Distinct regulation of two flagella by calcium during chemotaxis of male gametes in the brown alga Mutimo cylindricus (Cutleriaceae, Tilopteridales).

Brown algal male gametes show chemotaxis to the sex pheromone that is released from female gametes. The chemotactic behavior of the male gametes is controlled by the changes in the beating of two flagella known as the anterior and posterior flagellum. Our previous study using Mutimo cylindricus showed that the sex pheromone induced an increment in both the deflection angle of the anterior flagellum and sustained unilateral bend of the posterior flagellum, but the mechanisms regulating these two flagellar waveforms were not fully revealed. In this study, we analyzed the changes in swimming path and flagellar waveforms with a high-speed recording system under different calcium conditions. The extracellular Ca2+ concentration at 10-3 M caused an increment in the deflection angle of the anterior flagellum only when ionomycin was absent. No sustained unilateral bend of the posterior flagellum was induced either in the absence or presence of ionomycin in extracellular Ca2+ concentrations below 10-2 M. Real-time Ca2+ imaging revealed that there is a spot near the basal part of anterior flagellum showing higher Ca2+ than in the other parts of the cell. The intensity of the spot slightly decreased when male gametes were treated with the sex pheromone. These results suggest that Ca2+-dependent changes in the anterior and posterior flagellum are regulated by distinct mechanisms and that the increase in the anterior flagellar deflection angle and sustained unilateral bend of the posterior flagellum may not be primarily induced by the Ca2+ concentration.

Nexin-Dynein regulatory complex component DRC7 but not FBXL13 is required for sperm flagellum formation and male fertility in mice.

Flagella and cilia are evolutionarily conserved cellular organelles. Abnormal formation or motility of these organelles in humans causes several syndromic diseases termed ciliopathies. The central component of flagella and cilia is the axoneme that is composed of the '9+2' microtubule arrangement, dynein arms, radial spokes, and the Nexin-Dynein Regulatory Complex (N-DRC). The N-DRC is localized between doublet microtubules and has been extensively studied in the unicellular flagellate Chlamydomonas. Recently, it has been reported that TCTE1 (DRC5), a component of the N-DRC, is essential for proper sperm motility and male fertility in mice. Further, TCTE1 has been shown to interact with FBXL13 (DRC6) and DRC7; however, functional roles of FBXL13 and DRC7 in mammals have not been elucidated. Here we show that Fbxl13 and Drc7 expression are testes-enriched in mice. Although Fbxl13 knockout (KO) mice did not show any obvious phenotypes, Drc7 KO male mice were infertile due to their short immotile spermatozoa. In Drc7 KO spermatids, the axoneme is disorganized and the '9+2' microtubule arrangement was difficult to detect. Further, other N-DRC components fail to incorporate into the flagellum without DRC7. These results indicate that Drc7, but not Fbxl13, is essential for the correct assembly of the N-DRC and flagella.

Spawning-Induced pH Increase Activates Sperm Attraction and Fertilization Abilities in Eggs of the Ascidian, Phallusia philippinensis and Ciona intestinalis.

In Phlebobranchiata ascidians, oocytes and spermatozoa are stored in the oviduct and spermiduct, respectively, until spawning occurs. Gametes in the gonoducts are mature and fertilizable; however, it was found that the gametes of the ascidians Phallusia philippinensis and Ciona intestinalis could not undergo fertilization in the gonoductal fluids. The body fluids of the ascidians, especially in the gonoducts, were much more acidic (pH 5.5-6.8) than seawater (pH 8.2), and the fertilization rate was low under such acidic conditions. Hence, we examined the effect of pH on gametes. Pre-incubation of gonoductal eggs at pH 8.2 prior to insemination increased fertilization rates, even when insemination was performed under low pH conditions. Furthermore, an increase in ambient pH induced an increase in the intracellular pH of the eggs. It was also found that an increase in ambient pH triggered the release of sperm attractants from the egg and is therefore necessary for sperm chemotaxis. Hence, acidic conditions in the gonoductal fluids keep the gametes, especially eggs, infertile, and the release of eggs into seawater upon spawning induces an increase in ambient pH, which enables egg fertilization.

Single-cell genomics unveiled a cryptic cyanobacterial lineage with a worldwide distribution hidden by a dinoflagellate host.

Cyanobacteria are one of the most important contributors to oceanic primary production and survive in a wide range of marine habitats. Much effort has been made to understand their ecological features, diversity, and evolution, based mainly on data from free-living cyanobacterial species. In addition, symbiosis has emerged as an important lifestyle of oceanic microbes and increasing knowledge of cyanobacteria in symbiotic relationships with unicellular eukaryotes suggests their significance in understanding the global oceanic ecosystem. However, detailed characteristics of these cyanobacteria remain poorly described. To gain better insight into marine cyanobacteria in symbiosis, we sequenced the genome of cyanobacteria collected from a cell of a pelagic dinoflagellate that is known to host cyanobacterial symbionts within a specialized chamber. Phylogenetic analyses using the genome sequence revealed that the cyanobacterium represents an underdescribed lineage within an extensively studied, ecologically important group of marine cyanobacteria. Metagenomic analyses demonstrated that this cyanobacterial lineage is globally distributed and strictly coexists with its host dinoflagellates, suggesting that the intimate symbiotic association allowed the cyanobacteria to escape from previous metagenomic studies. Furthermore, a comparative analysis of the protein repertoire with related species indicated that the lineage has independently undergone reductive genome evolution to a similar extent as Prochlorococcus, which has the most reduced genomes among free-living cyanobacteria. Discovery of this cyanobacterial lineage, hidden by its symbiotic lifestyle, provides crucial insights into the diversity, ecology, and evolution of marine cyanobacteria and suggests the existence of other undiscovered cryptic cyanobacterial lineages.

The Roles of Two CNG Channels in the Regulation of Ascidian Sperm Chemotaxis.

Spermatozoa sense and respond to their environmental signals to ensure fertilization success. Reception and transduction of signals are reflected rapidly in sperm flagellar waveforms and swimming behavior. In the ascidian Ciona intestinalis (type A; also called C. robusta), an egg-derived sulfated steroid called SAAF (sperm activating and attracting factor), induces both sperm motility activation and chemotaxis. Two types of CNG (cyclic nucleotide-gated) channels, Ci-tetra KCNG (tetrameric, cyclic nucleotide-gated, K+-selective) and Ci-HCN (hyperpolarization-activated and cyclic nucleotide-gated), are highly expressed in Ciona testis from the comprehensive gene expression analysis. To elucidate the sperm signaling pathway to regulate flagellar motility, we focus on the role of CNG channels. In this study, the immunochemical analysis revealed that both CNG channels are expressed in Ciona sperm and localized to sperm flagella. Sperm motility analysis and Ca2+ imaging during chemotaxis showed that CNG channel inhibition affected the changes in flagellar waveforms and Ca2+ efflux needed for the chemotactic turn. These results suggest that CNG channels in Ciona sperm play a vital role in regulating sperm motility and intracellular Ca2+ regulation during chemotaxis.

14-3-3εa directs the pulsatile transport of basal factors toward the apical domain for lumen growth in tubulogenesis.

The Ciona notochord has emerged as a simple and tractable in vivo model for tubulogenesis. Here, using a chemical genetics approach, we identified UTKO1 as a selective small molecule inhibitor of notochord tubulogenesis. We identified 14-3-3εa protein as a direct binding partner of UTKO1 and showed that 14-3-3εa knockdown leads to failure of notochord tubulogenesis. We found that UTKO1 prevents 14-3-3εa from interacting with ezrin/radixin/moesin (ERM), which is required for notochord tubulogenesis, suggesting that interactions between 14-3-3εa and ERM play a key role in regulating the early steps of tubulogenesis. Using live imaging, we found that, as lumens begin to open between neighboring cells, 14-3-3εa and ERM are highly colocalized at the basal cortex where they undergo cycles of accumulation and disappearance. Interestingly, the disappearance of 14-3-3εa and ERM during each cycle is tightly correlated with a transient flow of 14-3-3εa, ERM, myosin II, and other cytoplasmic elements from the basal surface toward the lumen-facing apical domain, which is often accompanied by visible changes in lumen architecture. Both pulsatile flow and lumen formation are abolished in larvae treated with UTKO1, in larvae depleted of either 14-3-3εa or ERM, or in larvae expressing a truncated form of 14-3-3εa that lacks the ability to interact with ERM. These results suggest that 14-3-3εa and ERM interact at the basal cortex to direct pulsatile basal accumulation and basal-apical transport of factors that are essential for lumen formation. We propose that similar mechanisms may underlie or may contribute to lumen formation in tubulogenesis in other systems.

High potassium seawater inhibits ascidian sperm chemotaxis, but does not affect the male gamete chemotaxis of a brown alga.

Male gamete chemotaxis towards the female gamete is a general strategy to facilitate the sexual reproduction in many marine eukaryotes. Biochemical studies of chemoattractants for male gametes of brown algae have advanced in the 1970s and 1980s, but the molecular mechanism of male gamete responses to the attractants remains elusive. In sea urchin, a K+ channel called the tetraKCNG channel plays a fundamental role in sperm chemotaxis and inhibition of K+ efflux through this channel by high K+ seawater blocks almost all cell responses to the chemoattractant. This signalling mechanism could be conserved in marine invertebrates as tetraKCNG channels are conserved in the marine invertebrates that exhibit sperm chemotaxis. We confirmed that high K+ seawater also inhibited sperm chemotaxis in ascidian, Ciona intestinalis (robusta), in this study. Conversely, the male gamete chemotaxis towards the female gamete of a brown alga, Mutimo cylindricus, was preserved even in high K+ seawater. This result indicates that none of the K+ channels is essential for male gamete chemotaxis in the brown alga, suggesting that the signalling mechanism for chemotaxis in this brown alga is quite different from that of marine invertebrates. Correlated to this result, we revealed that the channels previously proposed as homologues of tetraKCNG in brown algae have a distinct domain composition from that of the tetraKCNG. Namely, one of them possesses two repeats of the six transmembrane segments (diKCNG) instead of four. The structural analysis suggests that diKCNG is a cyclic nucleotide-modulated and/or voltage-gated K+ channel.

Ttll9-/- mice sperm flagella show shortening of doublet 7, reduction of doublet 5 polyglutamylation and a stall in beating.

Nine outer doublet microtubules in axonemes of flagella and cilia are heterogeneous in structure and biochemical properties. In mammalian sperm flagella, one of the factors to generate the heterogeneity is tubulin polyglutamylation, although the importance of the heterogeneous modification is unclear. Here, we show that a tubulin polyglutamylase Ttll9 deficiency (Ttll9(-/-)) causes a unique set of phenotypes related to doublet heterogeneity. Ttll9(-/-) sperm axonemes had frequent loss of a doublet and reduced polyglutamylation. Intriguingly, the doublet loss selectively occurred at the distal region of doublet 7, and reduced polyglutamylation was observed preferentially on doublet 5. Ttll9(-/-) spermatozoa showed aberrant flagellar beating, characterized by frequent stalls after anti-hook bending. This abnormal motility could be attributed to the reduction of polyglutamylation on doublet 5, which probably occurred at a position involved in the switching of bending. These results indicate that mammalian Ttll9 plays essential roles in maintaining the normal structure and beating pattern of sperm flagella by establishing normal heterogeneous polyglutamylation patterns.

A Unique Seminal Plasma Protein, Zona Pellucida 3-Like Protein, has Ca2+ -Dependent Sperm Agglutination Activity.

Identification of seminal proteins provides a means of investigating their roles. Despite their importance in the study of protein function, such as regulation of sperm motility, it is difficult to select candidates from the large number of proteins. Analyzing the rate of molecular evolution is a useful strategy for selecting candidates, and expressing the protein allows the examination of its function. In the present study, we investigated seminal plasma proteins of the cichlid Oreochromis mossambicus, which exhibits a unique mode of fertilization and a rapidly evolving gene that encodes a seminal plasma protein, zona-pellucida 3-like (ZP3-like), which does not belong to the same molecular family as other ZPs. Seminal plasma proteins of O. mossambicus were separated by two-dimensional electrophoresis, and 19 major proteins were identified by mass spectrometry (MALDI-Tof Mass). Because proteins that are under positive selection often impact sperm function, the rates of molecular evolution of these proteins were analyzed in terms of non-synonymous/synonymous substitutions (ω). Among the 19 proteins, positive selection was supported for five genes; functional assays were carried out on four of the proteins encoded by these genes. Of the four positively selected proteins, only ZP3-like protein agglutinated sperm in a dose- and Ca2+ -dependent manner. The other three proteins did not affect sperm motility. Because of the unique fertilization type, in which fertilization occurs in the buccal cavity, the need to retain sperm within the cavity during spawning, and the agglutination of sperm, which may be partly assisted by ZP3-like protein, may contribute to fertilization success. Fertilization in the buccal cavity may be related to its rapid molecular evolution.

Release of Sticky Glycoproteins from Chlamydomonas Flagella During Microsphere Translocation on the Surface Membrane.

Chlamydomonas flagella display surface motility such that small polystyrene beads (microspheres) attached to the flagellar membrane move bidirectionally along the flagellum. This surface motility enables cells to glide on a solid substrate to which they are attached by the flagellar surface. Previous studies suggested that microsphere movement and gliding motility result from the movement of transmembrane glycoprotein(s) within the plane of the plasma membrane, driven by intraflagellar transport (IFT), which utilizes cytoplasmic dynein and kinesin-2. However, it is not well understood how a cell can continuously glide in one direction further than a single flagellar length. Here we show that, during microsphere translocation on the flagella of a non-motile mutant, pf18, some flagellar glycoproteins, including FMG-1B and FAP113, detach from the membrane and attach to the microspheres. We propose that such relocation of surface glycoproteins underlies the ability to glide over a long distance. Surface motility is likely common to cilia/flagella of various organisms, as a similar microsphere movement is observed in the apical ciliary tuft in sea urchin embryos.

The birth of quail chicks after intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) has been successfully used to produce offspring in several mammalian species including humans. However, ICSI has not been successful in birds because of the size of the egg and difficulty in mimicking the physiological polyspermy that takes place during normal fertilization. Microsurgical injection of 20 or more spermatozoa into an egg is detrimental to its survival. Here, we report that injection of a single spermatozoon with a small volume of sperm extract (SE) or its components led to the development and birth of healthy quail chicks. SE contains three factors - phospholipase Cζ (PLCZ), aconitate hydratase (AH) and citrate synthase (CS) - all of which are essential for full egg activation and subsequent embryonic development. PLCZ induces an immediate, transient Ca(2+) rise required for the resumption of meiosis. AH and CS are required for long-lasting, spiral-like Ca(2+) oscillations within the activated egg, which are essential for cell cycle progression in early embryos. We also found that co-injection of cRNAs encoding PLCZ, AH and CS support the full development of ICSI-generated zygotes without the use of SE. These findings will aid our understanding of the mechanism of avian fertilization and embryo development, as well as assisting in the manipulation of the avian genome and the production of transgenic and cloned birds.

Inverse relationship of Ca2+-dependent flagellar response between animal sperm and prasinophyte algae.

Symmetry/asymmetry conversion of eukaryotic flagellar waveform is caused by the changes in intracellular Ca2+. Animal sperm flagella show symmetric or asymmetric waveform at lower or higher concentration of intracellular Ca2+, respectively. In Chlamydomonas, high Ca2+ induces conversion of flagellar waveform from asymmetric to symmetry, resulting in the backward movement. This mirror image relationship between animal sperm and Chlamydomonas could be explained by the distinct calcium sensors used to regulate the outer arm dyneins (Inaba 2015). Here we analyze the flagellar Ca2+-response of the prasinophyte Pterosperma cristatum, which shows backward movement by undulating four flagella, the appearance similar to animal sperm. The moving path of Pterosperma shows relatively straight in artificial seawater (ASW) or ASW in the presence of a Ca2+ ionophore A23187, whereas it becomes circular in a low Ca2+ solution. Analysis of flagellar waveform reveals symmetric or asymmetric waveform propagation in ASW or a low Ca2+ solution, respectively. These patterns of flagellar responses are completely opposite to those in sperm flagella of the sea urchin Anthocidaris crassispina, supporting the idea previously proposed that the difference in flagellar response to Ca2+ attributes to the evolutional innovation of calcium sensors of outer arm dynein in opisthokont or bikont lineage.

Self-incompatibility response induced by calcium increase in sperm of the ascidian Ciona intestinalis.

Many hermaphroditic organisms possess a self-incompatibility system to avoid self-fertilization. Recently, we identified the genes responsible for self-sterility in a hermaphroditic primitive chordate (ascidian), Ciona intestinalis: sperm-side polycystin 1-like receptors s-Themis-A/B and egg-side fibrinogen-like ligands on the vitelline coat (VC) v-Themis-A/B. Here, we investigated the sperm behavior and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to self/nonself-recognition. We found that sperm motility markedly decreased within 5 min after attachment to the VC of self-eggs but not after attachment to the VC of nonself-eggs and that the apparent decrease in sperm motility was suppressed in low Ca(2+) seawater. High-speed video analysis revealed that sperm detached from the self-VC or stopped motility within 5 min after binding to the self-VC. Because s-Themis-B contains a cation channel domain in its C terminus, we monitored sperm [Ca(2+)](i) by real-time [Ca(2+)](i) imaging using Fluo-8H-AM (AAT Bioquest, Inc.). Interestingly, we found that sperm [Ca(2+)](i) rapidly and dramatically increased and was maintained at a high level in the head and flagellar regions when sperm interacted with the self-VC but not when the sperm interacted with the nonself-VC. The increase in [Ca(2+)](i) was also suppressed by low-Ca(2+) seawater. These results indicate that the sperm self-recognition signal triggers [Ca(2+)](i) increase and/or Ca(2+) influx, which elicits a self-incompatibility response to reject self-fertilization in C. intestinalis.

Calaxin drives sperm chemotaxis by Ca²âº-mediated direct modulation of a dynein motor.

Sperm chemotaxis occurs widely in animals and plants and plays an important role in the success of fertilization. Several studies have recently demonstrated that Ca(2+) influx through specific Ca(2+) channels is a prerequisite for sperm chemotactic movement. However, the regulator that modulates flagellar movement in response to Ca(2+) is unknown. Here we show that a neuronal calcium sensor, calaxin, directly acts on outer-arm dynein and regulates specific flagellar movement during sperm chemotaxis. Calaxin inhibition resulted in significant loss of sperm chemotactic movement, despite normal increases in intracellular calcium concentration. Using a demembranated sperm model, we demonstrate that calaxin is essential for generation and propagation of Ca(2+)-induced asymmetric flagellar bending. An in vitro motility assay revealed that calaxin directly suppressed the velocity of microtubule sliding by outer-arm dynein at high Ca(2+) concentrations. This study describes the missing link between chemoattractant-mediated Ca(2+) signaling and motor-driven microtubule sliding during sperm chemotaxis.

Roles of extracellular ions and pH in 5-HT-induced sperm motility in marine bivalve.

Factors that inhibit and stimulate the initiation of sperm motility were determined for Manila clam (Ruditapes philippinarum), Pacific oyster (Crassostrea gigas), and Japanese scallop (Patinopecten yessoensis). Compared with artificial seawater (ASW), serotonin (5-hydroxytryptamine creatinine sulfate, 5-HT) could fully trigger sperm motility and increase sperm velocity and motility duration. Sperm motility was decreased in ASW at pH 6.5-7.0 and suppressed at pH 4.0. In Manila clam and Pacific oyster, 5-HT could overcome the inhibitory effects of acidic pH on sperm motility. In the presence of nigericin (a K(+)/H(+) exchanger), sperm motility was only triggered at pH 8.3. Testicular fluid K(+) concentrations were two- to fourfold higher than that in ASW. Sperm motility and velocity were decreased in ASW or 5-HT containing ≥40  mM K(+) or ≥2.5  mM 4-aminopyridine, suggesting K(+) efflux requirement to initiate motility. Sperm motility and velocity were reduced in ASW or 5-HT containing EGTA or W-7, suggesting that extracellular Ca(2)(+) is required for Ca(2)(+)/calmodulin-dependent flagellar beating. Ca(2)(+) influx occurs via Ca(2)(+) channels because sperm motility and velocity were decreased in both ASW and 5-HT containing T-type and L-type Ca(2)(+) channel blockers. 5-HT-dependent initiation of sperm motility was associated with intracellular Ca(2)(+) rise, which was comparable to that seen in ASW but was not observed in the presence of EGTA or a Ca(2)(+) channel blocker. Extracellular Na(+) is also essential for sperm motility initiation via regulation of Na(+)/Ca(2)(+) exchange. Overall, 5-HT-dependent initiation of sperm motility in marine bivalve mollusks is an osmolality-independent mechanism and regulated by extracellular pH, K(+), Ca(2)(+), and Na(+).

Sperm activation by heat shock protein 70 supports the migration of sperm released from sperm storage tubules in Japanese quail (Coturnix japonica).

Systems for maintaining the viability of ejaculated sperm in the female reproductive tract are widespread among vertebrates and invertebrates. In birds, this sperm storage function is performed by specialized simple tubular invaginations called sperm storage tubules (SSTs) in the uterovaginal junction (UVJ) of the oviduct. Although the incidence and physiological reasons for sperm storage in birds have been reported extensively, the mechanisms of sperm uptake by the SSTs, sperm maintenance within the SSTs, and control of sperm release from the SSTs are poorly understood. In this study, we demonstrated that the highly conserved heat shock protein 70 (HSP70) stimulates sperm motility in vitro and also that HSP70 expressed in the UVJ may facilitate the migration of sperm released from the SSTs. Quantitative RT-PCR analysis demonstrated that the expression of HSP70 mRNA in the UVJ increases before ovulation/oviposition. Gene-specific in situ hybridization and immunohistochemical analysis with a specific antibody to HSP70 demonstrated that HSP70 is localized in the surface epithelium of the UVJ. Furthermore, injection of anti-HSP70 antibody into the vagina significantly inhibited fertilization in vivo. In addition, we found that recombinant HSP70 activates flagellar movement in the sperm and that the binding of recombinant HSP70 to the sperm surface is mediated through an interaction with voltage-dependent anion channel protein 2 (VDAC2). Our results suggest that HSP70 binds to the sperm surface by interacting with VDAC2 and activating sperm motility. This binding appears to play an important role in sperm migration within the oviduct.

Autonomous changes in the swimming direction of sperm in the gastropod Strombus luhuanus.

The sperm of the gastropod Strombus luhuanus show dimorphism. The eusperm have a nucleus and fertilize the egg, whereas the other type of sperm, parasperm, are anucleate and are thought to assist fertilization. Here we report the autonomous changes in the swimming pattern of S. luhuanus eusperm. In artificial seawater, the eusperm collected from S. luhuanus sperm ducts formed sperm bundles and initially swam backward with asymmetric flagellar waveforms to detach from the bundles. One hour later, the sperm began to swim forward and in a circle. After an additional 1 h incubation, the sperm swam straight, with a change in the flagellar waveforms from asymmetric to symmetric. Spontaneous backward swimming with symmetric waveforms was also observed. The eusperm stored in the female seminal receptacle were motile and showed forward symmetric swimming with spontaneous backward swimming, which appeared necessary for detachment from the wall of receptacle. All of these motility changes were observed in the absence of parasperm, suggesting that these changes autonomously occur in eusperm. Our waveform analysis of these swimming patterns revealed that only the swimming with symmetric waveform showed reverse propagation of the flagellar waveforms. Both types of backward swimming were diminished in Ca(2+)-free seawater and in seawater containing Ni(2+), indicating the regulation of swimming direction by Ca(2+)-dependent signal transduction.

Distinct roles of soluble and transmembrane adenylyl cyclases in the regulation of flagellar motility in Ciona sperm.

Adenylyl cyclase (AC) is a key enzyme that synthesizes cyclic AMP (cAMP) at the onset of the signaling pathway to activate sperm motility. Here, we showed that both transmembrane AC (tmAC) and soluble AC (sAC) are distinctly involved in the regulation of sperm motility in the ascidian Ciona intestinalis. A tmAC inhibitor blocked both cAMP synthesis and the activation of sperm motility induced by the egg factor sperm activating and attracting factor (SAAF), as well as those induced by theophylline, an inhibitor of phoshodiesterase. It also significantly inhibited cAMP-dependent phosphorylation of a set of proteins at motility activation. On the other hand, a sAC inhibitor does not affect on SAAF-induced transient increase of cAMP, motility activation or protein phosphorylation, but it reduced swimming velocity to half in theophylline-induced sperm. A sAC inhibitor KH-7 induced circular swimming trajectory with smaller diameter and significantly suppressed chemotaxis of sperm to SAAF. These results suggest that tmAC is involved in the basic mechanism for motility activation through cAMP-dependent protein phosphorylation, whereas sAC plays distinct roles in increase of flagellar beat frequency and in the Ca2+-dependent chemotactic movement of sperm.

X-ray diffraction recording from a small amount of fibrous protein materials oriented by a micro shear-flow cell.

This paper describes a method for recording X-ray diffraction patterns from a small amount of fibrous protein materials while being oriented by using a micro shear-flow cell. This cell consists of two concentrically arranged glass tubes. The inner tube is stationary, while the outer one rotates at a high speed. The gap between the two tubes is about 100 µm, into which the suspension of fibrous protein materials is injected. By using synchrotron-radiation X-ray microbeams (diameter, 10 µm), clear diffraction images from oriented protein materials can be recorded. The required volume of the sample is only about 10 µl. This method can also be combined with the laser-flash photolysis of caged compounds. Examples of application of this method to the flagella of a green alga Chlamydomonas, and sperm of a tunicate Ciona are presented.