Axonemal Movement Research Articles

Self-sustaining oscillation of two axonemal microtubules based on a stochastic bonding model between microtubules and dynein.

The motility of cilia and flagella plays important physiological roles, and there has been a great deal of research on the mechanisms underlying the motility of molecular motors. Although recent molecular structural analyses have revealed the components of the ciliary axoneme, the mechanisms involved in the regulation of dynein activity are still unknown, and how multiple dyneins coordinate their movements remains unclear. In particular, the mode of binding for axonemal dynein has not been elucidated. In this study, we constructed a thermodynamic stochastic model of microtubule-dynein coupling and reproduced the experiments of Aoyama and Kamiya on the minimal component of axonemal microtubule-dynein. We then identified the binding mode of axonemal dynein and clarified the relationship between dynein activity distribution and axonemal movement. Based on our numerical results, the slip-bond mechanism agrees quantitatively with the experimental results in terms of amplitude, frequency, and propagation velocity, implying that axial microtubule-dynein coupling may follow a slip-bond mechanism. Moreover, the frequency and propagation velocity decayed in proportion to the fourth power of microtubule length, and the critical load of the trigger for the oscillation agreed well with Euler's critical load.

Ciliary Dyneins and Dynein Related Ciliopathies

Although ubiquitously present, the relevance of cilia for vertebrate development and health has long been underrated. However, the aberration or dysfunction of ciliary structures or components results in a large heterogeneous group of disorders in mammals, termed ciliopathies. The majority of human ciliopathy cases are caused by malfunction of the ciliary dynein motor activity, powering retrograde intraflagellar transport (enabled by the cytoplasmic dynein-2 complex) or axonemal movement (axonemal dynein complexes). Despite a partially shared evolutionary developmental path and shared ciliary localization, the cytoplasmic dynein-2 and axonemal dynein functions are markedly different: while cytoplasmic dynein-2 complex dysfunction results in an ultra-rare syndromal skeleto-renal phenotype with a high lethality, axonemal dynein dysfunction is associated with a motile cilia dysfunction disorder, primary ciliary dyskinesia (PCD) or Kartagener syndrome, causing recurrent airway infection, degenerative lung disease, laterality defects, and infertility. In this review, we provide an overview of ciliary dynein complex compositions, their functions, clinical disease hallmarks of ciliary dynein disorders, presumed underlying pathomechanisms, and novel developments in the field.

Flagellar oscillation: a commentary on proposed mechanisms

Eukaryotic flagella and cilia have a remarkably uniform internal 'engine' known as the '9+2' axoneme. With few exceptions, the function of cilia and flagella is to beat rhythmically and set up relative motion between themselves and the liquid that surrounds them. The molecular basis of axonemal movement is understood in considerable detail, with the exception of the mechanism that provides its rhythmical or oscillatory quality. Some kind of repetitive 'switching' event is assumed to occur; there are several proposals regarding the nature of the 'switch' and how it might operate. Herein I first summarise all the factors known to influence the rate of the oscillation (the beating frequency). Many of these factors exert their effect through modulating the mean sliding velocity between the nine doublet microtubules of the axoneme, this velocity being the determinant of bend growth rate and bend propagation rate. Then I explain six proposed mechanisms for flagellar oscillation and review the evidence on which they are based. Finally, I attempt to derive an economical synthesis, drawing for preference on experimental research that has been minimally disruptive of the intricate structure of the axoneme. The 'provisional synthesis' is that flagellar oscillation emerges from an effect of passive sliding direction on the dynein arms. Sliding in one direction facilitates force-generating cycles and dynein-to-dynein synchronisation along a doublet; sliding in the other direction is inhibitory. The direction of the initial passive sliding normally oscillates because it is controlled hydrodynamically through the alternating direction of the propulsive thrust. However, in the absence of such regulation, there can be a perpetual, mechanical self-triggering through a reversal of sliding direction due to the recoil of elastic structures that deform as a response to the prior active sliding. This provisional synthesis may be a useful basis for further examination of the problem.

Geometry drives the "deviated-bending" of the bi-tubular structures of the 9 + 2 axoneme in the flagellum.

The axoneme "9 + 2" is basically a system constituted of a cylinder of 9 microtubule doublets surrounding a central pair of microtubules. These bi-tubular structures are considered as the support system of the active molecular complexes that generate and regulate the axonemal movement. Schoutens has calculated their moments of inertia [Schoutens, 1994: Journal of Theoretical Biology 171:163-177]. The results obtained allowed us to assume that these bi-tubular systems are endowed with dynamic properties that could be involved in the regulation of the axonemal machinery. For the first time, using the finite elements methods and the resistance of material principles, we have now calculated that the curvature of the axoneme induces the deviated-bending of the bi-tubular structures of the axoneme, because of their geometry only; they behave as beams in a framework. This approach is similar to the one used to measure the deflection of a single microtubule [Kasas et al., 2004: Chem Phys Chem 5:252-257]. These behaviors induce internal movement or constraints of either couples or triplets of doublets within the axonemal cylinder that could be directly involved in a constrained or a spontaneous "convergence/divergence" equilibrium of the cylindrical generatrices that they draw along the axonemal cylinder, which could apparently regulate the activity of the axonemal motors (the dynein arms). These results are discussed here, taking into consideration the dynamic propagation of the wave train along the flagellar axoneme, and the regulated balance between the activities of the two opposite sides of the axoneme during the beat. This study raises a few questions about the architecture-activity duo of the axonemal doublets.

Phosphorylation of axonemal 21 kDa and 26 kDa proteins modulates activation of sperm motility in the ascidian,Ciona intestinalis

Protein phosphorylation is highly coupled with sperm motility activation in several animal species. The micro-tubule based flagellar motor protein, dynein, is a candidate for a phosphoprotein related to sperm activation in many animal species (Morisawa & Hayashi, 1985; Hayashiet al., 1987; Dey & Brokaw, 1991; Stephens & Prior, 1992; Inabaet al., 1998, 1999). Sperm motility of the ascidiansCiona intestinalisandC. savignyiis activated by a factor derived from unfertilised eggs named sperm activating and attracting factor (SAAF). SAAF elevates the intracellular cyclic AMP (cAMP) level by a mechanism dependent on membrane hyperpolarisation and extracellular Ca2+(Yoshidaet al., 1994; Izumiet al., 1999). Experiments using demembranatedCionasperm showed that cAMP is required prior to ATP for the activation of axonemal movement (Opreska & Brokaw, 1983; Morisawaet al., 1984; Brokaw, 1985; Dey & Brokaw, 1991; Chaudhryet al., 1995) and that many sperm flagellar proteins including dynein light chain are phosphorylated during incubation of demembranated sperm with ATP and cAMP (Dey & Brokaw, 1991). However, there is no evidence of which proteins are phosphorylated during the SAAF-dependent activation ofCionasperm motility.

Role of the ionic environment and internal pH on sperm activity.

In most species, once formed in the testis, spermatozoa are bathed in a fluid where they remained immobile and with a very low level of metabolism. This immotile status is understandable in view of the need to preserve the sperm energy reserve and to decrease the risk of alteration to membranes, internal structures and biochemical compounds by endogenous oxidizing agents produced by mitochondrial activity. This quiescent phase can be of different lengths and finishes when the semen is released into the external environment where the spermatozoa become motile and metabolically active. For invertebrates, and some fish, sexual activity is generally seasonal and fertilization is external. Spermatozoa, once differentiated in the gonad, remain there completely quiescent until they are released into the external medium, which is either fresh water or sea water. Dilution of the testicular fluid surrounding the spermatozoa allows the initiation of motility and metabolism. In fact, this seminal fluid has an inhibitory effect on sperm activity. For birds and mammals (including humans), the situation is much more complex. In these species, sperm production is almost continuous although for some of them, seasonal variations occur. When spermatozoa are released from the Sertoli cells, they are rapidly exported from the testis to the epididymis where the composition of the surrounding medium is profoundly modified. For most species, the spermatozoa remain immobile in the lower part of the epididymis, even though they have gained the capability to be fully motile as shown by dilution in an adequate medium. In vivo, motility is activated when the spermatozoa are mixed with secretions from the different accessory glands during ejaculation. This paper will review the role played by environmental factors, such as ions, in the activation of sperm motility and metabolism of different species of invertebrates and vertebrates. Special attention is given to changes in sperm internal pH, its regulation and role in the activation of sperm axonemal movement.

Chapter 72 Modified Nucleotides as Probes for Dynein

Publisher Summary This chapter focuses on the ribose-modified analogs. Such analogs appear to be useful probes for axonemal dynein structure and function. Unlike many other analogs, these analogs have a high affinity for axonemal dyneins and support axonemal movement. Benzoyl benzoyl ATP (Bz 2 -ATP) is a photoaffinity label; it has been used to identify the ribose-binding site of myosin and may be useful for labeling dynein. Anthraniloyl ATP (antATP) and methylanthraniloyl ATP (mantATP) are fluorescent analogs that are easy to synthesize and have a large Stoke's shift. Axonemal dyneins have very high affinity and relatively high turnover with ATP as compared with other nucleoside triphosphates, and there are relatively few analogs that support axonemal motion. Among these are those with modifications on the 2-position of adenine, 2 ′ - and 3 ′ -positions on the ribose, and substitution of S for the α phosphate. Before discussing the use of these analogs in investigating axonemal dyneins, the chapter points out a couple of key differences between axonemal dyneins and cytoplasmic dyneins or other motor molecules. First, an axoneme is a very complicated structure that requires chemomechanical coupling of many dyneins that are functionally distinct. Second, even a single axonemal dynein complex is composed of more than one heavy chain, each of which potentially has multiple nucleotide binding sites. This complexity may be dissected by the judicious use of ribose-modified analogs.

Regulation of sperm motility at the axonemal level

With very few exceptions, the basic structure of the 9+2 axoneme has been well preserved over a very long period of evolution from protozoa to mammais. This stability indicates that the basic structural components of the axoneme visible by electron microscopy, as well as most of the other unidentified components, have withstood the passage of time. It also means that components of the 9+2 axoneme have sufficient diversity in function to accommodate the various types of motility patterns encountered in different species of flagella. Several of the 200 polypeptides that constitute the axoneme have been identified as components of the dynein arms, radial spokes etc. but many more remain to be identified and their function(s) remain to be determined. Because this review deals with the regulation of flagellar movement at the axonemal level, it does not include regulation of flagella by extracellular factors unless these factors have a direct action on axonemal components. In this context, it is very important firstly to understand the structural components of the axoneme and how they influence and regulate axonemal movement. Different primitive organisms are mentioned in this review since major breakthroughs in our understanding of how an axoneme generates different types of movement have been made through their study. Despite some variations in structure and function of axonemal components, the basic mechanisms involved in the regulation of flagella from Chlamydomonas or sea urchin spermatozoa should also apply to the more evolved mammalian species, including human spermatozoa.

Specificity of ATP for the initiation of flagellar motility of hamster sperm.

Hamster sperm collected from the cauda epididymis were washed and the pellet diluted in a medium containing Triton x 100 (Sigma, Saint Louis, MO, USA) to dissolve the cell membrane and then reactivated with various concentrations of ATP (0-3 mM). Spermatozoal axonemes were initially immotile but the maximal percentage motility was obtained almost immediately following addition of 1 mM ATP. A stepwise increase in the concentration of ATP caused a 5-min delay in development of maximal reactivation and a change in the beating pattern as indicated by a decrease in the percentage of reactivating sperm. Under the same experimental condition GPT, UTP, CTP, and AMP failed to initiate the axonemal motility. In demembranated sperm reactivated by ADP, however, the beat frequency was lower compared with that reactivated by ATP. Pretreatment of the sperm with the mitochondrial phosphorylation blockers oligomycin and 2'4'-DNP (dinitrophenol) failed to inhibit the axonemal movement, although the presence of the dynein ATPase inhibitor, vanadate, was able to inhibit the reactivation. These results suggest that the exogenous ATP, but not the mitochondrial ATP, is responsible for axonemal motility.

Sea urchin axonemal motion supported by fluorescent, ribose-modified analogues of ATP.

The axonemal motion supported by fluorescent ribose-modified analogues, anthraniloyl ATP (Ant-ATP) and methylanthraniloyl ATP (Mant-ATP), was investigated. Ant-ATP and Mant-ATP supported good vigorous motion. A detailed study of the movement shows that the maximum beat frequencies (Vmax) were significantly lower with the analogues. However, Michaelis constants (Km) for beat frequency were also significantly lower than with ATP. Thus the net effect of changes in these two parameters, Vmax/Km, was similar for ATP and Ant-ATP and higher with Mant-ATP. Thus these fluorescent analogues are good substrates for axonemal movement. The consistently higher value of Vmax/Km, a measure of substrate selectivity, with Mant-ATP over Ant-ATP suggests a feature of the ribose binding site. Other significant differences in the movement with the fluorescent analogues are quantified in terms of kinetic measures of sliding velocity and bend propagation velocity.

In vitro maturation of the potential for movement of carp spermatozoa

Carp semen obtained from isolated fish after hormonal stimulation was highly variable in terms of volume of semen, osmotic pressure of the seminal plasma, and sperm capacity to move. Moreover, this last parameter was unstable when the spermatozoa were kept within the seminal plasma, and the present work was designed to investigate and possibly correct this phenomenon. Sperm potential movement was the major parameter studied and was measured by the percentage of motile cells in a final 3.000-fold dilution in a medium of low osmotic pressure in which sperm movement is known to occur (Morisawa and Suzuki, Science 210:1145-1147, 1980). This was completed with occasional measurements of flagellar beat frequencies and demembranation-reactivation of axonemal movement. The results showed that sperm potential movement was preserved upon dilution of the semen into cold 200 mM KCl medium and that semen of initially "poor" quality or spermatozoa that had lost their capacity to move during storage in the semen recovered gradually their potential movement during incubation at 2 degrees C in the same medium. The K+ dependence for both the conservation and the regeneration of sperm capacity to move showed a minimal requirement of 50 mM KCl in media of high osmotic pressure. Na+ ions had similar properties but not divalent cations. The K+ activation was not pH dependent between pH 9.03 and 6.04. Whatever the functional state of live spermatozoa, demembranation-reactivation occurred in ATP-Mg2+. It is concluded that, with dilution of the semen in appropriate conditions, carp spermatozoa retain or acquire potential movement and therefore are a lower vertebrate spermatozoa model available year-round. In addition, obtaining potentially nonmotile sperm and reversion in vitro might be useful to study the control of in vitro maturation.

Ionic regulation of the plasma membrane potential of rainbow trout (Salmo gairdneri) spermatozoa: Role in the initiation of sperm motility

The ionic dependence of the trout sperm plasma membrane potential was analysed by measuring the accumulation of the lipophilic ions 3H-tetraphenylphosphonium (TPP) and 14C-thiocyanate (SCN) following dilution in artificial media isotonic to the seminal fluid. Our data showed that the trout sperm plasma membrane has a mixed conductance: the plasma membrane potential is sensitive upon the transmembrane gradients of K+, Na+, and H+. This potential is negative (less than -40 mV) in a 125 mM choline chloride media (ChM) at pH 8.5. Replacement of choline by sodium has a small depolarizing effect. The membrane potential is about -15 mV in a 125 mM potassium chloride and falls near zero mV only if valinomycin is added. In ChM changing the external pH (pHe) greatly affects the membrane potential: its value rises from less than -40 mV at pHe 9.0 to -17 mV at pHe 5.0. This pH effect is observed also in presence of sodium or potassium. A decrease in the transmembrane proton gradient produced by increasing internal pH without changing pHe induces also a depolarisation of the plasma membrane. In the different media in which trout sperm remain immotile after dilution (media with [K+] greater than 20-40 mM or a pH less than 7.5) the plasma membrane is more depolarized than in media allowing motility, suggesting a relationship between the state of membrane polarization and the intracellular effectors of the axonemal movement.

Mechanochemical coupling in eukaryotic flagella

Quantitative analyses of ATP hydrolysis coupled to movement of eukaryotic flagella is important for understanding the relationship between ATP hydrolysis and movement. The difference in ATPase activity between intact motile axonemes (that is the cytoskeletal core of flagella) and homogenized or immotile axonemes has been assumed to be coupled to movement. However, recent findings on rates of steps in the dynein ATPase cycle and the effect of interaction with microtubules on those steps call for reassessment of movement-coupled ATPase. From these studies, it is clear that dynein ATPase activity is not as tightly coupled to interaction with microtubules as myosin ATPase activity is coupled to interaction with actin. The method by which axonemal movement is inhibited will critically affect the interpretation of difference in ATPase activity. If the homogenization or similar methods uncouple dynein, the difference in ATPase activity is not a useful measurement. Greater understanding of the relationship between dynein kinetics and axonemal movement may be obtained by use of conditions and substrates with known effects at specific steps in the dynein mechanochemical cycle and quantitating their effects on movement.

2-Chloro adenosine triphosphate as substrate for sea urchin axonemal movement.

The 2-substituted ATP analog 2-Chloro ATP was tested for its capacity to support axonemal movement. The movement of sea urchin axonemes reactivated with 2-Cl ATP appeared very similar to that with ATP. Detailed waveform analysis indicated that bend angle and shear amplitude were not significantly different for ATP and 2-Cl ATP. Although wavelength differs at particular nucleotide concentrations, if normalized to the beat frequency, it is similar for ATP and 2-Cl ATP. The main difference in the movement with the two analogs was seen in beat frequency and sliding velocity. The Vmax for beat frequency and mean sliding velocity was lower for 2-Cl ATP. The apparent Km for beat frequency and sliding velocity was much lower for 2-Cl ATP. The ratio of these two effects, that is, (Vmax/Km) is higher for 2-Cl ATP. Thus 2-Cl ATP is a good substrate for axonemal movement. The significantly lower Km of 2-Cl ATP was also demonstrated by its ability to support oscillatory motion at concentrations below that for ATP. The observations identify the structures and conformation of substrate necessary to support axonemal movement.