Coordinated oscillations in cortical actin and Ca2+ correlate with cycles of vesicle secretion

Abstract

The actin cortex both facilitates and hinders the exocytosis of secretory granules. How cells consolidate these two opposing roles was not well understood. Here we show that antigen activation of mast cells induces oscillations in Ca(2+) and PtdIns(4,5)P(2) lipid levels that in turn drive cyclic recruitment of N-WASP and cortical actin level oscillations. Experimental and computational analysis argues that vesicle fusion correlates with the observed actin and Ca(2+) level oscillations. A vesicle secretion cycle starts with the capture of vesicles by actin when cortical F-actin levels are high, followed by vesicle passage through the cortex when F-actin levels are low, and vesicle fusion with the plasma membrane when Ca(2+) levels subsequently increase. Thus, cells employ oscillating levels of Ca(2+), PtdIns(4,5)P(2) and cortical F-actin to increase secretion efficiency, explaining how the actin cortex can function as a carrier as well as barrier for vesicle secretion.

Figure 1. Depolymerization of cortical F-actin increases total amount of secreted enzyme but with slower initial kinetics

a Population measurement of total secreted β-hexoamidase 30-min past pharmacological activation with Ionomycin (1μM) and PdbU (100ng/ml) addition to cells pre-treated with 4 μM Latrunculin (green) or DMSO control (blue). (P-value <0.001, two sample t-test, error-bars s.e.m, N=64). b Time courses of initial loss of secretory granule monitored with LysoTracker. Loss of SG is defined as the relative drop in fluorescent intensity from the intensity prior to antigen addition. Cells were stimulated with Ionomycin (1μM) and PdbU (100 ng/ml); control (DMSO) and Latrunculin A (4 μM) pre-treated (5 min prior to drug addition) cells shown in blue and green, respectively. Errorbars show the 95% confident intervals (N=1258 & 2623). c-d Disappearance of secretory granule marker LysoTracker corresponds to an increase in exocytosis marker VAMP7-pHlourin. c Time series of secretory granule marker (SG) and pH sensitive VAMP7-pHlourin. Black vertical line marks the addition of 1μM Ionomycin and 100 ng/ml PdBU. Dashed lines show time-points of snapshots shown in d Scale-bar 5 μm. Note that both panel a and c are based on LysoTracker marking of SG, with panel a shows the loss of SG and panel c shows remaining SG intensity to correspond to the images in d. e Time series of Calcium (orange) signals and dextran-FITC (red) release following activation of the mast cell receptor FcεRI. The increase in pH during vesicle fusion results in de-quenching of FITC, causing a transient fluorescent secretion signal.

Figure 2. Receptor-stimulated cell-wide and local F-actin oscillations

a Live measurements of local cortical F-actin oscillations using F-Tractin biosensor and TIRF optical sectioning (left). Distinct local cortical F-actin concentration changes are observed at different sites, two representative regions of interests are shown in blue in the periphery and green in the cell center (right). b Two types of F-actin oscillations with distinct power spectra. Amplitudes of the power spectra (decibel, dB) were converted to a color code (left panel, x-axis; period time labels) for each pixel in a cell (y-axis), and clustered into 2 groups. The two subtypes of oscillations map to center and peripheral cell adhesion regions, respectively. Map (right) shows the projection of the major period of each local F-Tractin power spectra onto the cell image. c Oscillations are synchronized between pixels across the entire center region but not in the peripheral region. Panel on left shows single site examples with exponential fits to the spatial correlation function for pixels in the center and peripheral regions, respectively. Image on right shows a map of the fitted spatial correlation length for each pixel. Scale-bar 5 μm.

Figure 3. Oscillations in PI(4,5)P2 lipid precede F-actin oscillations

a Secretion occurs exclusively in the center and not peripheral region. Left two panels show a cell loaded with the secretory marker dextran-FITC before (left) and 1 second (middle) after an exocytosis event, scale-bar 5 μm, exocytosis event is marked by a yellow box. Kymograph in middle is taken through the center of the yellow box. The right shows a map projecting 155 exocytosis events from 12 cells according to their distance from the periphery (monitored in the first 20 minutes after antigen addition). The red line marks the normalized area where central F-actin oscillations are observed. b Synchronized phase-shifted oscillations in PI(4,5)P2 (monitored with PH-PLCδ) and cortical F-actin. c Example of a time series of signals measured with Epi-fluorescent of Fura-2 (orange; Ca2+ signals) and a second PI(4,5)P2 biosensor, the Tubby domain from the Tubby protein tagged with YFP and imaged in TIRF (cyan). Raw data for panels b & c is presented in Supp Table 1 d Cross-correlation between the time series shown in c was used to estimate the lag and significance of the correlation between the two time series. e Comparison of the lag between calcium and PH-plcδ to that between calcium and Tubby domain. Bars show median lag and errorbars are the median absolute deviation. P-value = 0.57, Wilcoxon rank sum test, N=15.

Figure 4. Oscillation in PM levels of N-WASP connect PI(4,5)P2 and F-actin oscillations

a Synchronized phase-shifted oscillations in N-WASP (purple) and cortical F-actin (F-Tractin, green) concentration in center region b. PI(4,5)P2 signals are followed by N-WASP recruitment and then F-actin polymerization. Lags between N-WASP and PHplcd to F-Tractin were significantly bigger than zeros (P-values <0.05 N=8 and P-value < 0.01 N=7 respectively) with the average lag between PHplcd and F-Tractin significantly bigger than the lag between N-WASP and F-Tractin (P-value <0.05, N=15, errorbars show s.e.m.). c A reduction in PI(4,5)P2 by forced translocation of a 5′PIP2 phosphatase rapidly decreases N-WASP PM localization. Partial recruitment of YFP-FKBP-Inp54p to the plasma membrane (upper panel) by the addition of 0.5 μM rapamycin (to trigger binding to PM localized FRB) (T=620) triggers a small reduction in the levels of PI(4,5)P2 as reported by the PH-plcδ-mTurquoise biosensor (middle panel). A small relative reduction in PI(4,5)P2 was sufficient to cause a rapid reduction in the localization of mCherry-N-WASP to the plasma membrane (lower panel). Raw data for panels a & c is presented in Supp Table 1.

Figure 5. Inhibition of N-WASP dynamic inhibits F-actin oscillation without effecting Ca2+ and PI(4,5)P2 oscillations

a Wiskostatin-mediated inhibition of N-WASP and actin dynamics. Co-oscillation of pathway components after stimulation with antigen. The dashed line indicated addition of 8.33 μM N-WASP inhibitor Wiskostatin. b Inhibition of N-WASP dynamics via chemically induced FRB/FKBP dimerization of an inhibitory N-WASP construct. Co-oscillation of pathway components after stimulation with antigen. In this experiment, N-WASP was tagged with an FKBP and a non-flourescent plasma membrane targeted FRB domain was co-expressed. The dashed line indicated addition of 5 μM Rapamycin that induces dimerization of the FKBP and FRB domains which caused anchoring of N-WASP to the plasma membrane that resulted in inhibition of actin oscillations without any significant change in PI(4,5)P2 oscillations. Raw data for panels a & b is presented in Supp Table 1.

Figure 6. Synchronized phase-shifted oscillations of Calcium, PI(4,5)P2, N-WASP, and F-actin

Ca2+ oscillations drive cortical F-actin oscillations. a Example of a live-cell 5-channel analysis of Ca2+ (Fura2), PI(4,5)P2, N-WASP and F-actin signals shows synchronized oscillations. Stopping Ca2+ oscillations using the SERCA ER pump inhibitor thapsigargin blocks all oscillations. b Changes in the intensity of the four biosensor in a representative cell. The time is indicated when actin oscillations where inhibited using 4 μM Latrunculin. c Suppressing actin polymerization using cytocholasin D blocked F-actin oscillations but not the other oscillations. Raw data for panels a, b & c is presented in Supp Table 1. d Schematics of the Ca2+ pathway that generates coordinated cortical F-actin oscillations. P-value indicates probability that the lag between sequential biosensors is bigger than zero based on cross-correlation analysis of 83 cycles in 4 cells. e Average normalized cycle intensities of all cycles. Color codes are the same as in panels a and b.

Figure 7. Mathematical model of how phase-shifted oscillations in cortical F-actin and Calcium correlates with a repetitive cycle of vesicle capture, passage and fusion to enhance secretion rates

a. Scheme of the mathematical model. (Inset) The F-actin and Ca2+ dependencies of the three vesicle state transitions, Capture, Passage and Fusion. Vcortex and V_pm are the respective concentrations of vesicles bound to actin cortex and plasma membrane. b. Simulation output of the model. The upper panel shows the model input: Ca2+ and F-actin oscillations. The middle panel shows the changes in the concentration of actin bound (blue) and plasma membrane bound (green) vesicles. The lower panel shows the output, the secretion rate. c. Comparison of model predictions of secretion rates for oscillatory and constant of F-actin and Ca2+ input levels. Two levels of constant Ca2+ and F-actin are shown: average, where the F-actin and Ca2+ levels are the mean of the oscillatory inputs and optimal where the levels where chose to maximize total secretion. d. Monitoring antigen-triggered recruitment of myosin Va, a motor protein that binds vesicles to F-actin and transports them. Left image panels show that recruitment of myosin Va occurs in the center where fusion occurs (maximum projection before and 2 minutes after antigen addition; scale-bar 5 μm). Right bar graph shows that, during F-actin oscillation cycles, the recruitment preferentially occurs when F-actin levels are high. As a control average intensity of a GFP membrane marker (Lyn) is compared to GFP-Myosin Va (MyoVa) during low actin (light green) and high actin (dark green) phases during oscillations. Errorbars are s.e.m, n = 83, p-value < 0.001 student t-test.

Figure 8. Direct measurements of enhancement in secretion due to Ca2+ and F-actin oscillations

a Bar diagram showing that secretion rates are more efficient for cells that oscillate (blue) compared to cells with elevated but non-oscillating Ca2+ levels (red). Addition of thapsigargin or cyotocholasin D to oscillating cells abolish the enhanced secretion without significantly affecting secretion when added to non-oscillating cells. Addition of Jasplakinolide abolished the oscillating cell’s advantage and caused an additional overall reduction in secretion. Addition of DMSO is included as a control. (N-oscillating = 2891, N-non-oscillating = 4642 cells; errorbars are s.e.m. Result of t-tests comparing the exocytosis level of oscillating cells to non-oscillating, secretion before the addition of drug or secretion after addition of DMSO are presented above the bar plot., n.s. not statistically significant, * P-value<0.05 and *** P-value<0.001). b Example of an oscillating cell treated with Thapsigargin, showing that secretion slows even though the average Ca2+ level is higher. c Example of an oscillating cell treated with Jasplakinolide, showing that stabilizing the actin cortex inhibits secretion. d Schematics of the proposed secretion cycle based on phase shifted F-actin and Ca2+ oscillation cycles. In each cycle, Capture occurs at high F-actin, followed by Passage at low F-actin, followed by Fusion when Ca2+ levels subsequently increase. Note that changes in the cartoon are not to scale and for illustration purpose only.