Phylogeny and expression of axonemal and cytoplasmic dynein genes in sea urchins.

Transcripts approximately 14.5 kilobases in length from 14 different genes that encode for dynein heavy chains have been identified in poly(A)+ RNA from sea urchin embryos. Analysis of the changes in level of these dynein transcripts in response to deciliation, together with their sequence relatedness, suggests that 11 or more of these genes encode dynein isoforms that participate in regeneration of external cilia on the embryo, whereas the single gene whose deduced sequence closely resembles that of cytoplasmic dynein in other organisms appears not to be involved in this regeneration. The four consensus motifs for phosphate binding found previously in the beta heavy chain of sea urchin dynein are present in all five additional isoforms for which extended sequences have been obtained, suggesting that these sites play a significant role in dynein function. Sequence analysis of a approximately 400 amino acid region encompassing the putative hydrolytic ATP-binding site shows that the dynein genes fall into at least six distinct classes. Most of these classes in sea urchin have a high degree of sequence identity with one of the dynein heavy chain genes identified in Drosophila, indicating that the radiation of the dynein gene family into the present classes occurred at an early stage in the evolution of eukaryotes. Evolutionary changes in cytoplasmic dynein have been more constrained than those in the axonemal dyneins.

A cytoplasmic dynein heavy chain in sea urchin embryos.

By making the hypothesis that the pattern of conserved sequence residues in the vicinity of the hydrolytic ATP-binding site of dynein would resemble that in myosins from a broad variety of sources, we designed degenerate oligonucleotide primers capable of amplifying this region of multiple dynein isoforms from sea urchin embryo poly(A)+ RNA. Quantification of the expression of two of these dynein isoforms has shown that the level of mRNA encoding for the beta-heavy chain, like that of tubulin, increases 2-3-fold after deciliation of the embryos, whereas the expression of the second dynein isoform, like that of actin, is essentially unaffected. This second isoform is believed to be the cytoplasmic dynein of sea urchin embryos.

A PCR procedure to determine the sequence of large polypeptides by rapid walking through a cDNA library.

A procedure that uses the PCR to make rapid successive steps through a random-primed cDNA library has been developed to provide a method for sequencing very long genes that are difficult to obtain as a single clone. In each successive step, the portions of partial clones that extend out from the region of known DNA sequence are amplified by two stages of PCR with nested, outward-directed primers designed approximately 50 bases in from the end of the known sequence, together with a general primer based on the sequence of the vector. This procedure has been used to determine the coding sequence of the cDNA for the beta heavy chain of axonemal dynein from embryos of the sea urchin Tripneustes gratilla. By starting from a single parent clone, whose translated amino acid sequence overlapped the microsequence of a tryptic peptide of the beta heavy chain, and making 3 such walk steps downstream and 14 walk steps upstream, we obtained a sequence of 13,799 base pairs that had an open reading frame of 13,398 base pairs. This sequence encodes a polypeptide with 4466 residues of Mr 511,804 that is believed to correspond to the complete beta heavy chain of ciliary outer arm dynein.

Multiple nucleotide-binding sites in the sequence of dynein beta heavy chain.

Axonemal dyneins have two or three globular heads joined by flexible tails to a common base, with each head/tail unit consisting of a single heavy-chain polypeptide of relative molecular mass greater than 400,000. The sizes of the components have been deduced by electron microscopy. The isolated beta heavy chain of sea urchin sperm flagella, which is immunologically identical to that of the embryo cilia, is of particular interest as it retains the capability for microtubule translocation in vitro. Limited proteolysis of the beta heavy chain divides it into two fragments, A and B, which sediment separately at 12S and 6S, and possibly correspond to the head and tail domains of the molecule. Dynein ATPase is the energy-transducing enzyme that generates the sliding movement between tubules that underlies the beating of cilia and flagella of eukaryotes, and possibly also other large intracellular movements. Here we report that the deduced amino-acid sequence of the beta heavy chain of axonemal dynein from embryos of the sea urchin Tripneustes gratilla has 4,466 residues and contains the consensus motifs for five nucleotide-binding sites. The probable hydrolytic ATP-binding site can be identified by its location close to or at the V1 site of vanadate-mediated photo-cleavage. The general features of the map of photocleavage and proteolytic peptides reported earlier have been confirmed, except that the map's polarity is reversed. The predicted secondary structure of the beta heavy chain consists of an alpha/beta-type pattern along its whole length. The two longest regions of potential alpha helix, with unbroken heptad hydrophobic repeats 120 and 50 amino acids long, may be of functional importance. But dynein does not seem to contain an extended coiled-coil tail domain.

Transient behavior of sea urchin sperm flagella following an abrupt change in beat frequency.

Within the approximate range of 30-80 Hz, the flagellar beat frequency of a sea urchin sperm held by its head in the tip of a micropipet is governed by the vibration frequency of the micropipet. We have imposed abrupt changes in flagellar beat frequency by changing the vibration frequency of the micropipet within this range and used a high-speed video system to analyze the flagellar wave parameters during the first few cycles following the change. Our results demonstrate that the various flagellar beat parameters differ in the time they take to adjust to the new conditions. The initiation rate of new bends at the base is directly governed by the frequency of the vibration and changes immediately to the new frequency. The length and the propagation velocity of the developed bends become adjusted to the new conditions within approximately 1 beat cycle, whereas the bend angles take more than 4 beat cycles to attain their new steady-state value. Bends initiated shortly before the change in frequency occurs attain a final length and angle that depends on the relative durations of growth at the old and new frequencies. Our results suggest that the flagellar wavelength and bend angle are regulated by different mechanisms with the second not being directly dependent on bend initiation.

Bidirectional swimming in spermatozoa of Tephritid flies.

Our observations show that spermatozoa of the Mediterranean fruit fly Ceratitis capitata and of Dacus oleae and Dacus dorsalis are capable of swimming backwards as well as forwards, and that they can change direction abruptly. The preferred direction is backwards, observed in spermatozoa obtained from the male genitalia. Forwards swimming spermatozoa were frequently seen in the spermatheca and close to the eggs. The change in swimming direction appears to be effected solely by a change in the direction of bend propagation, with no significant change in other waveform parameters. In vitro reactivated spermatozoa swim forwards only and require a minimum free Ca++ concentration of about 10(-6) M for movement. A switching of wave propagation from one direction to the other under control of intracellular free Ca++ concentration is suggested. Perhaps the backwards movement allows easier delivery of spermatozoa from the common envelope embedding the heads in the male apparatus, and assures a more efficient movement of the sperm towards the egg, especially given the enormous relative length of the head. The forwards movement is favoured in order to orient the sperm for penetration of the micropile.

Vanadate-sensitized cleavage of dynein heavy chains by 365-nm irradiation of demembranated sperm flagella and its effect on the flagellar motility.

Irradiation of demembranated flagella of sea urchin sperm at 365 nm in the presence of 0.05-1 mM MgATP and 5-10 microM vanadate (Vi) cleaves the alpha and beta heavy chains of the outer arm dynein at the same site and at about the same rate as reported previously for the solubilized dynein (Gibbons, I. R., Lee-Eiford, A., Mocz, G., Phillipson, C. A., Tang, W.-J. Y., and Gibbons, B. H. (1987) J. Biol. Chem. 262, 2780-2786). The decrease in intact alpha and beta heavy chain material is biphasic, with about 80% being lost with a half-time of 8-10 min, and the remainder more slowly. Five other axonemal polypeptides of Mr greater than 350,000 are lost similarly, concomitant with the appearance of at least 9 new peptides of Mr 150,000-250,000. The motility of irradiated sperm flagella upon subsequent dilution into reactivation medium containing 1 mM ATP and 2.5 mM catechol shows a progressive decrease in flagellar beat frequency for irradiation times that produce up to about 50% cleavage of the dynein heavy chains; more prolonged irradiation causes irreversible loss of motility. Competition between photocleaved and intact outer arm dynein for rebinding to dynein-depleted sperm flagella shows that cleavage has little effect upon the ability for rebinding, although the cleaved dynein partially inhibits subsequent motility. Substitution of MnATP for the MgATP in the irradiation medium prevents the loss of all of the axonemal polypeptides during irradiation for up to 60 min and also protects the potential for subsequent flagellar motility. It is concluded that loss of the five axonemal polypeptides upon irradiation results from a Vi-sensitized photocleavage similar to that which occurs in the alpha and beta heavy chains of outer arm dynein and that these polypeptides represent Vi-inhibitable ATPase subunits of dyneins located in the inner arms and possibly elsewhere in the flagellar axoneme.

Photosensitized cleavage of dynein heavy chains. Cleavage at the "V1 site" by irradiation at 365 nm in the presence of ATP and vanadate.

Irradiation of soluble dynein 1 from sea urchin sperm flagella at 365 nm in the presence of MgATP and 0.05-50 microM vanadate (Vi) cleaves the alpha and beta heavy chains (Mr 428,000) at their V1 sites to give peptides of Mr 228,000 and 200,000, without the nonspecific side effects produced by irradiation at 254 nm as described earlier (Lee-Eiford, A., Ow, R. A., and Gibbons, I. R. (1986) J. Biol. Chem. 261, 2337-2342). The decrease in intact heavy chain material is biphasic; in 10 microM Vi, approximately 80% occurs with a half-time of 7 min and the remainder with a half-time of about 90 min, and the yield of cleavage peptides is better than 90%. Loss of dynein ATPase activity appears to be a direct result of the cleavage process and is not significantly affected by the presence of up to 0.1 M cysteamine (CA, 60-23-1) or 2-aminoethyl carbamimidothioic acid dihydrobromide (CA, 56-10-0) as free radical trapping agents. The concentration of Vi required for 50% maximal initial cleavage rate is 4.5 microM, while that for 50% ATPase inhibition is 0.8 microM, both in a 0.6 M NaCl medium. In the presence of 20 microM Vi, CTP and UTP support cleavage at about half the rate of ATP, whereas GTP and ITP support cleavage only if the Vi concentration is raised to about 200 microM. Substitution of any of the transition metal cations Cr2+, Mn2+, Fe2+, or Co2+ for the usual Mg2+ suppresses the photocleavage, presumably by quenching the excited chromophore prior to scission of the heavy chain. The photocleaved dynein 1 binds to dynein-depleted flagella similarly to intact dynein 1, but upon reactivation of the flagella with 1 mM ATP their motility is partially inhibited, rather than being augmented as with intact dynein. These results indicate that Vi acts as a photosensitizing catalyst and suggest that the cleavage proceeds through excitation of Vi bound to dynein at the hydrolytic ATP binding site on each heavy chain, probably in a dynein X MgADP X Vi complex. The exquisite specificity of Vi-sensitized photocleavage will aid the peptide mapping of dynein heavy chains and may be of broader use in studies of protein structure.

Organic anions stabilize the reactivated motility of sperm flagella and the latency of dynein 1 ATPase activity.

Substitution of any of a variety of organic anions, including acetate, propionate, lactate, gluconate, and succinate, for chloride in the reactivation medium improves the motility of demembranated sperm of Tripneustes gratilla. At the optimum concentration of 0.20 N, all of these anions improve the duration of motility, with lactate and gluconate being the best. The Michaelis constant for beat frequency (Kmf) is lower (0.11-0.14 mM at 22 degrees C) in most of the organic anions than it is in Cl- (0.20 mM), and the minimum ATP concentration required to support oscillatory beating is reduced from 10 microM in chloride to 2 microM in acetate, which together indicate a greater affinity of the axonemal ATPase for MgATP2- in the organic anions media. The maximal beat frequency, fmax, is as high as 42 Hz in 0.2 N succinate compared to 31 Hz in Cl-, whereas the mean bend angle averages 2.8 rad in acetate compared to 2.4 rad in Cl-; these values give a calculated average velocity of tubule sliding of approximately 15 micron/s in acetate and succinate, which is approximately 30% greater than the value of 11 micron/s observed in chloride. The reactivated sperm are sixfold more sensitive to vanadate inhibition in 0.2 M acetate than they are in 0.15 M Cl-. The specific ATPase activity of soluble dynein 1, which increases more than 15-fold between 0 and 1.0 N Cl-, undergoes only a twofold activation over the same range of organic anion concentration, and, like the reactivated motility, is up to 50-fold more sensitive to vanadate. This greater apparent mechanochemical efficiency and the increased sensitivity to vanadate inhibition in the organic anions suggest that they, unlike chloride, do not promote the spontaneous dissociation of ADP and PO4(3-) from the dynein-ADP-PO4 kinetic intermediate in the dynein crossbridge cycle. The use of organic anion media may lead to significant improvements in reactivation of other motile and transport systems.

Live and reactivated motility in the 9+0 flagellum of Anguilla sperm.

The sperm flagella of the eel, Anguilla anguilla, are capable of vigorous motion in spite of having an axoneme with reduced structure that lacks the outer dynein arms, radial spokes and spoke heads, the two central tubules and the central tubule projections that are all part of the standard "9+2" axoneme. These sperm progress forward rapidly as a result of the propagation of helicoidal waves distally along the flagellum. Their flagellar beat frequencies are high, 93 Hz at 21 degrees C, and they roll at a frequency of about 19 Hz. Eel sperm could be demembranated with Nonidet P-40 and reactivated with MgATP2- in 0.22 M K acetate at pH 8.1. The reactivated motility closely resembles that of the live sperm, with a beat frequency of 69 Hz, but the demembranated flagella are unusually fragile, and commonly disintegrate by a combination of splitting, coiling, and sliding within a few minutes. Little reactivation is obtained if acetate is replaced by Cl- in the reactivating medium. The Michaelis constant for beat frequency (0.2 mM) is similar to that obtained for several "9+2" flagella. These sperm, however, appear to lack the mechanism by which Ca2+ regulates waveform. Our results indicate that eel sperm flagella, which at rest are straight, are induced to bend helicoidally by ATP, as the result of sliding between tubules that is blocked at both the base and tip of the organelle. The flagellar waveform consists of a series of planar bends separated by short regions of right-handed twist, which give it an overall left-handed helicoidal form.

Lithium reversibly inhibits microtubule-based motility in sperm flagella.

The sliding-tubule hypothesis of flagellar movement has strong experimental support, but our present knowledge of the mechanism by which this sliding is coordinated and converted into flagellar oscillation and bend propagation is quite limited. Among the few facts known are: (1) calcium has a role in regulating the asymmetry of flagellar waveform, (2) the initiation or activation of motility in spermatozoa from several species involves a cyclic AMP-dependent phosphorylation reaction, and (3) the conversion of tubule sliding to bending does not require the radial spokes or central tubules. Inhibitors are valuable tools for investigating mechanisms involved in cellular function, and we report here that Li+ in low concentrations reversibly inhibits the microtubule-based movement of reactivated sea urchin sperm flagella. The evidence indicates that the action of Li+ is directed primarily towards one or more regulatory sites through which Ca2+ modulates the asymmetry of flagellar waveform, rather than towards dynein ATPase itself. Lithium also appears to inhibit the sperm adenylate cyclase, but this action does not seem to be relevant to its inhibition of normal motility. Our findings indicate the need for considerable caution when using ATP analogues supplied as the Li+ salt.

Structure and motility of the 9 + 0 flagellum of eel spermatozoa.

Scanning and electron microscopic study of the spermatozoon of the eel, Anguilla anguilla, has shown that the large oblong head is attached eccentrically by one end to the basal end of the true flagellum and contains a pseudoflagellum that splits into two groups of tubules as it passes around the nucleus. The 9+0 axoneme of the true flagellum lacks outer dynein arms and the radial spoke complex. The movement of Anguilla sperm is characterized by rapid forward progression that is the result of left-handed helicoidal waves propagated distally at a beat frequency at 21 degrees of about 95 Hz. Thus, these sperm flagella, although reduced in structure, propagate three dimensional rather than planar waves as in sea urchin sperm flagella, and beat at a frequency which so far as we know is the highest observed in eukaryotic flagella.

Effects of organic solvents on flagellar asymmetry and quiescence in sea urchin sperm.

Low concentrations of methanol, 2-propanol and ethylene glycol increase the asymmetry of the flagellar waveforms ad the turning rate of both live sperm and potentially symmetrical sperm reactivated with 1 mM-MgATP2-, while at the same time causing a decrease in the heat frequency. Similar effects are observed if the solvents are added to preparations of potentially symmetrical sperm reactivated in the presence of 1 mM free Ca2+, or to potentially asymmetrical sperm reactivated without added Ca2+, A second group of solvents, N,N-dimethylformamide, formamide and p-dioxane, also decrease the flagellar beat frequency, but have the opposite effect on symmetry, reducing the asymmetry of the waveforms and the turning rate of potentially symmetrical sperm reactivated in the presence of 1 mM free Ca2+. These effects of solvents are all reversible within about 5 min after initial exposure to solvent. Higher concentrations of methanol and 2-propanol (above approximately 5 and 0.8 mole %, respectively) induce quiescence in potentially asymmetrical sperm reactivated with concentrations of MgATP2- ranging from 10 microM to 1 mM. The quiescent flagella initially assume a bent form very similar to that seen in Ca2+-induced quiescence, and show a subsequent time-dependent distortion of the initial bent from with eventual disintegration and splitting off of bundles of microtubules. Dimethylformamide, formamide and dioxane have almost no effect on the intrinsic asymmetry of potentially asymmetrical sperm reactivated in the absence of added Ca2+, but addition of these solvents to potentially asymmetrical sperm that have been induced to become quiescent by addition of 0.1 mM free Ca2+ causes the sperm to resume swimming with flagellar waveforms that are substantially more symmetrical that those of the starting preparation before the addition of Ca2+. Mild digestion with trypsin of reactivated sperm that have been induced either to beat asymmetrically or to become quiescent by addition of methanol causes a gradual appearance of symmetrical flagellar beating, as in the case of Ca2+-induced quiescence. The flagellar beat frequency, however, remains low, at about 20 Hz. The results suggest that the solvents either mimic or block the action of CA2+ by interaction with a Ca2+-dependent regulatory protein, and may also induce alteration in the rate constants of dynein ATPase.

Transient flagellar waveforms during intermittent swimming in sea urchin sperm. I. Wave parameters.

Flagellar waveforms have been studied during the stopping and starting transients of light-induced, Ca2+-mediated, intermittent swimming of live sperm of the sea urchin Tripneustes gratilla. Tracings of successive frames of movie film made at about 200 frames s-1 were used to determine the bend propagation velocity, beat frequency, and bend angles during three stopping and four starting transients chosen as representative of the range of variation among sperm in the preparations. A stopping transient begins with a transitional stage in which the asymmetry of the bending waves increases steadily over 2-6 beat cycles (40-120 ms), with the angles of successive fully developed principal bends increasing and those of reverse bends decreasing. This is followed by a blocked stage, lasting one beat cycle (20 ms), in which a principal bend becomes arrested and then decays in the mid-region of the flagellum. The next principal bend forms but remains unpropagated at the base, apparently because no following reverse bend is initiated, and the flagellum becomes quiescent. Quiescent flagella have a characteristic, highly asymmetric waveform consisting of a sharp principal bend of about 3.2 rad at the basal end, a nearly straight mid-region and a gentle principal bend of about 0.4 rad near the tip. After a quiescent period of 0.2-2 s, motility is resumed with the initiation of a new reverse bend at the base. This bend and the proximal principal bend remaining from quiescence begin to propagate but they decay before passing more than halfway along the flagellum. In this blocked stage of the starting transient, which lasts 1-15 beat cycles (20-300 ms), successive principal and reverse bends are propagated progressively further along the flagellum but they decay before reaching the tip and the asymmetry remains at the high value characteristic of quiescence. The first propagation of a principal bend to the tip marks the beginning of the transitional stage of the transient, during which the asymmetry of the bending waves gradually decreases until after 2-5 beat cycles (40-100 ms) it reaches the value characteristic of steady-state beating. In both stopping and starting flagella the beat frequency and the mean of the principal and reverse bend angles remain constant throughout the transient (except for the beat cycle immediately pre- or post-quiescence), indicating that they are regulated by mechanisms almost completely independent of that regulating wave symmetry. The bend propagation velocity remains constant during stopping transients but it is diminished during the blocked stage of starting transients, indicating that the bend velocity, and hence the wavelength, can be altered by changes in the internal resistance to bend propagation.

Intermittent swimming in live sea urchin sperm.

Sperm of the sea urchin Tripneustes gratilla repeatedly start and stop swimming when suspended in seawater and observed by dark-field microscopy. While in the quiescent state, which usually lasts about a second, the sperm assume s shape resembling a cane, with a sharp bend of approximately 3.4 rad in the proximal region of the flagellum and very little curvature in the rest of the flagellum except for a slight curve near the tip. The occurrence of quiescence requires the presence of at least 2 mM Ca2+ in the seawater, and the percentage of sperm quiescent at any one time increases substantially when the sperm are illuminated with blue light. With intense illumination, close to 100% of the sperm become quiescent, and this percentage decreases gradually to approximately 0.3% over a 10(4)-fold decrease in light intensity. An increased concentration of K+ in the seawater also increases the percentage of quiescence, with a majority of the sperm being quiescent in seawater containing 80 mM KCl. The induction of quiescence by light or by increased KCl is completely inhibited by 10 micrometers chlorpromazine, and approximately 90% inhibited by 1 mM procaine or sodium barbital. Sperm treated with the divalent-cation ionophore A23187 swim quite normally, although for a relatively short period, in artificial seawater lacking divalent cations, but are abruptly arrested upon addition of 0.04--0.2 mM free Ca2%. The flagellar waveform of these arrested sperm is almost identical to that of light-induced quiescence in the live sperm. The results support the hypothesis that quiescence is induced by a rise in intracellular Ca2%, perhaps as a consequence of a membrane depolarization, and that it is similar to the arrest response in cilia.

Calcium-induced quiescence in reactivated sea urchin sperm.

Sperm flagella of the sea urchin Tripneustes gratilla beat with asymmetrical bending waves after demembranation with Triton X-100 in the presence of EGTA and reactivation at pH 8.1 with 1 mM ATP in the presence of 2 mM MgSO4. Addition of 0.1--0.2 mM free Ca2+ to these reactivated sperm induces 70--95% of them to become quiescent. This quiescence can be reversed by reduction of the free Ca2% concentration with EGTA, or by dilution to reduce the MgATP2- concentration below 0.3 mM. The quiescent waveform is characterized by a sharp principal bend of approximately 5.6 rad in the proximal region of the flagellum, a slight reverse bend in the midregion that averages approximately 0.3 rad, and a principal bend of approximately 1.1 rad in the tip. The quiescent sperm are highly fragile mechanically, and disruption, including microtubule sliding, occurs spontaneously at a slow rate upon standing or immediately upon gentle agitation. Mild digestion by trypsin causes a gradual appearance of normal, symmetrical flagellar beating. Addition of increasing concentrations of vanadate to quiescent sperm causes a graded decrease in the proximal bend angle, with 50 micrometers vanadate reducing it to approximately 2.6 rad. In the presence of 0.1 mM free Ca2% and 10 micrometers vanadate, a characteristic, crescented stationary bend is induced in the demembranated sperm, without intermediate oscillatory beating, by the addition of either 0.1 or 1 mM ATP. In the absence of vanadate, these two concentrations of ATP produce asymmetric beating and quiescence, respectively. The results support the hypothesis that quiescence in live sperm is induced by an elevated concentration of intracellular Ca2%. In addition, they demonstrate that bending can occur in flagella in which oscillatory beating is inhibited and emphasize the close relationship between asymmetric beating and quiescence.