WNK kinases sense molecular crowding and rescue cell volume via phase separation.

When challenged by hypertonicity, dehydrated cells must recover their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless condensates, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to restore cell volume. WNK1 condensate formation is driven by its intrinsically disordered C terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows kinase activity despite an inhibitory ionic milieu and permits cell volume recovery through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

Whole-body integration of gene expression and single-cell morphology.

Animal bodies are composed of cell types with unique expression programs that implement their distinct locations, shapes, structures, and functions. Based on these properties, cell types assemble into specific tissues and organs. To systematically explore the link between cell-type-specific gene expression and morphology, we registered an expression atlas to a whole-body electron microscopy volume of the nereid Platynereis dumerilii. Automated segmentation of cells and nuclei identifies major cell classes and establishes a link between gene activation, chromatin topography, and nuclear size. Clustering of segmented cells according to gene expression reveals spatially coherent tissues. In the brain, genetically defined groups of neurons match ganglionic nuclei with coherent projections. Besides interneurons, we uncover sensory-neurosecretory cells in the nereid mushroom bodies, which thus qualify as sensory organs. They furthermore resemble the vertebrate telencephalon by molecular anatomy. We provide an integrated browser as a Fiji plugin for remote exploration of all available multimodal datasets.

Dopamine Secretion Is Mediated by Sparse Active Zone-like Release Sites.

Dopamine controls essential brain functions through volume transmission. Different from fast synaptic transmission, where neurotransmitter release and receptor activation are tightly coupled by an active zone, dopamine transmission is widespread and may not necessitate these organized release sites. Here, we determine whether striatal dopamine secretion employs specialized machinery for release. Using super resolution microscopy, we identified co-clustering of the active zone scaffolding proteins bassoon, RIM and ELKS in ∼30% of dopamine varicosities. Conditional RIM knockout disrupted this scaffold and, unexpectedly, abolished dopamine release, while ELKS knockout had no effect. Optogenetic experiments revealed that dopamine release was fast and had a high release probability, indicating the presence of protein scaffolds for coupling Ca2+ influx to vesicle fusion. Hence, dopamine secretion is mediated by sparse, mechanistically specialized active zone-like release sites. This architecture supports spatially and temporally precise coding for dopamine and provides molecular machinery for regulation.

Ancestral Circuits for the Coordinated Modulation of Brain State.

Internal states of the brain profoundly influence behavior. Fluctuating states such as alertness can be governed by neuromodulation, but the underlying mechanisms and cell types involved are not fully understood. We developed a method to globally screen for cell types involved in behavior by integrating brain-wide activity imaging with high-content molecular phenotyping and volume registration at cellular resolution. We used this method (MultiMAP) to record from 22 neuromodulatory cell types in behaving zebrafish during a reaction-time task that reports alertness. We identified multiple monoaminergic, cholinergic, and peptidergic cell types linked to alertness and found that activity in these cell types was mutually correlated during heightened alertness. We next recorded from and controlled homologous neuromodulatory cells in mice; alertness-related cell-type dynamics exhibited striking evolutionary conservation and modulated behavior similarly. These experiments establish a method for unbiased discovery of cellular elements underlying behavior and reveal an evolutionarily conserved set of diverse neuromodulatory systems that collectively govern internal state.

Dense Circuit Reconstruction to Understand Neuronal Computation: Focus on Zebrafish.

The dense reconstruction of neuronal wiring diagrams from volumetric electron microscopy data has the potential to generate fundamentally new insights into mechanisms of information processing and storage in neuronal circuits. Zebrafish provide unique opportunities for dynamical connectomics approaches that combine reconstructions of wiring diagrams with measurements of neuronal population activity and behavior. Such approaches have the power to reveal higher-order structure in wiring diagrams that cannot be detected by sparse sampling of connectivity and that is essential for neuronal computations. In the brain stem, recurrently connected neuronal modules were identified that can account for slow, low-dimensional dynamics in an integrator circuit. In the spinal cord, connectivity specifies functional differences between premotor interneurons. In the olfactory bulb, tuning-dependent connectivity implements a whitening transformation that is based on the selective suppression of responses to overrepresented stimulus features. These findings illustrate the potential of dynamical connectomics in zebrafish to analyze the circuit mechanisms underlying higher-order neuronal computations.

Multivalent Proteins Rapidly and Reversibly Phase-Separate upon Osmotic Cell Volume Change.

Processing bodies (PBs) and stress granules (SGs) are prominent examples of subcellular, membraneless compartments that are observed under physiological and stress conditions, respectively. We observe that the trimeric PB protein DCP1A rapidly (within ∼10 s) phase-separates in mammalian cells during hyperosmotic stress and dissolves upon isosmotic rescue (over ∼100 s) with minimal effect on cell viability even after multiple cycles of osmotic perturbation. Strikingly, this rapid intracellular hyperosmotic phase separation (HOPS) correlates with the degree of cell volume compression, distinct from SG assembly, and is exhibited broadly by homo-multimeric (valency ≥ 2) proteins across several cell types. Notably, HOPS sequesters pre-mRNA cleavage factor components from actively transcribing genomic loci, providing a mechanism for hyperosmolarity-induced global impairment of transcription termination. Our data suggest that the multimeric proteome rapidly responds to changes in hydration and molecular crowding, revealing an unexpected mode of globally programmed phase separation and sequestration.

Molecular Crowding: Physiologic Sensing and Control.

The cytoplasm is densely packed with molecules that contribute to its nonideal behavior. Cytosolic crowding influences chemical reaction rates, intracellular water mobility, and macromolecular complex formation. Overcrowding is potentially catastrophic; to counteract this problem, cells have evolved acute and chronic homeostatic mechanisms that optimize cellular crowdedness. Here, we provide a physiology-focused overview of molecular crowding, highlighting contemporary advances in our understanding of its sensing and control. Long hypothesized as a form of crowding-induced microcompartmentation, phase separation allows cells to detect and respond to intracellular crowding through the action of biomolecular condensates, as indicated by recent studies. Growing evidence indicates that crowding is closely tied to cell size and fluid volume, homeostatic responses to physical compression and desiccation, tissue architecture, circadian rhythm, aging, transepithelial transport, and total body electrolyte and water balance. Thus, molecular crowding is a fundamental physiologic parameter that impacts diverse functions extending from molecule to organism.

A physicochemical perspective on cellular ageing.

Cellular ageing described at the molecular level is a multifactorial process that leads to a spectrum of ageing trajectories. There has been recent discussion about whether a decline in physicochemical homeostasis causes aberrant phase transitions, which are a driver of ageing. Indeed, the function of all biological macromolecules, regardless of their participation in biomolecular condensates, depends on parameters such as pH, crowding, and redox state. We expand on the physicochemical homeostasis hypothesis and summarise recent evidence that the intracellular milieu influences molecular processes involved in ageing.

Grain boundary plasticity initiated by excess volume.

Grain boundaries (GBs) serve not only as strong barriers to dislocation motion, but also as important carriers to accommodate plastic deformation in crystalline solids. During deformation, the inherent excess volume associated with loose atomic packing in GBs brings about a microscopic degree of freedom that can initiate GB plasticity, which is beyond the classic geometric description of GBs. However, identification of this atomistic process has long remained elusive due to its transient nature. Here, we use Au polycrystals to unveil a general and inherent route to initiating GB plasticity via a transient topological transition process triggered by the excess volume. This route underscores the general impact of a microscopic degree of freedom which is governed by a stress-triaxiality-based criterion. Our findings provide a missing perspective for developing a more comprehensive understanding of the role of GBs in plastic deformation.

Indications of flat bands driving the δ to α volume collapse of plutonium.

On cooling from the melt, plutonium (Pu) undergoes a series of structural transformations accompanied by a ≈ 28% reduction in volume from its δ phase to its α phase at low temperatures. While Pu's partially filled 5f-electron shells are known to be involved, their precise role in the transformations has remained unclear. By using calorimetry measurements on α-Pu and gallium-stabilized δ-Pu combined with resonant ultrasound and X-ray scattering data to account for the anomalously large softening of the lattice with temperature, we show here that the difference in electronic entropy between the α and δ phases dominates over the difference in phonon entropy. Rather than finding an electronic specific heat characteristic of broad f-electron bands in α-Pu, as might be expected to occur within a Kondo collapsed phase in analogy with cerium, we find it to be indicative of flatter subbands. An important role played by Pu's 5f electrons in the formation of its larger unit cell α phase comprising inequivalent lattice sites and varying bond lengths is therefore suggested.

The Hippo pathway drives the cellular response to hydrostatic pressure.

Cells need to rapidly and precisely react to multiple mechanical and chemical stimuli in order to ensure precise context-dependent responses. This requires dynamic cellular signalling events that ensure homeostasis and plasticity when needed. A less well-understood process is cellular response to elevated interstitial fluid pressure, where the cell senses and responds to changes in extracellular hydrostatic pressure. Here, using quantitative label-free digital holographic imaging, combined with genome editing, biochemical assays and confocal imaging, we analyse the temporal cellular response to hydrostatic pressure. Upon elevated cyclic hydrostatic pressure, the cell responds by rapid, dramatic and reversible changes in cellular volume. We show that YAP and TAZ, the co-transcriptional regulators of the Hippo signalling pathway, control cell volume and that cells without YAP and TAZ have lower plasma membrane tension. We present direct evidence that YAP/TAZ drive the cellular response to hydrostatic pressure, a process that is at least partly mediated via clathrin-dependent endocytosis. Additionally, upon elevated oscillating hydrostatic pressure, YAP/TAZ are activated and induce TEAD-mediated transcription and expression of cellular components involved in dynamic regulation of cell volume and extracellular matrix. This cellular response confers a feedback loop that allows the cell to robustly respond to changes in interstitial fluid pressure.

Interventional Pulmonology: Extending the Breadth of Thoracic Care.

Interventional pulmonary medicine has developed as a subspecialty focused on the management of patients with complex thoracic disease. Leveraging minimally invasive techniques, interventional pulmonologists diagnose and treat pathologies that previously required more invasive options such as surgery. By mitigating procedural risk, interventional pulmonologists have extended the reach of care to a wider pool of vulnerable patients who require therapy. Endoscopic innovations, including endobronchial ultrasound and robotic and electromagnetic bronchoscopy, have enhanced the ability to perform diagnostic procedures on an ambulatory basis. Therapeutic procedures for patients with symptomatic airway disease, pleural disease, and severe emphysema have provided the ability to palliate symptoms. The combination of medical and procedural expertise has made interventional pulmonologists an integral part of comprehensive care teams for patients with oncologic, airway, and pleural needs. This review surveys key areas in which interventional pulmonologists have impacted the care of thoracic disease through bronchoscopic intervention.

Cardiomyocyte PANX1 Controls Glycolysis and Neutrophil Recruitment in Hypertrophy.

BACKGROUND: PANX1 (pannexin 1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, the possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated. METHOD: We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1MyHC6). RESULTS: PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism and resulting glycolytic ATP production, with a concurrent decrease in oxidative phosphorylation, both in vivo and in vitro. In vitro, treatment of H9c2 cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knockdown of PANX1. To investigate nonischemic heart failure and the preceding cardiac hypertrophy, we administered isoproterenol, and we demonstrated that Panx1MyHC6 mice were protected from systolic and diastolic left ventricle volume increases as a result of cardiomyocyte hypertrophy. Moreover, we found that Panx1MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45+), particularly neutrophils (CD11b+, Ly6g+), to the myocardium. CONCLUSIONS: Together, these data demonstrate that PANX1 deficiency in cardiomyocytes increases glycolytic metabolism and protects against cardiac hypertrophy in nonischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in patients with heart failure.

Glucagon Receptor Antagonist for Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is an emerging major unmet need and one of the most significant clinic challenges in cardiology. The pathogenesis of HFpEF is associated with multiple risk factors. Hypertension and metabolic disorders associated with obesity are the 2 most prominent comorbidities observed in patients with HFpEF. Although hypertension-induced mechanical overload has long been recognized as a potent contributor to heart failure with reduced ejection fraction, the synergistic interaction between mechanical overload and metabolic disorders in the pathogenesis of HFpEF remains poorly characterized. METHOD: We investigated the functional outcome and the underlying mechanisms from concurrent mechanic and metabolic stresses in the heart by applying transverse aortic constriction in lean C57Bl/6J or obese/diabetic B6.Cg-Lepob/J (ob/ob) mice, followed by single-nuclei RNA-seq and targeted manipulation of a top-ranked signaling pathway differentially affected in the 2 experimental cohorts. RESULTS: In contrast to the post-trans-aortic constriction C57Bl/6J lean mice, which developed pathological features of heart failure with reduced ejection fraction over time, the post-trans-aortic constriction ob/ob mice showed no significant changes in ejection fraction but developed characteristic pathological features of HFpEF, including diastolic dysfunction, worsened cardiac hypertrophy, and pathological remodeling, along with further deterioration of exercise intolerance. Single-nuclei RNA-seq analysis revealed significant transcriptome reprogramming in the cardiomyocytes stressed by both pressure overload and obesity/diabetes, markedly distinct from the cardiomyocytes singularly stressed by pressure overload or obesity/diabetes. Furthermore, glucagon signaling was identified as the top-ranked signaling pathway affected in the cardiomyocytes associated with HFpEF. Treatment with a glucagon receptor antagonist significantly ameliorated the progression of HFpEF-related pathological features in 2 independent preclinical models. Importantly, cardiomyocyte-specific genetic deletion of the glucagon receptor also significantly improved cardiac function in response to pressure overload and metabolic stress. CONCLUSIONS: These findings identify glucagon receptor signaling in cardiomyocytes as a critical determinant of HFpEF progression and provide proof-of-concept support for glucagon receptor antagonism as a potential therapy for the disease.

Brain volume, energy balance, and cardiovascular health in two nonindustrial South American populations.

Little is known about brain aging or dementia in nonindustrialized environments that are similar to how humans lived throughout evolutionary history. This paper examines brain volume (BV) in middle and old age among two indigenous South American populations, the Tsimane and Moseten, whose lifestyles and environments diverge from those in high-income nations. With a sample of 1,165 individuals aged 40 to 94, we analyze population differences in cross-sectional rates of decline in BV with age. We also assess the relationships of BV with energy biomarkers and arterial disease and compare them against findings in industrialized contexts. The analyses test three hypotheses derived from an evolutionary model of brain health, which we call the embarrassment of riches (EOR). The model hypothesizes that food energy was positively associated with late life BV in the physically active, food-limited past, but excess body mass and adiposity are now associated with reduced BV in industrialized societies in middle and older ages. We find that the relationship of BV with both non-HDL cholesterol and body mass index is curvilinear, positive from the lowest values to 1.4 to 1.6 SDs above the mean, and negative from that value to the highest values. The more acculturated Moseten exhibit a steeper decrease in BV with age than Tsimane, but still shallower than US and European populations. Lastly, aortic arteriosclerosis is associated with lower BV. Complemented by findings from the United States and Europe, our results are consistent with the EOR model, with implications for interventions to improve brain health.

Transforming eukaryotic cell culture with macromolecular crowding.

In multicellular organisms, the intracellular and extracellular spaces are considerably packed with a diverse range of macromolecular species. Yet, standard eukaryotic cell culture is performed in dilute, and deprived of macromolecules culture media, that barely imitate the density and complex macromolecular composition of tissues. Essentially, we drown cells in a sea of media and then expect them to perform physiologically. Herein, we argue the use of macromolecular crowding (MMC) in eukaryotic cell culture for regenerative medicine and drug discovery purposes.

Impacts of altered exercise volume, intensity, and duration on the activation of AMPK and CaMKII and increases in PGC-1α mRNA.

The purpose of this review is to explore and discuss the impacts of augmented training volume, intensity, and duration on the phosphorylation/activation of key signaling protein - AMPK, CaMKII and PGC-1α - involved in the initiation of mitochondrial biogenesis. Specifically, we explore the impacts of augmented exercise protocols on AMP/ADP and Ca2+ signaling and changes in post exercise PGC - 1α gene expression. Although AMP/ADP concentrations appear to increase with increasing intensity and during extended durations of higher intensity exercise AMPK activation results are varied with some results supporting and intensity/duration effect and others not. Similarly, CaMKII activation and signaling results following exercise of different intensities and durations are inconsistent. The PGC-1α literature is equally inconsistent with only some studies demonstrating an effect of intensity on post exercise mRNA expression. We present a novel meta-analysis that suggests that the inconsistency in the PGC-1α literature may be due to sample size and statistical power limitations owing to the effect of intensity on PGC-1α expression being small. There is little data available regarding the impact of exercise duration on PGC-1α expression. We highlight the need for future well designed, adequately statistically powered, studies to clarify our understanding of the effects of volume, intensity, and duration on the induction of mitochondrial biogenesis by exercise.

LRRC8/VRAC volume-regulated anion channels are crucial for hearing.

Hearing crucially depends on cochlear ion homeostasis as evident from deafness elicited by mutations in various genes encoding cation or anion channels and transporters. Ablation of ClC­K/barttin chloride channels causes deafness by interfering with the positive electrical potential of the endolymph, but roles of other anion channels in the inner ear have not been studied. Here we report the intracochlear distribution of all five LRRC8 subunits of VRAC, a volume-regulated anion channel that transports chloride, metabolites, and drugs such as the ototoxic anti-cancer drug cisplatin, and explore its physiological role by ablating its subunits. Sensory hair cells express all LRRC8 isoforms, whereas only LRRC8A, D and E were found in the potassium-secreting epithelium of the stria vascularis. Cochlear disruption of the essential LRRC8A subunit, or combined ablation of LRRC8D and E, resulted in cochlear degeneration and congenital deafness of Lrrc8a-/- mice. It was associated with a progressive degeneration of the organ of Corti and its innervating spiral ganglion. Like disruption of ClC-K/barttin, loss of VRAC severely reduced the endocochlear potential. However, the mechanism underlying this reduction seems different. Disruption of VRAC, but not ClC-K/barttin, led to an almost complete loss of Kir4.1 (KCNJ10), a strial K+ channel crucial for the generation of the endocochlear potential. The strong downregulation of Kir4.1 might be secondary to a loss of VRAC-mediated transport of metabolites regulating inner ear redox potential such as glutathione. Our study extends the knowledge of the role of cochlear ion transport in hearing and ototoxicity.

Probing the in situ volumes of Arabidopsis leaf plastids using three-dimensional confocal and scanning electron microscopy.

Leaf plastids harbor a plethora of biochemical reactions including photosynthesis, one of the most important metabolic pathways on Earth. Scientists are eager to unveil the physiological processes within the organelle but also their interconnection with the rest of the plant cell. An increasingly important feature of this venture is to use experimental data in the design of metabolic models. A remaining obstacle has been the limited in situ volume information of plastids and other cell organelles. To fill this gap for chloroplasts, we established three microscopy protocols delivering in situ volumes based on: (i) chlorophyll fluorescence emerging from the thylakoid membrane, (ii) a CFP marker embedded in the envelope, and (iii) calculations from serial block-face scanning electron microscopy (SBFSEM). The obtained data were corroborated by comparing wild-type data with two mutant lines affected in the plastid division machinery known to produce small and large mesophyll chloroplasts, respectively. Furthermore, we also determined the volume of the much smaller guard cell plastids. Interestingly, their volume is not governed by the same components of the division machinery which defines mesophyll plastid size. Based on our three approaches, the average volume of a mature Col-0 wild-type mesophyll chloroplasts is 93 µm3 . Wild-type guard cell plastids are approximately 18 µm3 . Lastly, our comparative analysis shows that the chlorophyll fluorescence analysis can accurately determine chloroplast volumes, providing an important tool to research groups without access to transgenic marker lines expressing genetically encoded fluorescence proteins or costly SBFSEM equipment.

Bi-allelic LETM1 variants perturb mitochondrial ion homeostasis leading to a clinical spectrum with predominant nervous system involvement.

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.