Sea Urchin Sperm Research Articles

Investigation of Sea Urchin Sperm Motility: an Undergraduate Project

Cells that display self-motility are a very interesting case of active matter. In particular, sperm cells move in order to encounter and fertilize the egg. Individual and collective movements are crucial for fertilization, especially in species where this process is external, like in sea urchins. These organisms release sperm and eggs into sea water. Sperm cells propel themselves by rotating their flagella (1.5 cycles per second). In this work we experimentally study the collective and individual movement of sea urchin sperm (Echinometra vanbrunti).

Mechanical induction of oscillatory movement in demembranated, immotile flagella of sea urchin sperm at very low ATP concentrations.

Oscillation is a characteristic feature of eukaryotic flagellar movement. The mechanism involves the control of dynein-driven microtubule sliding under self-regulatory mechanical feedback within the axoneme. To define the essential factors determining the induction of oscillation, we developed a novel experiment by applying mechanical deformation of demembranated, immotile sea urchin sperm flagella at very low ATP concentrations, below the threshold of ATP required for spontaneous beating. Upon application of mechanical deformation at above 1.5 µmoll-1 ATP, a pair of bends could be induced and was accompanied by bend growth and propagation, followed by switching the bending direction. For an oscillatory, cyclical bending response to occur, the velocity of bend propagation towards the flagellar tip must be kept above certain levels. Continuous formation of new bends at the flagellar base was coupled with synchronized decay of the preceding paired bends. Induction of cyclical bends was initiated in a constant direction relative to the axis of the flagellar 9+2 structure, and resulted in the so-called principal bend. In addition, stoppage of the bending response occasionally occurred during development of a new principal bend, and in this situation, formation of a new reverse bend did not occur. This observation indicates that the reverse bend is always active, opposing the principal bend. The results show that mechanical strain of bending is a central component regulating the bend oscillation, and switching of the bend direction appears to be controlled, in part, by the velocity of wave propagation.

Lens Connexin Channels Have Differential Permeability to the Second Messenger cAMP.

PurposeGap junction channels exhibit connexin specific biophysical properties, including the selective intercellular passage of larger solutes, such as second messengers. Here, we have examined the cyclic nucleotide permeability of the lens connexins, which could influence events like epithelial cell division and differentiation.MethodsWe compared the cAMP permeability through channels composed of Cx43, Cx46, or Cx50 using simultaneous measurements of junctional conductance and intercellular transfer. For cAMP detection, the recipient cells were transfected with a cAMP sensor gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents were recorded from the other cell of a pair that expressed SpIH. cAMP permeability was also directly visualized in transfected cells using a chemically modified fluorescent form of the molecule.ResultscAMP transfer was observed for homotypic Cx43 channels over a wide range of junctional conductance. Homotypic Cx46 channels also transferred cAMP, but permeability was reduced compared with Cx43. In contrast, homotypic Cx50 channels exhibited extremely low permeability to cAMP, when compared with either Cx43, or Cx46.ConclusionsThese data show that channels made from Cx43 and Cx46 result in the intercellular delivery of cAMP in sufficient quantity to activate cyclic nucleotide-modulated channels. The data also suggest that the greatly reduced cAMP permeability of Cx50 channels could play a role in the regulation of cell division in the lens.

Flagellar ultrastructure suppresses buckling instabilities and enables mammalian sperm navigation in high-viscosity media.

Eukaryotic flagellar swimming is driven by a slender motile unit, the axoneme, which possesses an internal structure that is essentially conserved in a tremendous diversity of sperm. Mammalian sperm, however, which are internal fertilizers, also exhibit distinctive accessory structures that further dress the axoneme and alter its mechanical response. This raises the following two fundamental questions. What is the functional significance of these structures? How do they affect the flagellar waveform and ultimately cell swimming? Hence we build on previous work to develop a mathematical mechanical model of a virtual human sperm to examine the impact of mammalian sperm accessory structures on flagellar dynamics and motility. Our findings demonstrate that the accessory structures reinforce the flagellum, preventing waveform compression and symmetry-breaking buckling instabilities when the viscosity of the surrounding medium is increased. This is in agreement with previous observations of internal and external fertilizers, such as human and sea urchin spermatozoa. In turn, possession of accessory structures entails that the progressive motion during a flagellar beat cycle can be enhanced as viscosity is increased within physiological bounds. Hence the flagella of internal fertilizers, complete with accessory structures, are predicted to be advantageous in viscous physiological media compared with watery media for the fundamental role of delivering a genetic payload to the egg.

Kinetic and photonic techniques to study chemotactic signaling in sea urchin sperm.

Sperm from sea urchins are attracted by chemical cues released by the egg-a mechanism called chemotaxis. We describe here the signaling pathway and molecular components endowing sperm with single-molecule sensitivity. Chemotactic signaling and behavioral responses occur on a timescale of a few milliseconds to seconds. We describe the techniques and chemical tools used to resolve the signaling events in time. The techniques include rapid-mixing devices, rapid stroboscopic microscopy, and photolysis of caged second messengers and chemoattractants.

Targeting cell-bound MUC1 on myelomonocytic, monocytic leukemias and phenotypically defined leukemic stem cells with anti-SEA module antibodies

Cell surface molecules aberrantly expressed or overexpressed by myeloid leukemic cells represent potential disease-specific therapeutic targets for antibodies. MUC1 is a polymorphic glycoprotein, the cleavage of which yields two unequal chains: a large extracellular α subunit containing a tandem repeat array bound in a strong noncovalent interaction to a smaller β subunit containing the transmembrane and cytoplasmic domains. Because the α-chain can be released from the cell-bound domains of MUC1, agents directed against the α-chain will not effectively target MUC1+ cells. The MUC1 SEA (a highly conserved protein module so called from its initial identification in a sea urchin sperm protein, in enterokinase, and in agrin) domain formed by the binding of the α andβchains represents a stable structure fixed to the cell surface at all times. DMB-5F3, a partially humanized murine anti-MUC1 SEA domain monoclonal antibody, was used to examine MUC1 expression in acute myeloid leukemia (AML) and was found to bind acute myelomonocytic and monocytic leukemia (AML-M4 and AML-M5) cell lines. We also examined monocytic neoplasms freshly obtained from patients including chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia, which were found to uniformly express MUC1. CD34+/lin-/CD38- or CD38+ presumed leukemic stem cell populations from CD34+ AML and CD34-CD38- or CD38+ populations from CD34- AML were also found to express MUC1, although at low percentages. Based on these studies, we generated an anti-MUC1 immunotoxin to directly gauge the cytotoxic efficacy of targeting AML-bound MUC1. Using single-chain DMB-5F3 fused to recombinant gelonin toxin, the degree of AML cytotoxicity was found to correlate with MUC1 expression. Our data support the use of an anti-MUC1 SEA module-drug conjugates to selectively target and inhibit MUC1-expressing myelomonocytic leukemic cells.

Cryopreservation of sea urchin sperm and early life stages.

Sea urchins are excellent model organisms useful for several lines of biotechnological research. Sea urchins are typically collected from the sea and kept in research facilities all over the world for such purposes. Cryopreservation can be a very powerful tool to enhance the use of sea urchins as a model species for research. The development of cryopreservation protocols for different sea urchin gametes, embryos, and larvae allows year round access to high quality material outside the natural reproductive season. It also reduces the uncertainty and variability that may be caused by changing oceanic, meteorological, and environmental conditions. Access to cryopreserved gametes and embryos will allow using these model organisms in laboratories all around the world, regardless of their facilities or their proximity to a natural population of sea urchins. Cryopreservation is a very useful tool for aquaculture production, fisheries conservation, and wild stock enhancement allowing spat supply all year round without the need of conditioning broodstock for out of season reproduction-which is expensive, time consuming, and often unfruitful. It will also provide flexibility for selective breeding programs by allowing crossings of individuals with different reproductive seasons. Although cryopreservation protocols have been successfully developed for many valuable fisheries species such as sturgeons, salmonid fishes, and other marine invertebrates (e.g., oysters), only a small amount of research has been carried out regarding sea urchin cryopreservation. In this chapter, we outline protocols for the cryopreservation of sea urchin cells.

Turning dyneins off bends cilia.

Ciliary and flagellar motility is caused by the ensemble action of inner and outer dynein arm motors acting on axonemal doublet microtubules. The switch point or switching hypothesis, for which much experimental and computational evidence exists, requires that dyneins on only one side of the axoneme are actively working during bending, and that this active motor region propagate along the axonemal length. Generation of a reverse bend results from switching active sliding to the opposite side of the axoneme. However, the mechanochemical states of individual dynein arms within both straight and curved regions and how these change during beating has until now eluded experimental observation. Recently, Lin and Nicastro used high‐resolution cryo‐electron tomography to determine the power stroke state of dyneins along flagella of sea urchin sperm that were rapidly frozen while actively beating. The results reveal that axonemal dyneins are generally in a pre‐power stroke conformation that is thought to yield a force‐balanced state in straight regions; inhibition of this conformational state and microtubule release on specific doublets may then lead to a force imbalance across the axoneme allowing for microtubule sliding and consequently the initiation and formation of a ciliary bend. Propagation of this inhibitory signal from base‐to‐tip and switching the microtubule doublet subsets that are inhibited is proposed to result in oscillatory motion.

A novel cross-species inhibitor to study the function of CatSper Ca2+ channels in sperm.

Sperm from many species share the sperm-specific Ca2+ channel CatSper that controls the intracellular Ca2+ concentration and, thereby, the swimming behaviour. A growing body of evidence suggests that the mechanisms controlling the activity of CatSper and its role during fertilization differ among species. A lack of suitable pharmacological tools has hampered the elucidation of the function of CatSper. Known inhibitors of CatSper exhibit considerable side effects and also inhibit Slo3, the principal K+ channel of mammalian sperm. The compound RU1968 was reported to suppress Ca2+ signaling in human sperm by an unknown mechanism. Here, we examined the action of RU1968 on CatSper in sperm from humans, mice, and sea urchins. We resynthesized RU1968 and studied its action on sperm from humans, mice, and the sea urchin Arbacia punctulata by Ca2+ fluorimetry, single-cell Ca2+ imaging, electrophysiology, opto-chemistry, and motility analysis. RU1968 inhibited CatSper in sperm from invertebrates and mammals. The compound lacked toxic side effects in human sperm, did not affect mouse Slo3, and inhibited human Slo3 with about 15-fold lower potency than CatSper. Moreover, in human sperm, RU1968 mimicked CatSper dysfunction and suppressed motility responses evoked by progesterone, an oviductal steroid known to activate CatSper. Finally, RU1968 abolished CatSper-mediated chemotactic navigation in sea urchin sperm. We propose RU1968 as a novel tool to elucidate the function of CatSper channels in sperm across species.

Cytotoxicity and genotoxicity of CuO nanoparticles in sea urchin spermatozoa through oxidative stress

Copper oxide nanoparticles (CuO NPs) are extensively used in various industrial and commercial applications. Despite their wide application may lead to the contamination of marine ecosystem, their potential environmental effects remain to be determined. Toxicity assessment studies have primarily focused on investigating the effects of CuO NPs on fertilization success and embryo development of different sea urchin species while the impact on sperm quality have never been assessed. In this line, this study aims to assess the effects of CuO NPs on the spermatozoa of the sea urchin Paracentrotus lividus.After sperm exposure to CuO NPs, biomarkers of sperm viability, cytotoxicity, oxidative stress, and genotoxicity as well as morphology were evaluated. Results showed that CuO NPs exposure decreased sperm viability, impaired mitochondrial activity and increased the production of reactive oxygen species (ROS) and lipid peroxidation. Furthermore, CuO NPs exposure caused DNA damage and morphological alterations. Together with the antioxidant rescue experiments, these results suggest that oxidative stress is the main driver of CuO NP spermiotoxic effects. The mechanism of toxicity is here proposed: the spontaneous generation of ROS induced by CuO NPs and the disruption of the mitochondrial respiratory chain lead to production of ROS that, in turn, induce lipid peroxidation and DNA damage, and result in defective spermatozoa up to induce sperm cytotoxicity. Investigating the effects of CuO NPs on sea urchin spermatozoa, this study provides valuable insights into the mechanism of reproductive toxicity induced by CuO NPs.

Physical Forces Determining the Persistency and Centering Precision of Microtubule Asters.

In early embryos, microtubules form star-shaped aster structures that can measure up to hundreds of micrometres, and move at high speeds to find the geometrical centre of the cell. This process, known as aster centration, is essential for the fidelity of cell division and development, but how cells succeed in moving these large structures through their crowded and fluctuating cytoplasm remains unclear. Here, we demonstrate that the positional fluctuations of migrating sea urchin sperm asters are small, anisotropic, and associated with the stochasticity of dynein-dependent forces moving the aster. Using in vivo magnetic tweezers to directly measure aster forces inside cells, we derive a linear aster force-velocity relationship and provide evidence for a spring-like active mechanism stabilizing the transverse position of the asters. The large frictional coefficient and spring constant quantitatively account for the amplitude and growth characteristics of athermal positional fluctuations, demonstrating that aster mechanics ensure noise suppression to promote persistent and precise centration. These findings define generic biophysical regimes of active cytoskeletal mechanics underlying the accuracy of cell division and early embryonic development.

Colorimetric method for determining viability of sea urchin sperm applied in toxicity tests

The aim of this study was to improve the methodological procedure for the evaluation of sea urchin (Lytechinus variegatus) sperm sensitivity in MTT (3-(4,5-dimethylthiazol-2yl)-2,5 diphenyltetrazolium bromide) enzyme reduction assays with the formation of formazan (purple color) in the interior of viable cells. Assays were carried out with the reference toxicants sodium dodecyl sulfate (SDS), copper, zinc, cadmium and ammonium, using a sperm solution previously activated in sea water and a sperm solution prepared in sea water containing 400 μg L-1 verapamil, which enabled activation of the sperm to occur only when exposed to the toxicants. The assays performed with sperm in verapamil presented similar sensitivity to that shown in the fertilization tests with copper, zinc and SDS, while the assays carried out with the pre-activated sperm solution presented greater resistance to the action of the toxicants. It appears that the action of verapamil involves an intracellular effect on the distribution of Ca2+ ions and that the toxicants used prevent the metabolic reactivation of the sperm.

Decision making improves sperm chemotaxis in the presence of noise.

To navigate their surroundings, cells rely on sensory input that is corrupted by noise. In cells performing chemotaxis, such noise arises from the stochastic binding of signalling molecules at low chemoattractant concentrations. We reveal a fundamental relationship between the speed of chemotactic steering and the strength of directional fluctuations that result from the amplification of noise in a chemical input signal. This relation implies a trade-off between steering that is slow and reliable, and steering that is fast but less reliable. We show that dynamic switching between these two modes of steering can substantially increase the probability to find a target, such as an egg to be found by sperm cells. This decision making confers no advantage in the absence of noise, but is beneficial when chemical signals are detectable, yet characterized by low signal-to-noise ratios. The latter applies at intermediate distances from a target, where signalling molecules are diluted, thus defining a ‘noise zone’ that cells have to cross. Our results explain decision making observed in recent experiments on sea urchin sperm chemotaxis. More generally, our theory demonstrates how decision making enables chemotactic agents to cope with high levels of noise in gradient sensing by dynamically adjusting the persistence length of a biased random walk.

Regulatory mechanisms of sperm flagellar motility by metachronal and synchronous sliding of doublet microtubules.

What is the role of metachronal and synchronous sliding in sperm flagellar motility? Both metachronal and oscillatory synchronous sliding are essential for sperm flagellar motility, while the change in mode of synchronous sliding between the non-oscillatory synchronous sliding of a specific pair of the doublet microtubules and the oscillatory synchronous sliding between most pairs of doublet microtubules modulates the sperm flagellar motility. Metachronal and synchronous sliding of doublet microtubules are involved in sperm flagellar motility and regulation of these sliding movements controls flagellar bend formation. To study the regulatory mechanisms of metachronal and synchronous sliding in flagellar movement of golden hamster spermatozoa, changes in these sliding movements during hyperactivation were examined by measuring the angle of the tangent to the flagellar shaft with reference to the central axis of the sperm head (the shear angle) along the flagellum. Golden hamster spermatozoa were obtained from the caudal epididymis of five sexually mature golden hamsters. Results from three experiments were averaged. The number of spermatozoa analyzed is 15 activated sperm, 22 hyperactivated sperm and 20 acrosome-reacted sperm. For detailed field-by-field analysis, an individual flagellar image was tracked automatically using the Autotrace module of image analysis software. The coordinate values of the flagellar shaft were used to calculate the shear angle, which is proportional to the amount of microtubule sliding at any given position along the flagellum. The maximum shear angles of metachronal and synchronous sliding were obtained from the mean shear angles between the maximum shear angles of pro-hook bends and the absolute values of the minimum shear angles of anti-hook bends, which represent the amplitude of a set of successive shear angle curves, with 3-12 shear curves covering one beat cycle of sperm flagellar movement. Asymmetry of flagellar waves was expressed by the mean shear angle between the maximum shear angle of pro-hook bends and the minimum shear angle of anti-hook bends at 100 μm from the head-midpiece junction. The asymmetrical flagellar movements observed in the activated (non-hyperactivated) and hyperactivated spermatozoa were characterized by the non-oscillatory synchronous sliding of a specific pair of the doublets; the large asymmetrical flagellar movement in the hyperactivated spermatozoa was generated by the large non-oscillatory synchronous sliding. Both the metachronal and synchronous sliding increased during the hyperactivation; however, the large symmetrical flagellar movement of the acrosome-reacted spermatozoa was characterized by the oscillatory synchronous sliding between most pairs of doublets. These results demonstrated that the metachronal and synchronous sliding are involved in generation and modulation of sperm flagellar motility; however, two types of synchronous sliding, non-oscillatory and oscillatory sliding, modulate the sperm flagellar motility by enhancing the sliding of a specific pair of the doublets or the sliding between most pairs of the doublets. None. This is an indirect study of the metachronal and synchronous sliding of doublet microtubules. Studies based on the direct observation of behavior of dynein are needed to clarify the sliding microtubule theory of flagellar movement of spermatozoa. Both the metachronal and oscillatory synchronous sliding of doublet microtubule generate and modulate sperm flagellar motility, while the change in mode of synchronous sliding between the non-oscillatory synchronous sliding and oscillatory synchronous sliding modulates the sperm flagellar motility. The coordination between these sliding leads to various types of flagellar and ciliary motility, including the asymmetrical beating in flagellar and ciliary movement and planar or helical beating in sea urchin spermatozoa. Moreover, the finding that the metachronal sliding and two types of synchronous sliding generate and modulate the flagellar motility will open a new avenue for quantitative analysis of flagellar and ciliary motility. The authors have no conflict of interest and no funding to declare.

Abstract 3427: Targeting MUC13 to overcome the survival mechanisms for improved response to chemotherapy

Abstract Background: Pancreatic cancer (PanCa) is a third leading cause of cancer-related deaths in the US due to late diagnosis and development of chemoresistance. Therefore, understanding molecular mechanisms that confer survival benefit to PanCa cells may offer new therapeutic strategies for PanCa treatment.Mucin, MUC13 aberrantly expressed in PanCa,promoting cancer growth and progression and these effects are abrogated by microRNA-145 (miR-145) restoration. Unlike other cancer types, PanCa is highly resistant to Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) that emerges as one of the most promising experimental cancer therapeutic drugs.Herein, we demonstrate the integration of novel approach to overcome chemoresistance and offer TRAIL-based therapeutic strategies. Methods: MUC13 expressing and null stable PanCa cells were generated to investigate the role of MUC13 in cell survival.miR-145 mimics were used to investigate the effect of MUC13 silencing in promoting survival and inhibiting apoptosis in presence of TRAIL using Western blotting, cell proliferation (MTT), Acridine orange staining and flow cytometry apoptosis assays(cell cycle, Annexin V/7AAD staining). Various distinct domain specific constructs of MUC13 were constructed such as the mucin (α), Beta (β) sea urchin sperm protein enterokinase arginine (SEA) domain and cytoplasmic (CD) domains and transfected into MUC13 null Panc-1 cells to identify the role of different domains in eliciting survival benefit to PanCa cells. Results: Results demonstrate that MUC13 expression blocks activation of caspase-8 and death receptor-mediated apoptosis in PanCa cells in response toTRAIL treatment as observed through Western blotting and flow cytometer. Inhibition of MUC13 using shRNA knockdown or miR-145 restoration resulted in the TRAIL-mediated increase in apoptotic cell death as evidenced by AnnexinV/ 7AAD and sub G0 population, as well as rendered PanCa cells sensitive to treatment with drugs, such as paclitaxel. Additionally, cells treated with TRAIL in combination with paclitaxel or Abraxane showed enhanced apoptosis on inhibition of MUC13 expression using miR-145 restoration. Further investigation showed that cytoplasmic domain of MUC13 (MUC13-CD) is indispensable for blockingcaspase-8 activation and PARP cleavage, indicating that the MUC13-CD blocks TRAIL-induced signaling upstream to Bid by inhibiting caspase-8 activation. Conclusion: These observations suggest that MUC13 contributes to the survival advantage in PanCa cells in response to treatment with drugs or death inducing ligands such as, Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) which can be strategically overcome by miR-145 replenishment. These findings indicate that MUC13 silencing sensitizes PanCa cells towards TRAIL therapy and counteracts chemoresistance mechanisms in PanCa that may lead to novel combination therapies for PanCa treatment. Citation Format: Saini Setua, Sheema Khan, Murali M. Yallapu, Stephen W. Behrman, Nadeem Zafar, Meena Jaggi, Subhash C. Chauhan. Targeting MUC13 to overcome the survival mechanisms for improved response to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3427. doi:10.1158/1538-7445.AM2017-3427

Network model predicts that CatSper is the main Ca2+ channel in the regulation of sea urchin sperm motility

Spermatozoa sea urchin swimming behaviour is regulated by small peptides from the egg outer envelope. Speract, such a peptide, after binding to its receptor in Strongylocentrotus purpuratus sperm flagella, triggers a signaling pathway that culminates with a train of intracellular calcium oscillations, correlated with changes in sperm swimming pattern. This pathway has been widely studied but not fully characterized. Recent work on Arbacia punctulata sea urchin spermatozoa has documented the presence of the Ca2+ CatSper channel in their flagella and its involvement in chemotaxis. However, if other calcium channels participate in chemotaxis remains unclear. Here, based on an experimentally-backed logical network model, we conclude that CatSper is fundamental in the S. purpuratus speract-activated sea urchin sperm signaling cascade, although other Ca2+ channels could still be relevant. We also present for the first time experimental corroboration of its active presence in S. purpuratus sperm flagella. We argue, prompted by in silico knock-out calculations, that CatSper is the main generator of calcium oscillations in the signaling pathway and that other calcium channels, if present, have a complementary role. The approach adopted here allows us to unveil processes, which are hard to detect exclusively by experimental procedures.

Antibodies to sea urchin zygote particles and sperm vesicles reveal unexpected structures during cleavage and localize in known fate regions

Antibodies to sea urchin zygote particles and sperm vesicles reveal unexpected structures during cleavage and localize in known fate regions

Expansion of divergent SEA domains in cell surface proteins and nucleoporin 54.

SEA (sea urchin sperm protein, enterokinase, agrin) domains, many of which possess autoproteolysis activity, have been found in a number of cell surface and secreted proteins. Despite high sequence divergence, SEA domains were also proposed to be present in dystroglycan based on a conserved autoproteolysis motif and receptor-type protein phosphatase IA-2 based on structural similarity. The presence of a SEA domain adjacent to the transmembrane segment appears to be a recurring theme in quite a number of type I transmembrane proteins on the cell surface, such as MUC1, dystroglycan, IA-2, and Notch receptors. By comparative sequence and structural analyses, we identified dystroglycan-like proteins with SEA domains in Capsaspora owczarzaki of the Filasterea group, one of the closest single-cell relatives of metazoans. We also detected novel and divergent SEA domains in a variety of cell surface proteins such as EpCAM, α/ε-sarcoglycan, PTPRR, collectrin/Tmem27, amnionless, CD34, KIAA0319, fibrocystin-like protein, and a number of cadherins. While these proteins are mostly from metazoans or their single cell relatives such as choanoflagellates and Filasterea, fibrocystin-like proteins with SEA domains were found in several other eukaryotic lineages including green algae, Alveolata, Euglenozoa, and Haptophyta, suggesting an ancient evolutionary origin. In addition, the intracellular protein Nucleoporin 54 (Nup54) acquired a divergent SEA domain in choanoflagellates and metazoans.

Effects of external strain on the regulation of microtubule sliding induced by outer arm dynein of sea urchin sperm flagella.

Oscillatory bending movement of eukaryotic flagella is powered by orchestrated activity of dynein motor proteins that hydrolyse ATP and produce microtubule sliding. Although the ATP concentration within a flagellum is kept uniform at a few millimoles per litre level, sliding activities of dyneins are dynamically coordinated along the flagellum in accordance with the phase of bending waves. Thus at the organellar level the dynein not only generates force for bending but also modulates its motile activity by responding to bending of the flagellum. Single molecule analyses have suggested that dynein at the molecular level, even if isolated from the axoneme, could alter the modes of motility in response to mechanical strain. However, it still remains unknown whether the coordinated activities of multiple dyneins can be modulated directly by mechanical signals. Here, we studied the effects of externally applied strain on the sliding movement of microtubules interacted with an ensemble of dynein molecules adsorbed on a glass surface. We found that by bending the microtubules with a glass microneedle, three modes of motility that have not been previously characterized without bending can be induced: stoppage, backward sliding and dissociation. Modification in sliding velocities was also induced by imposed bending. These results suggest that the activities of dyneins interacted with a microtubule can be modified and coordinated through external strain in a quite flexible manner, and that such a regulatory mechanism may be the basis of flagellar oscillation.

Sperm as microswimmers – navigation and sensing at the physical limit

Many cells and microorganisms have evolved a motility apparatus to explore their surroundings. For guidance, these biological microswimmers rely on physical and chemical cues that are transduced by cellular pathways into directed movement – a process called taxis. Only few biological microswimmers have been studied as detailed as sperm from sea urchins. Sperm and eggs are released into the seawater. To enhance the chances of fertilization, eggs release chemical factors – called chemoattractants – that establish a chemical gradient and, thereby, guide sperm to the egg. Sea urchin sperm constitute a unique model system for understanding cell navigation at every level: from molecules to cell behaviours. We will outline the chemotactic signalling pathway of sperm from the sea urchin Arbacia punctulata and discuss how signalling controls navigation in a chemical gradient. Finally, we discuss recent insights into sperm chemotaxis in three dimensions (3D).