EPEC induces isolated Ca2+ responses of limited amplitude in epithelial cells.

HeLa cells were loaded with the fluorescent indicator Cal-520, challenged with the indicated bacteria and subjected to live-cell Ca2+ imaging at a frequency of one acquisition every 10 seconds (A-C, F) or fixed and processed for fluorescence microscopy analysis (D-E) (Materials and Methods). A, Representative traces of Ca2+ variations in single cells. The black arrowheads indicate the time of bacterial challenge. The blue arrowheads indicate stimulation with 3 µM histamine. B, Response amplitude expressed as a percent of the maximal histamine response amplitude (N = 3, n > 63). C, Percent of cells exhibiting Ca2+ responses (N =3, cells > 66). (D, E) Cells challenged with RFP-expressing bacteria for 1 hour. D, Representative confocal micrographs. Staining with DAPI (blue), phalloidin-Alexa 488 (green). The lower panels show a higher magnification of the insets in the top panels. Scale bar = 10 µm. E, Percentage of bacteria-associated actin-rich pedestals (N = 3, cells > 273). F, average number of responses per cell during the first 30 min (0-30) and last 30 min (30-60) of bacterial challenge. Low MOI: 10 bacteria / cell. High MOI: 50 bacteria / cell. Bar: mean. (N = 3, cells > 63). Mann-Whitney test. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

EPEC-induced Ca2+ responses are elicited by ATP released by the T3SS translocon

HeLa cells were loaded with the fluorescent indicator Cal-520 or with 200 μM suramin for 30 minutes, challenged with the indicated bacteria and subjected to live-cell Ca2+ imaging for a 60 min-duration at a frequency of one acquisition every 10 seconds. A, Representative traces of Ca2+ variations in single cells. The black arrowheads indicate the time of bacterial challenge. The blue arrowheads indicate stimulation with 3 µM histamine. B, Percent of cells exhibiting Ca2+ responses (N = 3, cells > 70). C, Average number of responses per cell. Low MOI: 10 bacteria / cell. High MOI: 50 bacteria / cell. Bar: mean. (N = 3, cells > 30). Mann-Whitney test. **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

EPEC induces rapid and Coordinated Elementary Ca2+ Responses.

HeLa cells were loaded with the fluorescent indicator Cal-520, challenged with the indicated bacteria and subjected to high speed Ca2+ imaging at a frequency of one acquisition every 57 ms for a duration of 110 seconds. A, Representative time-serie of pseudocolored fluorescent micrographs of cells challenged with wild-type EPEC. The numbers indicate the elapsed time in ms from an arbitrarily determined origin. Scale bar = 10 µm. B, D, traces of Ca2+ variations in 2 subcellular regions of the same cell. B, WT: traces corresponding to the regions depicted in the image 0 of panel A. The arrowheads point to the Ca2+ responses shown in Panel A with the corresponding color. C, Percent of cells exhibiting Ca2+ responses (N > 3, cells > 134). + Suramin: treatment with 200 μM Suramin. +U73122: treatment with 10 μM U73122. E, Response amplitude expressed as a percent of the maximal response amplitude induced by treatment with 3 μM histamine (N = 3, cells > 166). F, average number of responses per cell. C, E, Bar: mean. Mann-Whitney test. **: p < 0.01; ****: p < 0.0001. F, High speed Ca2+ imaging was performed every 5 min for 110 seconds following infection with the indicated bacterial strain as depicted the scheme. The average number of responses per cell is indicated (N > 3, cells > 29).

EPEC-induced Coordinated Elementary Ca2+ Responses are reproduced by low ATP levels.

A-C, HeLa cells were loaded with the fluorescent indicator Cal-520, challenged with 150 nM ATP and subjected to Ca2+ imaging. Image acquisition every 52 ms (A) or 22 ms (B, C). A, Traces of Ca2+ variations corresponding to a single cell (red trace), or subcellular regions within the same cell (inset). A, arrowhead: challenge with 2 μM ATP. B, Time serie of fluorescent micrographs pseudocolored using the “glow” Fiji lookup table, where the blue pixel correspond to an arbitrarily set threshold value. The numbers indicate the elapsed time in seconds. Blue: high intensity pixels showing the large top cell area with CCRICs and the local lower puff area. Scale bar = 10 µm. C, Traces corresponding to Ca2+ variations in the subcellular regions depicted in Panel B. The responses are labelled 1-4, with the response 1 corresponding to the puff (Panel B, red ROI) impulsing the response 3 in the same region. Responses 2 and 4 correspond to CCRICs in Panel B, blue and green ROIs. Note the diffusion of the responses from the initial release area in other area inferred from the dampening of the response amplitude.

Modeling of Coordinated Elementary Ca2+ Responses.

Top, Ca2+ variations in subcellular area within a single cell are represented in pseudocolor. Shown are the maximum values of Δ[Ca2+]/[Ca2+]b reached in each compartment during a 60s simulation. Empty white squares: IP3R clusters. Graphs, Traces correspond to Ca2+ variations in the region with the matching color. Colored arrows: Ca2+ response due to the activation of an IP3 cluster in the region with the matching color. Colored arrowhead and dashed red and blue lanes: Ca2+ variations due to the diffusion of a Ca2+ response from or nearby to the region with the matching color. Black arrows: Ca2+ response due Ca2+-activated Ca2+ release. A, low density of IP3R clusters with local responses detected. B, C, Empty black box: area with a high density IP3R clusters. C, similar to B, but following IP3R cluster sensitization due to increased Ca2+ responses.

Low ATP levels dampen NF-kappaB activation.

HeLa cells were stimulated with 10 ng/ml TNF-α alone or in the presence of 150 nM ATP or 20 μM BAPTA-AM (F-H). At the indicated time points, cell lysates were analyzed by Western blot using the indicated antibodies. A, D, F, G, Representative blots. B, C, E, H, Densitometry analysis of the indicated antibody signal normalized to that of HSP90 (B, C) or total P65 (E, H), expressed as fold-increase to basal levels of p-IκB (B), IκB (C) or p-p65 (E, H) at time = 0. Values correspond to the mean ± SEM of 3 or 4 independent experiments. p-p65: anti-phospho P65 antibody. p-IκB: anti-phospho IκB antibody. ANCOVA test. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Low ATP levels down-regulate NF-kB O-GlcNAcylation in a Ca2+-dependent manner.

HeLa cells were stimulated with 10 ng/ml TNF-α alone or in the presence of 150 nM ATP with or without 20 μM BAPTA-AM for 12 min. Cell lysates were subjected to P65 immunoprecipitation. A, Representative blots with the indicated antibodies. IP: immunoprecipitates; L: total cell lysates. B, Densitometry analysis of the O-GlucNAc signal in P65 immunoprecipitates normalized to that of TNF-α alone. Mann-Whitney test. N = 4. *: p < 0.05. ns: not significant.

Model describing the dynamics of a cluster of IP3Rs.

From Ornelas-Guevara et al. (2023). Ca²⁺ diffusion is modelled as a stochastic jump between adjacent compartments (Kraus et al., 1996). The propensity of a Ca²⁺ ion to move depends on the deterministic diffusion coefficient (100 µm²/s) and concentration gradients, assuming isotropic and homogeneous diffusion. All reaction and diffusion events are simulated using the Gillespie algorithm. At each step, an event is selected probabilistically based on its propensity (See propensity functions in Note1. Table 1)

Propensity functions and action of each process.

𝑁𝑐𝑎,𝑖 and 𝑁𝑐𝑎,𝑗 represent the number of ions in the current box (i) and in an adjacent box (j) selected randomly.

Parameter values used for the simulations shown in Figure 5.

A, Effective diffusion coefficient of Ca2+ (Deff) as a function of free cytosolic [Ca2+] (c) for a mixture of three buffers. Buffer 1 is immobile and low-affinity (Kd = 10 µM, total concentration 15 µM, diffusion coefficient 0 µm²/s). Buffer 2 is mobile and has moderate affinity (Kd = 10 µM, total concentration 85 µM, diffusion coefficient 15 µm²/s). Buffer 3 is highly mobile and has a high affinity (Kd = 0.1 µM, total concentration 15 µM, diffusion coefficient 120 µm2/s). B, C, Spatial profiles of free [Ca2+] 10 ms after a constant point-source release in the centre of a 10 × 10 µm2 domain representing a cell, obtained from a two-dimensional reaction–diffusion simulation with explicit Ca2+–buffer binding using the same buffer parameters as in (A). Only Ca2+ release and binding to buffers were included in the simulation. The rate of Ca2+ release is 10 times larger in C than in B. [Ca2+] peaks at 0.25 µM (B) and 2.5 µM (C).

Global Ca²⁺ response at higher [IP3].

A, Time course of the averaged cytosolic [Ca²⁺] obtained from the stochastic IP3R cluster model for a uniform [IP3] = 0.1 µM and an effective diffusion coefficient 𝐷eff = 40 µm2/s. All other parameters are identical to those used in the simulations shown in Fig. 5. Under these conditions, as opposed to CCRICs, the model produces a “classical” global Ca²⁺ response with larger amplitude and long duration. B, 2D cell geometry (10 × 10 µm²) used in the simulations. Squares indicate the random positions of IP₃R clusters.

EPEC induces Ca2+ responses with a frequency increasing during the time of infection.

Ca2+ imaging was performed on HeLa cells loaded with the Ca2+ fluorescent indicator Cal-520 and challenged with EPEC at a MOI of 100. A, Traces of Ca2+ variations in single cells. B, Trace corresponding to the average of traces shown in B (N = 2, cells = 43). Note that cells do not show an increase in basal Ca2+ levels that could be mistakenly interpreted from the averaging of Ca2+ responses over the cell population.

EPEC-induced Ca2+ responses does not depend on Ca2+ influx but on Ca2+ release.

HeLa cells were loaded with the fluorescent indicator Cal-520, challenged with a high MOI of EPEC wild-type (WT) or a low MOI of the ΔespC mutant and subjected to live-cell Ca2+ imaging at a frequency of one acquisition every 10 seconds (A-C). A, Representative traces of Ca2+ variations in single cells. The black arrowheads indicate the time of bacterial challenge. The blue arrowheads indicate stimulation with 3 µM histamine. B, Percent of cells exhibiting Ca2+ responses. Cells challenged with: ΔespC (N = 4, cells > 400); + U73122: ΔespC in the presence of 10 μM U73122 (N = 4, cells > 160); + U73343: ΔespC in the presence of 10 μM U73343 (N = 2, cells = 230); + PPADS: ΔespC in the presence of 20 μM PPADS (N = 4, cells > 400). C, cells challenged with wild-type EPEC in the absence or presence of + hexokinase (200 units/ml) and 5 mM glucose (+ HexoK, N = 3, cells > 120). D-F, + EGTA: cells treated with 4 mM EGTA. (N = 3, cells > 30). D, Percent of cells showing Ca2+ responses. E, F, Frequency of Ca2+ responses per cell. Mann-Whitney test. ns: not significant. **: p < 0.01; ****: p < 0.0001.

Suramin does not affect EPEC-Induced actin pedestals

HeLa cells were challenged with the indicated RFP-expressing bacterial strains in the presence or absence of 100 μM suramin. Samples were fixed and processed for fluorescence staining. A, Representative micrographs. Blue: DAPI; green: Phalloidin-Alexa488; red: bacteria. Scale bar = 10 µm. B, Percent of cells exhibiting actin-rich pedestals. High MOI: 50 bacteria / cell. Low MOI ΔespC: 10 bacteria / cell. (N=3, n > 150). Mann Whitney test. ns: not significant.

Low concentrations of histamine also induce small and fast coordinated Ca2+ responses.

HeLa cells were loaded with the fluorescent indicator Cal-520, challenged with 100 nM histamine and subjected to Ca2+ imaging, with image acquisition performed every 57 ms. Left: fluorescence micrograph of the single cell with black and red ROIs corresponding to the traces of Ca2+ variations in matching color. The box corresponds to a higher magnification of the inset in traces shown at the bottom. Arrowhead: challenge with 10 μM histamine. Traces are representative of 340 cells analyzed from 2 independent experiments, with a frequency of 4.5 ± 0.4 peaks·min⁻¹ (mean ± SEM) and peak duration of 4.45 ± 0.19 s (mean ± SEM).

EPEC induces CRICCs in polarized intestinal cells via eATP release

Polarized TC7 cells were loaded with the fluorescent indicator Cal-520 were challenged with the EPEC ΔespC strain expressing the Red Fluorescent Protein (RFP) (A-E) or with ATP (F, G). A, Representative confocal micrographs. Samples were, fixed and processed for fluorescence microscopy analysis. Green: ZO-1 staining. Red: RFP fluorescence. Scale bar = 10 µm. B-G, Samples were subjected to live-cell Ca2+ imaging at a frequency of one acquisition every 5 seconds. B, D, Representative traces of Ca2+ variations in single cells challenged with the indicated strain and inhibitor. The black arrowheads indicate the time of bacterial challenge. The blue arrowhead indicates stimulation with 3 µM histamine. C, E, Percent of cells exhibiting Ca2+responses (C) (N = 4, cells > 241) or CCRICs (E) (N =3, cells > 66) following challenge with the indicated bacterial strain and inhibitor. Each value corresponds to a replicate. Mann-Whitney test. ****: p < 0.0001. F, Representative traces of Ca2+ variations following challenge with 150 nM ATP (TC7 + ATP) or in buffer alone (TC7). The pink and green traces correspond to Ca2+ variations in ROIs within the same cell. G, Percent of cells exhibiting Ca2+responses following challenge with the indicated ATP concentration. Each value represents the mean ± SEM of at least 140 cells in 3 independent experiments.

CCRICs induced by EPEC result from Ca2+ released by highly transient or mobile IP3R clusters.

HeLa cells were loaded with Cal-520 and challenged with wild type EPEC at a MOI of 50 bacteria / cell. A, B, Globally Coordinated responses associated with small and highly mobile clusters. Time series micrographs, Cal-520 intensity depicted in pseudocolor. Numbers: time in ms. Lower Panels are magnification of the purple box in the upper panel, with a time interval of 57 ms. Scale bar = 5 μm. Note the high mobility of small clusters / channels. The black arrows point at larger and less mobile clusters. Traces, variations of Ca2+ in ROI depicted at T = 0 in the micrographs in the corresponding color. The black arrow points at the response peak illustrated by the time series. C, D, micrographs, Cal-520 fluorescence depicted in the Fiji red lute. Images were taken every 22 ms. Solid circles: ROIs where the variations of Ca2+ are shown in traces in the corresponding on the right. The blue ROI correspond to the Ca2+ release source based on the higher response amplitude, and the green ROI to a distal region. The number indicates the distance between the ROIs shown by the dashed line. C, Arrows point at the peak of the response in the ROI with the corresponding color. Note the delay in response elicitation between the source- (blue) and distal (green) ROI. D, Puff-like responses (black arrowhead). Note the absence of Ca2+ variations in the distal region (Top), and the simultaneous elicitation of a puff and CCRICs in different ROIs (Bottom).

Intracellular Ca2+ chelation inhibits CCRICs.

HeLa cells were loaded with the fluorescent indicator Cal-520 in the presence or absence of 20 μM EGTA-AM or BAPTA-AM. Samples were stimulated with 150 nM ATP and subjected to high speed Ca2+ imaging at a frequency of one acquisition every 22 ms. A, Representative traces of Ca2+ variations in 2 subcellular regions of the same cell in samples treated in the presence or absence of the indicated inhibitor. No Ca2+ responses were observed for cells treated with BAPTA-AM (N = 3, > 65 cells). EGTA-AM treatment led to an inhibition of Ca2+ responses, associated with small variations in the Ca2+ baseline that were arbitrarily scored as flattened Ca2+ pseudo-responses (ATP+EGTA-AM, red arrows). B, Percent of cells exhibiting Ca2+ responses (N > 3, cells > 62). C, average number of responses per cell. (N > 3, n > 62). Mann-Whitney test. **: p < 0.01; ***: p < 0.001.

Effects of ATP on TNF-α-induced profiles of O-GlcNacylation in HeLa cells.

HeLa cells were stimulated with 10 ng/ml TNF-α alone or co-stimulated with 10 ng/ml TNF-α and 150 nM ATP. Representative blots of cell lysates analyzed by Western blot at the time points indicated in minutes using the indicated antibodies.