The events that occur during chemotaxis of sperm are only partly known. As an essential step toward determining the underlying mechanism, we have recorded Ca 2 þ dynamics in swimming sperm of marine invertebrates. Stimulation of the sea urchin Arbacia punctulata by the chemoattractant or by intracellular cGMP evokes Ca 2 þ spikes in the flagellum. A Ca 2 þ spike elicits a turn in the trajectory followed by a period of straight swimming ('turn-andrun'). The train of Ca 2 þ spikes gives rise to repetitive loop-like movements. When sperm swim in a concentration gradient of the attractant, the Ca 2 þ spikes and the stimulus function are synchronized, suggesting that precise timing of Ca 2 þ spikes controls navigation. We identified the peptide asterosap as a chemotactic factor of the starfish Asterias amurensis. The Ca 2 þ spikes and swimming behavior of sperm from starfish and sea urchin are similar, implying that the signaling pathway of chemotaxis has been conserved for almost 500 million years.

Eggs attract sperm by chemical factors, a process called chemotaxis. Sperm from marine invertebrates use cGMP signalling to transduce incident chemoattractants into changes in the Ca2+ concentration in the flagellum, which control the swimming behaviour during chemotaxis. The signalling pathway downstream of the synthesis of cGMP by a guanylyl cyclase is ill-defined. In particular, the ion channels that are involved in Ca2+ influx and their mechanisms of gating are not known. Using rapid voltage-sensitive dyes and kinetic techniques, we record the voltage response that is evoked by the chemoattractant in sperm from the sea urchin Arbacia punctulata. We show that the chemoattractant evokes a brief hyperpolarization followed by a sustained depolarization. The hyperpolarization is caused by the opening of K+-selective cyclic-nucleotide-gated (CNG) channels in the flagellum. Ca2+ influx commences at the onset of recovery from hyperpolarization. The voltage threshold of Ca2+ entry indicates the involvement of low-voltage-activated Ca(v) channels. These results establish a model of chemosensory transduction in sperm whereby a cGMP-induced hyperpolarization opens Ca(v) channels by a 'recovery-from-inactivation' mechanism and unveil an evolutionary kinship between transduction mechanisms in sperm and photoreceptors.

Protein diffusion in lipid membranes is a key aspect of many cellular signaling processes. To quantitatively describe protein diffusion in membranes, several competing theoretical models have been proposed. Among these, the Saffman-Delbrück model is the most famous. This model predicts a logarithmic dependence of a protein's diffusion coefficient on its inverse hydrodynamic radius (D ∝ ln 1/R) for small radius values. For large radius values, it converges toward a D ∝ 1/R scaling. Recently, however, experimental data indicate a Stokes-Einstein-like behavior (D ∝ 1/R) of membrane protein diffusion at small protein radii. In this study, we investigate protein diffusion in black lipid membranes using dual-focus fluorescence correlation spectroscopy. This technique yields highly accurate diffusion coefficients for lipid and protein diffusion in membranes. We find that despite its simplicity, the Saffman-Delbrück model is able to describe protein diffusion extremely well and a Stokes-Einstein-like behavior can be ruled out.

The sea urchin sperm guanylyl cyclase chemoreceptor achieves ultrasensitive signal detection and precise signal modulation through high receptor density, subnanomolar ligand affinity, and sequential dephosphorylation.

Cytoplasmic dynein is a protein complex responsible for transporting cellular cargos on microtubules. Dynein's activities are regulated by other proteins including dynactin which mediates dynein-cargo interactions and increases dynein's processivity. The intermediate chain (IC) of dynein binds to dynactin p150Glued subunit, an interaction central to dynein regulation. The N-terminal domain of IC, N-IC is partially disordered, and contains binding site for a coiled-coil domain of p150Glued and a serine-rich region known to undergo phosphorylation in vivo. There are conflicting results to the effect of phosphorylation on dynactin binding. Using different techniques, one group showed phosphorylated S84 and a phosphomimetic mutant S84D abolished p150Glued binding in vitro while another group demonstrated S84D was still able to bind. We use mutagenesis, constructs of varying length, isothermal titration calorimetry (ITC) and NMR spectroscopy to identify the effect of phosphomimetic S84D on structure of IC and its ability to bind p150Glued. Backbone assignments of IC2C1-96 and secondary chemical shifts show residues 4-38 (helix 1) and 52-66 (helix 2) adopt helical structures and the rest of the protein is disordered. NMR and ITC titration studies identify the first 44 residues as sufficient for binding. The phosphomimetic mutant IC2CS84D abolishes p150Glued binding, without causing major changes in structure and dynamics as detected from NMR measurements. Chemical shift comparison between WT and S84D shows long-range effects on residues in the linker between helix 1 and 2, suggesting conformational changes in the linker. Mutations in the linker region restore p150Glued binding. Together, these results demonstrate for the first time phosphorylation in a disordered region affects binding at a distant site in the sequence, and is fine-tuned by changes in a disordered linker separating two structured domains. This regulatory control of dynein-dynactin interactions is key to cargo-and temporal-specific alterations in dynein transport.