Permeable Mechanosensitive Channel Research Articles

The local mechanosensitive response of primary cardiac fibroblasts is influenced by the microenvironment mechanics

Cardiac fibroblasts (CFs) are essential for preserving myocardial integrity and function. They can detect variations in cardiac tissue stiffness using various cellular mechanosensors, including the Ca2+ permeable mechanosensitive channel Piezo1. Nevertheless, how CFs adapt the mechanosensitive response to stiffness changes remains unclear. In this work we adopted a multimodal approach, combining the local mechanical stimulation (from 10 pN to 350 nN) with variations of culture substrate stiffness. We found that primary rat CFs cultured on stiff (GPa) substrates showed a broad Piezo1 distribution in the cell with particular accumulation at the mitochondria membrane. CFs displayed a force-dependent behavior in both calcium uptake and channel activation probability, showing a threshold at 300 nN, which involves both cytosolic and mitochondrial Ca2+ mobilization. This trend decreases as the myofibroblast phenotype within the cell population increases, following a possible Piezo1 accumulation at focal adhesion sites. In contrast, the inhibition of fibroblasts to myofibroblasts transition with soft substrates (kPa) considerably reduces both mechanically- and chemically-induced Piezo1 activation and expression. Our findings shed light on how Piezo1 function and expression are regulated by the substrate stiffness and highlight its involvement in the environment-mediated modulation of CFs mechanosensitivity.

Reprint of: Mechanosensitive ion channels in cell migration.

Cellular processes are initiated and regulated by different stimuli, including mechanical forces. Cell membrane mechanosensors represent the first step towards the conversion of mechanical stimuli to a biochemical or electrical response. Mechanosensitive (MS) ion channels form a growing family of ion gating channels that respond to direct physical force or plasma membrane deformations. A number of calcium (Ca2+) permeable MS channels are known to regulate the initiation, direction, and persistence of cell migration during development and tumour progression. While the evidence that links individual MS ion channels to cell migration is growing, a unified analysis of the molecular mechanisms regulated downstream of MS ion channel activation is lacking. In this review, we describe the MS ion channel families known to regulate cell migration. We discuss the molecular mechanisms that act downstream of MS ion channels with an emphasis on Ca2+ mediated processes. Finally, we propose the future directions and impact of MS ion channel activity in the field of cell migration.

MCAs in Arabidopsis are Ca2+-permeable mechanosensitive channels inherently sensitive to membrane tension

Mechanosensitive (MS) ion channels respond to mechanical stress and convert it into intracellular electric and ionic signals. Five MS channel families have been identified in plants, including the Mid1-Complementing Activity (MCA) channel; however, its activation mechanisms have not been elucidated in detail. We herein demonstrate that the MCA2 channel is a Ca2+-permeable MS channel that is directly activated by membrane tension. The N-terminal 173 residues of MCA1 and MCA2 were synthesized in vitro, purified, and reconstituted into artificial liposomal membranes. Liposomes reconstituted with MCA1(1-173) or MCA2(1-173) mediate Ca2+ influx and the application of pressure to the membrane reconstituted with MCA2(1-173) elicits channel currents. This channel is also activated by voltage. Blockers for MS channels inhibit activation by stretch, but not by voltage. Since MCA proteins are found exclusively in plants, these results suggest that MCA represent plant-specific MS channels that open directly with membrane tension.

Mechanosensitive ion channels in cell migration.

Cellular processes are initiated and regulated by different stimuli, including mechanical forces. Cell membrane mechanosensors represent the first step towards the conversion of mechanical stimuli to a biochemical or electrical response. Mechanosensitive (MS) ion channels form a growing family of ion gating channels that respond to direct physical force or plasma membrane deformations. A number of calcium (Ca2+) permeable MS channels are known to regulate the initiation, direction, and persistence of cell migration during development and tumour progression. While the evidence that links individual MS ion channels to cell migration is growing, a unified analysis of the molecular mechanisms regulated downstream of MS ion channel activation is lacking. In this review, we describe the MS ion channel families known to regulate cell migration. We discuss the molecular mechanisms that act downstream of MS ion channels with an emphasis on Ca2+ mediated processes. Finally, we propose the future directions and impact of MS ion channel activity in the field of cell migration.

Mix and match: Patchwork domain evolution of the land plant-specific Ca2+-permeable mechanosensitive channel MCA.

Multidomain proteins can have a complex evolutionary history that may involve de novo domain evolution, recruitment and / or recombination of existing domains and domain losses. Here, the domain evolution of the plant-specific Ca2+-permeable mechanosensitive channel protein, MID1-COMPLEMENTING ACTIVITY (MCA), was investigated. MCA, a multidomain protein, possesses a Ca2+-influx-MCAfunc domain and a PLAC8 domain. Profile Hidden Markov Models (HMMs) of domains were assessed in 25 viridiplantae proteomes. While PLAC8 was detected in plants, animals, and fungi, MCAfunc was found in streptophytes but not in chlorophytes. Full MCA proteins were only found in embryophytes. We identified the MCAfunc domain in all streptophytes including charophytes where it appeared in E3 ubiquitin ligase-like proteins. Our Maximum Likelihood (ML) analyses suggested that the MCAfunc domain evolved early in the history of streptophytes. The PLAC8 domain showed similarity to Plant Cadmium Resistance (PCR) genes, and the coupling of MCAfunc and PLAC8 seemed to represent a single evolutionary event. This combination is unique in MCA, and does not exist in other plant mechanosensitive channels. Within angiosperms, gene duplications increased the number of MCAs. Considering their role in mechanosensing in roots, MCA might be instrumental for the rise of land plants. This study provides a textbook example of de novo domain emergence, recombination, duplication, and losses, leading to the convergence of function of proteins in plants.

Aquaporins in platelet function

Structurally, aquaporins (AQPs) are small channel proteins with monomers of ~ 30 kDa that are assembled as tetramers to form pores on cell membranes. Aquaporins mediate the conduction of water but at times also small solutes including glycerol across cell membranes and along osmotic gradients. Thirteen isoforms of AQPs have been reported in mammalian cells, and several of these are likely expressed in platelets. Osmotic swelling mediated by AQP1 sustains the calcium entry required for platelet phosphatidylserine exposure and microvesiculation, through calcium permeable stretch-activated or mechanosensitive cation channels. Notably, deletion of AQP1 diminishes platelet procoagulant membrane dynamics in vitro and arterial thrombosis in vivo, independent of platelet granule secretion and without affecting hemostasis. Water entry into platelets promotes procoagulant activity, and AQPs may also be critical for the initiation and progression of venous thrombosis. Platelet AQPs may therefore represent valuable targets for future development of a new class of antithrombotics, namely, anti-procoagulant antithrombotics, that are mechanistically distinct from current antithrombotics. However, the structure of AQPs does not make for easy targeting of these channels, hence they remain elusive drug targets. Nevertheless, thrombosis data in animal models provide compelling reasons to continue the pursuit of AQP-targeted antithrombotics. In this review, we discuss the role of aquaporins in platelet secretion, aggregation and procoagulation, the challenge of drugging AQPs, and the prospects of targeting AQPs for arterial and venous antithrombosis.

The root growth reduction in response to mechanical stress involves ethylene-mediated microtubule reorganization and transmembrane receptor-mediated signal transduction in Arabidopsis.

We found that mutations in a Ca2+-permeable mechanosensitive channel MCA1, an ethylene-regulated microtubule-associated protein WDL5, and a versatile co-receptor BAK1 affect root growth response to mechanical stress. Plant root tips exposed to mechanical impedance show a temporal reduction in the elongation growth. The process involves a transient Ca2+ increase in the cytoplasm followed by ethylene signaling. To dissect the molecular mechanisms underlying this response, we examined the root growth of a series of Arabidopsis mutants with potentially altered response to mechanical stress after transfer from vertical to horizontal plates that were covered by dialysis membrane as an impedance. Among the plant hormone-response mutants tested, the ethylene-insensitive mutant ein3 was confirmed to show no growth reduction after the transfer. The root growth reduction was attenuated in a mutant of MCA1 encoding a Ca2+-permeable mechanosensitivechannel and that of WDL5 encoding an ethylene-regulated microtubule-associated protein. We also found that the growth reduction was enhanced in a mutant of BAK1 encoding a co-receptor that pairs with numerous leucine-rich repeat receptor kinases to modulate growth and immunity. These results suggest the root growth reduction in response to mechanical stress involves ethylene-mediated microtubule reorganization and also transmembrane receptor-mediated signal transduction.

The gravistimulation-induced very slow Ca2+ increase in Arabidopsis seedlings requires MCA1, a Ca2+-permeable mechanosensitive channel

Gravity is a critical environmental factor affecting the morphology and function of plants on Earth. Gravistimulation triggered by changes in the gravity vector induces an increase in the cytoplasmic free calcium ion concentration ([Ca2+]c) as an early process of gravity sensing; however, its role and molecular mechanism are still unclear. When seedlings of Arabidopsis thaliana expressing apoaequorin were rotated from the upright position to the upside-down position, a biphasic [Ca2+]c-increase composed of a fast-transient [Ca2+]c-increase followed by a slow [Ca2+]c-increase was observed. We find here a novel type [Ca2+]c-increase, designated a very slow [Ca2+]c-increase that is observed when the seedlings were rotated back to the upright position from the upside-down position. The very slow [Ca2+]c-increase was strongly attenuated in knockout seedlings defective in MCA1, a mechanosensitive Ca2+-permeable channel (MSCC), and was partially restored in MCA1-complemented seedlings. The mechanosensitive ion channel blocker, gadolinium, blocked the very slow [Ca2+]c-increase. This is the first report suggesting the possible involvement of MCA1 in an early event related to gravity sensing in Arabidopsis seedlings.

Plasma Membrane Ca2+ Permeable Mechanosensitive Channel OsDMT1 Is Involved in Regulation of Plant Architecture and Ion Homeostasis in Rice.

Plant architecture is an important factor for crop production. Plant height, tiller pattern, and panicle morphology are decisive factors for high grain yield in rice. Here, we isolated and characterized a T-DNA insertion rice mutant Osdmt1 (Oryza sativa dwarf and multi-tillering1) that exhibited a severe dwarf phenotype and multi-tillering. Molecular cloning revealed that DMT1 encodes a plasma membrane protein that was identified as a putative Ca2+ permeable mechanosensitive channel. The transcript expression level was significantly higher in the dmt1 mutant compared to wild type (WT). Additionally, the dmt1 homozygous mutant displayed a stronger phenotype than that of the WT and heterozygous seedlings after gibberellic acid (GA) treatment. RNA-seq and iTRAQ-based proteome analyses were performed between the dmt1 mutant and WT. The transcriptome profile revealed that several genes involved in GA and strigolactone (SL) biosyntheses were altered in the dmt1 mutant. Ca2+ and other ion concentrations were significantly enhanced in the dmt1 mutant, suggesting that DMT1 contributes to the accumulation of several ions in rice. Moreover, several EF-hand Ca2+ sensors, including CMLs (CaM-like proteins) and CDPKs (calcium-dependent protein kinases), displayed markedly altered transcript expression and protein levels in the dmt1 mutant. Overall, these findings aid in the elucidation of the multiply regulatory roles of OsDMT1/OsMCA1 in rice.

Transient Receptor Potential Vanilloid 4 Is Required for Foreign Body Response and Giant Cell Formation

The presence of biomaterials and devices implanted into soft tissue is associated with development of a foreign body response (FBR), a chronic inflammatory condition that can ultimately lead to implant failure, which may cause harm to or death of the patient. Development of FBR includes activation of macrophages at the tissue-implant interface, generation of destructive foreign body giant cells (FBGCs), and generation of fibrous tissue that encapsulates the implant. However, the mechanisms underlying the FBR remain poorly understood, as neither the materials composing the implants nor their chemical properties can explain triggering of the FBR. Herein, we report that genetic ablation of transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable mechanosensitive cation channel in the transient receptor potential vanilloid family, protects TRPV4 knockout mice from FBR-related events. The mice showed diminished collagen deposition along with reduced macrophage accumulation and FBGC formation compared with wild-type mice in a s.c. implantation model. Analysis of macrophage markers in spleen tissues and peritoneal cavity showed that the TRPV4 deficiency did not impair basal macrophage maturation. Furthermore, genetic deficiency or pharmacologic antagonism of TRPV4 blocked cytokine-induced FBGC formation, which was restored by lentivirus-mediated TRPV4 reintroduction. Taken together, these results suggest an important, previously unknown, role for TRPV4 in FBR.

Piezo1 Mediated Ca2+ Signaling Causes Nuclear Shrinkage under Fluid Shear Stress

Nuclei of cells respond to local mechanical cues with changes in their size, morphology, and motility, to maintain tissue homeostasis and proper functions. Previous studies indicated that the external forces are coupled to nuclei through the surrounding cytoskeleton network linked to the nuclear envelope, but the molecules that directly interact with outside environments are unclear. Here we show that the fluid shear stress applied to MDCK cells caused the nucleus to shrink through a Ca2+ dependent signaling pathway. Inhibiting Ca2+ permeable mechanosensitive Piezo1 channels, with the specific inhibitor GsMTx4, eliminated the nuclear changes. Piezo1 knockdown with miRNA that targets Piezo1 significantly reduced the nuclear shrinkage under shear stress. Activation of Piezo1 with Yoda1 caused similar nucleus shrinkage without shear stress. These results demonstrate that Piezo1 channel is accountable for transmitting the shear force input to nuclei via Ca2+ signaling. To assess the relative contributions of Ca2+ and cytoskeleton reorganization on force transduction, we examined F-actin reorganization under shear stress and static conditions, and showed that reorganization is not necessary for the nucleus shrinkage. These results emphasize the role of the mechanosensitive channels as primary transducers in force transmission to the nucleus.

Ca2+-permeable mechanosensitive channels MCA1 and MCA2 mediate cold-induced cytosolic Ca2+ increase and cold tolerance in Arabidopsis

Cold shock triggers an immediate rise in the cytosolic free calcium concentration ([Ca2+]cyt) in Arabidopsis thaliana and this cold-induced elevation of [Ca2+]cyt is inhibited by lanthanum or EGTA. It is suggested that intracellular calcium mainly contributes to the cold-induced [Ca2+]cyt response by entering into the cytosol. Two calcium-permeable mechanosensitive channels, MCA1 and MCA2 (mid1-complementing activity), have been identified in Arabidopsis. Here, we demonstrate that MCA1 and MCA2 are involved in a cold-induced increase in [Ca2+]cyt. The cold-induced [Ca2+]cyt increase in mca1 and mca2 mutants was markedly lower than that in wild types. The mca1 mca2 double mutant exhibited chilling and freezing sensitivity, compared to wild-type plants. Expression of At5g61820, At3g51660, and At4g15490, which are not regulated by the CBF/DREB1s transcription factor, was down-regulated in mca1 mca2. These results suggest that MCA1 and MCA2 are involved in the cold-induced elevation of [Ca2+]cyt, cold tolerance, and CBF/DREB1-independent cold signaling.

Polarity of varicosity initiation in central neuron mechanosensation.

Little is known about mechanical regulation of morphological and functional polarity of central neurons. In this study, we report that mechanical stress specifically induces varicosities in the axons but not the dendrites of central neurons by activating TRPV4, a Ca2+/Na+-permeable mechanosensitive channel. This process is unexpectedly rapid and reversible, consistent with the formation of axonal varicosities in vivo induced by mechanical impact in a mouse model of mild traumatic brain injury. In contrast, prolonged stimulation of glutamate receptors induces varicosities in dendrites but not in axons. We further show that axonal varicosities are induced by persistent Ca2+ increase, disassembled microtubules (MTs), and subsequently reversible disruption of axonal transport, and are regulated by stable tubulin-only polypeptide, an MT-associated protein. Finally, axonal varicosity initiation can trigger action potentials to antidromically propagate to the soma in retrograde signaling. Therefore, our study demonstrates a new feature of neuronal polarity: axons and dendrites preferentially respond to physical and chemical stresses, respectively.

Transmembrane Topologies of Ca2+-permeable Mechanosensitive Channels MCA1 and MCA2 in Arabidopsis thaliana

Sensing mechanical stresses, including touch, stretch, compression, and gravity, is crucial for growth and development in plants. A good mechanosensor candidate is the Ca(2+)-permeable mechanosensitive (MS) channel, the pore of which opens to permeate Ca(2+) in response to mechanical stresses. However, the structure-function relationships of plant MS channels are poorly understood. Arabidopsis MCA1 and MCA2 form a homotetramer and exhibit Ca(2+)-permeable MS channel activity; however, their structures have only been partially elucidated. The transmembrane topologies of these ion channels need to be determined in more detail to elucidate the underlying regulatory mechanisms. We herein determined the topologies of MCA1 and MCA2 using two independent methods, the Suc2C reporter and split-ubiquitin yeast two-hybrid methods, and found that both proteins are single-pass type I integral membrane proteins with extracellular N termini and intracellular C termini. These results imply that an EF hand-like motif, coiled-coil motif, and plac8 motif are all present in the cytoplasm. Thus, the activities of both channels can be regulated by intracellular Ca(2+) and protein interactions.

Mechanosensitive channel candidate MCA2 is involved in touch-induced root responses in Arabidopsis.

The Ca2+-permeable mechanosensitive (MS) channel is a mechanical stress sensor. We previously reported that Arabidopsis MCA1 and its paralog MCA2 functioned individually as Ca2+-permeable MS channels. In the present study, we showed that the primary roots of the mca2-null mutant behaved abnormally on the surface of hard medium. First, primary roots are known to exhibit a skewing growth pattern on the surface of vertically placed agar medium. On such surface, the primary roots of mca2-null skewed more than those of the wild type. Second, when seedlings were grown on a tilted agar surface, the primary root of mca2-null showed abnormal waving patterns. Third, wild-type seedlings eventually died when grown on horizontally placed 3.2% gelrite medium, which was too hard to allow the primary roots of the wild type to penetrate, because their primary roots sprang from the surface of the medium and may have been unable to absorb water and nutrients. In contrast, the primary roots of mca2-null, but not those of mca1-null, were able to creep over the surface of the medium and grow. Fourth, when grown on the surface of 3.2% agar medium supplemented with 30 mM CaCl2, only mca2-null grew with a root that coiled in a clockwise direction. Lastly, on the surface of vertically placed rectangular plates that allowed primary roots to grow vertically down to the frame of the plate, wild-type primary roots grew horizontally after touching the frame at an angle of 90∘. During the horizontal growth, only the extreme root tips maintained contact with the frame. In contrast, the primary roots of mca2-null allowed not only the extreme root tips, but also the meristem and elongation zones to maintain contact with the frame during horizontal growth. These results suggest that MCA2 is involved in touch-related root responses.

Glial cell-expressed mechanosensitive channel TRPV4 mediates infrasound-induced neuronal impairment

Vibroacoustic disease, a progressive and systemic disease, mainly involving the central nervous system, is caused by excessive exposure to low-frequency but high-intensity noise generated by various heavy transportations and machineries. Infrasound is a type of low-frequency noise. Our previous studies demonstrated that infrasound at a certain intensity caused neuronal injury in rats but the underlying mechanism(s) is still largely unknown. Here, we showed that glial cell-expressed TRPV4, a Ca(2+)-permeable mechanosensitive channel, mediated infrasound-induced neuronal injury. Among different frequencies and intensities, infrasound at 16Hz and 130dB impaired rat learning and memory abilities most severely after 7-14days exposure, a time during which a prominent loss of hippocampal CA1 neurons was evident. Infrasound also induced significant astrocytic and microglial activation in hippocampal regions following 1- to 7-day exposure, prior to neuronal apoptosis. Moreover, pharmacological inhibition of glial activation in vivo protected against neuronal apoptosis. In vitro, activated glial cell-released proinflammatory cytokines IL-1β and TNF-α were found to be key factors for this neuronal apoptosis. Importantly, infrasound induced an increase in the expression level of TRPV4 both in vivo and in vitro. Knockdown of TRPV4 expression by siRNA or pharmacological inhibition of TRPV4 in cultured glial cells decreased the levels of IL-1β and TNF-α, attenuated neuronal apoptosis, and reduced TRPV4-mediated Ca(2+) influx and NF-κB nuclear translocation. Finally, using various antagonists we revealed that calmodulin and protein kinase C signaling pathways were involved in TRPV4-triggered NF-κB activation. Thus, our results provide the first evidence that glial cell-expressed TRPV4 is a potential key factor responsible for infrasound-induced neuronal impairment.

Plant mechanosensing and Ca2+ transport

Mechanical stimuli generate Ca(2+) signals and influence growth and development in plants. Recently, candidates for Ca(2+)-permeable mechanosensitive (MS) channels have been identified. These channels are thought to be responsible for sensing osmotic shock, touch, and gravity. One candidate is the MscS-like (MSL) protein family, a homolog of the typical bacterial MS channels. Some of the MSL proteins are localized to plastids to maintain their shape and size. Another candidate is the mid1-complementing activity (MCA) protein family, which is structurally unique to the plant kingdom. MCA proteins are localized in the plasma membrane and are suggested to be involved in mechanosensing and to be functionally related to reactive oxygen species (ROS) signaling. Here, we review their structural features and role in planta.

Plasma membrane protein OsMCA1 is involved in regulation of hypo-osmotic shock-induced Ca2+influx and modulates generation of reactive oxygen species in cultured rice cells

BackgroundMechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca2+-permeable mechanosensitive channels. On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca2+ influx, protein phosphorylation and generation of reactive oxygen species (ROS). Pharmacological analyses show Ca2+ influx mediated by mechanosensitive Ca2+ channels to influence induction of osmotic signals, including ROS generation. However, molecular bases and regulatory mechanisms for early osmotic signaling events remain poorly elucidated.ResultsWe here identified and investigated OsMCA1, the sole rice homolog of putative Ca2+-permeable mechanosensitive channels in Arabidopsis (MCAs). OsMCA1 was specifically localized at the plasma membrane. A promoter-reporter assay suggested that OsMCA1 mRNA is widely expressed in seed embryos, proximal and apical regions of shoots, and mesophyll cells of leaves and roots in rice. Ca2+ uptake was enhanced in OsMCA1-overexpressing suspension-cultured cells, suggesting that OsMCA1 is involved in Ca2+ influx across the plasma membrane. Hypo-osmotic shock-induced ROS generation mediated by NADPH oxidases was also enhanced in OsMCA1-overexpressing cells. We also generated and characterized OsMCA1-RNAi transgenic plants and cultured cells; OsMCA1-suppressed plants showed retarded growth and shortened rachises, while OsMCA1-suppressed cells carrying Ca2+-sensitive photoprotein aequorin showed partially impaired changes in cytosolic free Ca2+ concentration ([Ca2+]cyt) induced by hypo-osmotic shock and trinitrophenol, an activator of mechanosensitive channels.ConclusionsWe have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Analyses of cultured cells with altered levels of this putative Ca2+-permeable mechanosensitive channel indicate that OsMCA1 is involved in regulation of plasma membrane Ca2+ influx and ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways.

Ca2+透過性機械受容チャネル候補遺伝子MCA1, MCA2がシロイヌナズナの根端成長に及ぼす影響の細胞動力学的解析

Ca2+透過性機械受容チャネル候補遺伝子MCA1, MCA2がシロイヌナズナの根端成長に及ぼす影響の細胞動力学的解析

Involvement of the putative Ca2+-permeable mechanosensitive channels, NtMCA1 and NtMCA2, in Ca2+ uptake, Ca2+-dependent cell proliferation and mechanical stress-induced gene expression in tobacco (Nicotiana tabacum) BY-2 cells

To gain insight into the cellular functions of the mid1-complementing activity (MCA) family proteins, encoding putative Ca²⁺-permeable mechanosensitive channels, we isolated two MCA homologs of tobacco (Nicotiana tabacum) BY-2 cells, named NtMCA1 and NtMCA2. NtMCA1 and NtMCA2 partially complemented the lethality and Ca²⁺ uptake defects of yeast mutants lacking mechanosensitive Ca²⁺ channel components. Furthermore, in yeast cells overexpressing NtMCA1 and NtMCA2, the hypo-osmotic shock-induced Ca²⁺ influx was enhanced. Overexpression of NtMCA1 or NtMCA2 in BY-2 cells enhanced Ca²⁺ uptake, and significantly alleviated growth inhibition under Ca²⁺ limitation. NtMCA1-overexpressing BY-2 cells showed higher sensitivity to hypo-osmotic shock than control cells, and induced the expression of the touch-inducible gene, NtERF4. We found that both NtMCA1-GFP and NtMCA2-GFP were localized at the plasma membrane and its interface with the cell wall, Hechtian strands, and at the cell plate and perinuclear vesicles of dividing cells. NtMCA2 transcript levels fluctuated during the cell cycle and were highest at the G1 phase. These results suggest that NtMCA1 and NtMCA2 play roles in Ca²⁺-dependent cell proliferation and mechanical stress-induced gene expression in BY-2 cells, by regulating the Ca²⁺ influx through the plasma membrane.