Trötschel C et al. (2020), Absolute proteomic quantification reveals desig...

ECB-ART-48688

EMBO J 2020 Feb 17;394:e102723. doi: 10.15252/embj.2019102723.

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Absolute proteomic quantification reveals design principles of sperm flagellar chemosensation.

Trötschel C , Hamzeh H , Alvarez L , Pascal R , Lavryk F , Bönigk W , Körschen HG , Müller A , Poetsch A , Rennhack A , Gui L , Nicastro D , Strünker T , Seifert R , Kaupp UB .

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Cilia serve as cellular antennae that translate sensory information into physiological responses. In the sperm flagellum, a single chemoattractant molecule can trigger a Ca2+ rise that controls motility. The mechanisms underlying such ultra-sensitivity are ill-defined. Here, we determine by mass spectrometry the copy number of nineteen chemosensory signaling proteins in sperm flagella from the sea urchin Arbacia punctulata. Proteins are up to 1,000-fold more abundant than the free cellular messengers cAMP, cGMP, H+ , and Ca2+ . Opto-chemical techniques show that high protein concentrations kinetically compartmentalize the flagellum: Within milliseconds, cGMP is relayed from the receptor guanylate cyclase to a cGMP-gated channel that serves as a perfect chemo-electrical transducer. cGMP is rapidly hydrolyzed, possibly via "substrate channeling" from the channel to the phosphodiesterase PDE5. The channel/PDE5 tandem encodes cGMP turnover rates rather than concentrations. The rate-detection mechanism allows continuous stimulus sampling over a wide dynamic range. The textbook notion of signal amplification-few enzyme molecules process many messenger molecules-does not hold for sperm flagella. Instead, high protein concentrations ascertain messenger detection. Similar mechanisms may occur in other small compartments like primary cilia or dendritic spines.

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Genes referenced: LOC100887844 LOC576733 pde5a

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	Figure 1. Signaling pathway controlling chemotactic navigation Schematic representation of signaling events. Left: time course of changes in cAMP (dotted line) and cGMP (solid line); right: time course of changes in V m (green), pHi (red); [Na+]i (orange), and [Ca2+]i (pink). Vertical dotted lines indicate time of stimulation.GC, receptor guanylate cyclase; CNGK, cyclic nucleotide‐gated K+‐selective channel; sNHE (SLC9C1), sperm‐specific Na+/H+ exchanger; HCN1, HCN2, hyperpolarization‐activated, and cyclic nucleotide‐gated channels; CatSper, cation channel of sperm; NCKX, Na+/Ca2+‐K+ exchanger; PMCA, plasma membrane Ca2+‐ATPase; PDE5, phosphodiesterase type 5; PDE10, phosphodiesterase type 10; sAC, soluble adenylate cyclase. For clarity, sAC is placed in the cytosol, although it is associated with the membrane; the same may apply to PDE10.
	Figure 2. Determination of flagellar volumes A–D. E. F′, F″.
	Figure 3. Dynamics of CNGK activation and cGMP hydrolysis Time course of the change in voltage V m evoked by a 2‐ms light flash in sperm that was loaded with caged cGMP (15 μM) and recorded in 100KASW. Inset: extended timescale showing the rise of V m. Fit of data (black) using two exponentials with time constants τrise = 17.4 ms and τrelax = 354 ms. Light flash in all panels is depicted as dashed magenta line.Time course of the changes in V m evoked by 100‐ms flash in 100KASW using caged cGMP (blue) and caged 8‐Br‐cGMP (black). The flashes released 260 nM cGMP or 2,600 nM 8‐Br‐cGMP.Time course of changes in V m in 100KASW evoked by 2‐s flashes at different light energy that corresponds to 440, 220, 75, and 8 nM/s of cGMP. Data were fitted using a heuristic model described by equation [Link] (Materials and Methods section) (black lines).Amplitude (red) and decay time τrelax (black) for different light energies and release rates for experiments as shown in panel C. τrelax was determined using a single exponential fit. The linear fit of the V m amplitude was restricted to low light levels; at higher levels, the V m response saturates (see panel C). Points indicate mean ± SD from five experiments.
	Figure 4. Discrete‐event simulations of cGMP dynamics Schemes illustrate three different situations defined by chemical reactions (2) to (5) in the Materials and Methods section. Colors of schemes refer to the simulations shown in panel (B).Characteristic decay of V m in experiments shown in Fig 3A and C (orange; τrelax = 0.384 s) is compared with cGMP hydrolysis by PDE5 in the absence of CNGK channels (blue; characteristic time τ = 0.131 s), dissociation of cGMP from CNGK at the rate k off (yellow; τ = 0.748 s), and cGMP hydrolysis in the presence of CNGK channels and PDE (violet; τ = 9.73 s). Simulations were done using those parameters listed in Appendix Table S3. cGMP is given in molecules.Simulations of the rate of cGMP hydrolysis upon varying the catalytic rate k cat of active PDE and the k on rate of cGMP binding to the CNBD of the CNGK channel. The gray plane indicates the experimental relaxation time (τrelax). The black dot represents k cat (24/s) and k on (5.2 × 107/M/s) compatible with previous studies (Appendix Table S3). The pairs of parameters compatible with the experimental τrelax are positioned at the intersection of the colored surface and the gray plane.Top view of panel C showing k off values compatible with k on rates used for simulations (assuming K D,CNGK = 25.7 nM; see Appendix Table S3).
	Figure 5. Recovery from hyperpolarization Time course of cGMP‐evoked changes in V m in 100KASW (red, 500 nM cGMP; pink, 9 nM cGMP) and normal ASW (blue, 500 nM cGMP; navy, 9 nM cGMP). The time constants of recovery after cGMP release (9 nM) were 289 ms (depolarization) and 303 ms (hyperpolarization). Signals in panels (A–C) were detected using the potentiometric probe FluoVolt; dashed magenta lines indicate the timing of the photolysis flash.Representative measurement of the dependence of recovery‐time constant on the amount of released cGMP. Inset: Decay time was constant for low stimulation levels equivalent to ρ 30 cGMP molecules or ≤ 3 resact molecules.Voltage response evoked by photolysis of DEACM‐caged cGMP (blue) or 8‐Br‐cGMP (red). The light energy was adjusted such that V m response amplitudes became similar; for 8‐Br‐cGMP, about five times more light energy was required. Light pulse 10 ms. V m responses evoked by cGMP in the presence (blue) and absence (red) of the HCN channel blocker ZD7288 (50 μM).cGMP‐evoked V m response in ASW (black), in the presence of IBMX (1 mM) (red), and in Ca2+‐free ASW ([Ca2+] ≤ 500 nM) in the presence of IBMX (1 mM) (blue). Signals in panels (D–F) were detected using the potentiometric probe Di‐8‐ANEPPS in the ratio mode.Normalized responses from panel (E).
	Figure 6. Compartmentalization of Ca2+ and pHi signals. Changes in relative fluorescence ∆F/F recorded from the head and the flagellum are shown in blue and black, respectively A, B. C. D.
	Figure 7. Channeling model of cGMP turnover cGMP molecules synthesized by the GC receptor rapidly bind to the CNGK channel. CNGK, PDE5, and cGMP form a ternary complex, and cGMP bound to CNGK is subsequently transferred to PDE5 for hydrolysis without being released to the cytosol.Relationship between cGMP synthesis rate and free cGMP concentration. We assume that a single GC molecule can synthesize 10 cGMP molecules during its lifetime of 150 ms (Pichlo et al, 2014). Inset: expanded scale.

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