Intercellular Structures Research Articles

The components of an electrical synapse as revealed by expansion microscopy of a single synaptic contact.

Most nervous systems combine both transmitter-mediated and direct cell-cell communication, known as 'chemical' and 'electrical' synapses, respectively. Chemical synapses can be identified by their multiple structural components. Electrical synapses are, on the other hand, generally defined by the presence of a 'gap junction' (a cluster of intercellular channels) between two neuronal processes. However, while gap junctions provide the communicating mechanism, it is unknown whether electrical transmission requires the contribution of additional cellular structures. We investigated this question at identifiable single synaptic contacts on the zebrafish Mauthner cells, at which gap junctions coexist with specializations for neurotransmitter release and where the contact unequivocally defines the anatomical limits of a synapse. Expansion microscopy of these single contacts revealed a detailed map of the incidence and spatial distribution of proteins pertaining to various synaptic structures. Multiple gap junctions of variable size were identified by the presence of their molecular components. Remarkably, most of the synaptic contact's surface was occupied by interleaving gap junctions and components of adherens junctions, suggesting a close functional association between these two structures. In contrast, glutamate receptors were confined to small peripheral portions of the contact, indicating that most of the synaptic area functions as an electrical synapse. Thus, our results revealed the overarching organization of an electrical synapse that operates with not one, but multiple gap junctions, in close association with structural and signaling molecules known to be components of adherens junctions. The relationship between these intercellular structures will aid in establishing the boundaries of electrical synapses found throughout animal connectomes and provide insight into the structural organization and functional diversity of electrical synapses.

Establishing the link between D-mannose and juvenile grass carp (Ctenopharyngodon idella): Improved growth and intestinal structure associated with endoplasmic reticulum stress, mitophagy, and apical junctional complexes

D-mannose, essential for protein glycosylation, has been reported to have immunomodulatory effects and to maintain intestinal flora homeostasis. In addition to evaluating growth performance, we examined the impact of D-mannose on the structure of epithelial cells and apical junction complexes in the animal intestine. All 1800 grass carp (16.20 ± 0.01 g) were randomly divided into six treatments with six replicates of 50 fish each and fed with six different levels of D-mannose (0.52, 1.75, 3.02, 4.28, 5.50 and 6.78 g/kg diet) for 70 d. The study revealed that D-mannose increased feed intake (P < 0.001) but did not affect the percent weight gain (PWG), special growth rate, and feed conversion ratio (P > 0.05). D-mannose supplementation at 1.75 g/kg increased crude protein content in fish and lipid production value (P < 0.05). D-mannose supplementation at 4.28 g/kg increased intestinal length, intestinal weight and fold height of grass carp compared to the control group (P < 0.05). This improvement may be attributed to the phosphomannose isomerase (PMI)-mediated enhancement of glycolysis. This study found that D-mannose supplementation at 4.28 or 3.02 g/kg reduced serum diamine oxidase activity or D-lactate content (P < 0.05) and improved cellular and intercellular structures for the first time. The improvement of cellular redox homeostasis involves alleviating endoplasmic reticulum (ER) stress through the inositol-requiring enzyme 1 (IRE1), RNA-dependent protein kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF6) signaling pathways. The alleviation of ER stress may be linked to the phosphomannomutase (PMM)-mediated enhancement of protein glycosylation. In addition, ubiquitin-dependent [PTEN-induced putative kinase 1 (PINK1)/Parkin] and ubiquitin-independent [BCL2-interacting protein 3-like (BNIP3L), BCL2-interacting protein 3 (BNIP3), and FUN14 domain containing 1 (FUNDC1)] mitophagy may play a role in maintaining cellular redox homeostasis. The enhancement of intercellular structures includes enhancing tight junction and adherent junction structures, which may be closely associated with the small Rho GTPase protein (RhoA)/the Rho-associated protein kinase (ROCK) signaling pathway. In conclusion, D-mannose improved intestinal cellular redox homeostasis associated with ER stress and mitophagy pathways, and enhanced intercellular structures related to tight junctions and adherent junctions. Furthermore, quadratic regression analysis of the PWG and intestinal reactive oxygen species content indicated that the optimal addition level of D-mannose for juvenile grass carp was 4.61 and 4.59 g/kg, respectively.

Capillarity in Interfacial Liquids and Marbles: Mechanisms, Properties, and Applications.

The mechanics of capillary force in biological systems have critical roles in the formation of the intra- and inter-cellular structures, which may mediate the organization, morphogenesis, and homeostasis of biomolecular condensates. Current techniques may not allow direct and precise measurements of the capillary forces at the intra- and inter-cellular scales. By preserving liquid droplets at the liquid-liquid interface, we have discovered and studied ideal models, i.e., interfacial liquids and marbles, for understanding general capillary mechanics that existed in liquid-in-liquid systems, e.g., biomolecular condensates. The unexpectedly long coalescence time of the interfacial liquids revealed that the Stokes equation does not hold as the radius of the liquid bridge approaches zero, evidencing the existence of a third inertially limited viscous regime. Moreover, liquid transport from a liquid droplet to a liquid reservoir can be prohibited by coating the droplet surface with hydrophobic or amphiphilic particles, forming interfacial liquid marbles. Unique characteristics, including high stability, transparency, gas permeability, and self-assembly, are observed for the interfacial liquid marbles. Phase transition and separation induced by the formation of nanostructured materials can be directly observed within the interfacial liquid marbles without the need for surfactants and agitation, making them useful tools to research the interfacial mechanics.

Co-clustering of EphB6 and ephrinB1 in trans restrains cancer cell invasion

EphB6 is an understudied ephrin receptor tyrosine pseudokinase that is downregulated in multiple types of metastatic cancers. Unlike its kinase-active counterparts which autophosphorylate and transmit signals upon intercellular interaction, little is known about how EphB6 functions in the absence of intrinsic kinase activity. Here, we unveil a molecular mechanism of cell-cell interaction driven by EphB6. We identify ephrinB1 as a cognate ligand of EphB6 and show that in trans interaction of EphB6 with ephrinB1 on neighboring cells leads to the formation of large co-clusters at the plasma membrane. These co-clusters exhibit a decreased propensity towards endocytosis, suggesting a unique characteristic for this type of cell-cell interaction. Using lattice light-sheet microscopy, 3D structured illumination microscopy and cryo-electron tomography techniques, we show that co-clustering of EphB6 and ephrinB1 promotes the formation of double-membrane tubular structures between cells. Importantly, we also demonstrate that these intercellular structures stabilize cell–cell adhesion, leading to a reduction in the invasive behavior of cancer cells. Our findings rationalize a role for EphB6 pseudokinase as a tumor suppressor when interacting with its ligands in trans.

Role of Cx43 on the Bone Cell Generation, Function, and Survival.

The presence of gap junction intercellular communication structures in bone cells has been known since the early 1970s, further confirmed by Doty and Marotti at the structural level in the 1980-1990s. Work by Civitelli, Donahue, and others showed the expression of Cx43 at the mRNA and protein levels in all bone cell types: osteoclasts (bone resorbing cells), osteoblasts (bone forming cells), and osteocytes (mature osteoblasts embedded in the bone matrix that regulate the function of both osteoclasts and osteoblasts). While Cx45, Cx46, and Cx37 were also shown to be expressed in bone cells, most studies have focused on Cx43, the most abundant member of the connexin (Cx) family of proteins expressed in bone. The role of Cx43 has been shown to be related to the formation of gap junction intercellular channels, to unopposed hemichannels, and to channel independent functions of the molecule. Cx43 participates in the response of bone cells to pharmacological, hormonal, and mechanical stimuli, and it is involved in the skeletal phenotype with old age. Human and murine studies have shown that mutations of Cx43 lead to oculodentodigital dysplasia and craniometaphyseal dysplasia, both conditions associated with abnormalities in the skeleton. However, whereas substantial advances have been made on the skeletal role of Cx43, further research is needed to understand the basis for the effects of mutated Cx43 and potential ways to prevent the effects of these mutations on bone.

X-ray radiation-induced intestinal barrier dysfunction in human epithelial Caco-2 cell monolayers

Radiation therapy and unwanted radiological or nuclear exposure, such as nuclear plant accidents, terrorist attacks, and military conflicts, pose serious health issues to humans. Dysfunction of the intestinal epithelial barrier and the leakage of luminal antigens and bacteria across the barrier have been linked to various human diseases. Intestinal permeability is regulated by intercellular structures, termed tight junctions (TJs), which are disrupted after radiation exposure. In this study, we investigated radiation-induced alterations in TJ-related proteins in an intestinal epithelial cell model. Caco-2 cells were irradiated with 2, 5, and 10 Gy and harvested 1 and 24 h after X-ray exposure. The trypan blue assay revealed that cell viability was reduced in a dose-dependent manner 24 h after X-ray exposure compared to that of non-irradiated cells. However, the WST-8 assay revealed that cell proliferation was significantly reduced only 24 h after radiation exposure to 10 Gy compared to that of non-irradiated cells. In addition, a decreased growth rate and increased doubling time were observed in cells irradiated with X-rays. Intestinal permeability was significantly increased, and transepithelial electrical resistance values were remarkably reduced in Caco-2 cell monolayers irradiated with X-rays compared to non-irradiated cells. X-ray irradiation significantly decreased the mRNA and protein levels of ZO-1, occludin, claudin-3, and claudin-4, with ZO-1 and claudin-3 protein levels decreasing in a dose-dependent manner. Overall, the present study reveals that exposure to X-ray induces dysfunction of the human epithelial intestinal barrier and integrity via the downregulation of TJ-related genes, which may be a key factor contributing to intestinal barrier damage and increased intestinal permeability.

Identification of time of death of cats according to histological changes in some organs

Determining the time of an animal’s death is an extremely relevant subject in the practice of modern forensic medicine, because the issue often arises when investigating crimes related to animal abuse, in particular, with domestic cats Felis silvestris catus (Linnaeus, 1758). Methods of identifying time of cats’ death are currently developed insufficiently and their introduction into the veterinary-forensic practice has been limited, including the histological method we have employed. We used five corpses of cats, from which we periodically, after certain time periods, gathered the material for histological study, sampling such organs as the liver, kidneys, lungs, heart (myocardium), and the skeletal muscles. From the collected material, we made histological sections and stained them with hematoxylin and eosin. We determined the general patterns in changes occurring in the internal organs of cats over time, namely changes in the staining intensity of the cell elements and intercellular structures on the histopreparations; formation of detritus-filled cavities; a number of distinctive changes in the connective-tissue stroma of the organs; emergence of baciliform bacteria in the tissues and formation of their colonies. In the muscle tissue, we found specific features such as loss of alignment and emergence of transversal grooves with subsequent fragmentation. The study revealed that the development of those changes in each organ clearly correlated with time that has passed since each animals had died. Thus, a complex evaluation of detected changes can give a more accurate – compared with other existing methods – assessment of the time of death of a corpse submitted to forensic veterinary examination. We recommend using the histological method for animals presumed to have been dead for no more than 18 days, because later the tissue disintegration reaches such a level that complicates the correlation of changes with time. We consider it promising to study the time of death of domestic cats using other methods for identifying criteria for postmortem intervals longer than 18 days, and also identifying time of death in other species of animals.

The shaping of onion seedlings performance through substrate formulation and co-inoculation with beneficial microorganism consortia.

Smart management in crop cultivation is increasingly supported by application of arbuscular mycorrhizal fungi (AMF) and plant growth-promoting microorganisms (PGPM), which sustain soil fertility and plant performance. The aim of this study was the evaluation of the effects of consortia composed of (Claroideoglomus claroideum BEG96, Claroideoglomus etunicatum BEG92, Funneliformis geosporum BEG199, Funneliformis mosseae BEG 95, and Rhizophagus irregularis BEG140) and PGPM (Azospirillum brasilense - AZ, or Saccharothrix sp. - S) on onion cultivated in growing media with a composition corresponding to a degraded soil. Three types of substrate formulations were used, with peat:sand ratios of 50:50, 70:30, 100:0 (v:v). The analysis of substrate parameters crucial for its fertility (pH, salinity, sorption complex capacity, and elements' content) and characteristics reflecting onion seedlings' performance (fresh weight, stress biomarkers, and elements' content) was performed. AMF colonized onion roots in all treatments, showing increasing potential to form intercellular structures in the substrates rich in organic matter. Additionally, co-inoculation with PGPM microorganisms accelerated arbuscular mycorrhiza establishment. Increased antioxidant activity and glutathione peroxidase (GPOX) activity of onion roots sampled from the formulations composed of peat and sand in the ratio of 100:0, inoculated with AMF+S, and positive correlation between GPOX, fresh weight and antioxidant activity of onion roots reflected the successful induction of plant acclimatization response. Total phenols content was the highest in roots and leaves of onion grown in substrates with 70:30 peat:sand ratio, and, in the case of roots, it was correlated with AMF colonization parameters but not with antioxidant activity. AMF and PGPM efficiency in supporting onion growth should be linked to the increased onion root system capacity in mineral salts absorption, resulting in more efficient aboveground biomass production. AMF and PGPM consortia were effective in releasing minerals to soluble fraction in substrates rich in organic matter, making elements available for uptake by onion root system, though this phenomenon depended on the PGPM species. Microorganism consortia enhanced onion seedlings' performance also in substrates with lower content of organic carbon through plant biofertilization and phytostimulation.

Modulation of Piezo1 influences human skin architecture and oxytocin expression.

Throughout our existence, the skin senses and analyses the mechanical forces imposed by the environment. In response to these environmental forces, skin can deform itself and achieve a biological response. The subsequent cutaneous plasticity emerges from mechanical properties arising from the collective action of the skin cells, particularly keratinocytes, that govern the tensile strength via cell-to-cell adhesions and via cell-matrix adhesion structures. In addition to serving as force-bearing entities, keratinocytes respond to forces by activating signalling pathways to control their own fate and function. To detect and adapt to mechanical signals, keratinocytes possess a panel of sensory receptors and junctional intercellular structures. Mechanically activated ion channel Piezo1 has been described as a force sensor and as being involved in pleasant touch perception. In this study, relationships between Piezo1 modulation and oxytocin synthesis were investigated. The expression of Piezo1 in the skin was studied and compared with the expression of TRPV1. Dooku1 antagonist and Jedi1 agonist were used to modulate Piezo1. The level of E-cadherin and oxytocin was monitored in ex vivo skin biopsies by immunodetection. Taken together, our results illustrate the major role of mechanosensitive ion channel Piezo1 in skin barrier integrity, and in peripheral oxytocin synthesis in the skin. In conclusion, this study highlights the relationships between pleasant touch, soft touch and local oxytocin synthesis.

Revealing the performance of phenoxyethanol-acid pretreatment on the intercellular structure of bamboo and its enzymatic hydrolysis

To understand the effect of sulfuric acid-based phenoxyethanol pretreatment on the enzymatic digestibility of bamboo, the changes of lignin/xylan content and their intercellular structures in pretreated bamboo were investigated by different spectrum technologies. The maximum removals of lignin and xylan achieved by pretreatment with phenoxyethanol-acid (1%, w/v) ratio of 4:1 at 120 °C resulted in pretreated bamboo with an enzymatic digestibility of 79.25%. Micro-computed tomography revealed that the macrostructure of bamboo cell wall was destroyed, exposing the macrofibers. Based on the fluorescence intensity of lignin and antibody-labeled xylan, measured by fluorescence microscopy, most of the lignin was removed from the intercellular complex layer in thin-walled cells, and the destruction of main chain of hemicellulose was more severe than that of branched chains during the pretreatment process. In addition, the number of sub-structures, functional groups, and molecular weights of dissolved lignin in the pretreatment hydrolysate was closely related to the phenoxyethanol-acid ratio employed during pretreatment. Compared with the control group, the structures of dissolved lignin changed more significantly as the percentage of phenoxyethanol in the system increased. When the proportion of phenoxyethanol in the system increased, the number of substructures (β-O-4, β-5, and β-β) and functional groups showed a decreasing trend.

Deep Learning to Detect the Function of Calmodulin in Alzheimer’s Disease

Alzheimer’s disease (AD) is marked by losses in cognitive functions. Hallmarks of AD, amyloid beta (Aβ) plaques and neurofibrillary tangles, are intercellular structures that are highly associated with AD. The accumulations of these molecules could be seen as results of other events in prior stages of AD. The calcium hypothesis was first proposed to explain the forming of these hallmarks while the calmodulin (CaM) hypothesis was proposed to provide more improved insight later. The CaM hypothesis states that the uncontrolled flux of calcium ions in the neurons is the cause of neurodegeneration. In addition, the critical role of CaM and CaM-binding proteins (CaMBPs) are identified in the hypothesis. In this project, the regulating role of CaM and CaM-related protein in AD are discussed, including CaMBPs, critical receptors and risk factors. Besides, the potential therapeutic target related to CaM are also mentioned to provide a promise direction of treatment for AD.

Multimodal study of CHI3L1 inhibition and its effect on angiogenesis, migration, immune response and refractive index of cellular structures in glioblastoma

Glioblastoma is one of the most aggressive tumours with a poor response to treatment and a poor prognosis for patients. One of the proteins expressed in glioblastoma tissue is CHI3L1 (YKL-40), which is upregulated and known for its angiogenesis-supporting and pro-tumour immunomodulatory effects in a variety of cancers. In this paper we present the anti-angiogenic, anti-migratory and immunomodulatory effects of the compound G721–0282, an inhibitor of CHI3L1. The inhibitor-induced changes were investigated using conventional techniques as well as the novel label-free digital holographic tomography (DHT), a quantitative phase imaging technique that allows the reconstruction of the refractive index (RI), which is used as an image contrast for 3D visualisation of living cells. DHT allowed digital staining of individual cells and intercellular structures based only on their specific RI. Quantitative spatially resolved analysis of the RI data shows that the concentration of G721–0282 leads to significant changes in the density of cells and their intracellular structures (in particular the cytoplasm and nucleus), in the volume of lipid droplets and in protein concentrations. Studies in the U-87 MG glioblastoma cell line, THP-1 monocytes differentiated into macrophages, human microvascular endothelial cells (HMEC-1) and in the spheroid model of glioblastoma composed of U-87 MG, HMEC-1 and macrophages suggest that inhibition of CHI3L1 may have potential in the antitumour treatment of glioblastoma. In this paper, we also propose a spheroid model for in vitro studies that mimics this type of tumour.

Conditional knockout of transient receptor potential melastatin 7 in the enamel epithelium: Effects on enamel formation.

Transient receptor potential melastatin 7 (TRPM7) is a unique ion channel connected to a kinase domain. We previously demonstrated that Trpm7 expression is high in mouse ameloblasts and odontoblasts, and that amelogenesis is impaired in TRPM7 kinase-dead mice. Here, we analyzed TRPM7 function during amelogenesis in Keratin 14-Cre;Trpm7fl/fl conditional knockout (cKO) mice and Trpm7 knockdown cell lines. cKO mice showed lesser tooth pigmentation than control mice and broken incisor tips. Enamel calcification and microhardness were lower in cKO mice. Electron probe microanalysis (EPMA) showed that the calcium and phosphorus contents in the enamel were lower in cKO mouse than in control mice. The ameloblast layer in cKO mice showed ameloblast dysplasia at the maturation stage. The morphological defects were observed in rat SF2 cells with Trpm7 knockdown. Compared with mock transfectants, the Trpm7 knockdown cell lines showed lower levels of calcification with Alizarin Red-positive staining and an impaired intercellular adhesion structures. These findings suggest that TRPM7 is a critical ion channel in enamel calcification for the effective morphogenesis of ameloblasts during amelogenesis.

Single-objective selective plane illumination microscopy for multi-well organoid imaging.

Current preclinical in vitro and non-human animal models fail to adequately reproduce human physiology and disease processes. While recent efforts have focused on using microfluidics to help create stem cell-derived organoids as a way to better model 3D vascularization and improve the delivery of therapeutics, there remain challenges especially in terms of long-term imaging. To address this, we report here on the development of single-objective Selective Plane Illumination Microscopy (SoSPIM) for high-content imaging. This platform is capable of imaging samples in different mounting formats, ranging from tissue slices, to conventional culture dishes and and multi-well plates. To demonstrate this platform's capabilities, we imaged cerebral organoid slices and fixed organoids cultured in multi-well microfluidic devices. Our future work will focus on applying SoSPIM for long-term live organoid imaging using machine learning approaches for tracking and mapping intercellular dynamics and structures.

Distribution of Domains Formed by Lateral Packing of Intercellular Lipid in the Stratum Corneum.

Intercellular lipids fill the interstices of corneocytes and serve a barrier function. The amount of transdermal water evaporation varies depending on the packing structure of intercellular lipids, as this structure is important for maintaining barrier efficacy. This packing structure consists of a mixture of crystals (orthorhombic and hexagonal) and liquid crystals (fluid phase), and the proportion of these phases is thought to affect barrier function. However, there have been no methods to visualize the actual distribution of the domains formed by packing structure in intercellular lipids. In this study, the planar distribution of intercellular lipid structures was determined using focal plane array (FPA)-based Fourier transform (FT) IR imaging analysis of stratum corneum cell units obtained by grid stripping. The lipid composition of ceramides was revealed by electrospray ionization tandem mass spectrometry (ESI-MS/MS)-based shotgun lipidomics. The distribution of domains formed by packing structures and the lipid composition of ceramides was compared in skin with high- or low-transepidermal water loss (TEWL). The orthorhombic proportion was lower in high-TEWL skin than in low-TEWL skin. ESI-MS/MS-based shotgun lipidomics analysis showed that the alpha-hydroxyceramide content in the low- and high-TEWL groups differed regarding the distribution of fatty acid chain lengths. The evaluation of stratum corneum cell units using FPA-based FTIR imaging is an innovative technology that can visualize the distribution of domains formed by intercellular lipid-packing structures. Increased proportions of alpha-hydroxyceramide subclasses such as alpha-hydroxy-sphingosine ceramide and alpha-hydroxy-phytosphingosine ceramide were associated with a reduced proportion of the orthorhombic packing structure domain.

Probing the interstitial calcium compartment.

Calcium in interstitial fluids is a crucial ion pool for entry into cells through a plethora of calcium-permeable channels. It is also sensed actively by dedicated receptors. While the mechanisms of global calcium homeostasis and regulation in body fluids appear well understood, more efforts and new technology are needed to elucidate local calcium handling in the small and relatively isolated interstitial spaces between cells. Here we review current methodology for monitoring interstitial calcium and highlight the potential of new approaches for its study. In particular, new generations of high-performance low-affinity genetically encoded calcium indicators could allow imaging of calcium in relatively inaccessible intercellular structures in live tissues and organisms.

SPROUT: spectral sparsification helps restore the spatial structure at single-cell resolution.

Single-cell RNA sequencing thoroughly quantifies the individual cell transcriptomes but renounces the spatial structure. Conversely, recently emerged spatial transcriptomics technologies capture the cellular spatial structure but skimp cell or gene resolutions. Ligand-receptor interactions reveal the potential of cell proximity since they are spatially constrained. Cell–cell affinity values estimated by ligand–receptor interaction can partially represent the structure of cells but falsely include the pseudo affinities between distant or indirectly interacting cells. Here, we develop a software package, SPROUT, to reconstruct the single-cell resolution spatial structure from the transcriptomics data through diminished pseudo ligand–receptor affinities. For spatial data, SPROUT first curates the representative single-cell profiles for each spatial spot from a candidate library, then reduces the pseudo affinities in the intercellular affinity matrix by partial correlation, spectral graph sparsification, and spatial coordinates refinement. SPROUT embeds the estimated interactions into a low-dimensional space with the cross-entropy objective to restore the intercellular structures, which facilitates the discovery of dominant ligand–receptor pairs between neighboring cells at single-cell resolution. SPROUT reconstructed structures achieved shape Pearson correlations ranging from 0.91 to 0.97 on the mouse hippocampus and human organ tumor microenvironment datasets. Furthermore, SPROUT can solely de novo reconstruct the structures at single-cell resolution, i.e., reaching the cell-type proximity correlations of 0.68 and 0.89 between reconstructed and immunohistochemistry-informed spatial structures on a human developing heart dataset and a tumor microenvironment dataset, respectively.

SIRT1 regulates cardiomyocyte alignment during maturation.

Cardiomyocyte elongation and alignment, a critical step in cardiomyocyte maturation starting from the perinatal stage, is crucial for formation of the highly organized intra- and inter-cellular structures for spatially and temporally ordered contraction in adult cardiomyocytes. However, the mechanism(s) underlying the control of cardiomyocyte alignment remains elusive. Here, we report that SIRT1, the most conserved NAD+-dependent protein deacetylase highly expressed in perinatal heart, plays an important role in regulating cardiomyocyte remodeling during development. We observed that SIRT1 deficiency impairs the alignment of cardiomyocytes/myofibrils and disrupts normal beating patterns at late developmental stages in an in vitro differentiation system from human embryonic stem cells. Consistently, deletion of SIRT1 at a late developmental stage in mouse embryos induced the irregular distribution of cardiomyocytes and misalignment of myofibrils, and reduced the heart size. Mechanistically, the expression of several genes involved in chemotaxis, including those in the CXCL12/CXCR4 and CCL2/CCR2/CCR4 pathways, was dramatically blunted during maturation of SIRT1-deficient cardiomyocytes. Pharmacological inhibition of CCL2 signaling suppressed cardiomyocyte alignment. Our study identifies a regulatory factor that modulates cardiomyocyte alignment at the inter-cellular level during maturation.

Side-Directed Release of Differential Extracellular Vesicle-associated microRNA Profiles from Bronchial Epithelial Cells of Healthy and Asthmatic Subjects.

Extracellular vesicles (EVs) are released by virtually all cells and may serve as intercellular communication structures by transmitting molecules such as proteins, lipids, and nucleic acids between cells. MicroRNAs (miRNAs) are an abundant class of vesicular RNA playing a pivotal role in regulating intracellular processes. In this work, we aimed to characterize vesicular miRNA profiles released in a side-directed manner by bronchial epithelial cells from healthy and asthmatic subjects using an air−liquid interface cell culture model. EVs were isolated from a culture medium collected from either the basolateral or apical cell side of the epithelial cell cultures and characterized by nano-flow cytometry (NanoFCM) and bead-based flow cytometry. EV-associated RNA profiles were assessed by small RNA sequencing and subsequent bioinformatic analyses. Furthermore, miRNA-associated functions and targets were predicted and miRNA network analyses were performed. EVs were released at higher numbers to the apical cell side of the epithelial cells and were considerably smaller in the apical compared to the basolateral compartment. EVs from both compartments showed a differential tetraspanins surface marker expression. Furthermore, 236 miRNAs were differentially expressed depending on the EV secretion side, regardless of the disease phenotype. On the apical cell side, 32 miRNAs were significantly altered in asthmatic versus healthy conditions, while on the basolateral cell side, 23 differentially expressed miRNAs could be detected. Downstream KEGG pathway analysis predicted mTOR and MAPK signaling pathways as potential downstream targets of apically secreted miRNAs. In contrast, miRNAs specifically detected at the basolateral side were associated with processes of T and B cell receptor signaling. The study proves a compartmentalized packaging of EVs by bronchial epithelial cells supposedly associated with site-specific functions of cargo miRNAs, which are considerably affected by disease conditions such as asthma.

Preparing Excitable Cardiac Papillary Muscle and Cardiac Slices for Functional Analyses.

While the reductionist approach has been fruitful in understanding the molecular basis of muscle function, intact excitable muscle preparations are still important as experimental model systems. We present here methods that are useful for preparing cardiac papillary muscle and cardiac slices, which represent macroscopic experimental model systems with fully intact intercellular and intracellular structures. The maintenance of these in vivo structures for experimentation in vitro have made these model systems especially useful for testing the functional effects of protein mutations and pharmaceutical candidates. We provide solutions recipes for dissection and recording, instructions for removing and preparing the cardiac papillary muscles, as well as instruction for preparing cardiac slices. These instructions are suitable for beginning experimentalists but may be useful for veteran muscle physiologists hoping to reacquaint themselves with macroscopic functional analyses.