An ultra-sensitive platinized nanocavity electrode for analysis of cytosolic catecholamines in one living cell.

The catecholamines, mainly dopamine (DA), are present in the cellular cytosol with low abundance, while, play key roles in various neurodegenerative disorders. Here, platinized nanocavity carbon electrodes are employed to analyze cytosolic catecholamines in a single living PC12 cell, which is not easily quantified using the classic electrodes. The confined structure and excellent conductivity in the platinized nanocavity accelerate the electron transfer of the DA, resulting in a low detection limit down to 50 nM. The sensitivity of DA detection is improved to be 10.73 pA mM-1 nm-1 in the response range of 50 nM-100 µM, which guarantees quantitative analysis of cytosolic catecholamines with low abundance. Eventually, the platinized nanocavity electrode is employed to detect cytosolic catecholamines in a single PC12 cell without an obvious interruption of cellular catecholamine level. The cytosolic catecholamines in a single PC12 cell is measured in situ to be 0.1 µM, which is achieved for the first time at the single cell level using the electrochemical method. The results demonstrate that the nanocavity electrode with a high sensitivity could offer a promising means to dynamically track catecholamines in a single cell.

Macromolecular condensation buffers intracellular water potential.

Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions1. Reciprocally, macromolecules restrict the movement of 'structured' water molecules within their hydration layers, reducing the available 'free' bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales2,3; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function.

Postsynaptic protein assembly in three and two dimensions studied by mesoscopic simulations.

Recently, cellular biomolecular condensates formed via phase separation have received considerable attention. While they can be formed either in cytosol (denoted as 3D) or beneath the membrane (2D), the underlying difference between the two has not been well clarified. To compare the phase behaviors in 3D and 2D, postsynaptic density (PSD) serves as a model system. PSD is a protein condensate located under the postsynaptic membrane that influences the localization of glutamate receptors and thus contributes to synaptic plasticity. Recent in vitro studies have revealed the formation of droplets of various soluble PSD proteins via liquid-liquid phase separation. However, it is unclear how these protein condensates are formed beneath the membrane and how they specifically affect the localization of glutamate receptors in the membrane. In this study, focusing on the mixture of a glutamate receptor complex, AMPAR-TARP, and a ubiquitous scaffolding protein, PSD-95, we constructed a mesoscopic model of protein-domain interactions in PSD and performed comparative molecular simulations. The results showed a sharp contrast in the phase behaviors of protein assemblies in 3D and those under the membrane (2D). A mixture of a soluble variant of the AMPAR-TARP complex and PSD-95 in the 3D system resulted in a phase-separated condensate, which was consistent with the experimental results. However, with identical domain interactions, AMPAR-TARP embedded in the membrane formed clusters with PSD-95, but did not form a stable separated phase. Thus, the cluster formation behaviors of PSD proteins in the 3D and 2D systems were distinct. The current study suggests that, more generally, stable phase separation can be more difficult to achieve in and beneath the membrane than in 3D systems.

Influence of Chemical and Genetic Manipulations on Cellular Organelles Quantified by Label-Free Optical Diffraction Tomography.

Label-free optical diffraction tomography provides three-dimensional imaging of cells and organelles, along with their refractive index (RI) and volume. These physical parameters are valuable for quantitative and accurate analysis of the subcellular microenvironment and its connections to intracellular biological properties. In biological and biochemical cell analysis, various invasive cell manipulations are used, such as temperature change, chemical fixation, live cell staining with fluorescent dye, and gene overexpression of exogenous proteins. However, it is not fully understood how these various manipulations affect the physicochemical properties of different organelles. In this study, we investigated the impact of these manipulations on the cellular properties of single HeLa cells. We found that after cell fixation and an increase in temperature, the RI value of organelles, such as the nucleus and cytoplasm, significantly decreased overall. Interestingly, unlike the cell nuclei, cytoplasmic RI values were hardly detected after membrane permeation, indicating that only intracytoplasmic components were largely lost. Additionally, our findings revealed that the expression of GFP and GFP-tagged proteins significantly increased the RI values of organelles in living cells compared to the less effective RI changes observed with chemical fluorescence staining for cell organelles. The result demonstrates that distinct types of invasive manipulations can alter the microenvironment of organelles in different ways. Our study sheds new light on how chemical and genetic manipulations affect organelles.

Pollution impact on metal and biomarker responses in intestinal cytosol of freshwater fish.

In the present study, essential and nonessential metal content and biomarker responses were investigated in the intestine of fish collected from the areas polluted by mining. Our objective was to determine metal and biomarker levels in tissue responsible for dietary intake, which is rarely studied in water pollution research. The study was conducted in the Bregalnica River, reference location, and in the Zletovska and Kriva Rivers (the Republic of North Macedonia), which are directly influenced by the active mines Zletovo and Toranica, respectively. Biological responses were analyzed in Vardar chub (Squalius vardarensis; Karaman, 1928), using for the first time intestinal cytosol as a potentially toxic cell fraction, since metal sensitivity is mostly associated with cytosol. Cytosolic metal levels were higher in fish under the influence of mining (Tl, Li, Cs, Mo, Sr, Cd, Rb, and Cu in the Zletovska River and Cr, Pb, and Se in the Kriva River compared to the Bregalnica River in both seasons). The same trend was evident for total proteins, biomarkers of general stress, and metallothioneins, biomarkers of metal exposure, indicating cellular disturbances in the intestine, the primary site of dietary metal uptake. The association of cytosolic Cu and Cd at all locations pointed to similar pathways and homeostasis of these metallothionein-binding metals. Comparison with other indicator tissues showed that metal concentrations were higher in the intestine of fish from mining-affected areas than in the liver and gills. In general, these results indicated the importance of dietary metal pathways, and cytosolic metal fraction in assessing pollution impacts in freshwater ecosystems.

Population-based heteropolymer design to mimic protein mixtures.

Biological fluids, the most complex blends, have compositions that constantly vary and cannot be molecularly defined1. Despite these uncertainties, proteins fluctuate, fold, function and evolve as programmed2-4. We propose that in addition to the known monomeric sequence requirements, protein sequences encode multi-pair interactions at the segmental level to navigate random encounters5,6; synthetic heteropolymers capable of emulating such interactions can replicate how proteins behave in biological fluids individually and collectively. Here, we extracted the chemical characteristics and sequential arrangement along a protein chain at the segmental level from natural protein libraries and used the information to design heteropolymer ensembles as mixtures of disordered, partially folded and folded proteins. For each heteropolymer ensemble, the level of segmental similarity to that of natural proteins determines its ability to replicate many functions of biological fluids including assisting protein folding during translation, preserving the viability of fetal bovine serum without refrigeration, enhancing the thermal stability of proteins and behaving like synthetic cytosol under biologically relevant conditions. Molecular studies further translated protein sequence information at the segmental level into intermolecular interactions with a defined range, degree of diversity and temporal and spatial availability. This framework provides valuable guiding principles to synthetically realize protein properties, engineer bio/abiotic hybrid materials and, ultimately, realize matter-to-life transformations.

Visualization of Cytosolic Galectin Accumulation Around Damaged Vesicles and Organelles.

Galectins are animal lectins that recognize ß-galactoside and bind glycans. Recent studies have indicated that cytosolic galectins recognize cytosolically exposed glycans and accumulate around endocytic vesicles or organelles damaged by various disruptive substances. Accumulated galectins engage other cytosolic proteins toward damaged vesicles, leading to cellular responses, such as autophagy. Disruptive substances include bacteria, viruses, particulate matters, and protein aggregates; thus, this process is implicated in the pathogenesis of various diseases. In this chapter, we describe methods for studying three disruptive substances: photosensitizers, Listeria monocytogenes, and Helicobacter pylori. We summarize the tools used for the detection of cytosolic galectin accumulation around damaged vesicles.

The yin and yang of intracellular delivery of amphipathic optical probes using n-butyl charge masking.

Monitoring and manipulation of ionized intracellular calcium concentrations within intact, living cells using optical probes with organic chromophores is a core method for cell physiology. Since all these probes have multiple negative charges, they must be smuggled through the plasma membrane in a transiently neutral form, with intracellular esterases used to deprotect the masked anions. Here we explore the ability of the synthetically easily accessible n-butyl ester protecting group to deliver amphipathic cargoes to the cytosol. We show that the size of the caging chromophore conditions the ability of intracellular probe delivery and esterase charge unmasking.

Assessment of the intracellular distribution of copper in liver specimens from cats.

The intracellular distribution of copper in the liver has been investigated in dogs and humans. However, this has not been reported in cats. This study aimed to assess the intracellular copper distribution in liver specimens from cats with a range of hepatic copper concentrations. Twenty-nine frozen liver specimens from cats were included. Each liver specimen was divided into two pieces for overall copper quantification and tissue fractionation. The copper concentrations in liver specimens and liver fractions were measured by flame atomic absorption spectroscopy. Five specimens had copper concentrations < 100 µg/g dry weight, eight had copper concentrations between 100 and 180 µg/g, 14 had copper concentrations between 181 and 700 µg/g, and two had copper concentrations >700 µg/g. Only one specimen had positive copper staining. Regardless of the overall concentrations, copper was mostly found in the cytosolic fraction followed by the nuclear, large granule, and microsomal fractions. Our findings indicate that similarly to other species, intracellular copper is predominantly found in the cytosolic and nuclear fractions in cats. The distribution in cats with copper-loaded conditions, such as primary copper hepatopathy, was not assessed but warrants evaluation.

Direct Cellular Delivery of Exogenous Genetic Material and Protein via Colloidal Nano-Assemblies with Biopolymer.

Direct cytosolic delivery of large biomolecules that bypass the endocytic pathways is a promising strategy for therapeutic applications. Recent works have shown that small-molecule, nanoparticle, and polymer-based carriers can be designed for direct cytosolic delivery. It has been shown that the specific surface chemistry of the carrier, nanoscale assembly between the carrier and cargo molecule, good colloidal stability, and low surface charge of the nano-assembly are critical for non-endocytic uptake processes. Here we report a guanidinium-terminated polyaspartic acid micelle for direct cytosolic delivery of protein and DNA. The polymer delivers the protein/DNA directly to the cytosol by forming a nano-assembly, and it is observed that <200 nm size of colloidal assembly with near-zero surface charge is critical for efficient cytosolic delivery. This work shows the importance of size and colloidal property of the nano-assembly for carrier-based cytosolic delivery of large biomolecules.

The discovery of potent small molecule cyclic urea activators of STING.

STING mediates innate immune responses that are triggered by the presence of cytosolic DNA. Activation of STING to boost antigen recognition is a therapeutic modality that is currently being tested in cancer patients using nucleic-acid based macrocyclic STING ligands. We describe here the discovery of 3,4-dihydroquinazolin-2(1H)-one based 6,6-bicyclic heterocyclic agonists of human STING that activate all known human variants of STING with high potency.

Liquid-Liquid Phase Separation at the Plasma Membrane-Cytosol Interface: Common Players in Adhesion, Motility, and Synaptic Function.

Networks of scaffold proteins and enzymes assemble at the interface between the cytosol and specific sites of the plasma membrane, where these networks guide distinct cellular functions. Some of these plasma membrane-associated platforms (PMAPs) include shared core components that are able to establish specific protein-protein interactions, to produce distinct supramolecular assemblies regulating dynamic processes as diverse as cell adhesion and motility, or the formation and function of neuronal synapses. How cells organize such dynamic networks is still an open question. In this review we introduce molecular networks assembling at the edge of migrating cells, and at pre- and postsynaptic sites, which share molecular players that can drive the assembly of biomolecular condensates. Very recent experimental evidence has highlighted the emerging role of some of these multidomain/scaffold proteins belonging to the GIT, liprin-α and ELKS/ERC families as drivers of liquid-liquid phase separation (LLPS). The data point to an important role of LLPS: (i) in the formation of PMAPs at the edge of migrating cells, where LLPS appears to be involved in promoting protrusion and the turnover of integrin-mediated adhesions, to allow forward cell translocation; (ii) in the assembly of the presynaptic active zone and of the postsynaptic density deputed to the release and reception of neurotransmitter signals, respectively. The recent results indicate that LLPS at cytosol-membrane interfaces is suitable not only for the regulation of active cellular processes, but also for the continuous spatial rearrangements of the molecular interactions involved in these dynamic processes.

Excitatory neurotransmission activates compartmentalized calcium transients in Müller glia without affecting lateral process motility.

Neural activity has been implicated in the motility and outgrowth of glial cell processes throughout the central nervous system. Here, we explore this phenomenon in Müller glia, which are specialized radial astroglia that are the predominant glial type of the vertebrate retina. Müller glia extend fine filopodia-like processes into retinal synaptic layers, in similar fashion to brain astrocytes and radial glia that exhibit perisynaptic processes. Using two-photon volumetric imaging, we found that during the second postnatal week, Müller glial processes were highly dynamic, with rapid extensions and retractions that were mediated by cytoskeletal rearrangements. During this same stage of development, retinal waves led to increases in cytosolic calcium within Müller glial lateral processes and stalks. These regions comprised distinct calcium compartments, distinguished by variable participation in waves, timing, and sensitivity to an M1 muscarinic acetylcholine receptor antagonist. However, we found that motility of lateral processes was unaffected by the presence of pharmacological agents that enhanced or blocked wave-associated calcium transients. Finally, we found that mice lacking normal cholinergic waves in the first postnatal week also exhibited normal Müller glial process morphology. Hence, outgrowth of Müller glial lateral processes into synaptic layers is determined by factors that are independent of neuronal activity.

When it comes to studying the nervous system, neurons often steal the limelight; yet, they can only work properly thanks to an ensemble cast of cell types whose roles are only just emerging. For example, 'glial cells' ­ their name derives from the Greek word for glue ­ were once thought to play only a passive, supporting function in nervous tissues. Now, growing evidence shows that they are, in fact, integrated into neural circuits: their activity is influenced by neurons, and, in turn, they help neurons to function properly. The role of glial cells is becoming clear in the retina, the thin, light-sensitive layer that lines the back of the eye and relays visual information to the brain. There, beautifully intricate Müller glial cells display fine protrusions (or 'processes') that intermingle with synapses, the busy space between neurons where chemical messengers are exchanged. These messengers can act on Müller cells, triggering cascades of molecular events that may influence the structure and function of glia. This is of particular interest during development: as Müller cells mature, they are exposed to chemicals released by more fully formed retinal neurons. Tworig et al. explored how neuronal messengers can influence the way Müller cells grow their processes. To do so, they tracked mouse retinal glial cells 'live' during development, showing that they were growing fine, highly dynamic processes in a region rich in synapses just as neurons and glia increased their communication. However, using drugs to disrupt this messaging for a short period did not seem to impact how the processes grew. Extending the blockade over a longer timeframe also did not change the way Müller cells developed, with the cells still acquiring their characteristic elaborate process networks. Taken together, these results suggest that the structural maturation of Müller glial cells is not impacted by neuronal signaling, giving a more refined understanding of how glia form in the retina and potentially in the brain.

Size-Dependent Electroporation of Dye-Loaded Polymer Nanoparticles for Efficient and Safe Intracellular Delivery.

Efficient and safe delivery of nanoparticles (NPs) into the cytosol of living cells constitutes a major methodological challenge in bio-nanotechnology. Electroporation allows direct transfer of NPs into the cytosol by forming transient pores in the cell membrane, but it is criticized for invasiveness, and the applicable particle sizes are not well defined. Here, in order to establish principles for efficient delivery of NPs into the cytosol with minimal cytotoxicity, the influence of the size of NPs on their electroporation and intracellular behavior is investigated. For this study, fluorescent dye-loaded polymer NPs with core sizes between 10 and 40 nm are prepared. Optimizing the electroporation protocol allows minimizing contributions of endocytosis and to study directly the effect of NP size on electroporation. NPs of <20 nm hydrodynamic size are efficiently delivered into the cytosol, whereas this is not the case for NPs of >30 nm. Moreover, only particles of core size <15 nm diffuse freely throughout the cytosol. While electroporation at excessive electric fields induces cytotoxicity, the use of small NPs <20 nm allows efficient delivery at mild electroporation conditions. These results give clear methodological and design guidelines for the safe delivery of NPs for intracellular applications.

Conformational dynamics of free and membrane-bound human Hsp70 in model cytosolic and endo-lysosomal environments.

The binding of the major stress-inducible human 70-kDa heat shock protein (Hsp70) to the anionic phospholipid bis-(monoacylglycero)-phosphate (BMP) in the lysosomal membrane is crucial for its impact on cellular pathology in lysosomal storage disorders. However, the conformational features of this protein-lipid complex remain unclear. Here, we apply hydrogen-deuterium exchange mass spectrometry (HDX-MS) to describe the dynamics of the full-length Hsp70 in the cytosol and its conformational changes upon translocation into lysosomes. Using wild-type and W90F mutant proteins, we also map and discriminate the interaction of Hsp70 with BMP and other lipid components of the lysosomal membrane. We identify the N-terminal of the nucleotide binding domain (residues 87-118) as the primary orchestrator of BMP interaction. We show that the conformation of this domain is significantly reorganized in the W90F mutant, explaining its inability to stabilize lysosomal membranes. Overall, our results reveal important new molecular details of the protective effect of Hsp70 in lysosomal storage diseases, which, in turn, could guide future drug development.

Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade.

Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades' response to desiccation. In this study, to elucidate these protein functions, we performed in vitro and in vivo characterizations of the reversible self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. We found that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased. We also demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol-gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into molecular strategies of organisms to adapt to extreme environments.

Cytotoxicity of snake venom Lys49 PLA2-like myotoxin on rat cardiomyocytes ex vivo does not involve a direct action on the contractile apparatus.

Viperid snake venoms contain a unique family of cytotoxic proteins, the Lys49 PLA2 homologs, which are devoid of enzymatic activity but disrupt the integrity of cell membranes. They are known to induce skeletal muscle damage and are therefore named 'myotoxins'. Single intact and skinned (devoid of membranes and cytoplasm but with intact sarcomeric proteins) rat cardiomyocytes were used to analyze the cytotoxic action of a myotoxin, from the venom of Bothrops asper. The toxin induced rapid hypercontraction of intact cardiomyocytes, associated with an increase in the cytosolic concentration of calcium and with cell membrane disruption. Hypercontraction of intact cardiomyocytes was abrogated by the myosin inhibitor para-aminoblebbistatin (AmBleb). No toxin-induced changes of key parameters of force development were observed in skinned cardiomyocytes. Thus, although myosin is a key effector of the observed hypercontraction, a direct effect of the toxin on the sarcomeric proteins -including the actomyosin complex- is not part of the mechanism of cytotoxicity. Owing to the sensitivity of intact cardiomyocytes to the cytotoxic action of myotoxin, this ex vivo model is a valuable tool to explore in further detail the mechanism of action of this group of snake venom toxins.

Delivery of Ultrasmall Nanoparticles to the Cytosolic Compartment of Pyroptotic J774A.1 Macrophages via GSDMDNterm Membrane Pores.

Endosome capture is a major physiological barrier to the successful delivery of nanomedicine. Here, we found a strategy to deliver ultrasmall nanoparticles (<10 nm) to the cytosolic compartment of pyroptotic cells with spontaneous endosomal escape. To mimic pathological pyroptotic cells, J774A.1 macrophages were stimulated with lipopolysaccharide (LPS) plus nigericin (Nig) or adenosine triphosphate (ATP) to form specific gasdermin D protein-driven membrane pores at an N-terminal domain (GSDMDNterm). Through GSDMDNterm membrane pores, both anionic and cationic nanoparticles (NPs) with diameters less than 10 nm were accessed into the cytosolic compartment of pyroptotic cells in an energy- and receptor-independent manner, while NPs larger than the size of GSDMDNterm membrane pores failed to enter pyroptotic cells. NPs pass through GSDMDNterm membrane pores via free diffusion and then access into the cytoplasm of pyroptotic cells in a microtubule-independent manner. Interestingly, we found that LPS-primed NPs may act as Trojan horse, deliver extracellular LPS into normal cells through endocytosis, and in turn induce GSDMDNterm membrane pores, which facilitate further internalization of NPs. This study presented a straightforward method of distinguishing normal and pyroptotic cells through GSDMD membrane pores, implicating their potential application in monitoring the delivery of desired nanomedicines in pyroptosis-related diseases and conditions.

Loss of major nutrient sensing and signaling pathways suppresses starvation lethality in electron transport chain mutants.

The electron transport chain (ETC) is a well-studied and highly conserved metabolic pathway that produces ATP through generation of a proton gradient across the inner mitochondrial membrane coupled to oxidative phosphorylation. ETC mutations are associated with a wide array of human disease conditions and to aging-related phenotypes in a number of different organisms. In this study, we sought to better understand the role of the ETC in aging using a yeast model. A panel of ETC mutant strains that fail to survive starvation was used to isolate suppressor mutants that survive. These suppressors tend to fall into major nutrient sensing and signaling pathways, suggesting that the ETC is involved in proper starvation signaling to these pathways in yeast. These suppressors also partially restore ETC-associated gene expression and pH homeostasis defects, though it remains unclear whether these phenotypes directly cause the suppression or are simply effects. This work further highlights the complex cellular network connections between metabolic pathways and signaling events in the cell and their potential roles in aging and age-related diseases.

Computational Study on the Function of Palmitoylation on the Envelope Protein in SARS-CoV-2.

SARS-CoV-2 that caused COVID-19 has spread since the end of 2019. Its major effects resulted in over four million deaths around the whole world by August 2021. Therefore, understanding virulence mechanisms is important to prevent future outbreaks and for COVID-19 drug development. The envelope (E) protein is an important structural protein, affecting virus assembly and budding. The E protein pentamer is a viroporin, serving as an ion transferring channel in cells. In this work, we applied molecular dynamic simulations and topological and electrostatic analyses to study the effects of palmitoylation on the E protein pentamer. The results indicate that the cation transferring direction is more from the lumen to the cytosol. The structure of the palmitoylated E protein pentamer is more stable while the loss of palmitoylation caused the pore radius to reduce and even collapse. The electrostatic forces on the two sides of the palmitoylated E protein pentamer are more beneficial to attract cations in the lumen and to release cations into the cytosol. The results indicate the importance of palmitoylation, which can help the drug design for the treatment of COVID-19.