Varicocoele-associated male infertility: Cellular and molecular perspectives of pathophysiology.

BACKGROUND: Varicocoele is a common risk factor associated with reduced male fertility potential. The current understanding of varicocoele pathophysiology does not completely explain the clinical manifestation of infertility. The present treatment options such as antioxidant supplementation and varicocoelectomy only help ≈35% of men to achieve spontaneous pregnancy. OBJECTIVE: This review aims to summarize the available knowledge on cellular and molecular alterations implicated to varicocoele-associated male infertility and also highlights the new knowledge generated by "omics" technologies. MATERIALS AND METHODS: PubMed, MEDLINE, Cochrane and Google Scholar databases are searched using different combinations of keywords (varicocoele, infertile/fertile men with varicocoele, cellular changes, molecular mechanisms, proteome, epigenome, transcriptome and metabolome). A total of 229 relevant human and animal studies published till 2021 were included in this review. RESULTS: Current understanding advocates oxidative stress (OS) as a major contributory factor to varicocoele-associated male infertility. Excessive OS causes alteration in testicular microenvironment and sperm DNA fragmentation, which further contributes to infertility. Molecular and omics studies have identified several promising biomarkers such as AAMP, SPINT1, MKI67 (genetic markers), sperm quality and function related protein markers, global sperm DNA methylation level (epigenetic marker), Hspa2, Protamine, Gadd7, Dynlt1 and Beclin1 (mRNA markers), PRDX2, HSPA, APOA2, YKL40 (seminal protein markers), total choline and PHGDH (metabolic markers). DISCUSSION: Mature spermatozoa harbours a plethora of molecular information in form of proteome, epigenome and transcriptome, which could provide very important clues regarding pathophysiology of varicocoele-associated infertility. Recent molecular and omics studies in infertile men with varicocoele have identified several promising biomarkers. Upon further validation with larger and well-defined studies, some of these biomarkers could aid in varicocoele management. CONCLUSION: The present evidences suggest that inclusion of OS and sperm DNA fragmentation tests could be useful to the diagnostic workup for men with varicocoele. Furthermore, including precise molecular markers may assist in diagnostics and prognostics of varicocoele-associated male infertility.

Dnah9 mutant mice and organoid models recapitulate the clinical features of patients with PCD and provide an excellent platform for drug screening.

Primary cilia dyskinesia (PCD) is a rare genetic disease caused by ciliary structural or functional defects. It causes severe outcomes in patients, including recurrent upper and lower airway infections, progressive lung failure, and randomization of heterotaxy. To date, although 50 genes have been shown to be responsible for PCD, the etiology remains elusive. Meanwhile, owing to the lack of a model mimicking the pathogenesis that can be used as a drug screening platform, thereby slowing the development of related therapies. In the current study, we identified compound mutation of DNAH9 in a patient with PCD with the following clinical features: recurrent respiratory tract infections, low lung function, and ultrastructural defects of the outer dynein arms (ODAs). Bioinformatic analysis, structure simulation assay, and western blot analysis showed that the mutations affected the structure and expression of DNAH9 protein. Dnah9 knock-down (KD) mice recapitulated the patient phenotypes, including low lung function, mucin accumulation, and increased immune cell infiltration. Immunostaining, western blot, and co-immunoprecipitation analyses were performed to clarify that DNAH9 interacted with CCDC114/GAS8 and diminished their protein levels. Furthermore, we constructed an airway organoid of Dnah9 KD mice and discovered that it could mimic the key features of the PCD phenotypes. We then used organoid as a drug screening model to identify mitochondrial-targeting drugs that can partially elevate cilia beating in Dnah9 KD organoid. Collectively, our results demonstrated that Dnah9 KD mice and an organoid model can recapture the clinical features of patients with PCD and provide an excellent drug screening platform for human ciliopathies.

Arsenic trioxide disturbs the LIS1/NDEL1/dynein microtubule dynamic complex by disrupting the CLIP170 zinc finger in head and neck cancer.

Cancer mortality is mainly caused by metastasis, which requires dynamic remodeling of cytoskeletal components such as microtubules. Targeting microtubules presents a promising antimetastatic strategy that could prevent cancer spreading and recurrence. It is known that arsenic trioxide (ATO) is able to inhibit the migration and invasion of solid malignant tumors, but its exact molecular mechanism remains unclear. Here, we report a novel molecular target and antimetastatic mechanism of ATO in head and neck squamous cell carcinoma (HNSCC). We found that cytoplasmic linker protein 170 (CLIP170) was overexpressed in HNSCC tissues and cells compared to normal controls. ATO at non-cytotoxic level (1 µM) inhibited the migration and invasion of HNSCC cells by displacing zinc in the zinc finger motif of CLIP170, which is a key protein that controls microtubule dynamics. The antimetastatic effects of ATO were equivalent to those of siRNA-mediated CLIP170 knockdown. Furthermore, ATO dysregulated microtubule polymerization via the CLIP170/LIS1/NDEL1/dynein signaling pathway in Cal27 cells as a functional consequence of CLIP170 zinc finger disruption. The effect was partially reversed by zinc supplementation. Taken together, these findings reveal that CLIP170 is a novel molecular target of ATO and demonstrate the capability and underlying mechanisms of ATO as a potential antimetastatic agent for HNSCC treatment.

Engineering with Biomolecular Motors.

Biomolecular motors, such as the motor protein kinesin, can be used as off-the-shelf components to power hybrid nanosystems. These hybrid systems combine elements from the biological and synthetic toolbox of the nanoengineer and can be used to explore the applications and design principles of active nanosystems. Efforts to advance nanoscale engineering benefit greatly from biological and biophysical research into the operating principles of motor proteins and their biological roles. In return, the process of creating in vitro systems outside of the context of biology can lead to an improved understanding of the physical constraints creating the fitness landscape explored by evolution. However, our main focus is a holistic understanding of the engineering principles applying to systems integrating molecular motors in general. To advance this goal, we and other researchers have designed biomolecular motor-powered nanodevices, which sense, compute, and actuate. In addition to demonstrating that biological solutions can be mimicked in vitro, these devices often demonstrate new paradigms without parallels in current technology. Long-term trends in technology toward the deployment of ever smaller and more numerous motors and computers give us confidence that our work will become increasingly relevant. Here, our discussion aims to step back and look at the big picture. From our perspective, energy efficiency is a key and underappreciated metric in the design of synthetic motors. On the basis of an analogy to ecological principles, we submit that practical molecular motors have to have energy conversion efficiencies of more than 10%, a threshold only exceeded by motor proteins. We also believe that motor and system lifetime is a critical metric and an important topic of investigation. Related questions are if future molecular motors, by necessity, will resemble biomolecular motors in their softness and fragility and have to conform to the "universal performance characteristics of motors", linking the maximum force and mass of any motor, identified by Marden and Allen. The utilization of molecular motors for computing devices emphasizes the interesting relationship among the conversion of energy, extraction of work, and production of information. Our recent work touches upon these topics and discusses molecular clocks as well as a Landauer limit for robotics. What is on the horizon? Just as photovoltaics took advantage of progress in semiconductor fabrication to become commercially viable over a century, one can envision that engineers working with biomolecular motors leverage progress in biotechnology and drug development to create the engines of the future. However, the future source of energy is going to be electricity rather than fossil or biological fuels, a fact that has to be accounted for in our future efforts. In summary, we are convinced that past, ongoing, and future efforts to engineer with biomolecular motors are providing exciting demonstrations and fundamental insights as well as opportunities to wander freely across the borders of engineering, biology, and chemistry.

Schistosoma japonicum calcium-binding tegumental protein SjTP22.4 immunization confers praziquantel schistosomulumicide and antifecundity effect in mice.

A family of platyhelminth tegument-specific proteins comprising of one or two calcium ion binding EF-hand and a dynein light chain-like domain, termed tegumental proteins, are considered as candidates of vaccine. In this study, we cloned and characterized SjTP22.4, a novel membrane-anchored tegumental protein in Schistosoma japonicum with theoretic MW of 22.4. The recombinant SjTP22.4 could be recognized by S. japonicum infected sera. Immunofluorescence revealed that this protein is not only located on the surface of tegument of adult and schistosomulum and cercaria, but also in the parenchymatous tissues and intestinal epithelium. Circular dichroism (CD) measurement demonstrated rSjTP22.4 had Ca(2+)-binding ability. The rSjTP22.4 vaccination without adjuvants produced comparable high level of antibody with that of immunization with adjuvants together indicated it was an antigen of strong antigenicity. The level of IgG1 is much higher than that of IgG2a and IgE is nearly negative in S. japonicum-infected and rSjTP22.4 immunized mice. In cercaria challenge experiment, mice vaccinated with SjTP22.4 showed no reduction in adult burden and egg production, comparing with the control mice, but 41% decrease in egg mature rate and 32% reduction in liver egg granuloma area. However, the SjTP22.4 immunized mice that received praziquantel treatment at 10d post infection caused 26% reduction in adult burden and 53% decrease in egg mature rate, comparing with the control mice only received praziquantel treatment. In conclusion, SjTP22.4 is a valuable vaccine candidate for S. japonicum of anti-pathogenesis and anti-transmission effect and plays a synergetic role in praziquantel to kill schistosomulum.

Dynein and dynactin components modulate neurodegeneration induced by excitotoxicity.

Glutamate excitotoxicity causes neuronal dysfunction and degeneration. It is implicated in chronic disorders, including Alzheimer's disease, and in acute CNS insults such as ischemia. These disorders share prominent morphological features, including axon degeneration and cell body death. However, the molecular mechanism underlying excitotoxicity-induced neurodegeneration remains poorly understood. A key molecular feature of neurodegeneration is deficits in microtubule-based cargo transport that plays a pivotal role in maintaining the balance of survival and stress signaling in the axon. We developed an excitotoxicity-induced neurodegeneration system in primary neuronal cultures. We find that excitotoxicity generates a C-terminal truncated form of p150Glued, a major component of the dynactin complex, which exacerbates axon degeneration. This p150Glued truncated form was identified in brain tissues of patients with Alzheimer's disease. Overexpression of wild-type (WT) dynein intermediate chain (DIC), a dynein component that interacts with p150Glued and links dynein and dynactin complexes, DIC (S84D) mutant, and WT p150Glued suppressed axon degeneration. These modulating effects of p150Glued and DIC on excitotoxicity-induced axon degeneration are also observed in apoptosis and cell body death. Thus, our findings identify retrograde transport proteins, p150Glued and DIC, as novel modulators of neurodegeneration induced by glutamate excitotoxicity.

Impacts of Usher syndrome type IB mutations on human myosin VIIa motor function.

Usher syndrome (USH) is a human hereditary disorder characterized by profound congenital deafness, retinitis pigmentosa, and vestibular dysfunction. Myosin VIIa has been identified as the responsible gene for USH type 1B, and a number of missense mutations have been identified in the affected families. However, the molecular basis of the dysfunction of USH gene, myosin VIIa, in the affected families is unknown to date. Here we clarified the effects of USH1B mutations on human myosin VIIa motor function for the first time. The missense mutations of USH1B significantly inhibited the actin activation of ATPase activity of myosin VIIa. G25R, R212C, A397D, and E450Q mutations abolished the actin-activated ATPase activity completely. P503L mutation increased the basal ATPase activity for 2-3-fold but reduced the actin-activated ATPase activity to 50% of the wild type. While all of the mutations examined, except for R302H, reduced the affinity for actin and the ATP hydrolysis cycling rate, they did not largely decrease the rate of ADP release from actomyosin, suggesting that the mutations reduce the duty ratio of myosin VIIa. Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa.

[High molecular weight protein detected in higher plant cells by antibodies against dynein is associated with vesicular organelles including Golgi apparatus].

The cytoplasmic dynein is a multisubunit complex driving organelles along microtubules to their minus-end. We used antibodies against two functional domains (motor and microtubule-binding) of one of principal components of the complex--dynein heavy chain of slime mould Dictyostelium discoideum--to test root meristem cells of wheat Triticum aestivum. The antibodies reacted with a high molecular weight protein (> 500 kDa) in the total cell extract and the band recognized by the antibodies in plant extracts had a lower electrophoretic mobility than the high molecular weight band of mammalian dynein. Antibodies coupled to protein A-Sepharose precipitated the high molecular weight protein from the purified cell extracts. Immunocytochemical analysis demonstrated that the antigen recognized by antibodies against dynein heavy chains is associated with the vesicles whose localization depends on the cell cycle stage. The antigen-positive vesicles were localized to the perinuclear region in interphase and early prophase, to the spindle periphery and to spindle pole region during mitosis, and to the interzonal region in the period of fragmoplast and cell plate formation. Some antigen-positive vesicles also reacted with antibodies against Golgi protein markers. The obtained data indicate that higher plant cells contain a high molecular weight protein interacting with antibodies against the motor and microtubules-binding domains of Dictyostelium dynein heavy chain. The revealed antigen was associated with the vesicular structures in the cytoplasm including the Golgi apparatus.

Shroom2, a myosin-VIIa- and actin-binding protein, directly interacts with ZO-1 at tight junctions.

Defects in myosin VIIa lead to developmental anomalies of the auditory and visual sensory cells. We sought proteins interacting with the myosin VIIa tail by using the yeast two-hybrid system. Here, we report on shroom2, a submembranous PDZ domain-containing protein that is associated with the tight junctions in multiple embryonic and adult epithelia. Shroom2 directly interacts with the C-terminal MyTH4-FERM domain of myosin VIIa and with F-actin. In addition, a shroom2 fragment containing the region of interaction with F-actin was able to protect actin filaments from cytochalasin-D-induced disruption in MDCK cells. Transfection experiments in MDCK and LE (L fibroblasts that express E-cadherin) cells led us to conclude that shroom2 is targeted to the cell-cell junctions in the presence of tight junctions only. In Ca(2+)-switch experiments on MDCK cells, ZO-1 (also known as TJP1) preceded GFP-tagged shroom2 at the differentiating tight junctions. ZO-1 directly interacts with the serine- and proline-rich region of shroom2 in vitro. Moreover, the two proteins colocalize in vivo at mature tight junctions, and could be coimmunoprecipitated from brain and cochlear extracts. We suggest that shroom2 and ZO-1 form a tight-junction-associated scaffolding complex, possibly linked to myosin VIIa, that bridges the junctional membrane to the underlying cytoskeleton, thereby contributing to the stabilization of these junctions.

How pollen tubes grow.

Sexual reproduction of flowering plants depends on delivery of the sperm to the egg, which occurs through a long, polarized projection of a pollen cell, called the pollen tube. The pollen tube grows exclusively at its tip, and this growth is distinguished by very fast rates and reaches extended lengths. Thus, one of the most fascinating aspects of pollen biology is the question of how enough cell wall material is produced to accommodate such rapid extension of pollen tube, and how the cell wall deposition and structure are regulated to allow for rapid changes in the direction of growth. This review discusses recent advances in our understanding of the mechanism of pollen tube growth, focusing on such basic cellular processes as control of cell shape and growth by a network of cell wall-modifying enzymes, molecular motor-mediated vesicular transport, and intracellular signaling by localized gradients of second messengers.

Vertebrate myosin VIIb is a high duty ratio motor adapted for generating and maintaining tension.

Kinetic adaptation of muscle and non-muscle myosins plays a central role in defining the unique cellular functions of these molecular motor enzymes. The unconventional vertebrate class VII myosin, myosin VIIb, is highly expressed in polarized cells and localizes to highly ordered actin filament bundles such as those found in the microvilli of the intestinal brush border and kidney. We have cloned mouse myosin VIIb from a cDNA library, expressed and purified the catalytic motor domain, and characterized its actin-activated ATPase cycle using quantitative equilibrium and kinetic methods. The myosin VIIb steady-state ATPase activity is slow (approximately 1 s(-1)), activated by very low actin filament concentrations (K(ATPase) approximately 0.7 microm), and limited by ADP release from actomyosin. The slow ADP dissociation rate constant generates a long lifetime of the strong binding actomyosin.ADP states. ADP and actin binding is uncoupled, which enables myosin VIIb to remain strongly bound to actin and ADP at very low actin concentrations. In the presence of 2 mm ATP and 2 microm actin, the duty ratio of myosin VIIb is approximately 0.8. The enzymatic properties of actomyosin VIIb are suited for generating and maintaining tension and favor a role for myosin VIIb in anchoring membrane surface receptors to the actin cytoskeleton. Given the high conservation of vertebrate class VII myosins, deafness phenotypes arising from disruption of normal myosin VIIa function are likely to reflect a loss of tension in the stereocilia of inner ear hair cells.

Assessment of differential gene expression in vestibular epithelial cell types using microarray analysis.

Current global gene expression techniques allow the evaluation and comparison of the expression of thousands of genes in a single experiment, providing a tremendous amount of information. However, the data generated by these techniques are context-dependent, and minor differences in the individual biological samples, methodologies for RNA acquisition, amplification, hybridization protocol and gene chip preparation, as well as hardware and analysis software, lead to poor correlation between the results. One of the significant difficulties presently faced is the standardization of the protocols for the meaningful comparison of results. In the inner ear, the acquisition of RNA from individual cell populations remains a challenge due to the high density of the different cell types and the paucity of tissue. Consequently, laser capture microdissection was used to selectively collect individual cells and regions of cells from cristae ampullares followed by extraction of total RNA and amplification to amounts sufficient for high throughput analysis. To demonstrate hair cell-specific gene expression, myosin VIIA, calmodulin and alpha9 nicotinic acetylcholine receptor subunit mRNAs were amplified using reverse transcription-polymerase chain reaction (RT-PCR). To demonstrate supporting cell-specific gene expression, cyclin-dependent kinase inhibitor p27kip1 mRNA was amplified using RT-PCR. Subsequent experiments with alpha9 RT-PCR demonstrated phenotypic differences between type I and type II hair cells, with expression only in type II hair cells. Using the laser capture microdissection technique, microarray expression profiling demonstrated 408 genes with more than a five-fold difference in expression between the hair cells and supporting cells, of these 175 were well annotated. There were 97 annotated genes with greater than a five-fold expression difference in the hair cells relative to the supporting cells, and 78 annotated genes with greater than a five-fold expression difference in the supporting cells relative to the hair cells.

A role for myosin VI in postsynaptic structure and glutamate receptor endocytosis.

Myosin VI (Myo6) is an actin-based motor protein implicated in clathrin-mediated endocytosis in nonneuronal cells, though little is known about its function in the nervous system. Here, we find that Myo6 is highly expressed throughout the brain, localized to synapses, and enriched at the postsynaptic density. Myo6-deficient (Snell's waltzer; sv/sv) hippocampus exhibits a decrease in synapse number, abnormally short dendritic spines, and profound astrogliosis. Similarly, cultured sv/sv hippocampal neurons display decreased numbers of synapses and dendritic spines, and dominant-negative disruption of Myo6 in wild-type hippocampal neurons induces synapse loss. Importantly, we find that sv/sv hippocampal neurons display a significant deficit in the stimulation-induced internalization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors (AMPARs), and that Myo6 exists in a complex with the AMPAR, AP-2, and SAP97 in brain. These results suggest that Myo6 plays a role in the clathrin-mediated endocytosis of AMPARs, and that its loss leads to alterations in synaptic structure and astrogliosis.

Molecular motors: Dynein's gearbox.

A new optical trapping study shows that the stepsize of cytoplasmic dynein varies according to the applied force, suggesting that this motor can change gear. Complementary biochemical kinetic work on yeast dynein mutants hints at the allosteric mechanisms involved.

A tektin homologue is decreased in chlamydomonas mutants lacking an axonemal inner-arm dynein.

In ciliary and flagellar axonemes, various discrete structures such as inner and outer dynein arms are regularly arranged on the outer doublet microtubules. Little is known about the basis for their regular arrangement. In this study, proteins involved in the attachment of inner-arm dyneins were searched by a microtubule overlay assay on Chlamydomonas mutant axonemes. A 58-kDa protein (p58) was found approximately 80% diminished in the mutants ida6 and pf3, both lacking one (species e) of the seven inner-arm species (a-g). Analysis of its cDNA indicated that p58 is homologous to tektin, a protein that was originally found in sea urchin and thought to be crucial for the longitudinal periodicity of the doublet microtubule. Unlike sea urchin tektin, which is a component of protofilament ribbons that occur after Sarkosyl treatment of axonemes, p58 was not contained in similar Sarkosyl-resistant ribbons from Chlamydomonas axonemes. Immunofluorescence microscopy showed that p58 was localized uniformly along the axoneme and on the basal body. The p58 signal was reduced in ida6 and pf3. These results suggest that a reduced amount of p58 is sufficient for the production of outer doublets, whereas an additional amount of it is involved in inner-arm dynein attachment.

Two approaches to isolate cytoplasmic dynein ATPase from Neurospora crassa.

Cytoplasmic dynein is a force-producing enzyme that, in association with dynactin, conducts minus-end directed transport of various organelles along microtubules. Biochemical analyses of cytoplasmic dynein and dynactin have been conducted primarily in vertebrate systems, whereas genetic analyses have been explored mainly in yeast and the filamentous fungi. To provide a complementary biochemical approach for the study of fungal dynein, we isolated/partially purified cytoplasmic dynein ATPase from the filamentous fungus Neurospora crassa. N. crassa dynein was partially purified by slightly modifying the existing procedures, described for mammalian cytoplasmic dynein that uses dynein-microtubule binding, followed by release with ATP and sucrose gradient fractionation. A novel approach was also used to isolate dynein-specific ATPase by gel filtration (Sepharose CL-4B). The K(m), ATP obtained by isolating dynein ATPase using gel filtration was similar to that obtained by using conventional method, suggests that contaminant proteins do not interfere with the dynein ATPase activity. Like vertebrate dynein, N. crassa dynein is a general NTPase with highest activity toward ATP, and only the ATPase activity is stimulated by microtubules. The K(m), ATP for N. crassa cytoplasmic dynein is 10- to 15-fold higher than that of the vertebrate enzyme.

High molecular weight polypeptides related to dynein heavy chains in Nicotiana tabacum pollen tubes.

Nicotiana tabacum pollen tubes contain two high molecular weight polypeptides (about 400 kDa), which are specifically expressed during pollen germination and pollen tube growth in BK medium. The high molecular weight doublet resembles the dynein heavy chains in some biochemical properties. Sedimentation profiles of pollen tube extracts show that the high molecular weight bands have sedimentation coefficients of 22 S and 12 S, respectively. ATPase assay of sedimentation fractions shows an activity ten times higher when stimulated by the presence of bovine brain microtubules in fractions containing the 22 S high molecular weight polypeptide. Both these high molecular weight polypeptides can bind microtubules in an ATP-dependent fashion. A mouse antiserum to a synthetic peptide reproducing the sequence of the most conserved ATP-binding site among dynein heavy chains recognized the two high molecular weight polypeptides. Therefore these polypeptides have sequences immunologically related to the ATP binding sites of dynein heavy chains.

ATP-insensitive interaction of the amino-terminal region of the beta heavy chain of dynein with microtubules.

The ATP-insensitive microtubule-binding site of dynein has been investigated by limited proteolysis of sea urchin sperm flagellar axonemes. Mild tryptic digestion cleaved the dynein beta chain at either of two principal cleavage sites, generating two sets of complementary peptides. Inclusion of ATP in the digestion medium had no effect on the generation of these primary fragments. Sucrose density gradient separation and immunostaining with monoclonal antibodies against epitopes on the beta chain showed that extraction of the digested axonemes with 1-3 mM ATP solubilizes the peptides located at the carboxy-terminal end of the original heavy chain. The solubilization of the peptides containing the amino end required the presence of 0.6 M NaCl and was not affected by ATP. While the outer arm dynein is in situ on the axoneme, the N-terminal 125-kDa domain of the beta chain was not digested by trypsin, whereas in soluble dynein this domain becomes rapidly degraded. These data suggest that the N-terminal domain of the beta chain is involved in its ATP-insensitive attachment to microtubules and support the hypothesis that the N-terminal 125-kDa peptide corresponds to the flexible tail of the dynein molecule seen in electron micrographs.

Four ATP-binding sites in the midregion of the beta heavy chain of dynein.

The 'motor' proteins of eukaryotic cells contain specialized domains that hydrolyse ATP to produce force and movement along a cytoskeletal polymer (actin in the case of the myosin family; microtubules in the case of the kinesin family and dyneins). There are motor-protein superfamilies in which each member has a conserved force-generating domain joined to a different 'tail' which conveys specific attachment properties. The minus-end-directed microtubule motors, the dyneins, may also constitute a superfamily of force-generating proteins with distinct attachment domains. Axonemal outer-arm dynein from sea urchin spermatozoa is a multimeric protein consisting of two heavy chains (alpha and beta) with ATPase activity, three intermediate chains and several light chains. Here I report the sequence of cloned complementary DNA encoding the beta heavy chain of a dynein motor molecule. The predicted amino-acid sequence reveals four ATP-binding consensus sequences in the central domain. The dynein beta heavy chain is thought to associate transiently with a microtubule during ATP hydrolysis, but the ATP-dependent microtubule-binding sequence common to the kinesin superfamily is not found in the dynein beta heavy chain. These unique features distinguish the dynein beta heavy chain from other motor protein superfamilies and may be characteristic of the dynein superfamily.

Some changes in the properties of dynein ATPase in situ and after extraction following heat treatment of cilia.

Glycerol-extracted cilia from Tetrahymena pyriformis were demembranated by treatment with Triton X-100 and then heated for up to 30 min at temperatures between 34-38 degrees C. Heat treatment caused an uncoupling of the ATPase from motility as indicated by an increase in ATPase activity and a loss of pellet height response. After heat treatment, the ATPase activity of the dynein in situ differed from that in unheated cilia as shown by an increased sensitivity to a lower temperature of assay (0 degrees C) and by a loss of the activation normally observed upon reaction with N-ethylmaleimide or p-phenylenedimaleimide. Upon extraction of the heat-treated cilia by Tris-EDTA, there was a large loss in ATPase activity so that the heat-treated cilia yielded a crude dynein fraction with a lower specific activity compared with that obtained from unheated controls. The difference was not due to a change in the amount of protein recovered or in the amount of ATPase activity which remained unextracted. Resolution of the crude dynein by sucrose density sedimentation indicated that activity was lost from both the 14S and 30S peaks but more so from the latter than from the former. Thus dynein in situ in cilia in which the ATPase has been uncoupled from motility by gentle heat treatment differs in several important respects from dynein inside unheated cilia.