Bull sperm 19S dynein polymerizes brain tubulin into microtubules

Crude dynein extracted from bull sperm flagella polymerized pure phosphocellulose tubulin isolated from brain tissues into microtubules. This effect was predominantly due to the 19S dynein particle in the extract. ATP stimulated up to five fold the polymerization of brain tubulin by bull sperm dynein. Hydrolysis of ATP was not required since vanadate at a concentration sufficient to block dynein ATPase activity did not interfere with ATP stimulation and because the non hydrolyzable ATP analogue adenylyl (beta-gamma-methylene) diphosphate (AMPPCP) had effects similar to those of ATP. These results suggest that, in addition to hydrolyzing ATP to generate the driving force necessary for microtubule sliding within the axoneme, dynein may also interact with ATP to polymerize tubulin into microtubules.     

An antiserum to the sea urchin 20 S egg dynein reacts with embryonic ciliary dynein but it does not react with the mitotic apparatus

Unfertilized sea urchin eggs contain one or more dynein-like enzymes which may be able to serve as microtubule translocators during embryonic development. There are at least two interesting possibilities for the function of the egg dynein: the enzyme may be involved in cytoplasmic microtubule movement such as mitotic spindle anaphase motion; or the enzyme may be a stored precursor for the dynein that functions in embryonic cilia, which are expressed and highly motile at the blastula stage of development. In order to determine directly the distribution and possible function of one of the previously described egg dyneins, the latent-activity 20 S egg dynein (Asai and Wilson, 1985), an antiserum was produced which was highly reactive with the important high Mr polypeptides of 20 S dynein. This antiserum reacted in "Western" immunoblots and in dot-blotting experiments with egg dynein and with embryonic ciliary dynein, but it did not react with any component of sperm flagella. Indirect double immunofluorescence microscopy demonstrated that the anti-20 S antiserum could brightly stain embryonic cilia but it did not stain the sperm flagella from the same sea urchin species. Under the same conditions that the antiserum stained cilia, anti-20 S did not stain the mitotic apparatus but it did appear to stain the cortical region of the dividing egg. In a time-course experiment, the antigen reactive with the anti-20 S antiserum gradually accumulated in the developing early sea urchin embryo. The most significant increase in the apparent concentration of the 20 S dynein occurred just prior to embryonic ciliation and during a period when the mitotic activity of the embryo was in decline. These results lead to two conclusions. First, ciliary dynein and sperm flagellar dynein, although derived from very similar organelles and from the same species of sea urchin, are immunologically distinct. Second, the 20 S egg dynein may be a stored precursor of embryonic ciliary dynein and does not appear to be a component of the mitotic apparatus.     

Properties of an antiserum against native dynein 1 from sea urchin sperm flagella

Effects of an antiserum against native dynein 1 from sperm flagella of the sea urchin Strongylocentrotus purpuratus were compared with effects of an antiserum previously obtained against an ATPase-active tryptic fragment (fragment 1A) of dynein 1 from sperm flagella of the sea urchin, Anthocidaris crassispina. Both antisera precipitate dynein 1 and do not precipitate dynein 2. Only the fragment 1A antiserum precipitates fragment 1A and produces a measurable inhibition of dynein 1 ATPase activity. Both antisera inhibit the movement and the movement-coupled ATP dephosphorylation of reactivated spermatozoa. The inhibition of movement by the antiserum against dynein 1 is much less than by the antiserum against fragment 1A, suggesting that a specific interference with the active ATPase site may be required for effective inhibition of movement. Both antisera reduce the bend angle as well as the beat frequency of reactivated S. purpuratus spermatozoa, suggesting that the bend angle may depend on the activity of the dynein arms which generate active sliding.     

Molecular characterization of Ciona sperm outer arm dynein reveals multiple components related to outer arm docking complex protein 2

Using proteomic and immunochemical techniques, we have identified the light and intermediate chains (IC) of outer arm dynein from sperm axonemes of the ascidian Ciona intestinalis. Ciona outer arm dynein contains six light chains (LC) including a leucine-rich repeat protein, Tctex1- and Tctex2-related proteins, a protein similar to Drosophila roadblock and two components related to Chlamydomonas LC8. No LC with thioredoxin domains is included in Ciona outer arm dynein. Among the five ICs in Ciona, three are orthologs of those in sea urchin dynein: two are WD-repeat proteins and the third one, unique to metazoan sperm flagella, contains both thioredoxin and nucleoside diphosphate kinase modules. The remaining two Ciona ICs have extensive coiled coil structure and show sequence similarity to outer arm dynein docking complex protein 2 (DC2) that was first identified in Chlamydomonas flagella. We recently identified a third DC2-like protein with coiled coil structure, Ci-Axp66.0 that is also associated in substoichiometric amounts with Ciona outer arm dynein. In addition, Oda5p, a component of an additional complex required for assembly of outer arm dynein in Chlamydomonas flagella, also groups with this family of DC2-like proteins. Thus, the assembly of outer arm dynein onto doublet microtubules involves multiple coiled-coil proteins related to DC2.     

Evidence that the Mg-ATPase in the cortical layer of sea urchin egg is dynein

Indirect immunofluorescence staining of cleaving sea urchin eggs with an antiserum against a tryptic fragment of dynein 1 (fragment 1A) from sea urchin sperm flagella suggested the presence of dynein in the cortex as well as in the mitotic apparatus. In the present study, we found that the Mg²⁺-ATPase activity of the isolated cortices from sea urchin eggs, which exhibited similar characteristics to those of flagellar dynein, was inhibited by 60–80% with the anti-fragment 1A serum. Faintly stained bands corresponding to the A-band (dynein 1) and the B-band of the sperm flagella was detected on sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis of the isolated cortices. Furthermore, the SDS-gel electrophoresis revealed the presence of a polypeptide band corresponding to dynein 1 in the antigen-antibody complex precipitated from the KCl-extract of the cortices with the antiserum.     

Substructure of sea urchin egg cytoplasmic dynein

The substructure of the cytoplasmic dynein molecule was studied using the quick-freeze, deep-etch technique. Cytoplasmic dynein purified as a 12 S form from the eggs of the sea urchin Hemicentrotus pulcherrimus was composed of a single high molecular weight polypeptide. Rotary shadowing images of cytoplasmic dynein either sprayed on to a mica surface or quick-frozen on mica flakes demonstrated a single-headed molecule, in contrast to the two-headed molecule of sea urchin sperm flagellar 21 S dynein. More detailed substructure was visualized by rotary shadowing after quick-freeze deep-etching. Cytoplasmic dynein consisted of a head and a stem. The head was pear-shaped (16 nm X 11 nm) and a little smaller than the pear-shaped head of 21 S dynein (18 nm X 14 nm). The form of the stem was irregular, and its apparent length varied from 0 to 32 nm. Binding of cytoplasmic dynein to brain microtubule in the solution was observed by negative staining, and that in the precipitate was examined by the quick-freeze, deep-etch method as well. Both methods revealed the presence of two kinds of microtubules, one a fully decorated microtubule and the other a non-decorated microtubule. Cytoplasmic dynein bound to microtubule also appeared as a globular particle. Neither the periodic binding nor the crossbridges that were observed with 21 S dynein were formed by cytoplasmic dynein, although cytoplasmic dynein appeared to bind to microtubules co-operatively.     

Inner arm dynein ATPase fraction of sea urchin sperm flagella causes active sliding of axonemal outer doublet microtubule.

In order to clarify the role of the inner arms of the axoneme in sperm flagellar movement, we prepared an ATPase fraction (12S) from the outer arm-depleted axonemes of sea urchin sperm flagella. When both arm-depleted axonemes were incubated with the 12S ATPase, they exhibited the sliding disintegration of outer doublet microtubules. Electron microscopy revealed that the ATPase rebound to the original inner arm sites of the axoneme. Therefore, it is quite likely that the 12S ATPase is one of the components of the inner arms. We referred to it as "inner arm dynein".     

Bending patterns of ATP-reactivated sea urchin sperm flagella following high salt extraction for removal of outer dynein arms

Two procedures were used for extraction of demembranated sea urchin sperm flagella with increased KCl concentrations, to remove outer dynein arms. Extraction with 0.55 M KCl in the Triton-demembranation solution produced a rapid fall in average sliding velocity to 50% of its unextracted value, with extensive changes in bending behavior of the distal half of the flagellum. Extraction with 0.42 M KCl following demembranation and activation by incubation with cAMP produced a more gradual fall in sliding velocity, reaching 50% of the unextracted value after 180 sec extraction. This procedure produced somewhat more normal bending patterns. In both cases, the bending pattern of the basal region of the flagellum is altered very little by extraction, in agreement with data from Chlamydomonas mutant flagella deficient in outer arm dyneins.     

Testing the geometric clutch hypothesis

The Geometric Clutch hypothesis is based on the premise that transverse forces (t-forces) acting on the outer doublets of the eukaryotic axoneme coordinate the action of the dynein motors to produce flagellar and ciliary beating. T-forces result from tension and compression on the outer doublets when a bend is present on the flagellum or cilium. The t-force acts to pry the doublets apart in an active bend, and push the doublets together when the flagellum is passively bent and thus could engage and disengage the dynein motors. Computed simulations of this working mechanism have reproduced the beating pattern of simple cilia and flagella, and of mammalian sperm. Cilia-like beating, with a clearly defined effective and recovery stroke, can be generated using one uniformly applied switching algorithm. When the mechanical properties and dimensions appropriate to a specific flagellum are incorporated into the model the same algorithm can simulate a sea urchin or bull sperm-like beat. The computed model reproduces many of the observed behaviors of real flagella and cilia. The model can duplicate the results of outer arm extraction experiments in cilia and predicted two types of arrest behavior that were verified experimentally in bull sperm. It also successfully predicted the experimentally determined nexin elasticity. Calculations based on live and reactivated sea urchin and bull sperm yielded a value of 0.5 nN/microm for the t-force at the switch-point. This is a force sufficient to overcome the shearing force generated by all the dyneins on one micron of outer doublet. A t-force of this magnitude should produce substantial distortion of the axoneme at the switch-point, especially in spoke or spoke-head deficient motile flagella. This concrete and verifiable prediction is within the grasp of recent advances in imaging technology, specifically cryoelectron microscopy and atomic force microscopy.     

Dynein 2. A new adenosine triphosphatase from sea urchin sperm flagella.

A new ATPase electrophoretically and immunologically distinct from the dynein ATPase studied previously has been solublized and purified from sea urchin sperm flagella. This ATPase has properties similar to those of dynein ATPase. Therefore, we propose that the two ATPases be considered as dynein isoenzymes, with previously studied dynein being known as dynein 1, and the newly discovered ATPase as dynein 2. Some physicochemical and enzymatic properties of dynein 2 have been determined. The molecular weight calculated from the sedimentation coefficient (12.3 "/- 1 S) and Stokes radius (12.8 "/- 0.4 nm) is 690,000 +/- 70,000. The molecular weight of the high molecular weight subunit of dynein 2 has been determined to be 325,000 +/- 40,000 by Na dodecyl-SO4-polyacrylamide gel electrophoresis. The enzymatic properties of dynein 1 and dynein 2 are similar in substrate specificity, pH optimum, and Mg2+ requirement for ATPase activity, but they differ in their Michaelis constant and in their dependence of ATPase activity upon salt concentration. Digestion of dynein 2 with trypsin yields an ATPase-containing protein fragment, similar to Fragment A obtained from dynein 1. An antiserum prepared against Fragment A from dynein 1 did not precipitate dynein 2 or inhibit its ATPase activity.     

The substructure of isolated and in situ outer dynein arms of sea urchin sperm flagella

Outer-arm dynein from the sperm of the sea urchin S. purpuratus was adsorbed to mica flakes and visualized by the quick-freeze, deep-etch technique. Replicas reveal particles comprised of two globular heads joined by two irregularly shaped stems which make contact along their length. One head is pear-shaped (18.5 X 12.5 nm) and the other is spherical (14.5-nm diam). The stems are decorated by a complex of bead-like subunits. The same two-headed protein is found in the 21S dynein-1 fraction of sucrose gradients. The beta-heavy chain/intermediate chain 1 (beta/IC-1) dynein subfraction, produced by low-salt dialysis and zonal centrifugation of the high-salt-extracted dynein-1, contains only single-headed molecules with single stems. These heads are predominantly pear-shaped (18.5 X 12.5 nm). Since 21S dynein-1 contains two heavy chains (alpha and beta), and the beta/IC-1 subfraction is comprised of only the beta-heavy chain (Tang et al., 1982, J. Biol. Chem. 257: 508-515), we conclude that each head is formed by a heavy chain, that the pear-shaped head contains the beta-heavy chain, and that the spherical head contains the alpha-heavy chain. The in situ outer dynein arms of demembranated sperm were also studied by the quick-freeze, deep-etch method. When frozen in reactivation buffer devoid of ATP, each arm consists of a large globular head that attaches to the A-microtubule by distally skewed subunits and attaches to the B-microtubule by a slender stalk. In ATP, this head shifts its orientation such that it can be seen to be constructed from two globular domains. We offer possible correlates between the in situ and the in vitro images, and we compare the structure of sea-urchin dynein with dynein previously described from Chlamydomonas and Tetrahymena.     

A monoclonal antibody against the dynein IC1 peptide of sea urchin spermatozoa inhibits the motility of sea urchin, dinoflagellate, and human flagellar axonemes.

To investigate the role of axonemal components in the mechanics and regulation of flagellar movement, we have generated a series of monoclonal antibodies (mAb) against sea urchin (Lytechinus pictus) sperm axonemal proteins, selected for their ability to inhibit the motility of demembranated sperm models. One of these antibodies, mAb D1, recognizes an antigen of 142 kDa on blots of sea urchin axonemal proteins and of purified outer arm dynein, suggesting that it acts by binding to the heaviest intermediate chain (IC1) of the dynein arm. mAb D1 blocks the motility of demembranated sea urchin spermatozoa by modifying the beating amplitude and shear angle without affecting the ATPase activity of purified dynein or of demembranated immotile spermatozoa. Furthermore, mAb D1 had only a marginal effect on the velocity of sliding microtubules in trypsin-treated axonemes. This antibody was also capable of inhibiting the motility of flagella of Oxyrrhis marina, a primitive dinoflagellate, and those of demembranated human spermatozoa. Localization of the antigen recognized by mAb D1 by immunofluorescence reveals its presence on the axonemes of flagella from sea urchin spermatozoa and O. marina but not on the cortical microtubule network of the dinoflagellate. These results are consistent with a dynamic role for the dynein intermediate chain IC1 in the bending and/or wave propagation of flagellar axonemes.     

A bipartite Ca2+-regulated nucleoside-diphosphate kinase system within the Chlamydomonas flagellum. The regulatory subunit p72

Regulation of flagellar activity in Chlamydomonas involves both Ca(2+) and cAMP-mediated signaling pathways. However, Chlamydomonas and sea urchin sperm flagella also exhibit nucleoside-diphosphate kinase (NDK) activity, suggesting a requirement for GTP within this highly conserved organelle. In sea urchin sperm, the NDK catalytic subunit is an integral component of the outer dynein arm. Here we describe a modular protein (p72) from the Chlamydomonas flagellum that consists of three domains closely related to the presumptive regulatory segment of rat NDK-7 followed by two EF-hands that are predicted to bind Ca(2+). There are close homologues of p72 in both mammalian and insect genomes. The p72 protein is tightly associated with the flagellar axoneme and is located along the entire length except at the transition zone. Cross-linking experiments suggest that p72 interacts with two or three additional axonemal polypeptides. The sensitivity of p72 to tryptic digestion differed considerably in the presence and the absence of Ca(2+), suggesting that it indeed binds this ligand. These studies indicate that the flagellar NDK system is bipartite with the regulatory and catalytic components residing on different polypeptides. We propose that Ca(2+) regulation of flagellar motility in Chlamydomonas may be achieved in part through a downstream GTP-mediated signaling pathway.     

Structure and mass of mammalian respiratory ciliary outer arm 19S dynein

Mammalian respiratory ciliary outer arm dyneins isolated as the major ATPase peak migrating at 19S on sucrose density gradients were examined by transmission electron microscopy of negatively stained samples and scanning transmission electron microscopy of unstained samples. The predominant discrete particle structure observed was composed of two globular heads apparently connected by amorphous or indistinct material. The heads were either circular or slightly elliptical of mean 13 +/- 1 X 10 +/- 2 nm dimensions. The mass of this structure averaged 1.22 +/- 0.34 million daltons with the individual globular heads averaging 310 +/- 77 kilodaltons (kD). Negative staining revealed that one or both of the globular heads often contained a central accumulation of stain measuring 2.5 +/- 1 nm across. A second type of structure, appearing with lesser frequency in the 19S fraction than in the unfractionated dynein preparation loaded onto the sucrose gradient, was a single globular head of 13 +/- 1 X 10 +/- 2 nm often with 2 +/- 1 nm centrally accumulated stain and with or without an appendage. This one-headed particle thus resembled one-half of the two-headed particle. Mass measurements were lower, however, for isolated, single globular heads, averaging 220 +/- 111 kD. A third type of particle observed was a ring-like structure with 4 +/- 1 nm centrally accumulated stain and without appendages. The ring structure was slightly larger in diameter, 14 +/- 1 nm, and had a greater peripheral accumulation of negative stain than either of the one- or two-headed particles, suggesting that it was not derived therefrom.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural-functional relationships of the dynein,spokes, and central-pair projections predicted from an analysis of the forces acting within a flagellum

In the axoneme of eukaryotic flagella the dynein motor proteins form crossbridges between the outer doublet microtubules. These motor proteins generate force that accumulates as linear tension, or compression, on the doublets. When tension or compression is present on a curved microtubule, a force per unit length develops in the plane of bending and is transverse to the long axis of the microtubule. This transverse force (t-force) is evaluated here using available experimental evidence from sea urchin sperm and bull sperm. At or near the switch point for beat reversal, the t-force is in the range of 0.25-1.0 nN/ micro m, with 0.5 nN/ micro m the most likely value. This is the case in both beating and arrested bull sperm and in beating sea urchin sperm. The total force that can be generated (or resisted) by all the dyneins on one micron of outer doublet is also approximately 0.5 nN. The equivalence of the maximum dynein force/ micro m and t-force/ micro m at the switch point may have important consequences. Firstly, the t-force acting on the doublets near the switch point of the flagellar beat is sufficiently strong that it could terminate the action of the dyneins directly by strongly favoring the detached state and precipitating a cascade of detachment from the adjacent doublet. Secondly, after dynein release occurs, the radial spokes and central-pair apparatus are the structures that must carry the t-force. The spokes attached to the central-pair projections will bear most of the load. The central-pair projections are well-positioned for this role, and they are suitably configured to regulate the amount of axoneme distortion that occurs during switching. However, to fulfill this role without preventing flagellar bend formation, moveable attachments that behave like processive motor proteins must mediate the attachment between the spoke heads and the central-pair structure.     

Proteins associated with soluble adenylyl cyclase in sea urchin sperm flagella

Adenylyl cyclases (ACs) synthesize cAMP and are present in cells as transmembrane AC and soluble AC (sAC). In sperm, the cAMP produced regulates ion channels and it also activates protein kinase-A that in turn phosphorylates specific axonemal proteins to activate flagellar motility. In mammalian sperm, sAC localizes to the midpiece of flagella, whereas in sea urchin sperm sAC is along the entire flagellum. Here we show that in sea urchin sperm, sAC is complexed with proteins of the plasma membrane and axoneme. Immunoprecipitation shows that a minimum of 10 proteins is tightly associated with sAC. Mass spectrometry of peptides derived from these proteins shows them to be: axonemal dynein heavy chains 7 and 9, sperm specific Na+/H+ exchanger, cyclic nucleotide-gated ion channel, sperm specific creatine kinase, membrane bound guanylyl cyclase, cyclic GMP specific phosphodiesterase 5A, the receptor for the egg peptide speract, and alpha- and beta-tubulins. The sAC-associated proteins could be important in linking membrane signal transduction to energy utilisation in the regulation of flagellar motility.     

Monoclonal antibodies to dynein subunits reveal the existence of cytoplasmic antigens in sea urchin egg

Monoclonal antibodies directed against subunits of a sea urchin flagellar dynein were used to test for the presence of cytoplasmic antigens in preparations of fertilized eggs and mitotic apparati . A 9-10 S complex composed of 330,000-, 134,000-, and 126,000-mol-wt subunits was isolated from outer arms of Strongylocentrotus purpuratus sperm flagella and used to characterize the antibodies. Seven monospecific antibodies to the 330,000 subunit and two against the 134,000 subunit of the 9-10 S complex were identified by binding to nitrocellulose blots of electrophoretograms resolving polypeptides from different dynein preparations. The antibodies were applied also to blots of polypeptides from fertilized sea urchin egg at the first metaphase and a cellular fraction of mitotic apparati . Three of the antibodies to the 330,000 subunit bound to a cytoplasmic polypeptide of approximately the same molecular weight and the two antibodies to the smaller subunits recognized a polypeptide of 124,000 apparent molecular weight. Both antigens appeared to be enriched in the fraction containing mitotic apparati . These results indicate that polypeptides similar to two subunits of the 9-10 S complex are present in eggs at metaphase, and they are apparently associated with the mitotic apparatus.     

Tctex2-related outer arm dynein light chain is phosphorylated at activation of sperm motility

When the motility of sperm is activated, only one light chain of flagellar outer arm dynein is phosphorylated in many organisms. We show here that the light chain to be phosphorylated was shown to be light chain 2 (LC2) in rainbow trout and chum salmon sperm and LC1 in sea urchin sperm. Molecular analyses of the phosphorylated light chains from sperm flagella of the salmonid fishes and sea urchin revealed that the light chains are homologs of the mouse t complex-encoded protein Tctex2, which is one of the putative t complex distorters. These results suggest that mouse Tctex2 might also be a light chain of flagellar outer arm dynein and that the abortive phosphorylation of Tctex2/outer arm dynein light chain might be related to the less progressive movement of sperm.     

Anthraniloyl ATP, a fluorescent analog of ATP, as a substrate for dynein ATPase and flagellar motility

We synthesized an anthraniloyl ATP (ant-ATP), which has a fluorescent anthraniloyl moiety at the OH group of ribose, to elucidate the mechanism of flagellar bend formation and its propagation in relation to the mechanochemical cycle of dynein ATPase. This fluorescent analog of ATP was efficiently hydrolyzed by 21 S dynein from sea urchin sperm flagella with Km = 7.6 microM, whereas the Km was 12 microM when ATP was used as the substrate. Similar Vmax values were obtained with both ATP and ant-ATP. Inhibition of the hydrolysis of ant-ATP by vanadate was a little smaller than that with ATP. Photosensitized cleavage of 21 S dynein heavy chains in the presence of ant-ATP and vanadate was also a little less efficient than that in the presence of ATP and vanadate. Ant-ATP also induced the disintegration of the trypsin-treated axoneme and the motility of demembranated sperm in a manner similar to ATP. When ATP was used as a substrate for the demembranated sperm, the apparent Michaelis constant for beat frequency (Km f) was 0.22 mM and the maximum frequency (fmax) was 36 Hz, whereas Km f) was 0.14 mM and fmax was 20 Hz for ant-ATP. Thus ant-ATP could be an efficient fluorescent analog of ATP for studying dynein ATPase and the mechanisms of flagellar motility.     

Two heavy chains of 21S dynein from sea urchin sperm flagella

The biochemical properties of 21S dynein derived from sea urchin sperm flagella and of its components dissociated by low salt treatment were studied. SDS-urea gel electrophoresis and two-dimensional gel electrophoresis showed that the 21S dynein preparation contains two distinct heavy chains. These two heavy chains, termed A alpha and A beta, had apparently the same molecular weight of 500,000 but showed different mobilities on SDS-urea gels. The isoelectric points of A alpha and A beta heavy chains were 5.7 and 5.2, respectively, in the presence of urea. Proteolytic digestion patterns of these two heavy chains were clearly different, but the amino acid compositions were similar. Low salt treatment and sucrose density gradient centrifugation could partially separate the components of 21S dynein into two fractions: the one with larger sedimentation coefficient contained the A alpha heavy chain, and the other with smaller sedimentation coefficient contained the A beta heavy chain and three intermediate chains. These two fractions showed distinctly different kinetic properties, and thus may play different roles in dynein-microtubule interaction.