morphine consumption in the periods between 0-4, 6-12 and 12-24 postoperative hours and total consumption within 24 h was lower than in the other two groups. Morphine consumption in the IA group was lower than in the control group in the period from 0 to 6 h and the same was true for total morphine consumption in 24 h. Postoperative VASr scores in the ISPB group were lower than both of the other groups in the first 2 h and lower than the control group in the 4th and 6th hours (p < 0.05). In the IA group, VASr and VASm scores in the 2nd, 4th and 6th hours were lower than in the control group (p < 0.05). CONCLUSION: Interscalene brachial plexus block was found to be more effective than intra-articular local anesthetic injection for postoperative analgesia. .

JUSTIFICATIVA E OBJETIVOS: Comparar os efeitos na analgesia no pós-operatório da administração de bloqueio do plexo braquial por via interescalênica guiado por ultrassom e bupivacaína intra-articular, feito com bupivacaína. MÉTODOS: No primeiro grupo de pacientes, 20 mL de bupivacaína a 0,25% e bloqueio do plexo braquial por via interescalênica guiado por ultrassom (BPBI) foram administrados, enquanto 20 mL de bupivacaína a 0,25% foram administrados por via intra-articular (IA) ao segundo grupo de pacientes após a cirurgia. Os pacientes do terceiro grupo foram considerados grupo controle e nenhum bloqueio foi feito. Analgesia controlada pelo paciente (ACP) com morfina foi usada nos três grupos para analgesia pós-operatória. RESULTADOS: No grupo BPBI, o consumo de morfina nos períodos entre 0-4, 6-12 e 12-24 horas após a cirurgia e o consumo total em 24 horas foram mais baixos do que nos outros dois grupos. O consumo de morfina no grupo IA foi menor do que no grupo controle no período de 0-6 horas, como também foi menor o consumo total de morfina em 24 horas. Os escores EVAr no pós-operatório do grupo BPBI foram menores do que os escores dos dois outros grupos nas primeiras duas horas e menores do que os do grupo controle nos períodos de 4 e 6 horas (p < 0,05). No grupo IA, os escores EVAr e EVAm nos períodos de 2, 4 e 6 horas foram menores do que no grupo controle (p < 0,05). CONCLUSÃO: O bloqueio do plexo braquial por via interescalênica mostrou ser mais eficaz do que a injeção intra-articular de anestésico local para analgesia pós-operatória. .

JUSTIFICACIÓN Y OBJETIVOS: En este estudio, nuestro objetivo fue comparar en el período postoperatorio los efectos analgésicos de la administración de la bupivacaína en el bloqueo del plexo braquial por vía interescalénica guiado por ecografía y bupivacaína intraarticular. MÉTODOS: En el primer grupo de pacientes se administraron 20 mL de bupivacaína al 0,25% y se llevó a cabo el bloqueo del plexo braquial por vía interescalénica (BPBI) guiado por ecografía, mientras que al segundo grupo de pacientes se le administraron 20 mL de bupivacaína al 0,25% por vía intraarticular (IA) tras la cirugía. Los pacientes del tercer grupo fueron considerados como grupo control y en ellos no se realizó ningún bloqueo. La analgesia controlada por el paciente con morfina se usó en los 3 grupos para la analgesia postoperatoria. RESULTADOS: En el grupo BPBI, el consumo de morfina en los períodos entre 0-4, 6-12 y 12-24 h del postoperatorio y el consumo total en 24 h fueron más bajos que en los otros 2 grupos. El consumo de morfina en el grupo IA fue menor que en el grupo control en el período de 0-6 h, como también fue menor el consumo total de morfina en 24 h. Las puntuaciones EVAr en el postoperatorio del grupo BPBI fueron menores que las de los otros 2 grupos en las primeras 2 h y menores que los del grupo control en los períodos de 4 y 6 h (p < 0,05). En el grupo IA, las puntuaciones EVAr y EVAm en los períodos de 2, 4 y 6 h fueron menores que en el grupo control (p < 0,05). CONCLUSIÓN: El BPBI mostró ser más eficaz que la inyección intraarticular de anestésico local para analgesia postoperatoria. .

Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains.

Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end-directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase associated with various cellular activities) per motor domain (AAA1-4). The main site of ATP hydrolysis, AAA1, is the only site considered by most dynein motility models. However, it remains unclear how ATPase activity and MT binding are coordinated within and between dynein's motor domains. Using optical tweezers, we characterize the MT-binding strength of recombinant dynein monomers as a function of mechanical tension and nucleotide state. Dynein responds anisotropically to tension, binding tighter to MTs when pulled toward the MT plus end. We provide evidence that this behavior results from an asymmetrical bond that acts as a slip bond under forward tension and a slip-ideal bond under backward tension. ATP weakens MT binding and reduces bond strength anisotropy, and unexpectedly, so does ADP. Using nucleotide binding and hydrolysis mutants, we show that, although ATP exerts its effects via binding AAA1, ADP effects are mediated by AAA3. Finally, we demonstrate "gating" of AAA1 function by AAA3. When tension is absent or applied via dynein's C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state. However, when tension is applied to the linker, ATP binding to AAA3 is sufficient to "open" the gate. These results elucidate the mechanisms of dynein-MT interactions, identify regulatory roles for AAA3, and help define the interplay between mechanical tension and nucleotide state in regulating dynein motility.

Reconstitution of dynein transport to the microtubule plus end by kinesin.

Cytoplasmic dynein powers intracellular movement of cargo toward the microtubule minus end. The first step in a variety of dynein transport events is the targeting of dynein to the dynamic microtubule plus end, but the molecular mechanism underlying this spatial regulation is not understood. Here, we reconstitute dynein plus-end transport using purified proteins from S. cerevisiae and dissect the mechanism using single-molecule microscopy. We find that two proteins-homologs of Lis1 and Clip170-are sufficient to couple dynein to Kip2, a plus-end-directed kinesin. Dynein is transported to the plus end by Kip2, but is not a passive passenger, resisting its own plus-end-directed motion. Two microtubule-associated proteins, homologs of Clip170 and EB1, act as processivity factors for Kip2, helping it overcome dynein's intrinsic minus-end-directed motility. This reveals how a minimal system of proteins transports a molecular motor to the start of its track.DOI: http://dx.doi.org/10.7554/eLife.02641.001.

WD60/FAP163 is a dynein intermediate chain required for retrograde intraflagellar transport in cilia.

Retrograde intraflagellar transport (IFT) is required for assembly of cilia. We identify a Chlamydomonas flagellar protein (flagellar-associated protein 163 [FAP163]) as being closely related to the D1bIC(FAP133) intermediate chain (IC) of the dynein that powers this movement. Biochemical analysis revealed that FAP163 is present in the flagellar matrix and is actively trafficked by IFT. Furthermore, FAP163 copurified with D1bIC(FAP133) and the LC8 dynein light chain, indicating that it is an integral component of the retrograde IFT dynein. To assess the functional role of FAP163, we generated an RNA interference knockdown of the orthologous protein (WD60) in planaria. The Smed-wd60(RNAi) animals had a severe ciliary assembly defect that dramatically compromised whole-organism motility. Most cilia were present as short stubs that had accumulated large quantities of IFT particle-like material between the doublet microtubules and the membrane. The few remaining approximately full-length cilia had a chaotic beat with a frequency reduced from 24 to ∼10 Hz. Thus WD60/FAP163 is a dynein IC that is absolutely required for retrograde IFT and ciliary assembly.

Protein-protein interactions between intermediate chains and the docking complex of Chlamydomonas flagellar outer arm dynein.

Outer arm dynein (OAD) is bound to specific loci on outer-doublet-microtubules by interactions at two sites: via intermediate chain 1 (IC1) and the outer dynein arm docking complex (ODA-DC). Studies using Chlamydomonas mutants have suggested that the individual sites have rather weak affinities for microtubules, and therefore strong OAD attachment to microtubules is achieved by their cooperation. To test this idea, we examined interactions between IC1, IC2 (another intermediate chain) and ODA-DC using recombinant proteins. Recombinant IC1 and IC2 were found to form a 1:1 complex, and this complex associated with ODA-DC in vitro. Binding of IC1 to mutant axonemes revealed that there are specific binding sites for IC1. From these data, we propose a novel model of OAD-outer doublet association.

The role of retrograde intraflagellar transport in flagellar assembly, maintenance, and function.

The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34°C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34°C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited.

Purification of Tetrahymena cytoskeletal proteins.

Like all eukaryotic cells, Tetrahymena thermophila contains a rich array of cytoskeletal proteins, some familiar and some novel. A detailed analysis of the structure, function, and interactions of these proteins requires procedures for purifying the individual protein components. Procedures for the purification of actin and tubulin from Tetrahymena are reviewed, followed by a description of a procedure that yields proteins from the epiplasmic layer and associated structures, including the tetrins. Finally, the challenges and opportunities for future advances are assessed.

Molecular cloning of the Robl gene from Bombyx mori and studies of its developmental and physicochemical regulation.

Dynein light chains function as motor acceptor to recruit cargos, which play vital roles in many cellular processes such as intracellular transport and mitosis. In this study, we cloned and expressed the dynein light chain LC7 gene BmRobl in silkworm. The full-length cDNA of the dynein light chain LC7 gene BmRobl is 757 bp and encoded 97 aa polypeptide. Its molecular weight was ~11 kDa confirmed by western blotting. The tissue and stage expression profile of BmRobl drafted by real time PCR revealed that presence of BmRobl transcript was examined in all tissue but prominent expression level was found in brain, wing disc, ovary and testis. In metamorphosis wing disc, BmRobl reached to peak during the prepupae stage compared with the larval and pupal stages. This indicated BmRobl might involve in wing discs development during metamorphosis. Besides, in vitro wing discs 20E cultivation was performed and BmRobl expression profile was detected. The results demonstrated that the BmRobl gene was significantly up-regulated with increase of 20E concentration; the mRNA level peaked at 2 µg/ml of 20E. However, the BmRobl expression nearly has no change cultivated by 20 µg/ml 20E compared with 0.02 µg/ml 20E. These indicated that BmRobl expression might directly or indirectly induced by 20E, besides, high concentration 20E was far too inducible, suggesting that low concentrations of ecdysteroid induce cell proliferation, whereas high concentrations inhibit cell proliferation. Moreover, the transport role of BmRobl was clarified by UV challenge and vanadate cultivation. Both the real time PCR and western blotting results showed that the BmRobl gene was degraded with increase in the concentration of sodium vanadate combined with elongation in the time of UV challenge. Interestingly, compared with the single treatment group and non-treatment group, the group treated by both sodium vanadate and UV have severe degradation. This indicated that UV and vanadate might down-regulate BmRobl synergetically. It was further speculated that BmRobl may function as a positive regulator of the dynein complex during cellular transport.

Three outer arm dynein heavy chains of Chlamydomonas reinhardtii operate in a coordinated fashion both in vitro and in vivo.

Outer arm dynein (OAD) in cilia and flagella contains two to three nonidentical heavy chains (HCs) that possess motor activity. In Chlamydomonas, flagellar OAD contains three HCs, alpha-, beta-, and gamma-HCs, each appearing to have a distinct role. To determine the precise molecular mechanism of their function, cross-sectional electron micrographs of wild-type and single HC-disruption mutants were compared and statistically analyzed. While the alpha-HC mutant displayed an OAD of lower density, which was attributed to a lack of alpha-HC, the OAD of beta- and gamma-HC mutants not only lacked the corresponding HC, but was also significantly affected in its structure, particularly with respect to the localization of alpha-HC. The lack of beta-HC induced mislocalization of alpha-HC, while a disruption of the gamma-HC gene resulted in the synchronized movement of alpha-HC and beta-HC in the manners for stacking. Interestingly, using cryo-electron microscopy, purified OADs were typically observed consisting of two stacked heads and an independent single head, which presumably corresponded to gamma-HC. This conformation is different from previous reports in which the three HCs displayed a stacked form in flagella observed by cryo-electron tomography and a bouquet structure on mica in deep-etch replica images. These results suggest that gamma-HC supports the tight stacking arrangement of inter or intra alpha-/beta-HC to facilitate the proper functioning of OAD.

The tail binds to the head-neck domain, inhibiting ATPase activity of myosin VIIA.

Myosin VIIA is an unconventional myosin, responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Here, we studied the molecular mechanism of regulation of myosin VIIA, which is currently unknown. Although it was originally thought that myosin VIIA is a dimeric myosin, our electron microscopic (EM) observations revealed that full-length Drosophila myosin VIIA (DM7A) is a monomer. Interestingly, the tail domain markedly inhibits the actin-activated ATPase activity of tailless DM7A at low Ca(2+) but not high Ca(2+). By examining various deletion constructs, we found that deletion of the distal IQ domain, the C-terminal region of the tail, and the N-terminal region of the tail abolishes the tail-induced inhibition of ATPase activity. Single-particle EM analysis of full-length DM7A at low Ca(2+) suggests that the tail folds back on to the head, where it contacts both the motor core domain and the neck domain, forming an inhibited conformation. We concluded that unconventional myosin that may be present a monomer in the cell can be regulated by intramolecular interaction of the tail with the head.

Purification of dyneins from sperm flagella.

Metazoan spermatozoa, especially those from marine invertebrates and fish, are excellent sources for isolating axonemal dyneins because of their cellular homogeneity and the large amounts that can be collected. Sperm flagella can be easily isolated by homogenization and subsequent centrifugation. Axonemes are obtained by demembranation of flagella with the nonionic detergent Triton X-100. The outer arm dyneins have been most widely studied because they are specifically extracted by a high-salt solution and can be isolated as a relatively pure fraction of ~20S two-headed dynein by sucrose density gradient centrifugation. Only a few reports have described the isolation of inner arm dyneins from sperm and the protocol has room for improvement. Sperm show clear changes in motility at fertilization, which are exerted through the regulation of axonemal dyneins by protein phosphorylation and Ca(2+) binding. Therefore dyneins from sperm flagella are an excellent biochemically tractable source for studying the regulation of axonemal dyneins. Here we describe protocols used for purification of flagellar dyneins from sperm of tunicates, sea urchins, and fish. The techniques described here could be applied to other species with appropriate modifications.

Analysis of redox-sensitive dynein components.

Redox-based regulation plays important roles in many cellular activities. Thioredoxins, one of the best characterized class of proteins involved in cellular redox regulation, are conserved components of eukaryotic ciliary/flagellar axonemal dyneins. Studies with Chlamydomonas showed that, under varying redox conditions, dynein-associated thioredoxins interact with different proteins through disulfide bonds and, as a consequence, flagella change their manner of beating. This chapter provides an overview of techniques for estimating and modulating the redox state of axonemal proteins, as well as for searching for redox-regulated proteins in the axoneme.