Cristiana Picco

Endosomal ClC-3 and ClC-4 Cl⁻/H⁺ antiporters are essential regulators of vesicular ion homeostasis and neuronal function, yet the mechanisms controlling their activity remain incompletely understood. TMEM9A has recently emerged as an accessory β-subunit of endosomal CLC transporters. Here, we define the functional and molecular specificity of TMEM9A across the CLC family. Electrophysiological recordings in Xenopus laevis oocytes and HEK293 cells demonstrated that TMEM9A strongly suppresses ClC-3- and ClC-4-mediated transport, while producing no detectable inhibition of ClC-2 or ClC-7 and only a modest effect on ClC-1. Consistent with these functional effects, colocalization microscopy and FLIM-FRET revealed close molecular association of TMEM9A with ClC-3 and ClC-4, with FRET efficiencies of 0.19 ± 0.02 and 0.15 ± 0.02, respectively, whereas substantially weaker interactions were detected with ClC-1 and ClC-7. In the mouse brain, Tmem9a, Tmem9b, Clcn3, and Clcn4 displayed broad and overlapping expression across cortex, hippocampus, and cerebellum. Moreover, immunofluorescence analysis showed moderate colocalization of TMEM9A with ClC-3 in primary neurons and substantial colocalization with ClC-3 and ClC-4 in heterologous cells. Together, these findings establish a preferential functional and molecular association of TMEM9A with ClC-3 and ClC-4, closely mirroring the selectivity of TMEM9B, and support the TMEM9 proteins as a regulatory β-subunit system for the neuronal endosomal CLC transporters.

Variants in CLCN3 and CLCN4, encoding the neuronal endosomal Cl⁻/H⁺ antiporters ClC-3 and ClC-4, are linked to neurodevelopmental disorders with broad phenotypic variability. Over sixty CLCN4 variants have been functionally characterized, showing gain- or loss-of-function (GoF or LoF) effects. While ClC-3 can function as a homodimer, ClC-4 depends on heterodimerization with ClC-3 for efficient endosomal trafficking. CLCN4, located on the X chromosome, exhibits diverse pathogenic outcomes: complete LoF variants often cause non-syndromic presentations in hemizygous males and are asymptomatic in heterozygous females, whereas certain missense variants with partial or complete LoF produce severe syndromic phenotypes in both sexes. Here, we demonstrate dominant effects of three CLCN4 variants within ClC-3/ClC-4 heterodimers using two-electrode voltage-clamp recordings in Xenopus laevis oocytes and whole-cell patch-clamp recordings in mammalian cells co-expressing both proteins via a bicistronic IRES construct. Our findings provide the first evidence of dominant-negative CLCN4 effects within ClC-3/ClC-4 complexes and establish a platform for functional analysis of additional disease-associated variants.

Despite a high sucrose accumulation in its taproot vacuoles, sugar beet (Beta vulgaris subsp. vulgaris) is sensitive to freezing. Earlier, a taproot‐specific accumulation of raffinose was shown to have beneficial effects on the freezing tolerance of the plant. However, synthesis of raffinose and other oligosaccharides of the raffinose family depends on the availability of myo‐inositol. Since inositol and inositol‐metabolising enzymes reside in different organelles, functional inositol metabolism and raffinose synthesis depend on inositol transporters. We identified five homologues of putative inositol transporters in the sugar beet genome, two of which, BvINT1;1 and BvINT1;2, are localised at the tonoplast. Among these, only the transcript of BvINT1;1 is highly upregulated in sugar beet taproots under cold. BvINT1;1 exhibits a high transport specificity for inositol and sugar beet mutants lacking functional BvINT1;1 contain increased inositol levels, likely accumulating in the vacuole, and decreased raffinose contents under cold treatment. Due to the quenching capacity of raffinose for Reactive Oxygen Species (ROS), which accumulate under cold stress, bvint1;1 sugar beet plants show increased expression of both, ROS marker genes and detoxifying enzymes. Based on these findings, we conclude that the vacuolar inositol transporter BvINT1;1 is contributing to ROS‐homoeostasis in the cold metabolism of sugar beet.

The nine-member CLC gene family of Cl⁻ chloride-transporting membrane proteins is divided into plasma membrane-localized Cl⁻ channels and endo-/lysosomal Cl⁻/H⁺ antiporters. Accessory proteins have been identified for ClC-K and ClC-2 channels and for the lysosomal ClC-7, but not the other CLCs. Here, we identified TMEM9 Domain Family Member B (TMEM9B), a single-span type I transmembrane protein of unknown function, to strongly interact with the neuronal endosomal ClC-3 and ClC-4 transporters. Co-expression of TMEM9B with ClC-3 or ClC-4 dramatically reduced transporter activity in Xenopus oocytes and transfected HEK cells. For ClC-3, TMEM9B also induced a slow component in the kinetics of the activation time course, suggesting direct interaction. Currents mediated by ClC-7 were hardly affected by TMEM9B, and ClC-1 currents were only slightly reduced, demonstrating specific interaction with ClC-3 and ClC-4. We obtained strong evidence for direct interaction by detecting significant Förster Resonance Energy Transfer (FRET), exploiting fluorescence lifetime microscopy-based (FLIM-FRET) techniques between TMEM9B and ClC-3 and ClC-4, but hardly any FRET with ClC-1 or ClC-7. The discovery of TMEM9B as a novel interaction partner of ClC-3 and ClC-4 might have important implications for the physiological role of these transporters in neuronal endosomal homeostasis and for a better understanding of the pathological mechanisms in CLCN3- and CLCN4-related pathological conditions.

The intestine is essential for the modulation of nutrient absorption and the removal of waste. Gut pathologies, such as cancer, inflammatory bowel diseases (IBD), irritable bowel syndrome (IBS), and celiac disease, which extensively impact gut functions, are thus critical for human health. Targeted drug delivery is essential to tackle these diseases, improve therapy efficacy, and minimize side effects. Recent strategies have taken advantage of both active and passive nanocarriers, which are designed to protect the drug until it reaches the correct delivery site and to modulate drug release via the use of different physical–chemical strategies. In this systematic review, we present a literature overview of the different nanocarriers used for drug delivery in a set of chronic intestinal pathologies, highlighting the rationale behind the controlled release of intestinal therapies. The overall aim is to provide the reader with useful information on the current approaches for gut targeting in novel therapeutic strategies.

To mobilize sparingly available phosphorus (P) in the rhizosphere, many plant species secrete malate to release P sorbed onto (hydr)oxides of aluminum and iron (Fe). In the presence of Fe, malate can provoke Fe over-accumulation in the root apoplast, triggering a series of events that inhibit root growth. Here, we identified HYPERSENSITIVE TO LOW P1 (HYP1), a CYBDOM protein constituted of a DOMON and a cytochrome b561 domain, as critical to maintain cell elongation and meristem integrity under low P. We demonstrate that HYP1 mediates ascorbate-dependent trans-plasma membrane electron transport and can reduce ferric and cupric substrates in Xenopus laevis oocytes and in planta. HYP1 expression is up-regulated in response to P deficiency in the proximal zone of the root apical meristem. Disruption of HYP1 leads to increased Fe and callose accumulation in the root meristem and causes significant transcriptional changes in roots. We further demonstrate that HYP1 activity overcomes malate-induced Fe accumulation, thereby preventing Fe-dependent root growth arrest in response to low P. Collectively, our results uncover an ascorbate-dependent metalloreductase that is critical to protect root meristems of P-deficient plants from increased Fe availability and provide insights into the physiological function of the yet poorly characterized but ubiquitous CYBDOM proteins.

Many studies highlighted the importance of the IK channel for the proliferation and the migration of different types of cancer cells, showing how IK blockers could slow down cancer growth. Based on these data, we wanted to characterize the effects of IK blockers on melanoma metastatic cells and to understand if such effects were exclusively IK-dependent. For this purpose, we employed two different blockers, namely clotrimazole and senicapoc, and two cell lines: metastatic melanoma WM266-4 and pancreatic cancer Panc-1, which is reported to have little or no IK expression. Clotrimazole and senicapoc induced a decrease in viability and the migration of both WM266-4 and Panc-1 cells irrespective of IK expression levels. Patch-clamp experiments on WM266-4 cells revealed Ca²⁺-dependent, IK-like, clotrimazole- and senicapoc-sensitive currents, which could not be detected in Panc-1 cells. Neither clotrimazole nor senicapoc altered the intracellular Ca²⁺ concentration. These results suggest that the effects of IK blockers on cancer cells are not strictly dependent on a robust presence of the channel in the plasma membrane, but they might be due to off-target effects on other cellular targets or to the blockade of IK channels localized in intracellular organelles.

Regulation of intracellular sugar homeostasis is maintained by regulation of activities of sugar import and export proteins residing at the tonoplast. We show here that the EARLY RESPONSE TO DEHYDRATION6-LIKE4 (ERDL4) protein, a member of the monosaccharide transporter family, resides in the vacuolar membrane in Arabidopsis (Arabidopsis thaliana). Gene expression and subcellular fractionation studies indicated that ERDL4 participates in fructose allocation across the tonoplast. Overexpression of ERDL4 increased total sugar levels in leaves due to a concomitantly induced stimulation of TONOPLAST SUGAR TRANSPORTER 2 (TST2) expression, coding for the major vacuolar sugar loader. This conclusion is supported by the finding that tst1-2 knockout lines overexpressing ERDL4 lack increased cellular sugar levels. ERDL4 activity contributing to the coordination of cellular sugar homeostasis is also indicated by two further observations. Firstly, ERDL4 and TST genes exhibit an opposite regulation during a diurnal rhythm, and secondly, the ERDL4 gene is markedly expressed during cold acclimation, representing a situation in which TST activity needs to be upregulated. Moreover, ERDL4-overexpressing plants show larger rosettes and roots, a delayed flowering time, and increased total seed yield. Consistently, erdl4 knockout plants show impaired cold acclimation and freezing tolerance along with reduced plant biomass. In summary, we show that modification of cytosolic fructose levels influences plant organ development and stress tolerance.

Summary: Ascorbate (Asc) is a major redox buffer of plant cells, whose antioxidant activity depends on the ratio with its one‐electron oxidation product monodehydroascorbate (MDHA). The cytoplasm contains millimolar concentrations of Asc and soluble enzymes that can regenerate Asc from MDHA or fully oxidized dehydroascorbate. Also, vacuoles contain Asc, but no soluble Asc‐regenerating enzymes. Here, we show that vacuoles isolated from Arabidopsis mesophyll cells contain a tonoplast electron transport system that works as a reversible, Asc‐dependent transmembrane MDHA oxidoreductase. Electron currents were measured by patch‐clamp on isolated vacuoles and found to depend on the availability of Asc (electron donor) and ferricyanide or MDHA (electron acceptors) on opposite sides of the tonoplast. Electron currents were catalyzed by cytochrome b561 isoform A (CYB561A), a tonoplast redox protein with cytoplasmic and luminal Asc binding sites. The K_m for Asc of the luminal (4.5 mM) and cytoplasmic site (51 mM) reflected the physiological Asc concentrations in these compartments. The maximal current amplitude was similar in both directions. Mutant plants with impaired CYB561A expression showed no detectable trans‐tonoplast electron currents and strong accumulation of leaf anthocyanins under excessive illumination, suggesting a redox‐modulation exerted by CYB561A on the typical anthocyanin response to high‐light stress.

Regulation of intracellular sugar homeostasis is maintained by regulation of activities of sugar import and export proteins residing at the tonoplast. We show here that the EARLY RESPONSE TO DEHYDRATION6-LIKE4 protein, being the closest homolog to the proton/glucose symporter ERDL6, resides in the vacuolar membrane. Gene expression and subcellular fractionation studies indicated that ERDL4 was involved in fructose allocation across the tonoplast. Overexpression of ERDL4 increased total sugar levels in leaves, due to a concomitantly induced stimulation of TST2 expression, coding for the major vacuolar sugar loader. This conclusion is supported by the finding that tst1-2 knockout lines overexpressing ERDL4 lack increased cellular sugar levels. ERDL4 activity contributing to the coordination of cellular sugar homeostasis is also indicated by two further observations. Firstly, ERDL4 and TST genes exhibit an opposite regulation during a diurnal rhythm, and secondly, the ERDL4 gene is markedly expressed during cold acclimation representing a situation in which TST activity needs to be upregulated. Moreover, ERDL4 -overexpressing plants show larger size of rosettes and roots, a delayed flowering time and increased total seed yield. Consistently, erdl4 knock-out plants show impaired cold acclimation and freezing tolerance along with reduced plant biomass. In summary, we show that modification of cytosolic fructose levels influences plant organ development and stress tolerance. One sentence summary: The activity of the vacuolar sugar porter ERDL4 is important for balanced cytosolic monosaccharide homeostasis and influences plant growth and cold response in Arabidopsis The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors is: Benjamin Pommerrenig ( pommerre@bio.uni-kl.de ).