Kashikar ND et al. (2012), Temporal sampling, resetting, and adaptation or...

ECB-ART-42580

J Cell Biol 2012 Sep 17;1986:1075-91. doi: 10.1083/jcb.201204024.

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Temporal sampling, resetting, and adaptation orchestrate gradient sensing in sperm.

Kashikar ND , Alvarez L , Seifert R , Gregor I , Jäckle O , Beyermann M , Krause E , Kaupp UB .

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Sperm, navigating in a chemical gradient, are exposed to a periodic stream of chemoattractant molecules. The periodic stimulation entrains Ca(2+) oscillations that control looping steering responses. It is not known how sperm sample chemoattractant molecules during periodic stimulation and adjust their sensitivity. We report that sea urchin sperm sampled molecules for 0.2-0.6 s before a Ca(2+) response was produced. Additional molecules delivered during a Ca(2+) response reset the cell by causing a pronounced Ca(2+) drop that terminated the response; this reset was followed by a new Ca(2+) rise. After stimulation, sperm adapted their sensitivity following the Weber-Fechner law. Taking into account the single-molecule sensitivity, we estimate that sperm can register a minimal gradient of 0.8 fM/µm and be attracted from as far away as 4.7 mm. Many microorganisms sense stimulus gradients along periodic paths to translate a spatial distribution of the stimulus into a temporal pattern of the cell response. Orchestration of temporal sampling, resetting, and adaptation might control gradient sensing in such organisms as well.

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Genes referenced: LOC100887844 LOC100893907 LOC115919910 LOC594261

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	Figure 1. Illustration of the experimental rationale. (A) Simulated stimulus function for a sperm exposed to repetitive pulses of resact: a time interval Δt = 1 s each. Stimulus function was calculated using Eq. 3. (B) Stimulus function for paired resact stimuli with longer Δt. (C and D) Time course of bound resact molecules per flagellum. (C, black line) Repetitive pulses of resact. (C, colored lines) Sperm, swimming on a circular path in a chemoattractant gradient, at different distances from the egg (in millimeter): 2.1, dark yellow; 2.2, red; 2.3, blue; 2.4, magenta; 2.5, green; 2.6, navy; 2.7, violet; 2.8, purple; 2.9, wine. (D) Two resact pulses at long Δt. (E) A second stimulus delivered during the latency (interrupted lines) of the Ca2+ signal probed temporal sampling. Latency of the Ca2+ signal (blue) matches the time to peak of the hyperpolarization Vm signal (red). (F) Complete time course of a typical Vm (red) and a corresponding Ca2+ signal (blue). In paired stimulus experiments, the second stimulus was delivered at different phases of the Ca2+ and Vm signals. Arrows indicate time of flashes.
	Figure 2. Sperm temporally sample chemoattractant molecules. (A and B) Identical paired stimuli of 12.5 pM resact were delivered by photolysis of 100 nM caged resact; stimulus functions (top), Vm signals (middle), and Ca2+ signals (bottom) are shown. Flashes in A: single 50% (blue), single 100% (black), and paired 50% with Δt = 50 ms (red). Flashes in B: single 50% (blue), single 100% (black), and paired 50% with Δt = 100 ms (red) or 200 ms (green). (C) Decrease of sampling efficacy of Vm (red) and Ca2+ (black) signals with increasing Δt. Three experiments as shown in A and B were analyzed. The Vm and Ca2+ signals evoked by a full intensity flash were normalized to 0 (baseline) and 1 (peak amplitude). The signal amplitudes evoked by paired half-intensity flashes were scaled to these two values. (D) Sampling efficacy depends on stimulus strength. The low stimulus strength regimen was achieved using 10 nM caged resact and flashes of 25 and 50% (black; n = 4). The high stimulus strength regimen was achieved using 100 nM caged resact and flashes of 50 and 100% (red; n = 3). (E) Sperm temporally sample changes in cGMP concentration. Ca2+ signals were evoked by photolysis of caged cGMP. Flashes: single 10% (blue), single 20% (black), and two 10% with a Δt of 30 ms (red), 75 ms (green), or 150 ms (magenta). (F) Vm signals evoked by paired stimuli of cGMP. Flashes: single 8% (blue), single 16% (black), and two 8% with Δt = 50 ms (red). (Inset) Vm signals of identical amplitude evoked by cGMP (green) and resact (orange). (G) Sperm count single resact molecules. Ca2+ signals were evoked by releasing ∼625 fM resact (blue), single full intensity flash (1.25 pM; black), and two half-intensity flashes (625 fM each) with Δt = 30 ms (red) or 75 ms (green). Error bars show SDs. Arrows indicate time of flashes.
	Figure 3. A second stimulus produces a Ca2+ drop followed by a new Ca2+ rise. (A) Ca2+ signals were evoked by cGMP. Single 25% flash (blue) and two identical 25% flashes (red; Δt = 1 s) are shown. (inset) The second Ca2+ signal was shifted by −Δt to the left and superposed on the first Ca2+ signal. (B) Vm signals evoked by paired cGMP stimuli (Δt = 1 s). (inset) Kinetics of the Ca2+ drop (blue) and the hyperpolarization (red) evoked by the second stimulus. Downward peaks of the Ca2+ drop and the hyperpolarization were normalized. (C and D) Ca2+ (C) and Vm (D) signals. Paired stimuli (caged resact of 100 nM) were delivered. Intensity of the first flash was kept constant at 50%, and intensity of the second flash (Δt = 1 s) was varied (in percentage): 10 (green), 25 (magenta), 50 (blue), and 100 (red). For comparison, a 50% flash signal without second flash is shown (black). (E) Relationship between ΔVm after the second flash and slope of the Ca2+ drop. The plot was constructed from experiments as in C and D (Vm signals, n = 4; Ca2+ signals, n = 3). Absolute values in millivolts for ΔVm were obtained from ΔR signals as previously described in Strünker et al. (2006). A linear regression was fitted through data points. Numbers near data points indicate the intensity of the second flash. Error bars show SDs. (F) Scheme of the delivery of a second stimulus at different Δt along a Ca2+ signal. (G) Paired cGMP flashes (10% each) were delivered with Δt (in seconds) of 2 (black), 4 (red), 6 (blue), 14 (green), and 28 (magenta). (H) Comparison of Ca2+ drops evoked by the second flash at various Δt as in G. The second flashes were aligned to t = 0 by shifting the x axis to the left by the respective −Δt. The fluorescence before t = 0 shows the [Ca2+]i at the time of the second flash. The Ca2+ signal evoked by the first flash is shown in orange. (I) Ca2+ drop amplitude (ΔFdrop) versus Ca2+ level (Fnorm) at the time of the second flash (blue symbols, n = 21). To compare across experiments, Ca2+ signals were normalized to 0 (baseline Ca2+ signal) and 1 (Ca2+ signal amplitude after the first flash). A linear regression was fitted (R2 = 0.98). Arrows indicate time of flashes.
	Figure 4. Sperm encode periodic stimuli. (A and B) Ca2+ (A) and Vm (B) signals upon repetitive stimuli of cGMP (25% flash; Δt = 1 s). (A, inset) Plot of peak amplitudes of Ca2+ rises after each flash. The Ca2+ signal amplitude after the first flash was taken as 1, and subsequent amplitudes were normalized (n = 6). (C) Superposition of Ca2+ drops as in A. Each trace represents the flash depicted in A. Flash number: second (red), third (green), fourth (blue), fifth (cyan), sixth (magenta), seventh (yellow), and eighth (dark yellow). Ca2+ drops were aligned to the time of the respective flash. (inset) Slope of Ca2+ rise after the drop after each flash (n = 6). Error bars are SDs. (D) Superposition of Vm signals as in B. Flash number: first (cyan), second (black), third (red), fourth (blue), fifth (green), sixth (magenta), seventh (dark yellow), and eighth (navy). For superposition, signals were aligned to the time of the respective flash. (inset) Plot of Vm amplitude (Δ(ΔR)) after each flash. To compare across different trials (n = 5), ΔVm after the first flash was taken as 1, and subsequent amplitudes were normalized. Arrows indicate time of flashes.
	Figure 5. Ca2+ drop in single cells. (A) Ca2+ signals from three different cells (depicted in three different colors) evoked by paired cGMP flashes (dashed lines; Δt = 800 ms). (B and C) Representative swimming paths and corresponding Ca2+ signals from single cells after a single flash (B, yellow box and black flashes) or two flashes (Δt = 800 ms; C, yellow boxes and black flashes). The head (blue traces) wiggles around the average path (black trace). Swimming direction is shown by red arrows. After the second flash (C), the swimming path transiently bent in a clockwise direction, whereas the control cell swam undeviated (B, gray flash).
	Figure 6. Functional implications of the Ca2+ drop. (A) Ca2+ signals derived from the data in Fig. 3 C. The amplitude of Ca2+ signals was rescaled to match the mean amplitude of single-cell Ca2+ signals. (B) Swimming paths reconstructed from Ca2+ signals in part A. Arrow indicates the swimming direction shortly before the second flash. (C) Sperm swimming in a gradient of resact. The UV profile used for uncaging resact is shown in shades of gray. At the foot and on the top of the gradient, the cell swims on smooth drifting circles (orange). In the region where the gradient is steep, the cell swims on looping trajectories (green). Arrows indicate the swimming direction.
	Figure 7. Adaptation of Vm and Ca2+ signals. (A) Adaptation and recovery of the Vm signal after an adapting stimulus. Sperm were mixed with 100 nM caged resact, equivalent to 100 pM resact because of the “residual” activity of caged resact. The final free resact concentration was ∼400 fM. Sperm were probed with a test stimulus (100% flash; releasing ∼30 pM resact) at different Δt (in seconds): 0.4 (black), 1.4 (red), 3.4 (green), 7.4 (blue), and 15.4 (cyan). (B) Recovery kinetics of the Vm signal as shown in A (n = 3; color coding as in A). The data points were fit with an exponential curve (R2 = 0.99). (C) Weber–Fechner plot. Sperm were adopted by background concentrations cb of 0.27, 2.1, 4.1, 10.1, 20.4, and 52.5 pM. Test stimuli cb of either resact or cGMP were given. 15 pM (black symbols) or 30 pM (red symbols) resact (cs) was released from caged resact. The voltage response S = ΔVm was normalized to the value in the absence of background S0 and plotted versus the concentration ratio R = cb/cs. Similarly, test stimuli of cGMP were probed (blue symbols). Solid lines were calculated using either Eqs. 1 or 2, respectively (resact, n = 3 and cGMP, n = 4). (D) Ca2+ signals evoked by a test stimulus of cGMP at different Δt of (in seconds): 1.2 (magenta), 3.2 (green), 7.2 (blue), 15.2 (red), and 29.2 (black). Ca2+ signal evoked by 4.1 pM background resact alone is shown in orange. (E) Shift of the dynamic range of Ca2+ signals. Test stimuli of cGMP (Δt = 15.2 s) were given either in the absence (black) or presence of adapting resact (in nanomolars): 0.25 (red), 2.5 (blue), and 25 (green). Ca2+ signal amplitudes produced by a test cGMP stimulus were plotted; in the presence of background resact, the Ca2+ signal amplitude was the difference between the value before the test stimulus and the value at the minimum of the Ca2+ drop. The data were fitted with the equation A=AmaxInIn+K1/2n, wherein A is the response amplitude, I denotes the flash intensity, and n is the Hill coefficient. (F and G) Apparent K1/2 (F) and response compression (G) at different background concentrations. Data are obtained from E. Error bars show SDs. Arrows indicate time of flashes.
	Figure 8. Range of sperm attraction. (A) Determination of the diffusion coefficient of resact by two-focus fluorescence correlation spectroscopy. Resact was labeled at Cys 1 with Alexa Fluor 488 dye. The autocorrelation function of the first focus (black), the second focus (red), and the cross correlation of both foci (yellow and green) are shown. Symbols are experimental data points, and solid lines are global fits using three fit parameters (see Materials and methods). Residuals are shown on the bottom. (B) Shape of the resact gradient from a single A. punctulata egg for different times (1–60 min) after egg release. (C, top) Peak resact concentration at the center of release for different times after egg release. (bottom) Distance from the egg at which a cell captures one resact molecule during a swimming circle. (D) Cartoon of circular swimming of sperm in a chemoattractant gradient. The concentration of resact (blue) increases linearly from c1 to c2 during a complete circle. The cell compares the concentration of resact bound along both semicircles and computes the difference ΔNabs. (E) Resact gradient (black) and the minimal gradient gmin (red) for the indicated times after egg release. For the effective range of sperm attraction, the resact gradient must be larger than gmin. (F) Effective range of sperm attraction versus time after egg release. At t > 30 min, gresact ≤ gmin in the very vicinity of the egg. Hence, we observe two boundaries, a short one and a long one for the effective gradient. (G) Resact gradient gresact (black) and minimal gradient gmin (red) for an egg that synthesizes 1,000 resact molecules/s. 60 min after egg release, the range of attraction is similar to that for an egg with no synthesis. However, near the egg, gresact > gmin even after 60 min.

References [+] :

Alvarez, The rate of change in Ca(2+) concentration controls sperm chemotaxis. 2012, Pubmed, Echinobase