Calcium Flickers Steer Cell Migration

In addition to extracellular chemoattractant stimuli, directional cell movement depends on an intracellular calcium signal that is well-organised in space, time and concentration^{6, 7, 9}. Over a decade ago, Fay and colleagues made the groundbreaking finding that intracellular calcium displays a rear-to-front gradient, with the lowest concentration at the front of a migrating cell⁶. However, this observation appears to be paradoxical, because the leading lamella, the signalling and motility centre of a migrating cell, contains numerous effector proteins that require high levels of calcium for activation^10–13. Although transient increases of calcium concentration have recently been observed in migrating cells, they are infrequent and mainly localised to the tail of the cell, and are thought to facilitate intermittent rear retraction⁷. Biochemical studies suggest that calcium entry is required to maintain ruffling structure, actin polymerisation, and the phosphatidylinositol-3,4,5-trisphosphate (PIP₃) signalling at the leading edge of macrophages¹⁴. To date, it remains perplexing how calcium regulates lamella dynamics during cell migration.

Using human embryonic lung fibroblasts (WI-38) as a model, we characterised the spatiotemporal organisation of intracellular calcium signals with the aid of real-time confocal microscopy. In migrating WI-38 fibroblasts that overtly displayed leading and trailing edges, we detected a shallow decreasing gradient of global calcium concentration (indexed by the fluo-4 to fura-red fluorescence ratio) that ran from the rear to the front (Fig. 1c, d), in agreement with previous findings^{6, 9}. Surprisingly, we found that discrete, local and short-lived high-calcium microdomains or “calcium flickers”, analogous to calcium sparks and puffs¹⁵, occurred against a quiescent background (supplementary video). High resolution linescan imaging revealed that the flickers occurred at a nearly constant rate of 1.92 ± 0.21 Hz/100 µm linescan (n = 18) (Fig. 1c). Individual events rapidly rose to about double the fluo-4 fluorescence (ΔF/F_o = 1.16 ± 0.02, n = 1,071), lasting variably from 10 ms to 4 s, and were confined to an area 5.27 ± 0.05 µm in diameter (Fig. 1c). Importantly, calcium flickers were abundant at the leading lamella (Fig. 1a), including in motile lamellipodia (Fig. 1b), but were sharply reduced elsewhere, resulting in an approximately 4:1 front-to-rear polarisation (Fig. 1d) that was opposite to the aforementioned global calcium gradient. Polarisation of flicker activity was common to fibroblasts undergoing migration, but it was not seen in stationary fibroblasts lacking morphological polarity and displaying a lower flicker incidence (0.57 ± 0.10 Hz/100 µm linescan, n = 12, p < 0.05 vs. migrating cells) (supplementary Fig. 1). Thus, flickers represent a distinctive, heretofore unappreciated modality of calcium signalling in migrating fibroblasts. Similar flickers were also evident in rat neonatal cardiac fibroblasts and 3T3-Swiss albino mouse embryonic fibroblasts (supplementary Fig. 2).

In search of the molecular basis of calcium flickers, we showed that calcium influx through the stretch-activated cation channel (SACC) was obligatory. Application of Ca²⁺-free medium containing 5 mM EGTA or streptomycin (200 µM), a SACC blocker¹⁶, immediately abolished flicker activity in WI-38 fibroblasts (Fig. 2a). On average, the signal mass of calcium flickers (space-time integral of the flicker signal) decreased by 98.3 ± 0.8% (EGTA; n = 9, p < 0.01 vs. control) or 93.1 ± 1.3% (streptomycin; n = 6, p < 0.01 vs. control). Likewise, Gd³⁺ (200 µM), a non-specific SACC blocker¹⁶, diminished the signal mass by 91.9 ± 1.2% (n = 6, p < 0.01 vs. control). To determine whether mechanical forces can directly trigger flickers, we showed that shear stress applied to the front of migrating fibroblasts immediately evoked a flurry of flicker activity (Fig. 2e). In a different approach, relaxing or stretching the cell by pushing or pulling the flexible substrate (with a needle tip) suppressed or enhanced the flicker activity, respectively (supplementary Fig. 3). Under cell-attached patch clamp conditions¹⁷, sudden application of negative pressure (~40 mm Hg) elicited bursting single-channel activity, while simultaneous confocal imaging visualised corresponding flicker-like events beneath the patch membrane (Fig. 2b). These results indicate that calcium flickers are triggered by calcium influx through SACCs.

SACCs belong to the transient receptor potential (TRP) ion channel superfamily. In mammals, about eight TRP channels in four subfamilies are thought to be sensitive to mechanical forces while being calcium-permeable⁸. Of these, TRPM7, TRPC6, TRPV2 and TRPP2 were expressed at relatively high mRNA levels in WI-38 fibroblasts (supplementary Fig. 4). Using RNA interference, we found that calcium flickers were virtually abolished by ~75% knockdown of TRPM7, but not that of the other three TRP channels (Fig. 2c, d, supplementary Fig. 5). More importantly, similar shear stress was unable to evoke flickers in TRPM7 knockdown cells, which displayed rare basal flicker activity (Fig. 2e). These data pinpointed TRPM7 as the specific SACC responsible for transducing mechanical signals into calcium flickers. A hallmark of TRPM7 is its sensitivity to inhibition by Mg²⁺ in addition to Gd³⁺ [¹⁸]. Raising extracellular Mg²⁺ from 1.0 to 10 mM largely abolished calcium flickers, while removing it enhanced flicker production (supplementary Fig. 6). Thus, TRPM7 acts as the mechanical sensor, the calcium flicker igniter, and the mechanochemical transducer in fibroblasts, revealing a novel role of this SACC in the regulation of cell migration (see below).

Since calcium release from the endoplasmic reticulum (ER) amplifies calcium influxes via the calcium-induced calcium release mechanism¹⁵, we next investigated whether ER calcium release participates in calcium flicker production. Inhibition of ER calcium recycling with the Ca²⁺ ATPase inhibitor thapsigargin (5 µM, 20 min incubation, after recession of the initial calcium transient) halved flicker amplitude without affecting flicker probability (Fig. 3a, c). Similar results were obtained by inhibiting the IP₃ receptor (IP₃R) with xestospongin C (5 µM), whereas IP₃-BM (2 µM), a membrane-permeable ester precursor of IP₃^[19], enhanced the flicker amplitude and ryanodine receptor inhibition (ryanodine, 25 µM) had no significant effect (Fig. 3a, c). Quantitative real-time PCR results showed that type 2 IP₃R (IP₃R2) and type 3 IP₃R (IP₃R3) are the primary IP₃R isoforms expressed in WI-38 fibroblasts (supplementary Fig. 7). In contrast to TRPM7, RNA interference knockdown of IP₃R2 (~80%), but not IP₃R3 (~60%), significantly decreased flicker amplitude but failed to alter flicker probability (Fig. 3b, c). This IP₃R isoform specificity is consistent with the fact that IP₃R2 is more sensitive to IP₃ and displays little calcium-dependent inactivation²⁰. Taking together, we concluded that calcium entry via TRPM7 is locally amplified by calcium release through IP₃R2 in the event of a calcium flicker. Coupling IP₃R2 to TRPM7 would enable flicker activity to decode IP₃-linked chemoattractant signal transduction.

Given the role of TRPM7 as a mechanical sensor and a calcium flicker igniter, we anticipated that flicker activity would be coupled to the migration-associated traction force. Indeed, the map of flicker ignition sites at the front of a cell largely overlapped, though with subtle differences, the matrix of focal adhesions (FAs) (Fig. 4a, supplementary Fig. 8), where traction force is created and transmitted²¹. Rapid local application of RGDS (2 mM, 1 min), which contains the RGD sequence that is recognized by integrins²², enhanced the flicker activity, while the control peptide RGES was ineffective (Fig. 4b, c), consistent with the finding that RGDS stimulates calcium transients in neuronal filopodia and growth cones²³. Disruption of protrusion by transient frontal application of cytochalasin D, inhibition of myosin ATPase by 2,3-butanedione monoxime or (−)blebbistatin²⁴ all inhibited lamella flicker production (Fig. 4b, c). These lines of evidence support the idea that decoding local membrane tension by flicker activity depends on cytoskeletal and morphological integrity.

Despite the low global calcium concentration, high-calcium microdomains created by mechanical stress in the leading lamella may activate a multitude of local calcium-dependent events critical to cell polarization and movement, including the PIP₃ signalling cascade^{25, 26}, a parallel phospholipase A2-mediated signalling mechanism²⁷, cytoskeleton dynamic such as actin remodelling¹⁰, FA detachment and relocation, and actin-myosin contraction. Next, we sought to determine the physiological role of calcium flickers in regulating cell migration, particularly turning behaviour that is almost entirely carried out within the leading lamella.

Platelet derived growth factor (PDGF) is a well-known chemoattractant that stimulates fibroblast migration during wound healing³. Its intracellular signalling pathways include generating traction force by Rac-dependent protrusion²⁸ and activation of the phospholipase C-PIP₂-IP₃ signaling cascade²⁹, both convergent on flicker production. When migrating fibroblasts were exposed to uniform PDGF (0.8 nM), increases in both flicker amplitude and probability were accompanied by a decrease in directional persistence (supplementary Fig. 9), the latter suggesting an enhanced propensity for turning of the cell³⁰. When a PDGF gradient was applied in the direction perpendicular to cell movement, migrating fibroblasts no longer moved persistently along the original path; rather, they turned towards the higher PDGF concentration (Fig. 5a). Time-lapse imaging revealed a rapid increase in lamella flicker activity and an accentuated front-to-rear polarisation (Fig. 5a). More importantly, a greater flicker signal mass was found in the portion facing the PDGF source (SM_α) than in the portion farther away from the source (SM_β), indicating the development of an asymmetry of flicker activity within the lamella (Fig. 5a). To demonstrate a linkage between lamella flicker asymmetry and turning behaviour, we examined the cumulative difference between SM_α and SM_β (ΣSM_α-β) and found that the time course of ΣSM_α-β nearly overlapped with that for the distance travelled in the direction of the PDGF gradient (y-distance) (Fig. 5b). On average, the correlation coefficient between ΣSM_α-β and y-distance was 0.72 in 25 migrating fibroblasts. Hence, patterned flicker activation in the leading lamella may translate directional sense and steer the cell to turn in response to PDGF gradients.

To further test the above hypothesis, we showed that impaired PDGF-induced lamella flicker asymmetry or diminishment of ΣSM_α-β by streptomycin and TRPM7 or IP₃R2 knockdown was accompanied by dwindling of the turning angle in a roughly proportional manner (Fig. 5c). Likewise, a robust match between the flicker activity and population chemotaxis was revealed by various pharmacological and molecular interventions including TRPM7 and IP₃R2 knockdown, and SACC blockade (Fig. 5d, supplementary Fig. 10). Furthermore, loading EGTA ester to disturb the flicker signal (supplementary Fig. 11) compromised the turning and chemotaxis abilities (Fig. 5c, d), while flicker activation by IP₃-BM enhanced both of them (Fig. 5c, d), suggesting a causal link between calcium flicker activity and fibroblast turning and chemotaxis.

In summary, we have demonstrated that calcium flickers arising from TRPM7 and IP₃R2 play an essential role in steering migrating fibroblasts. Despite that the global calcium gradient is opposite to the direction of cell migration, high calcium flicker activity would enable activation of calcium signalling cascades amidst a low calcium background at the leading edge, such that spatiotemporally patterned calcium flicker activity can orchestrate the complex turning behaviour of migrating cells (Fig. 5e). The coupling of TRPM7-mediated force-transducing calcium influx and local IP₃-induced calcium release would make this an ideal system for locomotion in response to chemoattractants (Fig. 5e). The present finding may have general ramifications because growth cones of neurons turn away from the side which filopodia displays higher local calcium signals²³ and calcium influx is essential to maintaining the leading-edge structure and activity in macrophages¹⁴. As such, unveiling calcium flickers in migrating cells opens a new avenue to investigate how local calcium signals orchestrate diverse biochemical pathways in the guidance of directional movement.