Distinct calcium regulation of TRPM7 mechanosensitive channels at plasma membrane microdomains visualized by FRET-based single cell imaging

Transient receptor potential subfamily M member 7 (TRPM7), a mechanosensitive Ca2+ channel, plays a crucial role in intracellular Ca2+ homeostasis. However, it is currently unclear how cell mechanical cues control TRPM7 activity and its associated Ca2+ influx at plasma membrane microdomains. Using two different types of Ca2+ biosensors (Lyn-D3cpv and Kras-D3cpv) based on fluorescence resonance energy transfer, we investigate how Ca2+ influx generated by the TRPM7-specific agonist naltriben is mediated at the detergent-resistant membrane (DRM) and non-DRM regions. This study reveals that TRPM7-induced Ca2+ influx mainly occurs at the DRM, and chemically induced mechanical perturbations in the cell mechanosensitive apparatus substantially reduce Ca2+ influx through TRPM7, preferably located at the DRM. Such perturbations include the disintegration of lipid rafts, microtubules, or actomyosin filaments; the alteration of actomyosin contractility; and the inhibition of focal adhesion and Src kinases. These results suggest that the mechanical membrane environment contributes to the TRPM7 function and activity. Thus, this study provides a fundamental understanding of how the mechanical aspects of the cell membrane regulate the function of mechanosensitive channels.

Transient receptor potential subfamily M member 7 (TRPM7), a mechanosensitive Ca 2+ channel, plays a crucial role in intracellular Ca 2+ homeostasis. However, it is currently unclear how cell mechanical cues control TRPM7 activity and its associated Ca 2+ influx at plasma membrane microdomains. Using two different types of Ca 2+ biosensors (Lyn-D3cpv and Kras-D3cpv) based on fluorescence resonance energy transfer, we investigate how Ca 2+ influx generated by the TRPM7-specific agonist naltriben is mediated at the detergent-resistant membrane (DRM) and non-DRM regions. This study reveals that TRPM7-induced Ca 2+ influx mainly occurs at the DRM, and chemically induced mechanical perturbations in the cell mechanosensitive apparatus substantially reduce Ca 2+ influx through TRPM7, preferably located at the DRM. Such perturbations include the disintegration of lipid rafts, microtubules, or actomyosin filaments; the alteration of actomyosin contractility; and the inhibition of focal adhesion and Src kinases. These results suggest that the mechanical membrane environment contributes to the TRPM7 function and activity. Thus, this study provides a fundamental understanding of how the mechanical aspects of the cell membrane regulate the function of mechanosensitive channels. com/) was used for the statistical evaluation of data. Unpaired t test and one-way analysis of variance were used for intergroup difference determination. All results were expressed as mean ± standard error of the mean, and a P value < 0.05 was considered significant.

TRPM7-induced Ca 2+ influx mainly occurs at the DRM domains.
To determine the TRPM7 channel activity in distinct domains of the cell PM, MCF-7 cells were treated with the selective TRPM7 activator naltriben 20 . Due to this activation, Ca 2+ concentration increased close to the TRPM7 channels. Transfected Lyn-D3cpv and Kras D3cpv allowed the monitoring of this concentration change at the DRM and non-DRM domains of the PM, respectively ( Fig. 1) 19 . As shown in Figs. 2 and 3, the increase in the FRET ratio signal level from Lyn-D3cpv was much higher than that from Kras-D3cpv, indicating that TRPM7 could be located mainly at the DRM domains of the PM. To confirm whether the increase in FRET signal following naltriben treatment is caused by Ca 2+ influx through TRPM7 channels, we examined it after blocking ER Ca 2+ ATPase pump under extracellular Ca 2+ free conditions. As a result, we observed that naltriben treatment did not trigger Ca 2+ influx in Lyn-D3cpv transfected cells under these conditions, indicating TRPM7-specific Ca 2+ signal ( Supplementary  Fig. 1).

TRPM7-induced Ca 2+ influx ceases after the disruption of DRM.
To determine how the destruction of lipid rafts affects the activity of TRPM7, pretreatment of MCF-7 cells with MβCD was carried out. Lipid rafts are enriched in cholesterol. MβCD captures cholesterol from lipid rafts, causing lipid rafts structure disintegration 21 . During subsequent live-cell imaging performed with transfected Lyn-D3cpv in MCF-7 cells, TRPM7-induced Ca 2+ influx in cells pretreated with MβCD was greatly diminished compared to that in cells without pretreatment (Figs. 2, 3). These results suggest that the presence of lipid rafts is necessary for the activation of TRPM7 channels.
MLCK inhibition with ML-7 diminishes TRPM7 activity. MLCK phosphorylates myosin II regulatory light chain and, consequently, myosin binds to actin, forming SFs. This process facilitates actomyosin contractility 11 . To investigate how lowered SF-dependent tension impacts on TRPM7 activity, we pretreated MCF-7 cells with the ML-7 drug, which is an MLCK inhibitor. During live-cell imaging, TRPM7-induced Ca 2+ influx in pretreated cells decreased significantly compared to that in samples that were not pretreated (Figs. 4, 5). These results show that impaired actomyosin contractility negatively affects TRPM7 channel activity. www.nature.com/scientificreports/ investigated whether RhoA mutants can alter TRPM7 channel activity. RhoA V14 is a constitutively active mutant because it binds to GTP constitutively, and RhoA N19 is a dominant-negative mutant because it has a low affinity to its activators (guanine nucleotide exchange factors) 22 . Plasmids encoding these RhoA variants were co-transfected with Lyn-D3cpv into MCF-7 cells. During live-cell imaging after the administration of naltriben, the FRET signal from cells co-transfected with RhoA N19 decreased slightly, and that from cells co-transfected with RhoA V14 remained at the base level (Figs. 4, 5). These results suggest that RhoA activity contributes to the regulation of TRPM7 activity.

RhoA affects TRPM7-induced
Cytochalasin-D pretreatment negatively altered the Ca 2+ flow through TRPM7. To further investigate the effect of actomyosin on TRPM7 activity, we examined how the violation of the actomyosin network structural integrity with mycotoxin cytochalasin-D affects the generated calcium ion flux. Cytochalasin-D www.nature.com/scientificreports/ breaks actin filaments, disrupts the cytoskeletal network, inhibits polymerization, and induces the depolymerization of actin filaments 23,24 . The cells were treated with cytochalasin-D prior to live-cell imaging. Consequently, the TRPM7 channels showed lower activity in the cells treated with mycotoxin than those that were not treated (Figs. 4, 5). Additionally, these cells changed their morphology, and this result is in accordance with that of a study by Schliwa 24 (Fig. 5). Thus, the ordered structure of the actomyosin network is important for TRPM7 channel activity.
Nocodazole pretreatment lowers the response of TRPM7 to naltriben. After examining the effect of actomyosin on TRPM7 activity, we investigated whether a similar response may be triggered by changes in another important component of the cytoskeleton; microtubules. Because nocodazole causes the depolymerization of microtubules 32 , we exposed the treatment group cells to it before imaging. Nocodazole-treated cells showed a reduction in TRPM7 activation induced by naltriben compared to the untreated cells (Fig. 6). To further investigate whether the activity of TRPM7 is reversed when microtubules are recovered, we examined TRPM7 activity in response to naltriben after nocodazole washout. As a result, we observed that there was no significant difference in TRPM7-induced Ca 2+ influx between the control (n = 7) and nocodazole washout group (n = 9), supporting that TRPM7 activity reverts after microtubule repolymerization ( Supplementary Fig. 2). Therefore, our data demonstrate that the structural support of microtubules is essential for TRPM7 function.

FAK inhibition with PF-573228 and Src inhibition with PP1 both depressed TRPM7-induced Ca 2+ influx. FAK is an important structural and functional component of FA and is involved in FA turnover 17 .
Reduced FA turnover can affect the organization of lipid rafts 6,8 . Since we identified that TRPM7 is mainly active in areas of lipid rafts, we suggest that a reduced FA turnover affects TRPM7 activity. To test this hypothesis, cells were treated with PF-573228 before imaging. PF-573228 inhibited FAK phosphorylation and turnover 33 .
The results showed reduced TRPM7 activity in cells treated with PF-573228 compared to that in untreated cells (Fig. 7). Src-induced FAK phosphorylation regulates both actin and adhesion dynamics 18 . To investigate whether Src influences TRPM7 activity, PP1, an Src inhibitor 34 , was used for cell pretreatment, and live-cell imaging was then performed. The Ca 2+ influx decreased, and this result was similar to the effect observed in cells treated with PF-573228 (Fig. 7).

Discussion
In this study, real-time live-cell imaging experiments were conducted, and, as a reporter, the FRET-based biosensor D3cpv and its derivatives were used to investigate the features of TRPM7 channel regulation by the mechanical cues of cells. To reveal how the TRPM7 channels were distributed relative to the PM domains, biosensors constructed prior to this study were used. Lyn-D3cpv, a biosensor designed to target lipid rafts, contains at its N-terminus a sequence from Lyn kinase, which undergoes post-translational acylation in the cells, such www.nature.com/scientificreports/ as myristoylation and palmitoylation. The residues of saturated and monounsaturated fatty acids attached in this process have a spatial affinity for densely packed lipid rafts. For Kras-D3cpv, the sequence from KRas was attached to the C-terminus of the biosensor and underwent prenylation in the cells. As a result, the attached bulky polyunsaturated chains were pushed into the non-DRM domains of the PM, which are arranged more spaciously. Because mutated calmodulin in the D3cpv construct binds available calcium ions quickly, it is possible to track the change in concentration of calcium ions precisely at the cell PM domains of interest (Fig. 1). We found that activation of TRPM7 with naltriben in MCF-7 cells occurred mostly for Lyn-D3cpv compared to Kras-D3cpv (Fig. 2). This indicates that TRPM7 is predominantly located at lipid rafts in the cell PM. TRPM7 may be a crucial regulator of Ca 2+ mobilization at DRM microdomains of the cell PM 19 , which is supported by the results of the present study. Live-cell imaging showed that such disruption halts TRPM7 activity at the DRM domains of the PM (Fig. 2). This suggests that the presence of lipid rafts is necessary for TRPM7 activity, which is consistent with the results of a previous study 19 .
We used various approaches to elucidate how altered actomyosin contractility affects TRPM7 channel activity. To influence the contractility of actomyosin by inhibiting MLCK, the cells were pretreated with the ML-7 drug. To study the effect of altered RhoA activity on TRPM7, mutant RhoA constructs were co-transfected with the Lyn-D3cpv biosensor into MCF-7 cells.
Before myosin can bind to actin to form SF filaments, the myosin II regulatory light chain needs to be phosphorylated by MLCK. The inhibition of MLCK lowers SF tension 11 . After pretreatment with ML-7, live-cell imaging showed that the TRPM7-induced Ca 2+ influx decreased compared to that of the control samples (Fig. 4). This indicates that impaired actomyosin contractility negatively affects TRPM7 channel activity. Additionally, actin filament disruption with cytochalasin-D significantly lowered Ca 2+ flow through TRPM7, which is in agreement with the aforementioned results (Fig. 4).
RhoA is the most expressed Rho-kinase in cells and can regulate actomyosin contractility but via a different mechanism than that described above. The difference is that, unlike with MLCK, the path through Rho kinases does not depend on Ca 2+ signaling. RhoA V14 is a constitutively active mutant due to constant GTP binding. Actomyosin is constantly polymerizing under its effect, and a constant SF tension is generated 13,14 . RhoA N19 has a lower affinity with guanine nucleotide exchange factors than that of the wild-type; thus, it functions as a dominant-negative mutant. As a result of the live-cell imaging of cells with co-transfected RhoA mutants, the absence of TRPM7-generated Ca 2+ influx in cells with constitutively active RhoA V14 mutant was found. RhoA V14 creates excess SF formation, which prevents FA disassembly 7 . In the absence of FA turnover, lipid rafts are not recruited in order around FA 8 ; thus, TRPM7 activity is lowered.
For co-transfected RhoA N19 in cells, where SF tension was weakened, the level of TRPM7-induced Ca 2+ influx decreased considerably (Fig. 4), which was consistent with the results of a previous study of the RhoA regulation effects on Ca 2+ oscillations 35 . The results of the present study are in accordance with those of a previous study 35 regarding the essential balanced RhoA/ROCK activity. Therefore, RhoA activity contributes to the regulation of TRPM7 activity.
In both the ML-7 pretreatment and the co-transfected RhoA N19, the SF tension was reduced. Therefore, a similar response from the channel was expected but did not occur. After pretreatment with ML-7, the strength www.nature.com/scientificreports/ of the signal indicating the influx of calcium ions through the TRPM7 channel was significantly lower than that of RhoA co-transfection. This apparent contradiction may be explained by the different mechanisms of the actomyosin contractility regulation by MLCK and RhoA. In a previous study regarding actomyosin contraction regulation 36 , it was concluded that the Ca 2+ -calmodulin-dependent pathway regulates the rapid contraction of peripheral SFs, and Rho-kinase maintains more finely-tuned SF contraction in cells. Similar conclusions were reached in another study regarding the membrane protrusions during fibroblast migration 37 . ML-7 rapidly changes the contraction of SFs at the cell periphery and may rupture lipid rafts around the FAs. It is possible that due to the violation of the optimal SF tension with ML-7 (or with cytochalasin-D SF destruction), the normal turnover of FA is hampered, which leads to the decreased activity of TRPM7. This may explain the lower level of TRPM7-induced Ca 2+ influx signal in the presence of ML-7 compared to that in the presence of RhoA N19. As in the studies discussed above, nocodazole treatment resulted in decreased TRPM7 activity. With the destruction of microtubules, FA turnover was hindered 7 and the recruitment of lipid rafts to nascent FA decreased, to which the decreased activity of TRPM7 was related. The hypothesis that the reduced FA turnover affects the TRPM7 activity was confirmed by the addition of PF-573228 or PP1, significantly reducing TRPM7 activity. FAK phosphorylation by Src is important for FA turnover 18 ; thus, inhibition of these kinases leads to the reduced recruitment of lipid rafts to FA and decreased TRPM7 activity. Additionally, TRPM7 not only localizes together with the calcium-dependent protease m-calpain near FA but also regulates m-calpain 38 . Without TRPM7 channel activity, m-calpain does not subject FAK to proteolysis, which leads to the reduced FA turnover and decreased recruitment of lipid rafts to FAs. This study confirms co-location of TRPM7 and peripheral focal adhesions, and the excessive activity of TRPM7 leads to cell rounding. As such, TRPM7-medicated Ca 2+ fluxes might be implicated in actomyosin remodeling and subsequent cell adhesion and migration by mediating focal adhesion complexes 4,41,42 . In addition, in the absence of a local influx of calcium ions, the activity of Ca 2+ -calmodulin-dependent MLCK should decrease. This should further reduce tension in the actomyosin network. Taken together, these processes should also lead to decreased TRPM7 activity, looping the sequence. In conclusion, in this study, we found that TRPM7 activity is dependent on the mechanical cues of the cell. TRPM7 activity was mainly detected at lipid rafts. In all cases of intervention in the considered system, the flow of calcium ions through the TRPM7 channel decreased. These results lay a foundation for the understanding of mechanosensitive channel regulation mechanisms and indicate that TRPM7 is part of a self-regulating mechanosensitive system (Fig. 8), carrying out constant adjustments for effective adaptation to extracellular conditions, which is necessary for cell survival.   39 . Inactive Src does not phosphorylate FAK 17 and allows RhoA to be excessively active, which leads to excess SF formation and contraction, which causes FA robustness and hampers turnover 7 . The destruction of microtubules has the same effect on FA turnover 16 . FAK influences both Rho and MLCK 40 . Inactive MLCK drastically lowers SF tension on the periphery 36 , which may interfere with FA turnover. All interventions in this system lowered TRPM7 activity. Restricted Ca 2+ influx affected Ca 2+calmodulin-dependent MLCK 11 and FA turnover 38 .