Face Of Membrane Research Articles

Differential effects of ganglioside lipids on the conformation and aggregation of islet amyloid polypeptide.

Despite causing over 1 million deaths annually, Type 2 Diabetes (T2D) currently has no curative treatments. Aggregation of the islet amyloid polypeptide (hIAPP) into amyloid plaques plays an important role in the pathophysiology of T2D and thus presents a target for therapeutic intervention. The mechanism by which hIAPP aggregates contribute to the development of T2D is unclear, but it is proposed to involve disruption of cellular membranes. However, nearly all research on hIAPP-lipid interactions has focused on anionic phospholipids, which are primarily present in the cytosolic face of plasma membranes. We seek here to characterize the effects of three gangliosides, the dominant anionic lipids in the outer leaflet of the plasma membrane, on the aggregation, structure, and toxicity of hIAPP. Our results show a dual behavior that depends on the molar ratio between the gangliosides and hIAPP. For each ganglioside, a low-lipid:peptide ratio enhances hIAPP aggregation and alters the morphology of hIAPP fibrils, while a high ratio eliminates aggregation and stabilizes an α-helix-rich hIAPP conformation. A more negative lipid charge more efficiently promotes aggregation, and a larger lipid headgroup improves inhibition of aggregation. hIAPP also alters the phase transitions of the lipids, favoring spherical micelles over larger tubular micelles. We discuss our results in the context of the available lipid surface area for hIAPP binding and speculate on a role for gangliosides in facilitating toxic hIAPP aggregation.

Anchorage of H3K9-methylated heterochromatin to the nuclear periphery helps mediate P-cell nuclear migration though constricted spaces in Caenorhabditis elegans.

Nuclei adjust their deformability while migrating through constrictions to enable structural changes and maintain nuclear integrity. The effect of heterochromatin anchored at the nucleoplasmic face of the inner nuclear membrane on nuclear morphology and deformability during in vivo nuclear migration through constricted spaces remains unclear. Here, we show that abolishing peripheral heterochromatin anchorage by eliminating CEC-4, a chromodomain protein that tethers H3K9-methylated chromatin to the nuclear periphery, disrupts constrained P-cell nuclear migration in Caenorhabditis elegans larvae in the absence of the established LINC complex-dependent pathway. CEC-4 acts in parallel to an actin and CDC-42-based pathway. We also demonstrate the necessity for the chromatin methyltransferases MET-2 and JMJD-1.2 during P-cell nuclear migration in the absence of functional LINC complexes. We conclude that H3K9-nethylated chromatin needs to be anchored to the nucleoplasmic face of the inner nuclear membrane to help facilitate nuclear migration through constricted spaces in vivo.

Optimization of laser-induced graphene membrane for simultaneous photo- and electro-thermal membrane distillation

To optimize the Laser-Induced Graphene (LIG) Janus membrane, this study investigated the effects of membrane pore structure, polydimethylsiloxane (PDMS) coating sequence and addition of silver (Ag) nanoparticles on membrane distillation (MD) performance. This study aimed to enhance the photothermal characteristics of graphene while using the intrinsic electrical conductivity for simultaneous photo- and electrothermal MD. Operating at the same photo- and electro-thermal power input, the LIG Janus membrane made by treating the membrane face with smaller pores (i.e., shiny side) gave an improved flux performance of up to 53.6% and a decrease in specific energy of 35.4% compared to that by treating the membrane face with larger pores (i.e., dull side). The effect of the PDMS coating sequence also depended on the pore structure. For the face with smaller pore structures, coating PDMS before laser irradiation (PDMS-BLSS) gave a flux improvement of up to 24.5% and a decrease in specific energy of 19.7%, compared to coating PDMS after laser irradiation (PDMS-ALSS). As for the face with larger pore structures, coating PDMS before laser irradiation (PDMS-BLDS) resulted in a flux reduction of up to 20.8% and an increase in specific energy of 27.1%, compared to coating PDMS after irradiation (PDMS-ALDS). The LIG Janus membranes embedded with Ag nanoparticles led to improved photothermal heating properties, improving flux by 43.1–65.8% and decreasing specific energy by 15.2–30.5% while maintaining similar electrothermal heating properties. Carrying out simultaneous photo- and electro-thermal MD indicates that only the Ag-doped Janus LIG membrane gave a synergistic effect whereby the flux of the combined heating mode was higher than the summation of the fluxes obtained when operating in the individual heating modes.

Human ESCRT-I and ALIX function as scaffolding helical filaments in vivo.

When cellular membranes are dissolved or breached, ESCRT is rapidly deployed to repair membranes to restore the integrity of intracellular compartments. Membrane sealing is ensured by ESCRT-III filaments assembled on the inner face of membrane; a mechanism termed inverse topology membrane scission. This mechanism, initiated by ESCRT-I and ALIX, is universally necessary for cytokinesis, wound repair, budding of enveloped viruses, and more. We show ESCRT-I and ALIX individually oligomerize into helical filaments that cluster newly discovered nucleic acid-binding interfaces and scaffold-in genomes within nascent virions and nuclear envelopes. These oligomers additionally appear to serve as ideal templates for ESCRT-III polymerization, as helical filaments of CHMP4B were found intertwined ESCRT-I or ALIX filaments in vivo . Similarly, corkscrew-like filaments of ALIX are also interwoven with ESCRT-I, supporting a model of inverse topology membrane scission that is synergistically reinforced by inward double filament scaffolding.

Superoxide signalling and antioxidant processing in the plant nucleus.

The superoxide anion radical (O2·-) is a one-electron reduction product of molecular oxygen. Compared with other forms of reactive oxygen species (ROS), superoxide has limited reactivity. Nevertheless, superoxide reacts with nitric oxide, ascorbate, and the iron moieties of [Fe-S] cluster-containing proteins. Superoxide has largely been neglected as a signalling molecule in the plant literature in favour of the most stable ROS form, hydrogen peroxide. However, superoxide can accumulate in plant cells, particularly in meristems, where superoxide dismutase activity and ascorbate accumulation are limited (or absent), or when superoxide is generated within the lipid environment of membranes. Moreover, oxidation of the nucleus in response to environmental stresses is a widespread phenomenon. Superoxide is generated in many intracellular compartments including mitochondria, chloroplasts, and on the apoplastic/cell wall face of the plasma membrane. However, nuclear superoxide production and functions remain poorly documented in plants. Accumulating evidence suggests that the nuclear pools of antioxidants such as glutathione are discrete and separate from the cytosolic pools, allowing compartment-specific signalling in the nucleus. We consider the potential mechanisms of superoxide generation and targets in the nucleus, together with the importance of antioxidant processing in regulating superoxide signalling.

Evaluation of the Microbial Profile on the Polydioxanone Membrane and the Collagen Membrane Exposed to Multi-Species Subgingival Biofilm: An In Vitro Study.

Dehiscence in surgeries involving membranes often leads to bacterial contamination, hindering the healing process. This study assessed bacterial colonization on various membrane materials. Polydioxanone (PDO) membranes, with thicknesses of 0.5 mm and 1 mm, and a collagen membrane were examined. Packages containing polystyrene pins were crafted using these membranes, attached to 24-well plates, and exposed to oral bacteria from supra and subgingival biofilm. After a week's anaerobic incubation, biofilm formation was evaluated using the DNA-DNA hybridization test. Statistical analysis employed the Kruskal-Wallis test with Dunn's post hoc test. The biofilm on the polystyrene pins covered by the 0.5 mm PDO membrane showed a higher count of certain pathogens. The collagen membrane had a greater total biofilm count on its inner surface compared to both PDO membranes. The external collagen membrane face had a higher total biofilm count than the 0.5 mm PDO membrane. Furthermore, the 1 mm PDO membrane exhibited a greater count of specific pathogens than its 0.5 mm counterpart. In conclusion, the collagen membrane presented more biofilm and pathogens both internally and on its inner surface.

The changing face of dialyzer membranes and dialyzers.

The key goals for dialysis treatments are to prevent the progressive accumulation of waste products of metabolism and volume overload. Traditionally uremic solutes have been classified according to molecular weight and termed small, middle sized, and large solutes. Solute clearance during dialysis sessions will potentially be by diffusion, convection and adsorption. Dialyzer membranes act as a semi-permeable membrane restricting solute removal predominantly by size. Small molecules move faster than large molecules, so small solutes are readily removed by diffusion. Increasing the size of the pores in the membrane will potentially allow middle and larger sized solutes to pass through the dialyzer membrane, although in practice there is a limit to increasing pore sizes to prevent the loss of albumin and other important proteins. Differences in membrane surface and charge will influence protein absorption. The removal of fluid during dialysis depends in part on the hydraulic permeability of the membrane. Combining higher hydraulic permeability and larger sized pores increases convective clearance with solutes moving across the membrane with the water movement. Depending upon dialyzer design, higher hydrostatic pressure as blood enters the dialyzer leads to a variable amount of internal diafiltration, so improving the clearance of middle sized solutes. Although the dialyzer membrane plays a key role in solute clearance, the design of the casing and header also play a role in directing the countercurrent blood and dialysate flows to maximize the surface area available for diffusive and convective clearances.

Orientation-controlled membrane anchoring of bioorthogonal catalysts on live cells via liposome fusion–based transport

An obstacle to conducting diverse bioorthogonal reactions in living systems is the sensitivity of artificial metal catalysts. It has been reported that artificial metallocatalysts can be assembled in "cleaner" environments in cells for stabilized performance, which is powerful but is limited by the prerequisite of using specific cells. We report here a strategy to establish membrane-anchored catalysts with precise spatial control via liposome fusion-based transport (MAC-LiFT), loading bioorthogonal catalytic complexes onto either or both sides of the membrane leaflets. We show that the inner face of the cytoplasmic membrane serves as a reliable shelter for metal centers, protecting the complexes from deactivation thus substantially lowering the amount of catalyst needed for effective intracellular catalysis. This MAC-LiFT approach makes it possible to establish catalyst-protective systems with exclusively exogenous agents in a wide array of mammalian cells, allowing convenient and wider use of diverse bioorthogonal reactions in live cellular systems.

Incorporating a Polyethyleneglycol Linker to Enhance the Hydrophilicity of Mitochondria-Targeted Triphenylphosphonium Constructs.

The targeting of bioactive molecules and probes to mitochondria can be achieved by coupling to the lipophilic triphenyl phosphonium (TPP) cation, which accumulates several hundred-fold within mitochondria in response to the mitochondrial membrane potential (Δψm ). Typically, a simple alkane links the TPP to its "cargo", increasing overall hydrophobicity. As it would be beneficial to enhance the water solubility of mitochondria-targeted compounds we explored the effects of replacing the alkyl linker with a polyethylene glycol (PEG). We found that the use of PEG led to compounds that were readily taken up by isolated mitochondria and by mitochondria inside cells. Within mitochondria the PEG linker greatly decreased adsorption of the TPP constructs to the matrix-facing face of the mitochondrial inner membrane. These findings will allow the distribution of mitochondria-targeted TPP compounds within mitochondria to be fine-tuned.

Vascular therapy for Duchenne muscular dystrophy (DMD).

Duchenne muscular dystrophy (DMD) is a progressive disease characterized by the wasting of the muscles that eventually lead to difficulty moving and, ultimately, premature death from heart and respiratory complications. DMD deficiency is caused by mutations in the gene encoding dystrophin, which prevents skeletal muscle, cardiac muscle, and other cells from producing the functional protein. Located on the cytoplasmic face of the plasma membrane of muscle fibers, dystrophin serves as a component of the dystrophin glycoprotein complex (DGC), mechanically reinforces the sarcolemma, and stabilizes the DGC, preventing it from contraction-mediated muscle degradation. In DMD muscle, dystrophin deficiency leads to progressive fibrosis, myofiber damage, chronic inflammation, and dysfunction of the mitochondria and muscle stem cells. Currently, DMD is incurable, and treatment involves the administration of glucocorticoids in order to delay disease progression. In the presence of developmental delay, proximal weakness, and elevated serum creatine kinase levels, a definitive diagnosis can usually be made after an extensive review of the patient's history and physical examination, as well as confirmation through muscle biopsy or genetic testing. Current standards of care include the use of corticosteroids to prolong ambulation and delay the onset of secondary complications, including respiratory muscle and cardiac functions. However, different studies have been carried out to show the relationship between vascular density and impaired angiogenesis in the pathogenesis of DMD. Several recent studies on DMD management are vascular targeted and focused on ischemia as a culprit for the pathogenesis of DMD. This review critically discusses approaches-such as modulation of nitric oxide (NO) or vascular endothelial growth factor (VEGF)-related pathways-to attenuate the dystrophic phenotype and enhance angiogenesis.

Identification of Compounds Preventing A. fumigatus Biofilm Formation by Inhibition of the Galactosaminogalactan Deacetylase Agd3.

The opportunistic fungus Aspergillus fumigatus causes a set of diseases ranging from allergy to lethal invasive mycosis. Within the human airways, A. fumigatus is embedded in a biofilm that forms not only a barrier against the host immune defense system, but also creates a physical barrier protecting the fungi from chemicals such as antifungal drugs. Novel therapeutic strategies aim at combining drugs that inhibit biofilm synthesis or disrupt existing biofilm with classical antimicrobials. One of the major constituents of A. fumigatus biofilm is the polysaccharide galactosaminogalactan (GAG) composed of α1,4-linked N-acetylgalactosamine, galactosamine, and galactose residues. GAG is synthesized on the cytosolic face of the plasma membrane and is extruded in the extracellular space, where it is partially deacetylated. The deacetylase Agd3 that mediates this last step is essential for the biofilm formation and full virulence of the fungus. In this work, a previously described enzyme-linked lectin assay, based on the adhesion of deacetylated GAG to negatively charged plates and quantification with biotinylated soybean agglutinin was adapted to screen microbial natural compounds, as well as compounds identified in in silico screening of drug libraries. Actinomycin X2, actinomycin D, rifaximin, and imatinib were shown to inhibit Agd3 activity in vitro. At a concentration of 100 µM, actinomycin D and imatinib showed a clear reduction in the biofilm biomass without affecting the fungal growth. Finally, imatinib reduced the virulence of A. fumigatus in a Galleria mellonella infection model in an Agd3-dependent manner.

Regulation of phosphoinositide metabolism in Apicomplexan parasites.

Phosphoinositides are a biologically essential class of phospholipids that contribute to organelle membrane identity, modulate membrane trafficking pathways, and are central components of major signal transduction pathways that operate on the cytosolic face of intracellular membranes in eukaryotes. Apicomplexans (such as Toxoplasma gondii and Plasmodium spp.) are obligate intracellular parasites that are important causative agents of disease in animals and humans. Recent advances in molecular and cell biology of Apicomplexan parasites reveal important roles for phosphoinositide signaling in key aspects of parasitosis. These include invasion of host cells, intracellular survival and replication, egress from host cells, and extracellular motility. As Apicomplexans have adapted to the organization of essential signaling pathways to accommodate their complex parasitic lifestyle, these organisms offer experimentally tractable systems for studying the evolution, conservation, and repurposing of phosphoinositide signaling. In this review, we describe the regulatory mechanisms that control the spatial and temporal regulation of phosphoinositides in the Apicomplexan parasites Plasmodium and T. gondii. We further discuss the similarities and differences presented by Apicomplexan phosphoinositide signaling relative to how these pathways are regulated in other eukaryotic organisms.

Extracellular Tau Oligomers Rapidly Induce Nuclear Invagination in Neurons

AbstractBackgroundNeuronal nuclei are normally spheroidal with a smoothly contoured surface. In Alzheimer’s disease (AD) and other tauopathies, however, neuronal nuclei often develop deep invaginations that include the nuclear lamina, a meshwork of intermediate filaments on the inner face of the inner nuclear membrane. The lamina is involved in numerous nuclear activities that might be adversely affected by invagination, including chromatin organization, nucleocytoplasmic trafficking of macromolecules, cell cycle regulation and apoptosis. Previous work showed that in vivo expression of pathogenically mutated human tau in Drosophila melanogaster neurons causes nuclear lamina invagination and that invaginated neuronal nuclei are found in human AD brain (Frost, et al. 2016. Current Biology 26; 129‐136). In light of this background we sought to shed further light on the mechanism of pathological nuclear invagination in mammalian neurons.MethodLamina invagination was assessed by immunofluorescence microscopy in human and mouse brain tissue, and in cultured mouse neurons using an antibody against lamin B1, a major subunit protein of the nuclear lamina. To ensure thorough neuroanatomical coverage in mouse brain, we stained lateral, medial, and middle sections from each brain, and imaged across the cortical region. To seek stimuli that would cause neuronal nuclear invagination, cultured mouse neurons were exposed to extracellular tau oligomers (xcTauOs).ResultWe detected nuclear invagination in neurons in brains of AD patients and CVN (APPSwDI;NOS2−/−) mice, in the latter of which nuclear invagination in cortical neurons was more frequent than in age‐matched wild type control neurons. In cultured mouse neurons, lamina invagination was induced by xcTauOs within one hour of exposure. This effect of xcTauOs was found to depend on intracellular tau, because nuclear invagination was not detected in neurons derived from tau knockout mice unless they expressed human tau by lentiviral transduction.ConclusionOur data confirm that nuclear invagination is common in both naturally occurring human AD and a transgenic mouse model of AD, and implicate xcTauOs as likely triggers of this phenomenon by a mechanism that also requires intracellular tau.

The impact of gender and peripheral blood parameters on the characteristics of L-PRF membranes.

The purpose of this study was to evaluate the impact of gender and peripheral blood parameters on the characteristics of Leucocyte-and Platelet-Rich Fibrin (L-PRF) membranes and to describe histologically three different zones of L-PRF membranes. Blood was collected from twenty healthy donors (10 men and 10 women). Peripheral blood parameters including leucocyte and platelet counts, and fibrinogen levels were recorded. L-PRF membranes were prepared to quantify the release of growth factors (PDGF, VEGF, BMP-2, and BMP-9) at 1, 2, 3 and 7 days and for histological examination. Three zones within each L-PRF membrane (face, body, and tail) were analysed separately, quantifying the area of leucocytes, platelets, and fibrin in percentage. The Young's modulus of the membranes was also considered (during tensile and compression tests). Women had significantly higher fibrinogen levels in their peripheral blood, and a higher release of BMP-9, whereas men showed a significantly higher Young's modulus in compression tests. The histology revealed significant differences in cellular content and fibrin concentration between the 3 areas, with the face being biologically the richest. Several factors influenced the final characteristics of L-PRF membranes. These need to be taken into consideration when interpreting the results of research, but especially in clinical practice.

Crosstalk between Oxidative Stress and Exosomes.

Mammals have several organs comprising various cells with different functions. Furthermore, eukaryotic cells are compartmentalized into functionally distinct organelles. Thus, for good organismal health, exosomes, which play an important role in cell-to-cell communication, interact closely with oxidative stress. Oxidative stress, which is recognized as a type of intracellular second signal, is aggravated by reactive species. As a subtype of reactive species, reactive oxygen species (ROS) can be produced on the extracellular face of the plasma membrane by NADPH oxidases, via the mitochondrial electron transport chain, in peroxisomes, and in the lumen of the endoplasmic reticulum. The scavenging of ROS is mainly dependent on peroxiredoxins, including GSH peroxidases, peroxiredoxins 3 and 5, and thioredoxin reductase. Intracellular ROS increase the number of intracellular multivesicular bodies (MVBs) by restraining their degradation in lysosomes, thereby enhancing the release of exosomes under the synergy of the depletion of exofacial GSH, which can be regulated by oxidative stress. In contrast, higher ROS levels can decrease the yield of exosomes by activating cellular autophagy to degrade MVBs. Moreover, exosomes can transfer the characteristics of parent cells to recipient cells. Here, we review the interaction between oxidative stress and exosomes in the hope of providing insights into their interplay.

ABCA1 is an extracellular phospholipid translocase

Production of high density lipoprotein (HDL) requires ATP-binding cassette transporter A1 (ABCA1) to drive phospholipid (PL) from the plasma membrane into extracellular apolipoprotein A-I. Here, we use simulations to show that domains of ABCA1 within the plasma membrane remove PL from the membrane’s outer leaflet. In our simulations, after the lipid diffuses into the interior of ABCA1’s outward-open cavity, PL extracted by the gateway passes through a ring-shaped domain, the annulus orifice, which forms the base of an elongated hydrophobic tunnel in the transporter’s extracellular domain. Engineered mutations in the gateway and annulus strongly inhibit lipid export by ABCA1 without affecting cell-surface expression levels. Our finding that ABCA1 extracts lipid from the outer face of the plasma membrane and forces it through its gateway and annulus into an elongated hydrophobic tunnel contrasts with the alternating access model, which proposes that ABCA1 flops PL substrate from the inner leaflet to the outer leaflet of the membrane. Consistent with our model, ABCA1 lacks the charged amino acid residues in the transmembrane domain found in the floppase members of the ABC transporter family.

Electrochemical Investigations of Ionic Transport through Synthetic Nanopore Membranes

Multiporous membrane systems have created a way for scientists to efficiently separate unwanted contaminates from wastewater and drinking water. In ion-exchange membranes the selectivity is typically governed by the surface charge. The ability for a membrane to reject ions of the same sign while simultaneously transporting ions of opposite sign is called permselectivity. In this work, 30 nm polycarbonate membranes are gold-plated using a well-known template synthesis method. The gold-plating time can be precisely controlled to give nanopores as small as 1 nm. By exposing this membrane to a chloride containing salt, a layer of adsorbed chloride will form along the pore walls and membrane faces. The adsorbed chloride gives this membrane a net negative surface charge which will reject anions and transport cations. Using a varying concentration cell, the permselectivity of these membranes can be obtained potentiometrically. The membrane is exposed to various chloride salt solutions to determine how the chemical identity of the cation affects permselectivity. The diameter of these nanopores is also changed to probe how the pore radius affects the permselectivity of these cations. These membranes show ideal cation permselectivity so long as the pore radius is smaller than the thickness of the electrical double layer. Membranes investigated in this work have effective diameters smaller than 10 nm.

The Cation Role in Ion Transport Selectivity of Gold-Plated Nanotubes

Ion permselectivity is an important ionic transport property exhibited in nanopores and nanotubes. Permselectivity develops due to the strong interactions between ions in solution and the nanopore/nanotube surfaces. Here, we study the ion permselectivity of gold-plated nanotube membranes. Gold-plated membranes were obtained by using an electroless gold plating method to deposit gold nanotubes into the pores and on the faces of track-etched polycarbonate membranes. These membranes have the potential to display cation permselectivity due to the excess surface charge density present on the membrane from chemisorbed Cl- anions. The cation permselectivity of membranes with 9.9 + 0.6 nm diameter nanotubes was studied potentiometrically by using a concentration cell. A new Debye-sphere based model was presented here to describe the extent of permselectivity. However, some ion specific effects with the gold-plated membrane are observed that cannot be interpreted by Debye theory. These ion-specific surface interactions lead to a reduction in the negative surface charge, causing no permselectivity for some cations as demonstrated by their lower than ideal cation transference numbers. Quantification of the surface charge density by XPS indicated the gold-plated membranes are highly charged due to Cl- chemisorption with a surface charge density of ~ 5.3 uC per cm2 corresponding to 3.3 x 1013 Cl--ions per cm2.

PvdM of fluorescent pseudomonads is required for the oxidation of ferribactin by PvdP in periplasmic pyoverdine maturation

Fluorescent pseudomonads such as Pseudomonas aeruginosa or Pseudomonas fluorescens produce pyoverdine siderophores that ensure iron-supply in iron-limited environments. After its synthesis in the cytoplasm, the nonfluorescent pyoverdine precursor ferribactin is exported into the periplasm, where the enzymes PvdQ, PvdP, PvdO, PvdN, and PtaA are responsible for fluorophore maturation and tailoring steps. While the roles of all these enzymes are clear, little is known about the role of PvdM, a human renal dipeptidase–related protein that is predicted to be periplasmic and that is essential for pyoverdine biogenesis. Here, we reveal the subcellular localization and functional role of PvdM. Using the model organism P. fluorescens, we show that PvdM is anchored to the periplasmic side of the cytoplasmic membrane, where it is indispensable for the activity of the tyrosinase PvdP. While PvdM does not share the metallopeptidase function of renal dipeptidase, it still has the corresponding peptide-binding site. The substrate of PvdP, deacylated ferribactin, is secreted by a ΔpvdM mutant strain, indicating that PvdM prevents loss of this periplasmic biosynthesis intermediate into the medium by ensuring the efficient transfer of ferribactin to PvdP in vivo. We propose that PvdM belongs to a new dipeptidase-related protein subfamily with inactivated Zn2+ coordination sites, members of which are usually genetically linked to TonB-dependent uptake systems and often associated with periplasmic FAD-dependent oxidoreductases related to d-amino acid oxidases. We suggest that these proteins are necessary for selective binding, exposure, or transfer of specific d- and l-amino acid–containing peptides and other periplasmic biomolecules in manifold pathways.

Genetically encoded fluorescent sensors adapted to acidic pH highlight subdomains within the plant cell apoplast.

Monitoring pH is one of the challenges in understanding diverse physiological regulations as well as ionic balance, especially in highly acidic environments such as the apoplast and the vacuole. To circumvent the poor efficiency of pH measurements below pH 5, we designed three genetically encoded sensors composed of two fluorescent proteins in tandem. We selected fluorescent protein pairs of low but sufficiently different pKa so that each protein could differentially sense the imposed pH. The generated tandems, named Acidin2, Acidin3, and Acidin4, were produced in Escherichia coli and extensively characterized. Altogether, these generated tandems cover a pH range of 3-8. The Acidins were targeted either for release in the apoplast (Apo) or for anchoring at the outer face of the plasma membrane (PM-Apo), with the fluorescent part exposed in the apoplast. Apoplastic Acidins in stably transformed Arabidopsis thaliana primary roots responded immediately and reversibly to pH changes, directly reporting physiological conditions related to cell elongation. In addition, membrane-anchored Acidins reveal a gradual acidification from the surface through the anticlinal wall of pavement cells, a process controlled at least partially by H+-ATPase activity.