tracking-single-proteins-lipid-bilayers-using-fluorescence

Tracking Single Proteins in Lipid Bilayers Using Fluorescence Microscopy

Direct observation is a fundamental method for scientific discovery and understanding. Time-lapse single-molecule fluorescence imaging allows the ...

University of Bari Aldo Moro

Astrocyte endfeet are endowed with aquaporin-4 (AQP4)-based assemblies called orthogonal arrays of particles (OAPs) whose function is still unclear. To investigate the function of OAPs and of AQP4 tetramers, we have generated a novel "OAP-null" mouse model selectively lacking the OAP forming M23-AQP4 isoform. We demonstrated that AQP4 transcript levels were not reduced by using qPCR. Blue native (BN)/SDS-PAGE and Western blot performed on OAP-null brain and primary astrocyte cultures showed the complete depletion of AQP4 assemblies, the selective expression of M1-AQP4-based tetramers, and a substantial reduction in AQP4 total expression level. Fluorescence quenching and super-resolution microscopy experiments showed that AQP4 tetramers were functionally expressed in astrocyte plasma membrane and their dimensions were reduced compared to wild-type assemblies. Finally, as shown by light and electron microscopy, OAP depletion resulted in a massive reduction in AQP4 expression and a loss of perivascular AQP4 staining at astrocyte endfeet, with only sparse labeling throughout the brain areas analyzed. Our study relies on the unique property of AQP4 to form OAPs, using a novel OAP-null mouse model for the first time, to show that (a) AQP4 assembly is essential for normal AQP4 expression level in the brain and (b) most of AQP4 is organized into OAPs under physiological conditions. Therefore, AQP4 tetramers cannot be used by astrocytes as an alternative to OAPs without affecting AQP4 expression levels, which is important in the physiological and pathological conditions in which OAP aggregation/disaggregation dynamics have been implicated.

Orthogonal arrays of particle assembly are essential for normal aquaporin-4 expression level in the brain.

The ability of astrocytes to control extracellular volume homeostasis is critical for brain function and pathology. Uncovering the mechanisms of cell volume regulation by astrocytes will be important for identifying novel therapeutic targets for neurological conditions, such as those characterized by imbalances to hydro saline challenges (as in edema) or by altered cell volume regulation (as in glioma). One major challenge in studying the astroglial membrane channels involved in volume homeostasis in cell culture model systems is that the expression patterns of these membrane channels do not resemble those observed in vivo. In our previous study, we demonstrated that rat primary astrocytes grown on nanostructured interfaces based on hydrotalcite-like compounds (HTlc) in vitro are differentiated and display molecular and functional properties of in vivo astrocytes, such as the functional expression of inwardly rectifying K channel (Kir 4.1) and Aquaporin-4 (AQP4) at the astrocytic microdomain. Here, we take advantage of the properties of differentiated primary astrocytes in vitro to provide an insight into the mechanism underpinning astrocytic cell volume regulation and its correlation with the expression and function of AQP4, Transient Receptor Potential Vanilloid 4 (TRPV4), and Volume Regulated Anion Channel (VRAC).

Cell Volume Regulation Mechanisms in Differentiated Astrocytes.

Astrocytes are key regulators of brain homeostasis, equilibrating ion, water, and neurotransmitter concentrations and maintaining essential conditions for proper cognitive function. Recently, it has been shown that the excitability of the actin cytoskeleton manifests in second-scale dynamic fluctuations and acts as a sensor of chemophysical environmental cues. However, it is not known whether the cytoskeleton is excitable in astrocytes and how the homeostatic function of astrocytes is linked to the dynamics of the cytoskeleton. Here it is shown that homeostatic regulation involves the excitable dynamics of actin in certain subcellular regions of astrocytes, especially near the cell boundary. The results further indicate that actin dynamics concentrate into "hotspot" regions that selectively respond to certain chemophysical stimuli, specifically the homeostatic challenges of ion or water concentration increases. Substrate topography makes the actin dynamics of astrocytes weaker. Super-resolution images demonstrate that surface topography is also associated with the predominant perpendicular alignment of actin filaments near the cell boundary, whereas flat substrates result in an actin cortex mainly parallel to the cell boundary. Additionally, coculture with neurons increases both the probability of actin dynamics and the strength of hotspots. The excitable systems character of actin thus makes astrocytes direct participants in neural cell network dynamics.

Decoding Natural Astrocyte Rhythms: Dynamic Actin Waves Result from Environmental Sensing by Primary Rodent Astrocytes.

ST2 is a member of interleukin 1 receptor family with soluble sST2 and transmembrane ST2L isoforms. The ligand of ST2 is IL-33, which determines the activation of numerous intracytoplasmic mediators following the binding with ST2L and IL-1RAcP, leading to nuclear signal and cardiovascular effect. Differently, sST2 is released in the blood and works as a decoy receptor, binding IL-33 and blocking IL-33/ST2L interaction. sST2 is mainly involved in maintaining homeostasis and/or alterations of different tissues, as counterbalance/activation of IL-33/ST2L axis is typically involved in the development of fibrosis, tissue damage, inflammation and remodeling. sST2 has been described in different clinical reports as a fundamental prognostic marker in patients with cardiovascular disease, as well as marker for the treatment monitoring of patients with heart failure; however, further studies are needed to better elucidate its role. In this review we reported the current knowledge about its role in coronary artery disease, heart failure, heart transplantation, heart valve disease, pulmonary arterial hypertension, and cardiovascular interventions.

The ST2/IL-33 Pathway in Adult and Paediatric Heart Disease and Transplantation.

Despite of the major role of aquaporin (AQP) water channels in controlling transmembrane water fluxes, alternative ways for modulating water permeation have been proposed. In the Central Nervous System (CNS), Aquaporin-4 (AQP4) is reported to be functionally coupled with the calcium-channel Transient-Receptor Potential Vanilloid member-4 (TRPV4), which is controversially involved in cell volume regulation mechanisms and water transport dynamics. The present work aims to investigate the selective role of TRPV4 in regulating plasma membrane water permeability in an AQP4-independent way. Fluorescence-quenching water transport experiments in  astrocytes revealed that cell swelling rate is significantly increased upon TRPV4 activation and in the absence of AQP4. The biophysical properties of TRPV4-dependent water transport were therefore assessed using the HEK-293 cell model. Calcein quenching experiments showed that chemical and thermal activation of TRPV4 overexpressed in HEK-293 cells leads to faster swelling kinetics. Stopped-flow light scattering water transport assay was used to measure the osmotic permeability coefficient (, cm/s) and activation energy (, kcal/mol) conferred by TRPV4. Results provided evidence that although the  measured upon TRPV4 activation is lower than the one obtained in AQP4-overexpressing cells ( of AQP4 = 0.01667 ± 0.0007;  of TRPV4 = 0.002261 ± 0.0004;  of TRPV4 + 4αPDD = 0.007985 ± 0.0006;  of WT = 0.002249 ± 0.0002), along with activation energy values ( of AQP4 = 0.86 ± 0.0006;  of TRPV4 + 4αPDD = 2.73 ± 1.9;  of WT = 8.532 ± 0.4), these parameters were compatible with a facilitated pathway for water movement rather than simple diffusion. The possibility to tune plasma membrane water permeability more finely through TRPV4 might represent a protective mechanism in cells constantly facing severe osmotic challenges to avoid the potential deleterious effects of the rapid cell swelling occurring via AQP channels.

AQP4-independent TRPV4 modulation of plasma membrane water permeability.

Left ventricular assist devices (LVADs) represent the final treatment for patients with end-stage heart failure (HF) not eligible for transplantation. Although LVAD design has been further improved in the last decade, their use is associated with different complications. Specifically, inflammation, fibrosis, bleeding events, right ventricular failure, and aortic valve regurgitation may occur. In addition, reverse remodeling is associated with substantial cellular and molecular changes of the failing myocardium during LVAD support with positive effects on patients' health. All these processes also lead to the identification of biomarkers identifying LVAD patients as having an augmented risk of developing associated adverse events, thus highlighting the possibility of identifying new therapeutic targets. Additionally, it has been reported that LVAD complications could cause or exacerbate a state of malnutrition, suggesting that, with an adjustment in nutrition, the general health of these patients could be improved.

Cellular and Molecular Mechanisms Activated by a Left Ventricular Assist Device.

Aquaporin-4 (AQP4) is a water channel protein found primarily in the central nervous system (CNS) that helps to regulate water-ion homeostasis. AQP4 exists in two major isoforms: M1 and M23. While both isoforms have a homotetrameric quaternary structure and are functionally identical when transporting water, the M23 isoform forms large protein aggregates known as orthogonal arrays of particles (OAPs). In contrast, the M1 isoform creates a peripheral layer around the outside of these OAPs, suggesting a thermodynamically stable interaction between the two. Structurally, the M1 isoform has an N-terminal tail that is 22 amino acids longer than the M23 isoform and contains two solvent-accessible cysteines available for -palmitoylation at cysteine-13 (Cys-13) and cysteine-17 (Cys-17) in the amino acid sequence. Earlier work suggests that the palmitoylation of these cysteines might aid in regulating AQP4 assemblies. This work discusses the thermodynamic driving forces for M1 protein-protein interactions and how the palmitoylation state of M1 affects them. Using temperature-dependent single-particle tracking, the standard state free energies, enthalpies, and entropies were measured for these interactions. Furthermore, we present a binding model based on measured thermodynamics and a structural modeling study. The results of this study demonstrate that the M1 isoform will associate with itself according to the following expressions: 2[AQP4-M1] ↔ [[AQP4-M1]] when palmitoylated and 3[AQP4-M1] ↔ [AQP4-M1] + [[AQP4-M1]] ↔ [[AQP4-M1]] when depalmitoylated. This is primarily due to a conformational change induced by adding the palmitic acid groups at Cys-13 and Cys-17 in the N-terminal tails of the homotetramers. In addition, a statistical mechanical model was developed to estimate the Gibbs free energy, enthalpy, and entropy for forming dimers and trimers. These results were in good agreement with experimental values.

Thermodynamics and -Palmitoylation Dependence of Interactions between Human Aquaporin-4 M1 Tetramers in Model Membranes.

In the adult brain, the water channel aquaporin-4 (AQP4) is expressed in astrocyte endfoot, in supramolecular assemblies, called "Orthogonal Arrays of Particles" (OAPs) together with the transient receptor potential vanilloid 4 (TRPV4), finely regulating the cell volume. The present study aimed at investigating the contribution of AQP4 and TRPV4 to CNS early postnatal development using WT and AQP4 KO brain and retina and neuronal stem cells (NSCs), as an in vitro model of astrocyte differentiation. Western blot analysis showed that, differently from AQP4 and the glial cell markers, TRPV4 was downregulated during CNS development and NSC differentiation. Blue native/SDS-PAGE revealed that AQP4 progressively organized into OAPs throughout the entire differentiation process. Fluorescence quenching assay indicated that the speed of cell volume changes was time-related to NSC differentiation and functional to their migratory ability. Calcium imaging showed that the amplitude of TRPV4 Ca transient is lower, and the dynamics are changed during differentiation and suppressed in AQP4 KO NSCs. Overall, these findings suggest that early postnatal neurodevelopment is subjected to temporally modulated water and Ca dynamics likely to be those sustaining the biochemical and physiological mechanisms responsible for astrocyte differentiation during brain and retinal development.

Aquaporin-4 and transient receptor potential vanilloid 4 balance in early postnatal neurodevelopment.

Bone health is the result of a tightly regulated balance between bone modeling and bone remodeling, and alterations of these processes have been observed in several diseases both in adult and pediatric populations. The imbalance in bone remodeling can ultimately lead to osteoporosis, which is most often associated with aging, but contributing factors can already act during the developmental age, when over a third of bone mass is accumulated. The maintenance of an adequate bone mass is influenced by genetic and environmental factors, such as physical activity and diet, and particularly by an adequate intake of calcium and vitamin D. In addition, it has been claimed that the integration of specific nutraceuticals such as resveratrol, anthocyanins, isoflavones, lycopene, curcumin, lutein, and β-carotene and the intake of bioactive compounds from the diet such as honey, tea, dried plums, blueberry, and olive oil can be efficient strategies for bone loss prevention. Nutraceuticals and functional foods are largely used to provide medical or health benefits, but there is an urge to determine which products have adequate clinical evidence and a strong safety profile. The aim of this review is to explore the scientific and clinical evidence of the positive role of nutraceuticals and functional food in bone health, focusing both on molecular mechanisms and on real-world studies.

Nutraceuticals and Functional Foods: A Comprehensive Review of Their Role in Bone Health.

Diabetes is a heterogeneous metabolic disease characterized by elevated blood glucose levels resulting from the destruction or malfunction of pancreatic β cells, insulin resistance in peripheral tissues, or both, and results in a non-sufficient production of insulin. To adjust blood glucose levels, diabetic patients need exogenous insulin administration together with medical nutrition therapy and physical activity. With the aim of improving insulin availability in diabetic patients as well as ameliorating diabetes comorbidities, different strategies have been investigated. The first approaches included enhancing endogenous β cell activity or transplanting new islets. The protocol for this kind of intervention has recently been optimized, leading to standardized procedures. It is indicated for diabetic patients with severe hypoglycemia, complicated by impaired hypoglycemia awareness or exacerbated glycemic lability. Transplantation has been associated with improvement in all comorbidities associated with diabetes, quality of life, and survival. However, different trials are ongoing to further improve the beneficial effects of transplantation. Furthermore, to overcome some limitations associated with the availability of islets/pancreas, alternative therapeutic strategies are under evaluation, such as the use of mesenchymal stem cells (MSCs) or induced pluripotent stem cells for transplantation. The cotransplantation of MSCs with islets has been successful, thus providing protection against proinflammatory cytokines and hypoxia through different mechanisms, including exosome release. The use of induced pluripotent stem cells is recent and requires further investigation. The advantages of MSC implantation have also included the improvement of diabetes-related comorbidities, such as wound healing. Despite the number of advantages of the direct injection of MSCs, new strategies involving biomaterials and scaffolds have been developed to improve the efficacy of mesenchymal cell delivery with promising results. In conclusion, this paper offered an overview of new alternative strategies for diabetes management while highlighting some limitations that will need to be overcome by future approaches.

Alternative therapeutic strategies in diabetes management.

Diabetic cardiomyopathy (DCM) represents one of the typical complications associated with diabetes. It has been described as anomalies in heart function and structure, with consequent high morbidity and mortality. DCM development can be described by two stages; the first is characterized by left ventricular hypertrophy and diastolic dysfunction, and the second by heart failure (HF) with systolic dysfunction. The proposed mechanisms involve cardiac inflammation, advanced glycation end products (AGEs) and angiotensin II. Furthermore, different studies have focused their attention on cardiomyocyte death through the different mechanisms of programmed cell death, such as apoptosis, autophagy, necrosis, pyroptosis and ferroptosis. Exosome release, adipose epicardial tissue and aquaporins affect DCM development. This review will focus on the description of the mechanisms involved in DCM progression and development.

Diabetic Cardiomyopathy: Role of Cell Death, Exosomes, Fibrosis and Epicardial Adipose Tissue.

Aquaporin-4 (AQP4) is a water-selective channel expressed in glial cells throughout the central nervous system (CNS). It serves as the primary water channel in the neuropil and plays roles in physiological functions, including regulating water homeostasis by adjusting cell volume and modulating neuronal activity. Different isoforms of AQP4 are expressed in glial-like cells known as sustentacular cells (SUSs) of the olfactory epithelium (OE). Notably, mice lacking all AQP4 isoforms exhibit impaired olfactory abilities. Therefore, we aim to uncover the physiological role of AQP4 isoforms, particularly the AQP4ex isoforms (AQP4M1ex, AQP4M23ex) and the orthogonal array of particles (OAPs)-forming isoform (AQP4M23) in the OE. We investigated the impact of AQP4 isoforms on the OE, observing a reduced number of mature olfactory sensory neurons (OSNs), SUSs, and globose basal cells (GBCs) in mice lacking AQP4ex (AQP4ex-KO) or OAPs (OAP-null). This suggests that AQP4 isoforms are involved in maintaining an optimal microenvironment in the OE, preserving cell density. Next, we explored the role of AQP4 in modulating odorant-evoked responses through electro-olfactogram recordings, where we found reduced odorant responses in mice lacking AQP4 isoforms. Assessments of olfactory ability revealed deficits in odor-guided food-seeking behavior in both AQP4ex-KO and OAP-null mice. Furthermore, AQP4ex-KO mice exhibited a diminished ability to discriminate between different odorants, while OAP-null mice were unable to recognize them as distinct. Overall, our data highlight the role of AQP4 isoforms in modulating neuronal homeostasis, influencing odorant-evoked responses and cell density in the OE, with AQP4ex emerging as a key regulator despite its low abundance.

The Aquaporin-4 Expression and Localization in the Olfactory Epithelium Modulate the Odorant-Evoked Responses and Olfactory-Driven Behavior.

Human aquaporin-4 (AQP4) is a water-channel protein crucial for water-ion homeostasis in the central nervous system. Dysregulation of AQP4 function is linked to neurological conditions like brain edema, neuromyelitis optica, and epilepsy. AQP4 has two isoforms, M1 and M23. The M23 isoform forms large structures known as Orthogonal Arrays of Particles (OAPs), while M1 coats the outer surface of OAPs. The dynamics of OAP aggregation may modulate water permeability and ion homeostasis in the brain. This study shows that the palmitoylation state of M1 influences OAP self-assembly, with depalmitoylated M1 producing OAPs 20 % larger than those formed by palmitoylated M1 at the same concentration. This, along with prior research on M1 aggregation, suggests palmitoylated M1 forms a single outer layer arounf OPAs, whereas depalmitoylated M1 creates a double layer. Single-particle tracking was used to study M23 OAP formation in lipid bilayers under equilibrium conditions. Our data support the idea that M1 regulates M23 OAP size, showing that average OAP size decreases as M1 concentration increases. This study explores the inhibitory effects of M1 on AQP4 M23 assembly and how M1's palmitoylation state affects OAP size regulation. These insights could lead to new therapeutic approaches for managing AQP4 assembly and function in related conditions.

Article	Year
Orthogonal arrays of particle assembly are essential for normal aquaporin-4 expression level in the brain. Glia\| PubMed ID: 32946135	2021
Cell Volume Regulation Mechanisms in Differentiated Astrocytes. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology\| PubMed ID: 34740285	2021
Decoding Natural Astrocyte Rhythms: Dynamic Actin Waves Result from Environmental Sensing by Primary Rodent Astrocytes. Advanced biology\| PubMed ID: 36709481	2023
The ST2/IL-33 Pathway in Adult and Paediatric Heart Disease and Transplantation. Biomedicines\| PubMed ID: 37371771	2023
AQP4-independent TRPV4 modulation of plasma membrane water permeability. Frontiers in cellular neuroscience\| PubMed ID: 37720545	2023
Cellular and Molecular Mechanisms Activated by a Left Ventricular Assist Device. International journal of molecular sciences\| PubMed ID: 38203459	2023
Thermodynamics and -Palmitoylation Dependence of Interactions between Human Aquaporin-4 M1 Tetramers in Model Membranes. The journal of physical chemistry. B\| PubMed ID: 38212942	2024
Aquaporin-4 and transient receptor potential vanilloid 4 balance in early postnatal neurodevelopment. Glia\| PubMed ID: 38362923	2024
Nutraceuticals and Functional Foods: A Comprehensive Review of Their Role in Bone Health. International journal of molecular sciences\| PubMed ID: 38892062	2024
Alternative therapeutic strategies in diabetes management. World journal of diabetes\| PubMed ID: 38983831	2024
Diabetic Cardiomyopathy: Role of Cell Death, Exosomes, Fibrosis and Epicardial Adipose Tissue. International journal of molecular sciences\| PubMed ID: 39273428	2024
The Aquaporin-4 Expression and Localization in the Olfactory Epithelium Modulate the Odorant-Evoked Responses and Olfactory-Driven Behavior. Glia\| PubMed ID: 40251831	2025
Size of aquaporin-4 orthogonal arrays of particles are affected by the palmitoylation state of the M1 isoform. Biochimica et biophysica acta. General subjects\| PubMed ID: 41077234	2025

Barbara Barile

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