A Multiparametric and High-Throughput Platform for Host-Virus Binding Screens.

Speed is key during infectious disease outbreaks. It is essential, for example, to identify critical host binding factors to pathogens as fast as possible. The complexity of host plasma membrane is often a limiting factor hindering fast and accurate determination of host binding factors as well as high-throughput screening for neutralizing antimicrobial drug targets. Here, we describe a multiparametric and high-throughput platform tackling this bottleneck and enabling fast screens for host binding factors as well as new antiviral drug targets. The sensitivity and robustness of our platform were validated by blocking SARS-CoV-2 particles with nanobodies and IgGs from human serum samples.

Advanced optical imaging reveals preferred spatial orientation of podocyte processes along the axis of glomerular capillaries.

Mammalian kidneys filter enormous volumes of water and small solutes, a filtration driven by the hydrostatic pressure in glomerular capillaries, which is considerably higher than in most other tissues. Interdigitating cellular processes of podocytes form the slits for fluid filtration connected by the membrane-like slit diaphragm cell junction containing a mechanosensitive ion channel complex and allow filtration while counteracting hydrostatic pressure. Several previous publications speculated that podocyte processes may display a preferable orientation on glomerular capillaries instead of a random distribution. However, for decades, the controversy over spatially oriented filtration slits could not be resolved due to technical limitations of imaging technologies. Here, we used advanced high-resolution, three-dimensional microscopy with high data throughput to assess spatial orientation of podocyte processes and filtration slits quantitatively. Filtration-slit-generating secondary processes preferentially align along the capillaries' longitudinal axis while primary processes are preferably perpendicular to the longitudinal direction. This preferential orientation required maturation in development of the mice but was lost in mice with kidney disease due to treatment with nephrotoxic serum or with underlying heterologous mutations in the podocyte foot process protein podocin. Thus, the observation that podocytes maintain a preferred spatial orientation of their processes on glomerular capillaries goes well in line with the role of podocyte foot processes as mechanical buttresses to counteract mechanical forces resulting from pressurized capillaries. Future studies are needed to establish how podocytes establish and maintain their orientation and why orientation is lost under pathological conditions.

Amyloid-ß accumulation in human astrocytes induces mitochondrial disruption and changed energy metabolism.

BACKGROUND: Astrocytes play a central role in maintaining brain energy metabolism, but are also tightly connected to the pathogenesis of Alzheimer's disease (AD). Our previous studies demonstrate that inflammatory astrocytes accumulate large amounts of aggregated amyloid-beta (Aß). However, in which way these Aß deposits influence their energy production remain unclear. METHODS: The aim of the present study was to investigate how Aß pathology in astrocytes affects their mitochondria functionality and overall energy metabolism. For this purpose, human induced pluripotent cell (hiPSC)-derived astrocytes were exposed to sonicated Aß42 fibrils for 7 days and analyzed over time using different experimental approaches. RESULTS: Our results show that to maintain stable energy production, the astrocytes initially increased their mitochondrial fusion, but eventually the Aß-mediated stress led to abnormal mitochondrial swelling and excessive fission. Moreover, we detected increased levels of phosphorylated DRP-1 in the Aß-exposed astrocytes, which co-localized with lipid droplets. Analysis of ATP levels, when blocking certain stages of the energy pathways, indicated a metabolic shift to peroxisomal-based fatty acid ß-oxidation and glycolysis. CONCLUSIONS: Taken together, our data conclude that Aß pathology profoundly affects human astrocytes and changes their entire energy metabolism, which could result in disturbed brain homeostasis and aggravated disease progression.

Super-Resolution Imaging of the Filtration Barrier Suggests a Role for Podocin R229Q in Genetic Predisposition to Glomerular Disease.

BACKGROUND: Diseases of the kidney's glomerular filtration barrier are a leading cause of end stage renal failure. Despite a growing understanding of genes involved in glomerular disorders in children, the vast majority of adult patients lack a clear genetic diagnosis. The protein podocin p.R229Q, which results from the most common missense variant in NPHS2, is enriched in cohorts of patients with FSGS. However, p.R229Q has been proposed to cause disease only when transassociated with specific additional genetic alterations, and population-based epidemiologic studies on its association with albuminuria yielded ambiguous results. METHODS: To test whether podocin p.R229Q may also predispose to the complex disease pathogenesis in adults, we introduced the exact genetic alteration in mice using CRISPR/Cas9-based genome editing (PodR231Q ). We assessed the phenotype using super-resolution microscopy and albuminuria measurements and evaluated the stability of the mutant protein in cell culture experiments. RESULTS: Heterozygous PodR231Q/wild-type mice did not present any overt kidney disease or proteinuria. However, homozygous PodR231Q/R231Q mice developed increased levels of albuminuria with age, and super-resolution microscopy revealed preceding ultrastructural morphologic alterations that were recently linked to disease predisposition. When injected with nephrotoxic serum to induce glomerular injury, heterozygous PodR231Q/wild-type mice showed a more severe course of disease compared with Podwild-type/wild-type mice. Podocin protein levels were decreased in PodR231Q/wild-type and PodR231Q/R231Q mice as well as in human cultured podocytes expressing the podocinR231Q variant. Our in vitro experiments indicate an underlying increased proteasomal degradation. CONCLUSIONS: Our findings demonstrate that podocin R231Q exerts a pathogenic effect on its own, supporting the concept of podocin R229Q contributing to genetic predisposition in adult patients.

A missense mutation converts the Na+,K+-ATPase into an ion channel and causes therapy-resistant epilepsy.

The ion pump Na+,K+-ATPase is a critical determinant of neuronal excitability; however, its role in the etiology of diseases of the central nervous system (CNS) is largely unknown. We describe here the molecular phenotype of a Trp931Arg mutation of the Na+,K+-ATPase catalytic α1 subunit in an infant diagnosed with therapy-resistant lethal epilepsy. In addition to the pathological CNS phenotype, we also detected renal wasting of Mg2+. We found that membrane expression of the mutant α1 protein was low, and ion pumping activity was lost. Arginine insertion into membrane proteins can generate water-filled pores in the plasma membrane, and our molecular dynamic (MD) simulations of the principle states of Na+,K+-ATPase transport demonstrated massive water inflow into mutant α1 and destabilization of the ion-binding sites. MD simulations also indicated that a water pathway was created between the mutant arginine residue and the cytoplasm, and analysis of oocytes expressing mutant α1 detected a nonspecific cation current. Finally, neurons expressing mutant α1 were observed to be depolarized compared with neurons expressing wild-type protein, compatible with a lowered threshold for epileptic seizures. The results imply that Na+,K+-ATPase should be considered a neuronal locus minoris resistentia in diseases associated with epilepsy and with loss of plasma membrane integrity.

A fast and simple clearing and swelling protocol for 3D in-situ imaging of the kidney across scales.

In recent years, many light-microscopy protocols have been published for visualization of nanoscale structures in the kidney. These protocols present researchers with new tools to evaluate both foot process anatomy and effacement, as well as protein distributions in foot processes, the slit diaphragm and in the glomerular basement membrane. However, these protocols either involve the application of different complicated super resolution microscopes or lengthy sample preparation protocols. Here, we present a fast and simple, five-hour long procedure for three-dimensional visualization of kidney morphology on all length scales. The protocol combines optical clearing and tissue expansion concepts to produce a mild swelling, sufficient for resolving nanoscale structures using a conventional confocal microscope. We show that the protocol can be applied to visualize a wide variety of pathologic features in both mouse and human kidneys. Thus, our fast and simple protocol can be beneficial for conventional microscopic evaluation of kidney tissue integrity both in research and possibly in future clinical routines.

Post-metaphase correction of aberrant kinetochore-microtubule attachments in mammalian eggs.

The accuracy of the two sequential meiotic divisions in oocytes is essential for creating a haploid gamete with a normal chromosomal content. Here, we have analysed the 3D dynamics of chromosomes during the second meiotic division in live mouse oocytes. We find that chromosomes form stable kinetochore-microtubule attachments at the end of prometaphase II stage that are retained until anaphase II onset. Remarkably, we observe that more than 20% of the kinetochore-microtubule attachments at the metaphase II stage are merotelic or lateral. However, < 1% of all chromosomes at onset of anaphase II are found to lag at the spindle equator and < 10% of the laggards missegregate and give rise to aneuploid gametes. Our results demonstrate that aberrant kinetochore-microtubule attachments are not corrected at the metaphase stage of the second meiotic division. Thus, the accuracy of the chromosome segregation process in mouse oocytes during meiosis II is ensured by an efficient correction process acting at the anaphase stage.

Sexual dimorphism in the width of the mouse synaptonemal complex.

Sexual dimorphism has been used to describe morphological differences between the sexes, but can be extended to any biologically related process that varies between males and females. The synaptonemal complex (SC) is a tripartite structure that connects homologous chromosomes in meiosis. Here, aided by super-resolution microscopy techniques, we show that the SC is subject to sexual dimorphism, in mouse germ cells. We have identified a significantly narrower SC in oocytes and have established that this difference does not arise from a different organization of the lateral elements nor from a different isoform of transverse filament protein SYCP1. Instead, we provide evidence for the existence of a narrower central element and a different integration site for the C-termini of SYCP1, in females. In addition to these female-specific features, we speculate that post-translation modifications affecting the SYCP1 coiled-coil region could render a more compact conformation, thus contributing to the narrower SC observed in females.

Extracellular vesicles from mast cells induce mesenchymal transition in airway epithelial cells.

BACKGROUND: In the airways, mast cells are present in close vicinity to epithelial cells, and they can interact with each other via multiple factors, including extracellular vesicles (EVs). Mast cell-derived EVs have a large repertoire of cargos, including proteins and RNA, as well as surface DNA. In this study, we hypothesized that these EVs can induce epithelial to mesenchymal transition (EMT) in airway epithelial cells. METHODS: In this in-vitro study we systematically determined the effects of mast cell-derived EVs on epithelial A549 cells. We determined the changes that are induced by EVs on A549 cells at both the RNA and protein levels. Moreover, we also analyzed the rapid changes in phosphorylation events in EV-recipient A549 cells using a phosphorylated protein microarray. Some of the phosphorylation-associated events associated with EMT were validated using immunoblotting. RESULTS: Morphological and transcript analysis of epithelial A549 cells indicated that an EMT-like phenotype was induced by the EVs. Transcript analysis indicated the upregulation of genes involved in EMT, including TWIST1, MMP9, TGFB1, and BMP-7. This was accompanied by downregulation of proteins such as E-cadherin and upregulation of Slug-Snail and matrix metalloproteinases. Additionally, our phosphorylated-protein microarray analysis revealed proteins associated with the EMT cascade that were upregulated after EV treatment. We also found that transforming growth factor beta-1, a well-known EMT inducer, is associated with EVs and mediates the EMT cascade induced in the A549 cells. CONCLUSION: Mast cell-derived EVs mediate the induction of EMT in epithelial cells, and our evidence suggests that this is triggered through the induction of protein phosphorylation cascades.

Spontaneous calcium activity in metanephric mesenchymal cells regulates branching morphogenesis in the embryonic kidney.

The central role of calcium signaling during development of early vertebrates is well documented, but little is known about its role in mammalian embryogenesis. We have used immunofluorescence and time-lapse calcium imaging of cultured explanted embryonic rat kidneys to study the role of calcium signaling for branching morphogenesis. In mesenchymal cells, we recorded spontaneous calcium activity that was characterized by irregular calcium transients. The calcium signals were dependent on release of calcium from intracellular stores in the endoplasmic reticulum. Down-regulation of the calcium activity, both by blocking the sarco-endoplasmic reticulum Ca2+-ATPase and by chelating cytosolic calcium, resulted in retardation of branching morphogenesis and a reduced formation of primitive nephrons but had no effect on cell proliferation. We propose that spontaneous calcium activity contributes with a stochastic factor to the self-organizing process that controls branching morphogenesis, a major determinant of the ultimate number of nephrons in the kidney.-Fontana, J. M., Khodus, G. R., Unnersjö-Jess, D., Blom, H., Aperia, A., Brismar, H. Spontaneous calcium activity in metanephric mesenchymal cells regulates branching morphogenesis in the embryonic kidney.

Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+, K+-ATPase control of cell adhesion, proliferation, and survival.

The ion pump Na+, K+-ATPase (NKA) is a receptor for the cardiotonic steroid ouabain. Subsaturating concentration of ouabain triggers intracellular calcium oscillations, stimulates cell proliferation and adhesion, and protects from apoptosis. However, it is controversial whether ouabain-bound NKA is considered a signal transducer. To address this question, we performed a global analysis of protein phosphorylation in COS-7 cells, identifying 2580 regulated phosphorylation events on 1242 proteins upon 10- and 20-min treatment with ouabain. Regulated phosphorylated proteins include the inositol triphosphate receptor and stromal interaction molecule, which are essential for initiating calcium oscillations. Hierarchical clustering revealed that ouabain triggers a structured phosphorylation response that occurs in a well-defined, time-dependent manner and affects specific cellular processes, including cell proliferation and cell-cell junctions. We additionally identify regulation of the phosphorylation of several calcium and calmodulin-dependent protein kinases (CAMKs), including 2 sites of CAMK type II-γ (CAMK2G), a protein known to regulate apoptosis. To verify the significance of this result, CAMK2G was knocked down in primary kidney cells. CAMK2G knockdown impaired ouabain-dependent protection from apoptosis upon treatment with high glucose or serum deprivation. In conclusion, we establish NKA as the coordinator of a broad, tightly regulated phosphorylation response in cells and define CAMK2G as a downstream effector of NKA.-Panizza, E., Zhang, L., Fontana, J. M., Hamada, K., Svensson, D., Akkuratov, E. E., Scott, L., Mikoshiba, K., Brismar, H., Lehtiö, J., Aperia, A. Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+, K+-ATPase control of cell adhesion, proliferation, and survival.

Glial origin of mesenchymal stem cells in a tooth model system.

Mesenchymal stem cells occupy niches in stromal tissues where they provide sources of cells for specialized mesenchymal derivatives during growth and repair. The origins of mesenchymal stem cells have been the subject of considerable discussion, and current consensus holds that perivascular cells form mesenchymal stem cells in most tissues. The continuously growing mouse incisor tooth offers an excellent model to address the origin of mesenchymal stem cells. These stem cells dwell in a niche at the tooth apex where they produce a variety of differentiated derivatives. Cells constituting the tooth are mostly derived from two embryonic sources: neural crest ectomesenchyme and ectodermal epithelium. It has been thought for decades that the dental mesenchymal stem cells giving rise to pulp cells and odontoblasts derive from neural crest cells after their migration in the early head and formation of ectomesenchymal tissue. Here we show that a significant population of mesenchymal stem cells during development, self-renewal and repair of a tooth are derived from peripheral nerve-associated glia. Glial cells generate multipotent mesenchymal stem cells that produce pulp cells and odontoblasts. By combining a clonal colour-coding technique with tracing of peripheral glia, we provide new insights into the dynamics of tooth organogenesis and growth.

Prompt apoptotic response to high glucose in SGLT-expressing renal cells.

It is generally believed that cells that are unable to downregulate glucose transport are particularly vulnerable to hyperglycemia. Yet, little is known about the relation between expression of glucose transporters and acute toxic effects of high glucose exposure. In the present ex vivo study of rat renal cells, we compared the apoptotic response to a moderate increase in glucose concentration. We studied cell types that commonly are targeted in diabetic kidney disease (DKD): proximal tubule cells, which express Na+-dependent glucose transporter (SGLT)2, mesangial cells, which express SGLT1, and podocytes, which lack SGLT and take up glucose via insulin-dependent glucose transporter 4. Proximal tubule cells and mesangial cells responded within 4-8 h of exposure to 15 mM glucose with translocation of the apoptotic protein Bax to mitochondria and an increased apoptotic index. SGLT downregulation and exposure to SGLT inhibitors abolished the apoptotic response. The onset of overt DKD generally coincides with the onset of albuminuria. Albumin had an additive effect on the apoptotic response. Ouabain, which interferes with the apoptotic onset, rescued from the apoptotic response. Insulin-supplemented podocytes remained resistant to 15 and 30 mM glucose for at least 24 h. Our study points to a previously unappreciated role of SGLT-dependent glucose uptake as a risk factor for diabetic complications and highlights the importance of therapeutic approaches that specifically target the different cell types in DKD.

SMLocalizer, a GPU accelerated ImageJ plugin for single molecule localization microscopy.

Summary: SMLocalizer combines the availability of ImageJ with the power of GPU processing for fast and accurate analysis of single molecule localization microscopy data. Analysis of 2D and 3D data in multiple channels is supported. Availability and implementation: Plugin freely available for Fiji and ImageJ2.0 through https://sourceforge.net/projects/smlocalizer/. Plugin also available for continuous updates through ImageJ update system, add http://sites.imagej.net/Cellular-Biophysics-KTH/ as update site in ImageJ. Java and CUDA source code freely available on the web at https://github.com/KristofferBernhem/SMlocalizer. Contact: brismar@kth.se. Supplementary information: Supplementary data are available at Bioinformatics online.

Confocal super-resolution imaging of the glomerular filtration barrier enabled by tissue expansion.

The glomerular filtration barrier, has historically only been spatially resolved using electron microscopy due to the nanometer-scale dimensions of these structures. Recently, it was shown that the nanoscale distribution of proteins in the slit diaphragm can be resolved by fluorescence based stimulated emission depletion microscopy, in combination with optical clearing. Fluorescence microscopy has advantages over electron microscopy in terms of multiplex imaging of different epitopes, and also the amount of volumetric data that can be extracted from thicker samples. However, stimulated emission depletion microscopy is still a costly technique commonly not available to most life science researchers. An imaging technique with which the glomerular filtration barrier can be visualized using more standard fluorescence imaging techniques is thus desirable. Recent studies have shown that biological tissue samples can be isotropically expanded, revealing nanoscale localizations of multiple epitopes using confocal microscopy. Here we show that kidney samples can be expanded sufficiently to study the finest elements of the filtration barrier using confocal microscopy. Thus, our result opens up the possibility to study protein distributions and foot process morphology on the effective nanometer-scale.

High density of REC8 constrains sister chromatid axes and prevents illegitimate synaptonemal complex formation.

During meiosis, cohesin complexes mediate sister chromatid cohesion (SCC), synaptonemal complex (SC) assembly and synapsis. Here, using super-resolution microscopy, we imaged sister chromatid axes in mouse meiocytes that have normal or reduced levels of cohesin complexes, assessing the relationship between localization of cohesin complexes, SCC and SC formation. We show that REC8 foci are separated from each other by a distance smaller than 15% of the total chromosome axis length in wild-type meiocytes. Reduced levels of cohesin complexes result in a local separation of sister chromatid axial elements (LSAEs), as well as illegitimate SC formation at these sites. REC8 but not RAD21 or RAD21L cohesin complexes flank sites of LSAEs, whereas RAD21 and RAD21L appear predominantly along the separated sister-chromatid axes. Based on these observations and a quantitative distribution analysis of REC8 along sister chromatid axes, we propose that the high density of randomly distributed REC8 cohesin complexes promotes SCC and prevents illegitimate SC formation.

Regulation of Neuronal Na,K-ATPase by Extracellular Scaffolding Proteins.

Neuronal activity leads to an influx of Na⁺ that needs to be rapidly cleared. The sodium-potassium ATPase (Na,K-ATPase) exports three Na⁺ ions and imports two K⁺ ions at the expense of one ATP molecule. Na,K-ATPase turnover accounts for the majority of energy used by the brain. To prevent an energy crisis, the energy expense for Na⁺ clearance must provide an optimal effect. Here we report that in rat primary hippocampal neurons, the clearance of Na⁺ ions is more efficient if Na,K-ATPase is laterally mobile in the membrane than if it is clustered. Using fluorescence recovery after photobleaching and single particle tracking analysis, we show that the ubiquitous α1 and the neuron-specific α3 catalytic subunits as well as the supportive ß1 subunit of Na,K-ATPase are highly mobile in the plasma membrane. We show that cross-linking of the ß1 subunit with polyclonal antibodies or exposure to Modulator of Na,K-ATPase (MONaKA), a secreted protein which binds to the extracellular domain of the ß subunit, clusters the α3 subunit in the membrane and restricts its mobility. We demonstrate that clustering, caused by cross-linking or by exposure to MONaKA, reduces the efficiency in restoring intracellular Na⁺. These results demonstrate that extracellular interactions with Na,K-ATPase regulate the Na⁺ extrusion efficiency with consequences for neuronal energy balance.

AT1-receptor response to non-saturating Ang-II concentrations is amplified by calcium channel blockers.

BACKGROUND: Blockers of angiotensin II type 1 receptor (AT1R) and the voltage gated calcium channel 1.2 (CaV1.2) are commonly used for treatment of hypertension. Yet there is little information about the effect of physiological concentrations of angiotensin II (AngII) on AT1R signaling and whether there is a reciprocal regulation of AT1R signaling by CaV1.2. METHODS: To elucidate these questions, we have studied the Ca2+ signaling response to physiological and pharmacological AngII doses in HEK293a cells, vascular smooth muscle cells and cardiomyocytes using a Ca2+ sensitive dye as the principal sensor. Intra-cellular calcium recordings were performed in presence and absence of CaV1.2 blockers. Semi-quantitative imaging methods were used to assess the plasma membrane expression of AT1R and G-protein activation. RESULTS: Repeated exposure to pharmacological (100 nM) concentrations of AngII caused, as expected, a down-regulation of the Ca2+ response. In contrast, repeated exposure to physiological (1 nM) AngII concentration resulted in an enhancement of the Ca2+ response. The up-regulation of the Ca2+ response to repeated 1 nM AngII doses and the down-regulation of the Ca2+ response to repeated 100 nM Angll doses were not accompanied by a parallel change of the AT1R plasma membrane expression. The Ca2+ response to 1 nM of AngII was amplified in the presence of therapeutic concentrations of the CaV1.2 blockers, nifedipine and verapamil, in vascular smooth muscle cells, cardiomyocytes and HEK293a cells. Amplification of the AT1R response was also observed following inhibition of the calcium permeable transient receptor potential cation channels, suggesting that the activity of AT1R is sensitive to calcium influx. CONCLUSIONS: Our findings have implications for the understanding of hyperactivity of the angiotensin system and for use of Ca2+ channel blockers as mono-therapy in hypertension.

Na+-K+-ATPase, a new class of plasma membrane receptors.

The Na(+)-K(+)-ATPase (NKA) differs from most other ion transporters, not only in its capacity to maintain a steep electrochemical gradient across the plasma membrane, but also as a receptor for a family of cardiotonic steroids, to which ouabain belongs. Studies from many groups, performed during the last 15 years, have demonstrated that ouabain, a member of the cardiotonic steroid family, can activate a network of signaling molecules, and that NKA will also serve as a signal transducer that can provide a feedback loop between NKA and the mitochondria. This brief review summarizes the current knowledge and controversies with regard to the understanding of NKA signaling.

Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue.

The glomerular filtration barrier, consisting of podocyte foot processes with bridging slit diaphragm, glomerular basement membrane, and endothelium, is a key component for renal function. Previously, the subtlest elements of the filtration barrier have only been visualized using electron microscopy. However, electron microscopy is mostly restricted to ultrathin two-dimensional samples, and the possibility to simultaneously visualize multiple different proteins is limited. Therefore, we sought to implement a super-resolution immunofluorescence microscopy protocol for the study of the filtration barrier in the kidney. Recently, several optical clearing methods have been developed making it possible to image through large volumes of tissue and even whole organs using light microscopy. Here we found that hydrogel-based optical clearing is a beneficial tool to study intact renal tissue at the nanometer scale. When imaging samples using super-resolution STED microscopy, the staining quality was critical in order to assess correct nanoscale information. The signal-to-noise ratio and immunosignal homogeneity were both improved in optically cleared tissue. Thus, STED of slit diaphragms in fluorescently labeled, optically cleared, intact kidney samples is a new tool for studying the glomerular filtration barrier in health and disease.