Dynein ATPase Research Articles

Arresting calcium-regulated sperm metabolic dynamics enables prolonged fertility in poultry liquid semen storage

The preservation of liquid semen is pivotal for both industrial livestock production and genetic management/conservation of species with sperm that are not highly cryo-tolerant. Nevertheless, with regard to poultry semen, even brief in vitro storage periods can lead to a notable decline in fertility, despite the in vivo capacity to maintain fertility for several weeks when within the hen’s sperm storage tubules. For fertility in sperm, intracellular calcium ions ([Ca2+]i) play a key role in signaling towards modifying energy metabolism. While reducing [Ca2+]i has been found to enhance the preservation of sperm fertility in some mammals, the connection between semen fertility and calcium availability in avian sperm has received limited attention. In this study, we demonstrate that the use of extracellular and intracellular calcium chelators in liquid semen extenders, specifically EGTA and EGTA-AM, has distinct effects on prolonging the fertility of chicken sperm. These results were validated through in vivo fertility tests. Mechanistically, the effects observed were linked to coordination of mitochondrial metabolism and ATP catabolism. Despite both calcium chelators inducing hypoxia, they differentially regulated mitochondrial respiration and ATP accumulation. This regulation was closely linked to a bimodal control of dynein ATPase activity; a direct initial activation with reduction in [Ca2+]i, and subsequent suppression by cytoplasmic acidification caused by lactic acid. These findings not only contribute to advancing poultry liquid semen preservation techniques, but also elucidates biologically relevant mechanisms that may underlie storage within the female reproductive tract in birds.

Microtubule acetylation induced by oxidative stress regulates subcellular distribution of lysosomal vesicles for amyloid-beta secretion.

Excessive production and accumulation of amyloid-beta (Aβ) in the brain are one of the hallmarks of Alzheimer's disease (AD). Although oxidative stress is known to trigger and promote the progression of AD, the molecular relationship between oxidative stress and Aβ production is not yet fully understood. In this study, we demonstrate that microtubule acetylation induced by oxidative stress plays a critical role in Aβ production and secretion by altering the subcellular distribution of Aβ precursor protein (APP)-containing lysosomal vesicles. Under oxidative stress, both H4-APPSwe/Ind and HEK293T-APPSwe/Ind cell lines showed increased microtubule acetylation and Aβ secretion. Knockdown (KD) of alpha-tubulin N-acetyltransferase 1 (ATAT1) by using a lentiviral shRNA not only inhibited the generation of intermediate APP fragments, such as β-CTF and AICD, but also suppressed Aβ secretion. Oxidative stress promoted the dispersion of LAMP1-positive vesicles to the periphery of the cell through microtubule acetylation, leading to the formation of neutralized lysosomal vesicles (NLVs), which was inhibited by ATAT1 KD. Treatment of the cells with the dynein ATPase inhibitor EHNA or downregulation of LIS1, a regulator of dynein-mediated intracellular transport, increased the peripheral localization of NLVs and promoted Aβ secretion, whereas KD of ADP ribosylation factor like GTPase 8B showed the opposite result. ATAT1 KD in the hippocampal region of the 5×FAD AD mouse model also showed significant reductions in Aβ plaque accumulation and memory loss. Taken together, these findings suggest that oxidative stress-induced microtubule acetylation promotes the peripheral localization of lysosomal vesicles to form NLVs, thereby enhancing Aβ secretion.

The cilia and flagella associated protein CFAP52 orchestrated with CFAP45 is required for sperm motility in mice

Asthenozoospermia characterized by decreased sperm motility is a major cause of male infertility, but the majority of the etiology remains unknown. Here, we showed that the cilia and flagella associated protein 52 (Cfap52) gene was predominantly expressed in testis and its deletion in a Cfap52 knockout mouse model resulted in decreased sperm motility and male infertility. Cfap52 knockout also led to the disorganization of the midpiece-principal piece junction of the sperm tail but had no effect on the axoneme ultrastructure in spermatozoa. Furthermore, we found that CFAP52 interacted with the cilia and flagella associated protein 45 (CFAP45) and knockout of Cfap52 decreased the expression level of CFAP45 in sperm flagellum, which further disrupted the microtubule sliding produced by dynein ATPase. Together, our studies demonstrate that CFAP52 plays an essential role in sperm motility by interacting with CFAP45 in sperm flagellum, providing insights into the potential pathogenesis of the infertility of the human CFAP52 mutations.

Peter Satir (1936–2022), cell biology pioneer and mentor

Peter Satir (1936–2022), cell biology pioneer and mentor

Ocean acidification impacts sperm swimming performance and pHi in the New Zealand sea urchin Evechinus chloroticus.

In sea urchins, spermatozoa are stored in the gonads in hypercapnic conditions (pH<7.0). During spawning, sperm are diluted in seawater of pH>8.0, and there is an alkalinization of the sperm's internal pH (pHi) through the release of CO2 and H+. Previous research has shown that when pHi is above 7.2-7.3, the dynein ATPase flagellar motors are activated, and the sperm become motile. It has been hypothesized that ocean acidification (OA), which decreases the pH of seawater, may have a narcotic effect on sea urchin sperm by impairing the ability to regulate pHi, resulting in decreased motility and swimming speed. Here, we used data collected from the same individuals to test the relationship between pHi and sperm motility/performance in the New Zealand sea urchin Evechinus chloroticus under near-future (2100) and far-future (2150) atmospheric PCO2conditions (RCP 8.5: pH 7.77, 7.51). Decreasing seawater pH significantly negatively impacted the proportion of motile sperm, and four of the six computer-assisted sperm analysis (CASA) sperm performance measures. In control conditions, sperm had an activated pHi of 7.52. Evechinus chloroticus sperm could not defend pHi in future OA conditions; there was a stepped decrease in the pHi at pH 7.77, with no significant difference in mean pHi between pH 7.77 and 7.51. Paired measurements in the same males showed a positive relationship between pHi and sperm motility, but with a significant difference in the response between males. Differences in motility and sperm performance in OA conditions may impact fertilization success in a future ocean.

Swarming Aqua Sperm Micromotors for Active Bacterial Biofilms Removal in Confined Spaces.

Microscale self‐propelled robots show great promise in the biomedical field and are the focus of many researchers. These tiny devices, which move and navigate by themselves, are typically based on inorganic microstructures that are not biodegradable and potentially toxic, often using toxic fuels or elaborate external energy sources, which limits their real‐world applications. One potential solution to these issues is to go back to nature. Here, the authors use high‐speed Aqua Sperm micromotors obtained from North African catfish (Clarias gariepinus, B. 1822) to destroy bacterial biofilm. These Aqua Sperm micromotors use water‐induced dynein ATPase catalyzed adenosine triphosphate (ATP) degradation as biocompatible fuel to trigger their fast speed and snake‐like undulatory locomotion that facilitate biofilm destruction in less than one minute. This efficient biofilm destruction is due to the ultra‐fast velocity as well as the head size of Aqua Sperm micromotors being similar to bacteria, which facilitates their entry to and navigation within the biofilm matrix. In addition, the authors demonstrate the real‐world application of Aqua Sperm micromotors by destroying biofilms that had colonized medical and laboratory tubing. The implemented system extends the biomedical application of Aqua Sperm micromotors to include hybrid robots for fertilization or cargo tasks.

CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45−/− mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

Sperm dynein ATPase and ATPases associated with various cellular activities (AAA+): regulation in oligo-astheno-teratozoospermia and necrozoospermia as extreme sperm motility disorders

Asthenozoospermia is the most frequent sperm motility disorder, but there are other more extreme sperm motility disorders, namely oligo-astheno-teratozoospermia (OAT) and necrozoospermia. There are several cellular mechanisms known for OAT and necrozoospermia, but there are limited data on dynein ATPase and ATPases associated with various cellular activities (AAA+). AAA1 is involved in ATP hydrolysis, while AAA2 is entangled in ATP-binding pocket. This study was conducted to investigate the role of dynein ATPase activity and quantification of AAA dynein. Spermatozoa from 14 men with OAT, 11 men with necrozoospermic and 17 men with normoozspermic samples were used in this study. Makler chamber was used to determine sperm concentration and motility, while Papanicolaou stained semen smears using World Health Organization-fifth edition criteria was performed to determine sperm morphology, and dynein ATPase was quantified by calculation of released inorganic phosphate. AAA was quantified by enzyme-linked immunosorbent assay, whereas the distribution was determined by immunocytochemistry. This study showed that the dynein ATPase activity in OAT and necrozoospermia was lower than in the normozoospermic group (2.68±0.76, 1.01±0.31, 7.22±1.08 μmol Pi/mg protein/h, respectively, P&lt;0.05), as well as the amounts of AAA1 and AAA2. In addition, staining for AAA in the sperm tail paralleled the dynein ATPase activity and quantity of AAA, being the highest in sperm from normozoospermic samples, lower in sperm from OAT samples, and almost undetectable in sperm from necrozoospermic samples. The structure and function of damaged sperm dynein may alter dynein ATPase activity and levels of AAA1 and AAA2.

Spermatozoa motility in bivalves: Signaling, flagellar beating behavior, and energetics

Though bivalve mollusks are keystone species and major species groups in aquaculture production worldwide, gamete biology is still largely unknown. This review aims to provide a synthesis of current knowledge in the field of sperm biology, including spermatozoa motility, flagellar beating, and energy metabolism; and to illustrate cellular signaling controlling spermatozoa motility initiation in bivalves. Serotonin (5-HT) induces hyper-motility in spermatozoa via a 5-HT receptor, suggesting a serotoninergic system in the male reproductive tract that might regulate sperm physiology. Acidic pH and high concentration of K+ are inhibitory factors of spermatozoa motility in the testis. Motility is initiated at spawning by a Na+-dependent alkalization of intracellular pH mediated by a Na+/H+ exchanger. Increase of 5-HT in the testis and decrease of extracellular K+ when sperm is released in seawater induce hyperpolarization of spermatozoa membrane potential mediated by K+ efflux and associated with an increase in intracellular Ca2+ via opening of voltage-dependent Ca2+ channels under alkaline conditions. These events activate dynein ATPases and Ca2+/calmodulin-dependent proteins resulting in flagellar beating. It may be possible that 5-HT is also involved in intracellular cAMP rise controlling cAMP-dependent protein kinase phosphorylation in the flagellum. Once motility is triggered, flagellum beats in asymmetric wave pattern leading to circular trajectories of spermatozoa. Three different flagellar wave characteristics are reported, including “full”, “twitching”, and “declining” propagation of wave, which are described and illustrated in the present review. Mitochondrial respiration, ATP content, and metabolic pathways producing ATP in bivalve spermatozoa are discussed. Energy metabolism of Pacific oyster spermatozoa differs from previously studied marine species since oxidative phosphorylation synthetizes a stable level of ATP throughout 24-h motility period and the end of movement is not explained by a low intracellular ATP content, revealing different strategy to improve oocyte fertilization success. Finally, our review highlights physiological mechanisms that require further researches and points out some advantages of bivalve spermatozoa to extend knowledge on mechanisms of motility.

Effect of inhibition of axonemal dynein ATPases on the regulation of flagellar and ciliary waveforms in Leishmania parasites

Trypanosomes of the genus Leishmania swim by undulating motions of a single flagellum driven by axonemal dynein ATPases, essential for parasite survival and infectivity. The flagellum possesses two waveforms; flagellar (tip-to-base) responsible for forward movements and ciliary (base-to-tip) possibly responsible for reorientation in response to changes in surroundings. However, the role of dyneins in regulating the two waveforms remains unknown. Moreover, the unpredictable nature of the parasite ciliary waveform makes it difficult to study. We have previously reported a detergent-extracted, ATP-reactivated model ideal for investigating flagellar motility regulation in Leishmania that allows one to generate reactivated Leishmania flagella with constitutively beating ciliary waves in presence of cyclic-AMP. Here, using three dynein inhibitors [erythro-9-(2-hydroxy-3-nonyl) adenine, ciliobrevin A and vanadate] we investigated the role of dyneins in regulating the two waveforms of Leishmania. Using high speed videomicroscopy we observed differential inhibition of beat frequencies and waveforms of flagellar and ciliary beats in live (in vivo) and ATP-reactivated (in vitro) parasites. Beat frequency of flagellar waveform was more strongly reduced than ciliary waveform. Surprisingly, inhibition of the ciliary waveform led to an altered phenotype with the distal half of the flagellum paralysed. ATPase assays confirmed that dynein activity of flagellar cells was more strongly inhibited compared to ciliary cells irrespective of the mechanism of inhibition. Possibly the two different waveforms are an outcome of changes in the mechanical properties of axonemal dyneins present at the tip of the flagellum that contains a sliding resistive structure. Our study suggests that dyneins responsible for the two waveforms in Leishmania bear different structural and functional conformations. Moreover, during ciliary beating, there is heterogeneity along the flagellum.

150 Runs of Homozygosity Analysis and their Possible Influence on Sperm Motility in Highly Consanguineous Bulls

Inbreeding depression is associated with emergence of deleterious effects and loss of genetic variability. Widespread use of genotyping technologies and new approaches for identification of runs-of-homozygosity (ROH) provide valuable tools to better understand the effects of inbreeding depression. We have previously demonstrated that inbreeding affects sperm motility patterns in cattle (Dorado et al. 2016 Reprod. Fertil. Dev. 29, 712-720; 10.1071/RD15324), with an increase in individuals presenting a hyperactivated-like motility. In this study, we characterised ROH patterns and performed gene ontology analysis of a large, highly consanguineous cattle population. Thirty-three Retinta bulls [average inbreeding percentage FPED = 16.57% (10.25 to 30.62%)] were genotyped using the Axiom® BOS 1 High-Density SNP Array (Thermo Fisher Scientific, Waltham, MA, USA). The ROH were estimated using CGATOH package and classified upon their length into 5 categories: 1-2, 2-4, 4-8, 8-16, and &gt;16 Mb, which are inversely related to inbreeding events occurring 50 to 3 generations before, respectively. The ROH showed an average length of 3.73 Mb (1.48 to 6.71 Mb). Total FROH was partially explained by the increase in ROH fragments longer than 8 Mb, which is consistent with recent inbreeding events that occurred in this population in the last 6 generations. Additionally, the distribution of ROH varied notably between chromosomes. For instance, &gt;16 Mb runs (very recent inbreeding event) were absent in BTA26 and BTA29, whereas certain loci on BTA7, BTA13, and BTA24 showed &gt;16 Mb runs in 9 animals. To identify candidate biological functions affected by inbreeding, we performed functional analysis of the genome areas covered by ROH &gt;8 Mb (our pedigree data covered 5.87 equivalent complete generations) using the Functional Annotation Clustering tool implemented in DAVID. Candidate regions were defined by occurring in ROH &gt;8 in more than 6 animals and by a distance &lt;1 Mb between adjacent single nucleotide polymorphisms. Eight significant gene clusters (enrichment score &gt;1.30; P &lt; 0.05) were identified, with 2 of these clusters related to sperm motility. One of these clusters (score 1.72) contained 23 genes coding microtubule-related proteins, which are associated with cellular movement structures such as flagella. The other cluster (score 1.42), included 4 genes related to dynein and motile cilium assembly ATPase complexes, associated with movement of eukaryotic flagella. We observed an additional cluster representing an ATP-binding feature, which included 77 genes (score 1.43). These results indicate that inbreeding could affect sperm motility by altering microtubule structure and motility. However, the fact that ROH were unevenly distributed across the genome, even in a highly inbred cattle population, also suggests that different metabolic pathways could be affected in individuals with similar inbreeding values. Therefore, the use of inbreeding coefficients as predictors for sperm quality should be approached carefully.

Characterization of ciliobrevin A mediated dynein ATPase inhibition on flagellar motility of Leishmania donovani

Axonemal dyneins are members of AAA+ proteins involved in force generation and are responsible for flagellar motility in eukaryotes. In this study, we characterized the effects of ciliobrevin A (CbA), a dynein ATPase inhibitor, on flagella driven motility of the protozoan parasite Leishmania donovani. Using fast–capture video microscopy, we observed that CbA decreased flagellar beat frequency of swimming parasites in a concentration-dependent manner. Beat frequency of live and reactivated L. donovani decreased by approximately 89% and 41% respectively in the presence of 250μM CbA. This inhibition was lost when CbA was removed, suggesting its effects were reversible. CbA also altered wavelength and amplitude of the flagellum of live parasites. Waveform analysis of live and reactivated L. donovani revealed that CbA significantly affected flagellar waveform by inducing non-uniform bends with the flagellum beating away from the cell axis. These results suggest that CbA sensitive dynein ATPases possibly are responsible for power generation and waveform maintenance of the flagellum of L. donovani. This ability to inhibit axonemal dyneins also emphasizes the use of dynein inhibitors as valuable tools in studying functional roles of axonemal dyneins of flagellated eukaryotes.

Effects of a Protein Kinase Inhibitor on Sperm Motility in the Japanese Quail.

Sperm motility is an essential trait for successful fertilization in animals. In birds, ejaculated sperm migrate into sperm storage tubules before fertilization and are stored in a quiescent state. We previously reported that this type of sperm's flagellar quiescence was induced by lactic acid through flagellar dynein ATPase inactivation following cytoplasmic acidification (<pH 6.0). However, signal transduction in the sperm cells leading to motility inactivation is not well understood. The aim of the present study was to investigate the role of protein kinases in putative signal transduction in quail spermatozoa motility in vitro. Following incubation with bisindolylmaleimide II (BisII), a potent-competitive protein kinase C (PKC) inhibitor, sperm motility decreased in a dose related-manner. However, no such inhibitory effect was found in sperm exposed to bisindolylmaleimide V, H-89, or LY294002, a weak inhibitor of PKC, a potent inhibitor of protein kinase A (PKA) and a selective inhibitor of phosphatidylinositol 3-kinase, respectively. BisII-treated sperm exhibited no significant differences in pHi, [Ca2+]i, mitochondrial activity, intracellular cAMP or ATP concentration, as well as dynein ATPase activity, compared to the control sperm. However, when the phosphorylated substrate proteins by PKC were detected by Western blot analysis, the intensity of the band in sperm incubated in the presence of BisII decreased. Moreover, immunoreactive PKCι and µ isoforms in the sperm lysates were also detected. These results indicated that the PKC signaling pathway may be involved in sperm motility regulation, and protein phosphorylation by PKC may be required to maintain flagellar movement in the Japanese quail.

Axoneme Structure from Motile Cilia.

The axoneme is the main extracellular part of cilia and flagella in eukaryotes. It consists of a microtubule cytoskeleton, which normally comprises nine doublets. In motile cilia, dynein ATPase motor proteins generate sliding motions between adjacent microtubules, which are integrated into a well-orchestrated beating or rotational motion. In primary cilia, there are a number of sensory proteins functioning on membranes surrounding the axoneme. In both cases, as the study of proteomics has elucidated, hundreds of proteins exist in this compartmentalized biomolecular system. In this article, we review the recent progress of structural studies of the axoneme and its components using electron microscopy and X-ray crystallography, mainly focusing on motile cilia. Structural biology presents snapshots (but not live imaging) of dynamic structural change and gives insights into the force generation mechanism of dynein, ciliary bending mechanism, ciliogenesis, and evolution of the axoneme.

Lactic acid is a sperm motility inactivation factor in the sperm storage tubules.

Although successful fertilization depends on timely encounters between sperm and egg, the decoupling of mating and fertilization often confers reproductive advantages to internally fertilizing animals. In several vertebrate groups, postcopulatory sperm viability is prolonged by storage in specialized organs within the female reproductive tract. In birds, ejaculated sperm can be stored in a quiescent state within oviductal sperm storage tubules (SSTs), thereby retaining fertilizability for up to 15 weeks at body temperature (41 °C); however, the mechanism by which motile sperm become quiescent within SSTs is unknown. Here, we show that low oxygen and high lactic acid concentrations are established in quail SSTs. Flagellar quiescence was induced by lactic acid in the concentration range found in SSTs through flagellar dynein ATPase inactivation following cytoplasmic acidification (<pH 6.0). The long-term preservation of sperm morphology under hypoxic and high temperature conditions indicates that a combination of these factors enables sperm cells to survive during the ovulation cycles. Our findings suggested a novel physiological role for lactic acid in promoting sperm quiescence in SSTs and opened up a new opportunity for technological improvement in prolonging sperm longevity at ambient or body temperature.

Enzyme activity in energy supply of spermatozoon motility in two taxonomically distant fish species (sterlet Acipenser ruthenus, Acipenseriformes and common carp Cyprinus carpio, Cypriniformes)

As spermatozoon motility duration differs significantly among fish species, the mechanism of ATP generation-regeneration and its distribution along the flagellum may be species-dependent. The present study compared the role of creatine kinase (CK) with that of adenylate kinase (AK) in ATP regeneration during motility of demembranated spermatozoa of taxonomically distant fish species, sterlet, and common carp, allowing investigation for the presence of the creatine-phosphocreatine (PCr) shuttle in sterlet spermatozoa. The flagellar beat frequency of demembranated spermatozoa was measured in reactivating media in the presence or absence of ATP, ADP, PCr, and CK and AK inhibitors. After demembranation, AK, CK, and total ATPase activity was measured in spermatozoon extracts. Beat frequency of demembranated spermatozoa was found to be positively correlated with ATP levels in reactivating medium and to reach a plateau at 0.8 mM and 0.6 mM ATP for carp and sterlet, respectively. It was shown for the first time that sterlet axonemal dynein ATPases have a higher affinity for ATP than do those of carp. Supplementation of reactivating medium with ADP and PCr without ATP resulted in beat frequencies comparable to that measured with 0.3 to 0.5-mM ATP for both studied species. The presence of the PCr-CK phosphagen system and its essential role in ATP regeneration were first confirmed for sturgeon spermatozoa. The inhibition of CK exerted a high impact on spermatozoon energy supply in both species, whereas the inhibition of AK was more pronounced in sterlet than in carp. This was confirmed by the quantification of enzyme activity in spermatozoon extracts. We concluded that spermatozoa of these taxonomically distant species use similar systems to supply energy for flagella motility, but with different efficacy.

A flipped ion pair at the dynein-microtubule interface is critical for dynein motility and ATPase activation.

Dynein is a motor protein that moves on microtubules (MTs) using the energy of adenosine triphosphate (ATP) hydrolysis. To understand its motility mechanism, it is crucial to know how the signal of MT binding is transmitted to the ATPase domain to enhance ATP hydrolysis. However, the molecular basis of signal transmission at the dynein-MT interface remains unclear. Scanning mutagenesis of tubulin identified two residues in α-tubulin, R403 and E416, that are critical for ATPase activation and directional movement of dynein. Electron cryomicroscopy and biochemical analyses revealed that these residues form salt bridges with the residues in the dynein MT-binding domain (MTBD) that work in concert to induce registry change in the stalk coiled coil and activate the ATPase. The R403-E3390 salt bridge functions as a switch for this mechanism because of its reversed charge relative to other residues at the interface. This study unveils the structural basis for coupling between MT binding and ATPase activation and implicates the MTBD in the control of directional movement.

A Mechanical Switch from Diffusion to Directional Motion Activates ATPase in Dynein Motor

Dynein is a motor protein that moves along microtubule tracks via the energy from ATP hydrolysis. Unlike other processive cytoskeletal motors, the dynein step size is highly variable with a significant level of diffusion. To investigate the molecular basis of the stochastic nature of dynein stepping, we here characterized the structure, physical properties, and effects of site-directed mutations of the dynein-microtubule interface.We found that mutation of either the R403 or E416 residue of α-tubulin to alanine changed the directional movement of the microtubules on a dynein-coated surface to undirected thermal diffusion, resulting in a loss of dynein ATPase activity. Biochemical and cryo-electron microscopy analyses of the microtubule binding domain (MTBD)-microtubule complex revealed that these tubulin residues switch dynein from diffusional to stationary binding by forming salt bridges with the residue in H1 and H6 of the MTBD. The formation of two salt bridges then triggers a registry change in the stalk coiled coil required for ATPase activation, and thus leads to directional movement. In this mechanism, the previously undescribed interaction between α-R403 and E3390 in H1 of the MTBD plays a key role, and is likely to explain the fact that the equivalent tubulin mutation in mammals (R402) can cause lissencephaly (Keays et al., Cell 128, 45-).Compared to kinesin-microtubule interactions, where the weak-to-strong state transition is mediated by several contact sites involving a few tubulin residues (Uchimura et al., EMBO. J., 29, 1167-), for dynein, the mechanical switch from diffusional to stationary binding is controlled by only two salt bridges. Because of this pinpoint regulation, the stepping motion of dynein might be only loosely coupled with the reaction of ATP hydrolysis, resulting in the variable step size.

Pigment granule translocation in red ovarian chromatophores from the palaemonid shrimp Macrobrachium olfersi (Weigmann, 1836): Functional roles for the cytoskeleton and its molecular motors

The binding of red pigment concentrating hormone (RPCH) to membrane receptors in crustacean chromatophores triggers Ca2+/cGMP signaling cascades that activate cytoskeletal motors, driving pigment granule translocation. We investigate the distributions of microfilaments and microtubules and their associated molecular motors, myosin and dynein, by confocal and transmission electron microscopy, evaluating a functional role for the cytoskeleton in pigment translocation using inhibitors of polymer turnover and motor activity in vitro. Microtubules occupy the chromatophore cell extensions whether the pigment granules are aggregated or dispersed. The inhibition of microtubule turnover by taxol induces pigment aggregation and inhibits re-dispersion. Phalloidin-FITC actin labeling, together with tannic acid fixation and ultrastructural analysis, reveals that microfilaments form networks associated with the pigment granules. Actin polymerization induced by jasplaquinolide strongly inhibits RPCH-induced aggregation, causes spontaneous pigment dispersion, and inhibits pigment re-dispersion. Inhibition of actin polymerization by latrunculin-A completely impedes pigment aggregation and re-dispersion. Confocal immunocytochemistry shows that non-muscle myosin II (NMMII) co-localizes mainly with pigment granules while blebbistatin inhibition of NMMII strongly reduces the RPCH response, also inducing spontaneous pigment dispersion. Myosin II and dynein also co-localize with the pigment granules. Inhibition of dynein ATPase by erythro-9-(2-hydroxy-3-nonyl) adenine induces aggregation, inhibits RPCH-triggered aggregation, and inhibits re-dispersion. Granule aggregation and dispersion depend mainly on microfilament integrity although microtubules may be involved. Both cytoskeletal polymers are functional only when subunit turnover is active. Myosin and dynein may be the molecular motors that drive pigment aggregation. These mechanisms of granule translocation in crustacean chromatophores share various features with those of vertebrate pigment cells.

Enhancement of dynein-mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes.

Dynein-mediated autophagosome (AP) trafficking was recently demonstrated to contribute to the formation of autophagolysosomes (APLs) and autophagic flux process in coronary arterial myocytes (CAMs). However, it remains unknown how the function of dynein as a motor protein for AP trafficking is regulated under physiological and pathological conditions. The present study tested whether the dynein-mediated autophagy maturation is regulated by a redox signalling associated with lysosomal Ca2+ release machinery. In primary cultures of CAMs, reactive oxygen species (ROS) including H2O2 and O2−. (generated by xanthine/xanthine oxidase) significantly increased dynein ATPase activity and AP movement, which were accompanied by increased lysosomal fusion with AP and APL formation. Inhibition of dynein activity by (erythro-9-(2-hydroxy-3-nonyl)adenine) (EHNA) or disruption of the dynein complex by dynamitin (DCTN2) overexpression blocked ROS-induced dynein activation, AP movement and APL formation, and resulted in an accumulation of AP along with a failed breakdown of AP. Antagonism of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ signalling with NED-19 and PPADS abolished ROS-enhanced lysosomal Ca2+ release and dynein activation in CAMs. In parallel, all these changes were also enhanced by overexpression of NADPH oxidase-1 (Nox1) gene in CAMs. Incubation with high glucose led to a marked O2−. production compared with normoglycaemic CAMs, while Nox1 inhibitor ML117 abrogated this effect. Moreover, ML117 and NED-19 and PPADS significantly suppressed dynein activity and APL formation caused by high glucose. Taken together, these data suggest that ROS function as important players to regulate dynein-dependent AP trafficking leading to efficient autophagic maturation in CAMs.