Huang JH et al. (2024), Robust trigger wave speed in Xenopus cytoplasmi...

XB-ART-60771

Nat Commun 2024 Jul 10;151:5782. doi: 10.1038/s41467-024-50119-0.

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Robust trigger wave speed in Xenopus cytoplasmic extracts.

Huang JH, Chen Y, Huang WYC, Tabatabaee S, Ferrell JE.

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Self-regenerating trigger waves can spread rapidly through the crowded cytoplasm without diminishing in amplitude or speed, providing consistent, reliable, long-range communication. The macromolecular concentration of the cytoplasm varies in response to physiological and environmental fluctuations, raising the question of how or if trigger waves can robustly operate in the face of such fluctuations. Using Xenopus extracts, we find that mitotic and apoptotic trigger wave speeds are remarkably invariant. We derive a model that accounts for this robustness and for the eventual slowing at extremely high and low cytoplasmic concentrations. The model implies that the positive and negative effects of cytoplasmic concentration (increased reactant concentration vs. increased viscosity) are nearly precisely balanced. Accordingly, artificially maintaining a constant cytoplasmic viscosity during dilution abrogates this robustness. The robustness in trigger wave speeds may contribute to the reliability of the extremely rapid embryonic cell cycle.

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Species referenced: Xenopus Xenopus laevis
Genes referenced: cdk1
GO keywords: cell cycle

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	Fig. 1: Mitotic trigger wave speed is robust to change in cytoplasmic concentration. a Schematic view of the mitotic control network. Note the multiple interconnected positive and double-negative feedback loops. b Preparation of cycling Xenopus egg extracts and the workflow of monitoring spontaneous mitotic trigger waves in thin PTFE tubes by epifluorescence microscopy. c Measurements of protein concentrations in the original and concentrated extract (retentate) as well the filtrate from the ultrafiltration filters. n = 4 for extracts, 6 for retentates, and 5 for filtrates. d A montage (left) and its corresponding kymograph (right) of a single tube undergoing 3 rounds of mitosis in the course of ~2 h. The bright signal is polymerized microtubules stained with SiR-tubulin and the dark bands correspond to the mitotic state in which most microtubules are depolymerized. e Representative kymographs (right column) of extracts of different cytoplasmic concentrations prepared from 1 x extracts (top 2 rows) or from 2 x retentate (bottom 3 rows). Left column shows the volume fractions of buffer (XB buffer without sucrose) and extracts that went into the samples. f Duration of the first completely observable cell cycle under the microscope, starting from interphase. Means ± S.E.M. are shown, n = 3 (three independent experiments). g Speeds of mitotic trigger waves at different cytoplasmic concentrations. Means ± S.E.M. are shown. Data is compiled from n = 8 independent experiments. Source data are provided as a Source Data file.
	Fig. 2: Apoptotic trigger wave speed is robust to change in cytoplasmic concentration. a Schematic view of the apoptotic control system. Note the multiple positive feedback loops. b A representative montage of an apoptotic trigger wave in a PTFE tube induced from the lower end. Bright signal is rhodamine 110 released by caspase 3/7 cleavage of (Z-DEVD)2-R110, which reports the activation of caspase 3/7. c Representative kymographs (middle and right columns) of extracts at different cytoplasmic concentrations prepared from either 1 x extract (top 2 rows) or retentate (bottom 3 rows). Rhodamine 110 fluorescence is shown in gray scale (middle column) or pseudo-color (right column). Pseudo-coloring demonstrates that a range of fluorescence thresholds would give very similar estimates of trigger wave velocity. d Speeds of apoptotic trigger waves at different cytoplasmic concentrations. Means ± S.E.M. are shown. Data are compiled from n = 9 independent experiments. Source data are provided as a Source Data file.
	Fig. 3: Mitotic and apoptotic activities are well-approximate by the logistic equation. a Relative CDK1 activity as a function of time at mitotic onset, normalized to a maximal activity of 1, as measured by H1 kinase activity assay, can be well-fitted by a logistic model. Data are taken from Pomerening et al.41. b Apoptosis as a function of time, measured at one point in a Teflon tube of extract. Fluorescence was followed as a function of time by microscopy. c Kinetics of (Z-DEVD)2-R110 cleavage. Open circles are data at each time point. The red curve is based on the ODE model for caspase 3/7 activation and (Z-DEVD)2-R110 cleavage (Eq. 16) fitted to the data. d Kinetics of caspase 3/7 activation. Open circles are concentrations of active caspase 3/7 calculated based on the ODE model (Eq. 15). The red curve is the logistic equation fitted to the data. Source data are provided as a Source Data file. a Relative CDK1 activity as a function of time at mitotic onset, normalized to a maximal activity of 1, as measured by H1 kinase activity assay, can be well-fitted by a logistic model. Data are taken from Pomerening et al.41. b Apoptosis as a function of time, measured at one point in a Teflon tube of extract. Fluorescence was followed as a function of time by microscopy. c Kinetics of (Z-DEVD)2-R110 cleavage. Open circles are data at each time point. The red curve is based on the ODE model for caspase 3/7 activation and (Z-DEVD)2-R110 cleavage (Eq. 16) fitted to the data. d Kinetics of caspase 3/7 activation. Open circles are concentrations of active caspase 3/7 calculated based on the ODE model (Eq. 15). The red curve is the logistic equation fitted to the data. Source data are provided as a Source Data file.
	Fig. 4: The effective diffusion coefficient of AF488-BSA and apparent activation rate constant of caspase 3/7 decrease exponentially with cytoplasmic concentration. a Representative fluorescence correlation spectroscopy (FCS) autocorrelation functions for AF488-BSA in extracts with different cytoplasmic concentrations. (See paper for formula) is the autocorrelation function and (See paper for formula) is the time delay. b Effective diffusion coefficient of AF488-BSA at different cytoplasmic concentrations. Effective diffusion coefficients were calculated by fitting the autocorrelation data from FCS measurements to a Brownian diffusion model. A 60 s fluorescence intensity time course was registered for each FCS measurement. Means ± 90% CI calculated from 3 measurements (n = 1) are shown. Solid black curve is an exponential curve fitted to the means. c (Z-DEVD)2-R110 cleavage kinetics can be monitored as apoptotic trigger waves sweep through a tube of extract. In this example, fluorescence from cleaved (Z-DEVD)2-R110 at 11 positions (left) are shown on the right. Cleaved (Z-DEVD)2-R110 concentration was inferred from fluorescence. d (Z-DEVD)2-R110 cleavage kinetics in a kymograph (upper) can be represented as a series of traces (lower). e Traces shown in (d) were aligned by time (upper) and the model for caspase 3/7 activation and (Z-DEVD)2-R110 cleavage was fitted to the data. Individual fitted traces are shown in the bottom panel. f The effective positive feedback rate constant at different cytoplasmic concentrations was extracted from the fitted model. Shown are means ± S.E.M. compiled from the same 9 experiments as the ones shown in Fig. 2d. The black solid curve is an exponential curve fitted to the means. Source data are provided as a Source Data file.
	Fig. 5: Mitotic and apoptotic trigger wave speeds at different cytoplasmic concentrations follow the generalized Luther’s formula. a Apoptotic wave speeds at different cytoplasmic concentrations. The solid black curve is the generalized Luther’s formula fitted to the means. Error bars are means ± S.E.M. Data are taken from the same 9 experiments as the ones shown in Fig. 2d. b The value of (See paper for formula), calculated as the product of the experimentally determined parameters (See paper for formula), (See paper for formula), and (See paper for formula), is compared to the fitted value (left panel). Likewise, the value of (See paper for formula) calculated as the sum of the experimentally determined parameters (See paper for formula), and (See paper for formula), is compared to the fitted value (right panel). (See paper for formula), (See paper for formula), (See paper for formula), and (See paper for formula) are from the exponential fits shown in Fig. 4. Error bars are S.E.M.s calculated directly from the fittings or propagated from the individual experimentally determined parameters. c Apoptotic wave speeds at low cytoplasmic concentrations. Dashed line shows the same fitted curve as in (a), which was obtained from only higher concentration data. Shown are means ± S.E.M. from 3 independent samples. d Mitotic wave speeds at different cytoplasmic concentrations, replotted from Fig. 1g and fitted to the generalized Luther’s formula (black curve). Error bars are means ± S.E.M. Data are from 8 independent experiments. Source data are provided as a Source Data file.
	Fig. 6: Opposing kinetic and physical effects give rise to robustness in trigger wave speeds. a Comparing the effect sizes of changes in rate constant, effective diffusion coefficient, and concentration. Effect size is defined as fold change relative to 1 x cytoplasmic concentration. For simplicity, the data for cytoplasmic concentrations < 0.3x, where the concentration effect dominates, are omitted. b, e Effective diffusion coefficients of AF488-BSA in XB buffers of various sucrose concentrations (b; no BSA present) or BSA concentrations (e; no sucrose present) as determined by FCS. Means ± 90% CI (n = 1) calculated from 3 measurements are shown. AF488-BSA diffusion in 1 x extract can be mimicked by ~0.8 M sucrose or ~150 mg mL−1 BSA. c, f Apoptotic trigger wave speeds with extracts diluted with buffer (black data points) or a viscogen (red data points) and compared with theoretical curves (dashed lines). Crowding effects are quantified by the parameter (See paper for formula). For apoptotic trigger waves, (See paper for formula) is ~1.3 (Fig. 5b) and is 0 if crowding effects are absent. We note that, in the case of (See paper for formula), wave speed (See paper for formula) is proportional to the square root of total caspase 3/7 concentration (See paper for formula). Means ± S.E.M. (n = 3) are shown for sucrose-containing buffers (c). Means (n = 2) are shown for BSA-containing buffers (f). The curves were set to equal the measured wave speeds at 1 x cytoplasmic concentration for both sucrose-containing and BSA-containing buffers. Apoptotic wave speeds at 0.5 x cytoplasmic concentration are also plotted. We note the scaling can be approximated by a horizontal line for (See paper for formula) between 0.5 x and 1 x cytoplasmic extract, whereas for (See paper for formula), a straight line with a positive slope. d, g Apoptotic trigger wave speeds are plotted for a range of sucrose-containing (d) or BSA-containing buffers (g) at different cytoplasmic concentrations. Means ± S.E.M. (n = 3) are shown for sucrose-containing buffers (d) whereas means (n = 2) are shown for BSA-containing buffers (g). Straight lines were fitted to each sucrose (d) or BSA (g) concentration. Only buffer without sucrose or BSA manifest straight lines with slightly negative slopes. Viscogen-containing buffers, be it sucrose or BSA, manifest positive slopes. Source data are provided as a Source Data file.
	Supplementary Fig. 1. Mitotic trigger wave speed is robust to dilution using either filtrate or buffer. Speeds of mitotic trigger waves at different cytoplasmic concentrations. The cycling extracts were diluted using either filtrate, XB buffer without sucrose, or water. Note that dilution with filtrate and buffer produced comparable wave speeds, whereas dilution with water resulted in a drop in speed. For extracts diluted with water below 0.6x, cycle progression was not observed. Means ± S.E.M. compiled from n = 3 independent samples are shown.
	Supplementary Fig. 2. Cell cycle and mitotic trigger waves at different cytoplasmic concentrations. a, b Representative kymographs depict cell cycle and mitotic trigger waves at various cytoplasmic concentrations, prepared from 2x retentate (a) and 1x extract (b). c Two additional instances showcase 0.5x extracts prepared from 2x retentate, highlighting the variability in the first complete cell cycle across different extracts. d In contrast to the continuous cycling observed in Fig. 1e, these two examples of 2x retentate underwent mitotic arrest, occurring either in the first mitosis or the second.
	Supplementary Fig. 3. Apoptotic trigger wave speed is robust to dilution using filtrate, buffer, and water. Speeds of apoptotic trigger wave at different cytoplasmic concentrations. The interphase extracts were diluted using either filtrate, XB buffer without sucrose, or water. Means from n = 2 independent experiments are shown.
	Supplementary Fig. 4. Anomalous diffusion fit to AF488-BSA diffusion in extracts. a, b FCS autocorrelation functions of AF488-BSA in extracts analyzed using the anomalous diffusion framework. Original data is the same as in Figure 4. Mean ± 90 CI from 3 measurements are shown (n = 1). Diffusion time 𝜏𝐷 (a) and anomalous diffusion exponent 𝛼 (b) at different cytoplasmic concentrations.
	Supplementary Fig. 5. Measuring (Z-DEVD)2-R110 cleavage rate coefficient ��. a Kinetics of (Z-DEVD)2-R110 cleavage in freshly prepared apoptotic extracts at various cytoplasmic concentration. These extracts were prepared from 1x extract (top) or 2x retentate (bottom). Data from 1 experiment are shown. b Cleavage rates of (Z-DEVD)2-R110 at different cytoplasmic concentrations were estimated based on the initial timepoints. c Second-order rate coefficients at different cytoplasmic concentrations were computed by accounting for (ZDEVD)2-R110 concentration and nominal active caspase 3/7 concentrations. Data are presented as means ± S.E.M. compiled from n = 3 experiments.