Reconstruction of the three-dimensional beat pattern underlying swimming behaviors of sperm.

 The eukaryotic flagellum propels sperm cells and simultaneously detects physical and chemical cues that modulate the waveform of the flagellar beat. Most previous studies have characterized the flagellar beat and swimming trajectories in two space dimensions (2D) at a water/glass interface. Here, using refined holographic imaging methods, we report high-quality recordings of three-dimensional (3D) flagellar bending waves. As predicted by theory, we observed that an asymmetric and planar flagellar beat results in a circular swimming path, whereas a symmetric and non-planar flagellar beat results in a twisted-ribbon swimming path. During swimming in 3D, human sperm flagella exhibit torsion waves characterized by maxima at the low curvature regions of the flagellar wave. We suggest that these torsion waves are common in nature and that they are an intrinsic property of beating axonemes. We discuss how 3D beat patterns result in twisted-ribbon swimming paths. This study provides new insight into the axoneme dynamics, the 3D flagellar beat, and the resulting swimming behavior.

Author Correction: The solute carrier SLC9C1 is a Na+/H+-exchanger gated by an S4-type voltage-sensor and cyclic-nucleotide binding.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The steering gaits of sperm.

Sperm are highly specialized cells, which have been subject to substantial evolutionary pressure. Whereas some sperm features are highly conserved, others have undergone major modifications. Some of these variations are driven by adaptation to mating behaviours or fitness at the organismic level. Others represent alternative solutions to the same task. Sperm must find the egg for fertilization. During this task, sperm rely on long slender appendages termed flagella that serve as sensory antennas, propellers and steering rudders. The beat of the flagellum is periodic. The resulting travelling wave generates the necessary thrust for propulsion in the fluid. Recent studies reveal that, for steering, different species rely on different fundamental features of the beat wave. Here, we discuss some examples of unity and diversity across sperm from different species with a particular emphasis on the steering mechanisms. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

Synergistic activation of CatSper Ca2+ channels in human sperm by oviductal ligands and endocrine disrupting chemicals.

STUDY QUESTION: Does the chemosensory activation of CatSper Ca2+ channels in human sperm give rise to additive, sub-additive or even synergistic actions among agonists? SUMMARY ANSWER: We show that oviductal ligands and endocrine disrupting chemicals (EDCs) activate human CatSper highly synergistically. WHAT IS KNOWN ALREADY: In human sperm, the sperm-specific CatSper channel controls the intracellular Ca2+ concentration and, thereby, several crucial stages toward fertilization. CatSper is activated by oviductal ligands and structurally diverse EDCs. The chemicals mimic the action of the physiological ligands, which might interfere with the precisely coordinated sequence of events underlying fertilization. STUDY DESIGN, SIZE, DURATION: For both oviductal ligands and EDCs, we examined in quantitative terms whether stimulation of human sperm in vitro with mixtures results in additive, sub-additive or synergistic actions. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied activation of CatSper in sperm of healthy volunteers, using kinetic Ca2+ fluorimetry and patch-clamp recordings. The combined action of progesterone and prostaglandins and of the EDCs benzylidene camphor sulfonic acid (BCSA) and α-Zearalenol was evaluated by curve-shift analysis, curvilinear isobolographic analysis and the combination-index method. MAIN RESULTS AND THE ROLE OF CHANCE: Analysis of the action of progesterone/prostaglandin and BCSA/α-Zearalenol mixtures in human sperm by fluorimetry revealed that the oviductal ligands and EDCs both evoke Ca2+ influx via CatSper in a highly synergistic fashion. Patch-clamp recordings of CatSper currents in human sperm corroborated the synergistic ligand-activation of the channel. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study. Future studies have to assess the physiological relevance in vivo. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that the fertilization process is orchestrated by multiple oviductal CatSper agonists that act in concert to control the behavior of sperm. Moreover, our results substantiate the concerns regarding the negative impact of EDCs on male reproductive health. So far, safety thresholds like the "No Observed Adverse Effect Level (NOAEL)" or "No Observed Effect Concentration (NOEC)" are set for individual EDCs. Our finding that EDCs act synergistically in human sperm challenges the validity of this procedure. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the German Research Foundation (SFB 645; CRU326), the Cells-in-Motion (CiM) Cluster of Excellence, Münster, (FF-2016-17), the 'Innovative Medical Research' of the University of Münster Medical School (BR121507), an EDMaRC research grant from the Kirsten and Freddy Johansen's Foundation, and the Innovation Fund Denmark (InnovationsFonden; 14-2013-4). The authors have no competing financial interests.

The solute carrier SLC9C1 is a Na+/H+-exchanger gated by an S4-type voltage-sensor and cyclic-nucleotide binding.

Voltage-sensing (VSD) and cyclic nucleotide-binding domains (CNBD) gate ion channels for rapid electrical signaling. By contrast, solute carriers (SLCs) that passively redistribute substrates are gated by their substrates themselves. Here, we study the orphan sperm-specific solute carriers SLC9C1 that feature a unique tripartite structure: an exchanger domain, a VSD, and a CNBD. Voltage-clamp fluorimetry shows that SLC9C1 is a genuine Na+/H+ exchanger gated by voltage. The cellular messenger cAMP shifts the voltage range of activation. Mutations in the transport domain, the VSD, or the CNBD strongly affect Na+/H+ exchange, voltage gating, or cAMP sensitivity, respectively. Our results establish SLC9C1 as a phylogenetic chimaera that combines the ion-exchange mechanism of solute carriers with the gating mechanism of ion channels. Classic SLCs slowly readjust changes in the intra- and extracellular milieu, whereas voltage gating endows the Na+/H+ exchanger with the ability to produce a rapid pH response that enables downstream signaling events.

Signaling in Sperm: More Different than Similar.

For a given sensory cell type, signaling motifs are rather uniform across phyla. By contrast, sperm from different species use diverse repertoires of sperm-specific signaling molecules and even closely related protein isoforms feature different properties and serve different functions. This surprising diversity has consequences for strategies in fertilization research and it will take some time to get the big picture. We discuss the function of receptors, ion channels, and exchangers embedded in cellular pathways from different sperm species.

At the physical limit - chemosensation in sperm.

Many cells probe their environment for chemical cues. Some cells respond to picomolar concentrations of neuropeptides, hormones, pheromones, or chemoattractants. At such low concentrations, cells encounter only a few molecules. The mechanistic underpinnings of single-molecule sensitivity are not known for any eukaryotic cell. Sea urchin sperm offer a unique model to unveil in quantitative terms the principles underlying chemosensation at the physical limit. Here, we discuss the mechanisms of such exquisite sensitivity and the computational operations performed by sperm during chemotactic steering. Moreover, we highlight commonalities and differences between signalling in sperm and photoreceptors and among sperm from different species.

Binding of Ca2+ to glutamic acid-rich polypeptides from the rod outer segment.

Rod photoreceptors contain three different glutamic acid-rich proteins (GARPs) that have been proposed to control the propagation of Ca(2+) from the site of its entry at the cyclic nucleotide-gated channel to the cytosol of the outer segment. We tested this hypothesis by measuring the binding of Ca(2+) to the following five constructs related to GARPs of rod photoreceptors: a 32-mer peptide containing 22 carboxylate groups, polyglutamic acid, a recombinant segment comprising 73 carboxylate groups (GLU), GARP1, and GARP2. Ca(2+) binding was investigated by means of a Ca(2+)-sensitive electrode. In all cases, Ca(2+) binds with low affinity; the half-maximum binding constant K(1/2) ranges from 6 to 16 mM. The binding stoichiometry between Ca(2+) ions and carboxylic groups is approximately 1:1; an exception is GARP2, where a binding stoichiometry of approximately 1:2 was found. Hydrodynamic radii of 1.6, 2.8, 3.3, 5.7, and 6.7 nm were determined by dynamic light scattering for the 32-mer, polyglutamic acid, GLU, GARP2, and GARP1 constructs, respectively. These results suggest that the peptides as well as GARP1 and GARP2 do not adopt compact globular structures. We conclude that the structures should be regarded as loose coils with low-affinity, high-capacity Ca(2+) binding.

Sperm chemotaxis in marine invertebrates--molecules and mechanisms.

Sperm are attracted by chemical substances which are released by the egg. This process is called chemotaxis. Several molecules that are involved in chemotactic signaling of sperm from marine invertebrates are described and a model of the signaling pathway is presented. We discuss the motor response during chemotaxis and propose a model of the navigation strategy of sperm.

Sperm chemotaxis: a primer.

Sperm become attracted by chemical substances that are released from the outer coating of the egg, a process called chemotaxis. In this paper the cellular pathway and the motor response during chemotaxis of sperm from sea urchin and starfish are briefly outlined.

Hyperpolarization-activated channels HCN1 and HCN4 mediate responses to sour stimuli.

Sour taste is initiated by protons acting at receptor proteins or channels. In vertebrates, transduction of this taste quality involves several parallel pathways. Here we examine the effects of sour stimuli on taste cells in slices of vallate papilla from rat. From a subset of cells, we identified a hyperpolarization-activated current that was enhanced by sour stimulation at the taste pore. This current resembled Ih found in neurons and cardio-myocytes, a current carried by members of the family of hyperpolarization-activated and cyclic-nucleotide-gated (HCN) channels. We show by in situ hybridization and immunohistochemistry that HCN1 and HCN4 are expressed in a subset of taste cells. By contrast, gustducin, the G-protein involved in bitter and sweet taste, is not expressed in these cells. Lowering extracellular pH causes a dose-dependent flattening of the activation curve of HCN channels and a shift in the voltage of half-maximal activation to more positive voltages. Our results indicate that HCN channels are gated by extracellular protons and may act as receptors for sour taste.

Ligand sensitivity of the 2 subunit from the bovine cone cGMP-gated channel is modulated by protein kinase C but not by calmodulin.

1. Homomeric cyclic nucleotide-gated (CNG) channels composed of alpha2 subunits from bovine cone photoreceptors were heterologously expressed in the human embryonic kidney (HEK) 293 cell line. Modulation of cGMP sensitivity by protein kinase C (PKC)-mediated phosphorylation and by binding of calmodulin (CaM) was investigated in inside-out patches. 2. A peptide encompassing the putative CaM-binding site within the N-terminus of the channel protein binds Ca(2+)-CaM with high affinity, yet the ligand sensitivity of alpha2 channels is not modulated by CaM. 3. PKC-mediated phosphorylation increased the activation constant (K(1/2)) for cGMP from 19 to 56 microM and decreased the Hill coefficient (from 2.5 to 1.5). The change in ligand sensitivity involves phosphorylation of the serine residues S577 and S579 in the cGMP-binding domain. The increase in K(1/2) was completely abolished in mutant channels in which the two serine residues were replaced by alanine. 4. An antibody specific for the delta isoform of PKC strongly labels the cone outer segments. 5. Modulation of cGMP affinity of bovine alpha2 CNG channels by phosphorylation could play a role in the regulation of photoreceptor sensitivity.

Molecular diversity of pacemaker ion channels.

Ionic currents activated by hyperpolarization and regulated by cyclic nucleotides were first discovered more than 20 years ago. Recently the molecular identity of the underlying channels has been unveiled. The structural features of the protein sequences are discussed and related to the mechanisms of activation, selectivity for cyclic nucleotides, and ion permeation. Coverage includes a comparison of the biophysical properties of recombinant and native channels and their significance for the physiological functions of these channels.

Fraction of the dark current carried by Ca(2+) through cGMP-gated ion channels of intact rod and cone photoreceptors.

The selectivity for Ca(2+) over Na(+), PCa/PNa, is higher in cGMP-gated (CNG) ion channels of retinal cone photoreceptors than in those of rods. To ascertain the physiological significance of this fact, we determined the fraction of the cyclic nucleotide-gated current specifically carried by Ca(2+) in intact rods and cones. We activated CNG channels by suddenly (<5 ms) increasing free 8Br-cGMP in the cytoplasm of rods or cones loaded with a caged ester of the cyclic nucleotide. Simultaneous with the uncaging flash, we measured the cyclic nucleotide-dependent changes in membrane current and fluorescence of the Ca(2+)-binding dye, Fura-2, also loaded into the cells. The ratio of changes in fura-2 fluorescence and the integral of the membrane current, under a restricted set of experimental conditions, is a direct measure of the fractional Ca(2+) flux. Under normal physiological salt concentrations, the fractional Ca(2+) flux is higher in CNG channels of cones than in those of rods, but it differs little among cones (or rods) of different species. Under normal physiological conditions and for membrane currents

A cGMP-signaling pathway in a subset of olfactory sensory neurons.

It is well established that signal transduction in sensory neurons of the rat olfactory epithelium involves a cAMP-signaling pathway. However, a small number of olfactory neurons specifically express cGMP-signaling components, namely a guanylyl cyclase (GC-D) and a cGMP-stimulated phosphodiesterase (PDE2). Here, we show that this subset of olfactory neurons expressing GC-D and PDE2 does also express the subunit of a cGMP-selective cyclic nucleotide-gated (CNG) channel that has been previously identified in cone photoreceptors. Further, components of the prototypical cAMP-signaling pathway could not be detected in this subpopulation of cells. These results imply that these neurons use an alternative signaling pathway, with cGMP as the intracellular messenger, and that, in these cells, the receptor current is initiated by the opening of cGMP-gated channels.

A point mutation in the pore region alters gating, Ca(2+) blockage, and permeation of olfactory cyclic nucleotide-gated channels.

Upon stimulation by odorants, Ca(2+) and Na(+) enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotide-gated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the alpha subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotide-gated channels play a similar role. We studied the corresponding glutamate (E340) of the alpha subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca(2+) and Mg(2+). E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca(2+) and Mg(2+) powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca(2+) relative to Na(+) was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca(2+) and Mg(2+), and affects channel gating by cAMP more than by cGMP.

Cell biology. A universal bicarbonate sensor.

The life-span of sperm may be short but it is certainly busy. The three principal molecular events that prepare sperm for fertilization are all controlled by the intracellular nucleotide adenosine 3',5'-monophosphate (cAMP). One of these, capacitation, is also regulated by bicarbonate ions. The elusive connection between cAMP and bicarbonate ions now appears to be solved as Kaupp and Weyand explain in their Perspective. Bicarbonate ions enter sperm through the anion transporter in the sperm plasma membrane and activate the soluble form of adenylyl cyclase, the enzyme that synthesizes cAMP (Chen et al.)

Permeation and activation of the M2 ion channel of influenza A virus.

The M(2) ion channel protein of influenza A virus is essential for mediating protein-protein dissociation during the virus uncoating process that occurs when the virus is in the acidic environment of the lumen of the secondary endosome. The difficulty of determining the ion selectivity of this minimalistic ion channel is due in part to the fact that the channel activity is so great that it causes local acidification in the expressing cells and a consequent alteration of reversal voltage, V(rev). We have confirmed the high proton selectivity of the channel (1.5-2.0 x 10(6)) in both oocytes and mammalian cells by using four methods as follows: 1) comparison of V(rev) with proton equilibrium potential; 2) measurement of pH(in) and V(rev) while Na(+)(out) was replaced; 3) measurements with limiting external buffer concentration to limit proton currents specifically; and 4) comparison of measurements of M(2)-expressing cells with cells exposed to a protonophore. Increased currents at low pH(out) are due to true activation and not merely increased [H(+)](out) because increased pH(out) stops the outward current of acidified cells. Although the proton conductance is the biologically relevant conductance in an influenza virus-infected cell, experiments employing methods 1-3 show that the channel is also capable of conducting NH(4)(+), probably by a different mechanism from H(+).