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# Acidification on the plasma membrane

Ewan Macdonald, Ludger Johannes, Christian Wunder

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Ewan Macdonald, Ludger Johannes, Christian Wunder. Acidification on the plasma membrane. Current Opinion in Cell Biology, 2025, 95, pp.102531. 10.1016/j.ceb.2025.102531. hal-05328086

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# Acidification on the plasma membrane

Ewan MacDonald¹, Ludger Johannes²,³ and Christian Wunder²,³

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The pH balance between extracellular and intracellular space is crucial for a multitude of cellular processes. Real-time observation of pH fluctuations in the range 4–9 in live cells and tissues in a sensitive, non-invasive manner has become feasible with advances in pH quantification by organic dyes, genetically encoded fluorescent proteins, and DNA-based probes. We discuss mechanisms through which pH affects cell cycle, transcription, senescence, neurotransmission, glycolipid-lectin driven endocytosis, tissue remodelling, immune responses, and GPCR signalling. Growth factor-stimulated acidification of the extracellular space notably triggers enzymatic reactions like desialylation at the plasma membrane that control processes involving cell migration and bone resorption. Research into the role of pH in cellular physiology continues to be a fertile ground for discovery that underscores its fundamental importance.

## Addresses

¹ Montpellier Cell Biology Research Center, CRBM, Université 40 Montpellier, CNRS, Montpellier, France

² Institut Curie, PSL Research University, U1339 INSERM, UMR3666 CNRS, Chemical Biology of Cancer Unit, Paris, France

³ Inria Center at University of Rennes, SAIRPICO Team, U1339 INSERM, Institut Curie, Chemical Biology of Cancer Unit, Paris, France

Corresponding authors: MacDonald, Ewan (ewan.macdonald@crbm.cnrs.fr); Wunder, Christian (christian.wunder@curie.fr); Johannes, Ludger (ludger.johannes@curie.fr)

**Current Opinion in Cell Biology 2025, 95:102531**

This review comes from a themed issue on **Membrane Trafficking (2025)**

Edited by Lois Weisman and Rob Parton

For complete overview of the section, please refer the article collection - **Membrane Trafficking (2025)**

Available online 15 May 2025

https://doi.org/10.1016/j.ceb.2025.102531

0955-0674/© 2025 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

The plasma membrane serves as a critical interface between the cell and its environment, playing a pivotal role in maintaining cellular homeostasis. Modulation of acidity across the plasma membrane is a key aspect of this regulation, with extracellular pH influencing

enzymatic activity, metabolic processes, cellular signaling, and interactions with the extracellular matrix [1]. Acid regulation is particularly vital in various physiological contexts, including immune responses [2], neuronal signalling [3], tissue repair, and endocytosis [4].

Recent advances in our understanding of dysregulation of extracellular pH, including how membrane lipids are able to sense pH changes have illuminated the complex mechanisms underlying acid regulation at this cellular interface [5]. Alterations in extracellular pH have been implicated in numerous pathophysiological conditions, including cancer metastasis [6,7], ischemic injury, inflammation, bone remodelling [8], neurodegenerative diseases [9], and chronic kidney disease [10].

In these conditions, an altered acid-base balance can promote disease progression or exacerbate tissue damage. As research continues to uncover the intricate pathways involved in extracellular acid regulation, a comprehensive understanding of these processes becomes increasingly essential for developing targeted therapeutic strategies [11]. This review elucidates recent developments in acid regulation at the plasma membrane, exploring advances in tool development, molecular mechanisms and physico-chemical principles, as well as potential physiological implications. By summarizing these recent discoveries, we are aiming to provide a brief overview of the current state of knowledge in this rapidly evolving field.

## Mechanistic insights into pH regulation and its biological functions

Recent research has uncovered new mechanisms of pH regulation in cellular systems, especially at the plasma membrane. These findings have deepened our understanding of how cells maintain acid-base balance and how low extracellular pH affects cellular functions.

**Plasma membrane pH regulation and endocytosis.** Heterogeneity in the pH at the exoplasmic leaflet of the plasma membrane has been documented at the nanoscale, with areas adjacent to proton transporters having values as low as pH 5.5 [12]. The extracellular pH is regulated primarily by membrane transporters such as Na⁺/H⁺ exchangers and monocarboxylate transporters, which extrude protons and acidic metabolites like
lactate, as well as by proton pumps like V-ATPase that actively export H⁺ ions to the extracellular space. An interesting hypothesis is that pH nanodomains around acid/base transporters may be stabilised by the glycolyx. However, since this claim relies on nonspecific tunicamycin treatment, more targeted experiments are needed for further verification [13]. In relation to endocytosis, a recent study reports on the discovery of a pH sensing mechanism at the cell surface that was termed desialylation glycoswitch [14]. Here, EGF triggers the Na+/H+ antiporter NHE1, by EGFR-mediated phosphorylation via the MAP kinase/RSK pathway (Fig. 1). Extracellular acidification activates the neuraminidases 1 and 3 at the plasma membrane, whose pH optimum is at 5.5. Enzymatic removal of α2-6 linked sialic acids on glycoproteins such as integrins enables the interaction with the glycan binding protein galectin-3, which drives the subsequent uptake of the glycoproteins via GlycoLipid-Lectin (GL-Lect) driven endocytosis [15]. Whether this mechanism explains earlier observations on proton and lipid phase

order-induced endocytosis remains to be addressed directly [4]. GL-Lect driven endocytosis is followed by retrograde trafficking to the Golgi apparatus. Here, glycoproteins are re-sialylated and undergo secretion to specialized areas of the plasma membrane such as the leading edge to sustain persistent cell migration. Of note, other glycosidases are also found at the plasma membrane or in the extracellular space [16,17]. Whether these participate in reversible de/re-glycosylation events as shown for de/re-sialylation in Ref. [14] remains to be addressed directly.

**Macropinocytosis.** Ras activation in cancer promotes macropinocytosis, facilitating the uptake of nutrients essential for cell growth [18]. The complexity of pH regulation at various membranes and its impact on this biological process is particularly evident in macropinocytosis. Ras activation triggers the translocation of vATPase to the cell surface and endosomes, which drives the transport of cholesterol from intracellular stores to the plasma membrane. This cholesterol is

<figure><img src="image_4.png" /><figcaption>**Figure 1**</figcaption></figure>

**Control of membrane dynamics by pH.** (I) Extracellular acidification via NHE1 controls clathrin-independent GL-Lect driven endocytosis through the remodelling of cell surface glycans. The activity of NHE1 is controlled through the MAPK pathway. (II) NHE1 is localised to invadopodia, and extracellular acidification is necessary for extracellular matrix degradation. (III) The activity of NHE1 is required for the induction of macropinocytosis by maintaining the optimal submembranous pH that is needed for the activity of Rac1 and Cdc42.
crucial for the Rac1 association with the plasma mem-
brane, a key step for macropinocytosis [19]. Both
vATPase and SLC4A7 do not influence submembranous
pH, while plasma membrane-localized NHE1 does, and
modulates the activity of Rac1 and Cdc42. These
GTPases require a neutral pH and are sensitive to local
decreases in submembranous pH [20] (Fig. 1).

**Proton sensing by GPCRs.** The discovery of proton and lysolipid sensing GPCRs (GPR4, GPR65, and GPR68) [21] has provided another insight on how pH affects protein function. The combination of deep mutational scanning, cryo-EM, and constant-pH molecular dynamics simulations provided molecular indications on how protonation-induced receptor activation is achieved [22]. An extracellular pocket lined with several polar and charged amino acids (histidine) seems to be critical for this. Interestingly, the regulation of these G-proteins is not just restricted to changes in extracellular pH but can also be operated by the acidic environment of the endosomal system [23].

**Acid sensing in the brain.** Neuronal activity is generally associated with a decrease in pH at the synaptic cleft [24]. Neurotransmitters are loaded into highly acidic synaptic vesicles through vATPase activity, and upon fusion with the plasma membrane protons are released into the extracellular environment. Following this, a transient increase in cytosolic pH occurs, in a mechanism that while incompletely understood, involves the Ca²⁺/H⁺-ATPase and incorporation of the vATPase into the presynaptic membrane [25,26]. Detection of pH by neurons is involved in mechanosensation, detection of CO₂ levels in blood, and acidosis associated pain [27].

Ion channels present at the synapse display differential sensitivity to extracellular pH. Decreases in pH facilitate the activation of the NMDA receptor [28,29], while GABA<sub>A</sub> activity is augmented by low extracellular pH [30]. The discovery of acid sensing ion channels (ASICs), of which there are at least 4 that belong to the degenerin/epithelial Na<sup>+</sup> ion channel super family [31], underlies mechanisms by which protons can act directly as fast neurotransmitters. Yamada et al. demonstrated that protons released from the presynaptic terminal can engage the activity of ASICs1-3 in synapses between Merkel cell and neurite cells [32]. These synapses are highly present at tactile areas such as fingertips and whisker follicles and function in complex sensory tasks such as identification of texture and shape. It remains to be explored whether this mechanism is operational everywhere where ASIC channels are expressed.

**Intracellular pH is mirrored by cell surface pH.** The global intracellular pH is regulated by powerful mechanisms using a fast Na⁺-dependent and Na⁺-independent Cl⁻/HCO₃⁻ exchange and a slow Na⁺/HCO₃⁻ symport. However, the cytosolic pH close to the plasma

membrane is often mirrored by the pH close to the
extracellular leaflet (acidic extracellular pH and alkaline
intracellular pH), which has a profound effect on actin
polymerization and cell migration (see below). The pH-
dynamics cannot be seen as a simple global transport
rate, especially if one considers the cytosol as separated
into local non-membrane-bound "organelles" or con-
densates [33].

**Intracellular pH and transcriptional regulation.** A 2024 study uncovered the role of intracellular pH in regulating global transcriptional changes in normal human breast epithelial cells [34]. 176 genes were differentially expressed upon changes of intracellular pH. High intracellular pH increased expression of glycolytic enzymes and regulators of pyruvate fate, shifting epithelial cells toward a glycolytic metabolism. How the mirroring of extracellular and intracellular pH controls transcription remains to be explored. This is particularly relevant given that extracellular acidosis is a key feature of the tumor microenvironment and chronic acid exposure or acid priming results in profound transcriptional regulation, including epithelial-mesenchymal transition (EMT).

These discoveries reveal the complexity of pH regula-
tion across cellular compartments. Below, we discuss
how pH regulation plays a crucial role in various cellular
processes, also offering new insights for biomed-
ical research.

**Pathophysiological processes**

Recent research has provided novel insights into the role of pH regulation in various physiological and pathological processes, highlighting its importance beyond basic homeostasis.

**Invadopodia and drug resistance.** The function of invadopodia critically depends on precise pH regulation in both the extracellular and intracellular spaces. In the extracellular environment, acidification is necessary to activate matrix metalloproteinases and other proteases that degrade the extracellular matrix [35]. Intracellular alkalinization, mediated by Na⁺/H⁺ exchangers, is crucial to control actin dynamics for invadopodia formation and maturation in cancer cells [36] (Fig. 1). The Na⁺/H⁺ exchanger NHE1 creates focused pH nano-domains at invadopodia sites, driving extracellular acidification for matrix degradation while simultaneously promoting local intracellular alkalization. The reversed pH gradient due to extracellular acidification in cancer has also been shown to favour the drug efflux pump activity of the hedgehog pathway component patched-1 [37].

**Cell migration** is a complex process that depends heavily
on precise pH regulation both inside and outside the
cell. Cells establish and maintain a local extracellular pH
gradient during migration, with more acidic pH at the leading edge and more alkaline pH at the rear [38]. These pH differences are essential for proper cell movement by affecting protein activity, cytoskeleton dynamics, and focal adhesion turnover [39,40]. Furthermore, an acidic pH at the leading edge of migrating cells might trigger the desialylation glycoswitch, GL-Lect driven endocytosis, retrograde trafficking, and polarized secretion for the dynamic localization of adhesion factors towards the leading edge and persistent cell migration through complex 3D environments [14,41]. NHE1 expression correlates with invasiveness of cancer cells and has emerged as a critical factor of cancer cell migration [42,43]. Interestingly, the localization of NHE1 to leading edge and rear of cells is dependent on the viscosity of the cell medium [44]. Changes in extracellular pH mediated through NHE1 activate aquaporins [45], which drive water flux into the cell inducing changes in cell size. This is used by cells for propulsion in a process termed the ‘osmotic engine’ [46]. Whether and how ‘glycoswitch’ and ‘osmotic engine’ processes are intertwined remains to be explored.

**Immune cell function.** Acidosis is often observed in cancer due to a shift from aerobic to anaerobic respiration, the so-called ‘Warburg effect’ [47]. This leads to decrease in extracellular pH due to the increased secretion of lactate. The impact of extracellular acidification on tumor infiltrating immune cells has emerged as an area of intense research, with important implications for the development of immunotherapy [7]. The exposure of T cells to lactate impacts upon their metabolism, which is critical for their proliferation and reduces their ability to produce factors such as interleukin-2 and interferon-γ [48,49]. Additionally, lactate favours the M2-polarization of tumour associated macrophages, which in turn leads to the expression of arginase 1 by macrophages promoting tumour growth [50]. Neutrophil extracellular trap (NET) formation is pH-dependent, with alkaline conditions enhancing NET release [51].

**Stroke.** During stroke, ischemia deprives the affected area of oxygen and glucose, causing a switch to anaerobic respiration and an accumulation of lactate acid. Extracellular pH can fall as low as 6.0 and the levels of glutamate can rise 10–100 fold [52]. A recent study by Lai et al. demonstrates a cooperativity of protons and glutamate to allosterically activate ASIC channels, inducing neurotoxicity associated with stroke [53].

**Bone remodelling.** Osteoblasts and osteoclasts rely on precise pH regulation for their functions [54]. Osteoclast-mediated bone resorption requires acidification of the resorption lacuna (or sealing zone), driven by V-ATPase activity and NHE family members. Osteoblast differentiation and mineralization are pH-sensitive processes, with alkaline conditions favouring bone

formation. Very recent data show that the activity of NHE family members, neuraminidases, and galectins are required for the integrity of the sealing zone [14] suggesting that the dynamic localization of integrins, CD44, and other adhesion factors to the sealing zone might depend on pH-induced desialylation, GL-Lect driven endocytosis, retrograde trafficking, and polarized secretion.

These recent findings underscore the complex and multifaceted roles of pH regulation in cellular physiology, extending far beyond simple maintenance of homeostasis. Understanding these pH-dependent processes may lead to new therapeutic strategies for various pathological conditions, including cancer, immune disorders, and bone diseases.

## Physiological function of pH regulation across other membranes

**Vacuolar pH dynamics and cell cycle regulation.** A groundbreaking study in 2023 revealed a previously unrecognized link between vacuolar pH dynamics and the cell cycle in *Saccharomyces cerevisiae* [55]. The lysosome-like vacuole alkalinizes before cell division and re-acidifies as cells divide. This process requires extracellular amino acid uptake, TORC1 activity, v-ATPase function, and cycling of vacuolar-specific lipid phosphatidylinositol 3,5-bisphosphate. Therefore, vacuolar pH regulation enables amino acid sequestration and mobilization, impacting mitochondrial function, ribosome homeostasis, and cell size control.

**Cellular senescence and pH regulation.** A connection between cellular senescence and intracellular acidification in cancer cells was also discovered in 2023 [56]. Strong positive correlations were observed between senescence and intracellular pH in breast cancer cell lines. The key protein involved in this process is the renin receptor, ATP6AP2, which is needed for the assembly of the lysosomal proton-transporting V-type ATPase. Induced senescence leads to intracellular acidification, lysosomal alkalization and dysfunction, accompanied by altered inflammatory and immune transcriptional profiles.

## Tools to measure pH

Precise pH measurement with high spatial and temporal resolution is crucial for understanding the mechanisms and functions of cellular pH regulation. While classical methods, such as spectrophotometric dyes and electrophysiological techniques, have been fundamental in characterizing bulk proton dynamics and pH regulation [57], recent advances in chemical biology, electrochemistry, and protein engineering have enriched pH sensing technology with more sensitive and versatile pH probes, enabling the detection of subtle pH fluctuations across various cellular compartments, including the
plasma membrane [58,59]. This chapter explores the latest developments in pH measurement tools, highlighting their unique capabilities and applications in cellular research (see Table 1).

## Genetically encoded pH sensors

Genetically encoded pH (GEpH) sensors have advanced the field by enabling long-term, non-invasive pH measurements through genetic coupling to specific proteins, allowing for continuous pH monitoring without disrupting cellular functions. These sensors offer unprecedented resolution and real-time monitoring, but need to be coupled to organelle or plasma membrane localized proteins for spatial applicability.

In general, pH sensors based on green fluorescent protein (GFP) and its variants face the problem of decreasing signal-to-noise ratio at lower pH values, as their pKa values of around 6.0–7.3 make them less effective in acidic environments. To overcome this issue of poor efficiency for measurements below pH 5, low pKa fluorescent proteins, like TagBFP (pKa 2.7) and Gamillus (pKa 3.4) have been introduced. These proteins maintain their fluorescence in highly acidic

environments, making them useful as stable components in tandem fluorescent protein sensors.

**pH-Lemon** is based on the principal design of pHalamemeon by combining the pH-sensitive EYFP and the pH-stable mTurquoise2. This resulted in an extended dynamic range. pH-Lemon has been validated at the plasma membrane as a GPI fusion protein or in cytosol, mitochondria, autophagosomes, and the ER lumen [60].

**SypHer3 and SypHerExtra** are recent members of the SypHer family, capable of measuring alkaline pH in a range of pH 6.5–9.0 and 7.5–9.5, respectively, using a fluorescence lifetime imaging microscopy (FLIM) approach [61].

A comprehensive study of 10 different fluorescent proteins and their pH sensitivity from pH 4 to 8 identified mTurquoise2, Gamillus, mNeonGreen, and TagRFP as particular suitable for the acidic pH range [62].

**CoGFP-mTagBFP2** offers a remarkable 200-fold change in fluorescence intensity by switching from pH 5 to 7.5. This was accomplished by combining the pH-

<caption>**Table 1**</caption>

**Fluorescent dyes and genetic pH indicators. See text for details.**

<table><thead><tr><th>Name</th><th></th><th>Excitation (nm)</th><th>Emission (nm)</th><th>pH range</th><th>Fold change/ pH unit</th><th>Comment</th><th>Reference</th></tr></thead><tbody><tr><td colspan="8"><strong>Genetic encoded pH sensors</strong></td></tr><tr><td>Acidin-2</td><td>mRFP-TagBFP2</td><td>561/405</td><td>610/460</td><td>2.5–5.5</td><td>1.4</td><td>FRET probe as well</td><td>[64]</td></tr><tr><td>Acidin-3</td><td>mRFP-Gamillus</td><td>561/488</td><td>610/515</td><td>3.0–6.0</td><td>0.9</td><td>FRET probe as well</td><td>[64]</td></tr><tr><td>Acidin-4</td><td>mRFP-SYFP2</td><td>561/514</td><td>610/525</td><td>4.5–7.0</td><td>2.1</td><td>FRET probe as well</td><td>[64]</td></tr><tr><td>mBeRFP (D162T)</td><td>Monomeric with dual excitation</td><td>440/580</td><td>500/625</td><td>5.2–7.0</td><td>15</td><td>Chlorid independent pH-sensor</td><td>[78]</td></tr><tr><td>EGF-CoGFP-mTagBFP2</td><td>CoGFP(Vo)-mTagBFP2</td><td>405/488</td><td>455/500</td><td>5.0–7.5</td><td>60</td><td>FRET probe</td><td>[63]</td></tr><tr><td>pH-Lemon</td><td>EYFP-mTurquoise2</td><td>430</td><td>525/475</td><td>4.5–7.0</td><td>0.25</td><td>FRET and FLIM probe</td><td>[60]</td></tr><tr><td>Superecliptic pHluorin (SEP)</td><td></td><td>491</td><td>511</td><td>5.5–7.4</td><td>25</td><td></td><td>[79]</td></tr><tr><td>LIME</td><td></td><td>488</td><td>510</td><td>5.5–7.4</td><td>51</td><td>Broad emission spectrum</td><td>[80]</td></tr><tr><td>SypHer3s</td><td></td><td>427<br>504</td><td>498<br>525</td><td>6.5–9.0</td><td>3</td><td>Ratiometric measurement</td><td>[81]</td></tr><tr><td>SypHer3s (2-photon)</td><td></td><td>890</td><td>525</td><td>6.5–9.0</td><td>3</td><td>Live cell, deep tissue imaging</td><td>[82]</td></tr><tr><td>SypHerExtra</td><td></td><td>445</td><td>515</td><td>7.5–9.5</td><td>3</td><td>FLIM, extracellular pH</td><td>[61]</td></tr><tr><td>pHLARE</td><td>sfGFP-mCherry</td><td>488<br>561</td><td>510<br>610</td><td>4.0–6.5</td><td>4</td><td>Lysosomal pH</td><td>[83]</td></tr><tr><td>pHmScarlet</td><td></td><td>562</td><td>585</td><td>5.5–7.4</td><td>13</td><td></td><td>[84]</td></tr><tr><td colspan="8"><strong>Fluorescent dyes</strong></td></tr><tr><td>I2-IR783-Mpip</td><td></td><td>405nm/490 nm &amp; 450 nm</td><td></td><td>5.0–9.0</td><td>2–3</td><td>Limitation: ROS generation</td><td>[65]</td></tr><tr><td>SiRpH5</td><td></td><td>580<br>663</td><td>690</td><td>4.5–7.5</td><td>2–3</td><td>High temporal resolution; suitable for in vitro and in vivo.</td><td>[71]</td></tr><tr><td>Acidifluor Orange</td><td></td><td>538</td><td>560</td><td>4.5–7.0</td><td>10</td><td></td><td>[70]</td></tr></tbody></table>
sensitive, dual-colour-emitting CoGFP_V0 from the sea cactus *Cavernularia obesa* with mTagBFP2. An EGF fused version was recombinantly expressed, purified, and used as a plasma membrane pH-sensor. However, EGF-CoGFP-mTagBFP2 should be further improved, e.g., through a cysteine-free variant such as cgfTagRFP [63].

Among red fluorescent pH sensors like pHuji and pHRed, none match the pH sensitivity or brightness of green-spectrum fluorescent proteins, due to suboptimal sensitivity, lower fluorescence, or pronounced photo-chromic behaviour.

Other genetic probes mentioned in Table 1 offer clear strengths as well, but have not been tested as fusion proteins targeting the plasma membrane or need to be adapted from plant research to the mammalian system (Acidin-2 to -4 [64]).

## pH sensitive fluorescent dyes

Fluorescent dyes are fundamental for pH monitoring, offering advantages over genetically encoded sensors such as smaller size, easier implementation, chemical coupling to ligands or antibodies, and ratiometric measurement for improved accuracy. Recent advances have further enhanced their specificity, sensitivity, and applicability.

### Near-infrared (NIR) pH-probes

enable deep tissue penetration, making them suitable for in vivo imaging in animal models [65]. In addition, NIR imaging minimizes interference from autofluorescence of the biological sample, thus enabling higher signal-to-noise ratios. A promising group of environmental sensitive molecules are Xanthene hybrids, i.e., Changsha dyes [66]. Optical properties of pwu show a pH sensitive emission in the range of 5–7. However, intracellular reactive oxygen species (ROS) might be generated upon strong 850 nm irradiation, which reduces their suitability for life cell imaging experiments [65].

### Nanoparticle (NP)-based sensors

include recently developed polystyrene or silica-based NPs, using an incorporated non-pH-responsive red reference dye (rhodamine), and a green emissive naphthalimide pH probe on the surface of the NPs. These pH-sensors allow for ratiometric pH sensing at the cell surface, addressing the challenge of extracellular pH measurement [67,68]. However, they are not monofunctionalized yet.

### Nanobody fused FRET sensors

bind to SPOT-tagged plasma membrane proteins for monitoring local pH in the range of 6–8. A recombinant anti-SPOT nanobody-fused biosensor was validated using a SPOT-tagged GPI-anchored protein [69]. A critical limitation of the sensor is its size, ranging from 70 to 100 kDa.

### AcidiFluor Orange

remains a pH-sensitive dye of choice for live-cell imaging, showing strong fluorescence at low pH and weak fluorescence at neutral pH, making it ideal for visualizing acidic organelles, local pH changes at the plasma membrane, and endocytic or exocytic dynamics [70].

Asymmetric **silicon-substituted rhodamines** (SiRs) were used to develop SiRpH5 and Me-pEPPR for the imaging of organelle pH and extracellular pH, respectively. Those probes are very photo resistant, and therefore suitable for monitoring endocytic events and for long-term tracking [71].

Despite these advancements, challenges remain in effectively targeting fluorescent dyes to specific compartments.

## DNA-based fluorescent pH sensors

Nanoscale engineering of DNA-based fluorescent sensors exploits the unique property of nucleic acids to undergo conformational changes in response to pH in the range of 5.5–7 [72,73]. These DNA-origami tools are highly specific, sensitive, responsive, and can be tailored for multiplexed measurements at the plasma membrane and in endocytic compartments. Compared to traditional dyes and genetically encoded sensors, they offer greater sensitivity, improved spatial resolution, and reliable performance in complex biological environments. Recent advancements in DNA-based pH sensors include:

### Aptamer-based sensors.
The combination of “chemical antibodies” — aptamers — and DNA nanotechnology enables targeting of pH-responsive DNA tools to specific regions at the plasma membrane [74].

### I-motif structures.
These pH-sensitive DNA structures undergo significant conformational changes in response to protonation, providing a basis for highly sensitive pH detection without any chemical modifications [75].

### DNA-enzyme conjugates.
Combining pH-sensitive DNA structures with enzymes has led to the development of amplified sensing systems with improved sensitivity and dynamic range [76].

### Nanomaterial-DNA hybrids.
Integration of DNA sensors with nanomaterials like gold nanoparticles or graphene oxide has resulted in enhanced signal output and improved spatial resolution [77].

## Conclusions and future perspectives

The study of how pH is regulated and how it affects cellular functions has gained significant momentum in recent years, revealing its critical role in various cellular processes. As research continues to evolve, several key
questions and areas for future exploration emerge,
particularly concerning intracellular pH dynamics and
plasma membrane pH regulation.

**Outstanding questions**

**Mechanisms of pH sensing.** Despite advances in un-
derstanding how cells sense and respond to pH changes,
e.g., through activation of pH-sensitive metal-
loproteinases and neuraminidases on the plasma mem-
brane, precise molecular mechanisms remain in many
cases poorly defined. Future research should focus on
elucidating the roles of specific ion channels, trans-
porters, and receptors involved in pH sensing, particu-
larly in relation to GPCRs and their interactions with
intracellular signalling pathways.

**Dynamic pH regulation during the cell cycle.** Recent findings indicate that intracellular pH dynamics play a crucial role during the cell cycle, with distinct fluctuations observed at different stages (e.g., decreases at G1/S transition and increases during G2/M). Further investigation is needed to determine how these transient changes affect cell cycle progression and whether dysregulated intracellular pH dynamics contribute to malignant transformations [85].

**Role of pH in cell migration and invasion.** The relationship between pH sensing and cell migration remains a fruitful area of exploration. Understanding how extracellular acidification influences invadopodia formation, matrix degradation, endocytic trafficking, and cell migration is expected to provide insights into cancer metastasis mechanisms. Investigating the interplay between local pH changes at the plasma membrane, glycan desialylation, and cytoskeletal dynamics may reveal novel mechanistic insights. Many important questions remain, such as: Are Na⁺/H⁺ antiporters such as NHE family members organized in protein complexes or membrane nanodomains with proton-driven enzymes, e.g., neuraminidases? How steep are proton gradients around antiporters within the immobilized layer of water on membranes [86]? Are glycolipids also desialylated?

**pH regulation in immune responses.** The effects of the tumour acidosis on tumour infiltrating immune cells are an important field of exploration for the development of immunotherapy. Understanding how acidic pH helps cancer cells evade immune surveillance is critical.

**Intracellular acidification and signalling pathways.** Emerging evidence shows that intracellular acidification is crucial in signalling networks, especially alongside calcium signalling [87], but its initiation and maintenance remain unclear. Recent findings suggest that lysosome-localized STAT3 helps keeping the cytosol alkaline even under acidic external conditions [88], highlighting the need for further studies on these interconnected pathways.

**Conclusion**

As the field of pH regulation continues to advance,
addressing these key questions will enhance our un-
derstanding of cellular physiology and its implications
for health and disease. By integrating insights from cell
biology with innovative experimental approaches, re-
searchers are expected to uncover new therapeutic av-
enues for managing conditions associated with
dysregulated pH dynamics.

**Credit author statement**

Conceptualization and writing: EM, LJ, CW.

**Funding**

We acknowledge the funding from Labex Cell(n)Scale (11-LABX-0038) and Idex Paris Sciences et Lettres (ANR-10-IDEX-0001-02 PSL), Mizutani Foundation for Glycosciences n° 200014, ERC Proof of Concept project 101062030, Q-Life ANR-17-CONV-0005, Agence Nationale pour la Recherche ANR-20-CE18-0016-03, ANR-22-CE11-0030-03, ANR-22-CE18-0048-04, PEPR ANR-22-PEBI-0007, Fondation pour la Recherche Médicale (FRM) EQU202103012926, Institut National du Cancer (INCa) n° 2018-1-PLBIO-01-ICR-1, n° 2019-1-PLBIO-05-1, La Ligue National contre le Cancer n° AAPARN 2021.LCC/ChP, and a Curie-NCBS Campus fellowship and ARC passerelle grant to EM.

**Declaration of competing interest**

The authors declare that they have no known competing
financial interests or personal relationships that could
have appeared to influence the work reported in
this paper.

**Data availability**

No data was used for the research described in
the article.

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