Specialized Junctions Research Articles

Mouse-derived Synaptosomes Trypsin Cleavage Assay to Characterize Synaptic Protein Sub-localization.

Neurons communicate through neurotransmission at highly specialized junctions called synapses. Each neuron forms numerous synaptic connections, consisting of presynaptic and postsynaptic terminals. Upon the arrival of an action potential, neurotransmitters are released from the presynaptic site and diffuse across the synaptic cleft to bind specialized receptors at the postsynaptic terminal. This process is tightly regulated by several proteins at both presynaptic and postsynaptic sites. The localization, abundance, and function of these proteins are essential for productive neurotransmission and are often affected in neurological and neurodegenerative disorders. Here, we outline a method for purifying mouse synaptosomes and using limited tryptic digestion to assess the subcellular localization of synaptic proteins. During synaptosomes purification, presynaptic terminals reseal and are protected from proteolysis, while postsynaptic proteins remain susceptible to tryptic cleavage. These changes can easily be evaluated by western blot analysis. This approach offers a straightforward and reliable method to evaluate the subcellular localization of synaptic proteins based on their proteolytic sensitivity, providing valuable insights into synaptic physiology and pathology. Key features • Builds upon the method developed by Boyken et al. [1] and introduces the use of isolated mouse synaptosomes to assess synaptic protein sub-localization. • Limited tryptic digestion differentiates between presynaptic and postsynaptic proteins based on proteolytic sensitivity. • Requires standard biochemical reagents and western blotting equipment and can be completed in two/three days, including synaptosome purification and western blot analysis. Graphical overview Overview of the synaptosomes trypsin cleavage assay. This protocol describes the isolation of synaptosomes from mouse brain tissue, followed by limited trypsin digestion to assess the compartmental localization of synaptic proteins. Synaptosomes, which are isolated via differential centrifugation, consist of resealed presynaptic terminals that are protected from proteolysis, while the exposed postsynaptic compartments are accessible to trypsin and undergo proteolytic cleavage. Following digestion, samples are analyzed using SDS-PAGE and western blotting. Proteins of interest are probed using specific antibodies to determine whether they are presynaptic or postsynaptic. Presynaptic proteins (e.g., synaptophysin, SNAP-25) remain intact, while postsynaptic proteins (e.g., GluA1, GluN2A) are cleaved.

Anatomical design and production of a novel three-dimensional co-culture system replicating the human flexor digitorum profundus enthesis.

The enthesis, the specialized junction between tendon and bone, is a common site of injury. Although notoriously difficult to repair, advances in interfacial tissue engineering techniques are being developed for restorative function. Most notably are 3D invitro co-culture models, built to recreate the complex heterogeneity of the native enthesis. While cell and matrix properties are often considered, there has been little attention given to native enthesis anatomical morphometrics and replicating these to enhance clinical relevance. This study focuses on the flexor digitorum profundus (FDP) tendon enthesis and, by combining anatomical morphometrics with computer-aided design, demonstrates the design and construction of an accurate and scalable model of the FDP enthesis. Bespoke 3D-printed mould inserts were fabricated based on the size, shape and insertion angle of the FDP enthesis. Then, silicone culture moulds were created, enabling the production of bespoke anatomical culture zones for an invitro FDP enthesis model. The validity of the model has been confirmed using brushite cement scaffolds seeded with osteoblasts (bone) and fibrin hydrogel scaffolds seeded with fibroblasts (tendon) in individual studies with cells from either human or rat origin. This novel approach allows a bespoke anatomical design for enthesis repair and should be applied to future studies in this area.

Synaptic alterations as a common phase in neurological and neurodevelopmental diseases: JNK is a key mediator in synaptic changes.

Synaptic alterations as a common phase in neurological and neurodevelopmental diseases: JNK is a key mediator in synaptic changes.

Identification of a novel mechanism for LFA-1 organization during NK cytolytic response.

The elimination of transformed and viral infected cells by natural killer (NK) cells requires a specialized junction between NK and target cells, denominated immunological synapse (IS). After initial recognition, the IS enables the directed secretion of lytic granules content into the susceptible target cell. The lymphocyte function-associated antigen (LFA)-1 regulates NK effector function by enabling NK-IS assembly and maturation. The pathways underlying LFA-1 accumulation at the IS in NK cells remained uncharacterized. A kinase anchoring protein 350 (AKAP350) is a centrosome/Golgi-associated protein, which, in T cells, participates in LFA-1 activation by mechanisms that have not been elucidated. We first evaluated AKAP350 participation in NK cytolytic activity. Our results showed that the decrease in AKAP350 levels by RNA interference (AKAP350KD) inhibited NK-YTS cytolytic activity, without affecting conjugate formation. The impairment of NK effector function in AKAP350KD cells correlated with decreased LFA-1 clustering and defective IS maturation. AKAP350KD cells that were exclusively activated via LFA-1 showed impaired LFA-1 organization and deficient lytic granule translocation as well. In NK AKAP350KD cells, activation signaling through Vav1 was preserved up to 10 min of interaction with target cells, but significantly decreased afterwards. Experiments in YTS and in ex vivo NK cells identified an intracellular pool of LFA-1, which partially associated with the Golgi apparatus and, upon NK activation, redistributed to the IS in an AKAP350-dependent manner. The analysis of Golgi organization indicated that the decrease in AKAP350 expression led to the disruption of the Golgi integrity in NK cells. Alteration of Golgi function by BFA treatment or AKAP350 delocalization from this organelle also led to impaired LFA-1 localization at the IS. Therefore, this study characterizes AKAP350 participation in the modulation of NK effector function, revealing the existence of a Golgi-dependent trafficking pathway for LFA-1, which is relevant for LFA-1 organization at NK-lytic IS.

Development and evaluation of ligand Binded Phytoactive extract loaded SLN for brain targeting

Drug delivery to the brain is the process of passing therapeutically active molecules across the Blood Brain Barrier for the purpose of treating brain disease. This is a complex process that must take into account the complex anatomy of the brain as well as the restrictions imposed by the special junctions of the Blood Brain Barrier. In response to the insufficiency in conventional delivery mechanisms, aggressive research efforts have recently focused on the development of new strategies to more efficiently deliver drug molecules to the CNS.The objective of present work deals with the preparation ofdevelopment and evaluation of ligand binded phytoactive extract loaded SLN for brain targeting.Solid lipid nanoparticles (SLNs) are spherical solid lipid nanoparticles in nanorange, easiy dispersibe in water or in aqueous surfactant solution present themselves as suitable candidate for drug delivery to the brain. These nanoparticles can easily penetrate blood brain barrier (BBB) and has good stability & lesser toxicity. In present work, phytoactive loaded SLN conjugated with lactoferrin was prepared by solvent evaporation method and was characterized for particle size, drug loading entrapment, ligend binding & targetability to brain cells.

Control of Synapse Structure and Function by Actin and Its Regulators.

Neurons transmit and receive information at specialized junctions called synapses. Excitatory synapses form at the junction between a presynaptic axon terminal and a postsynaptic dendritic spine. Supporting the shape and function of these junctions is a complex network of actin filaments and its regulators. Advances in microscopic techniques have enabled studies of the organization of actin at synapses and its dynamic regulation. In addition to highlighting recent advances in the field, we will provide a brief historical perspective of the understanding of synaptic actin at the synapse. We will also highlight key neuronal functions regulated by actin, including organization of proteins in the pre- and post- synaptic compartments and endocytosis of ion channels. We review the evidence that synapses contain distinct actin pools that differ in their localization and dynamic behaviors and discuss key functions for these actin pools. Finally, whole exome sequencing of humans with neurodevelopmental and psychiatric disorders has identified synaptic actin regulators as key disease risk genes. We briefly summarize how genetic variants in these genes impact neurotransmission via their impact on synaptic actin.

Neuronal functions of clathrin-associated endocytic sorting adaptors – from molecules to disease

Abstract Communication in the central nervous system is based on the transmission of electrical signals at specialized junctions between nerve cells termed synapses. During chemical neurotransmission, tiny membrane spheres called synaptic vesicles that are packed with neurotransmitters elicit a postsynaptic response by fusing with the presynaptic membrane and releasing their content into the synaptic cleft. Synaptic vesicle fusion is followed by the reuptake of the membrane by endocytosis and the local reformation of functional synaptic vesicles within the presynaptic compartment to sustain further rounds of neurotransmitter release. Here, we provide an overview of the clathrin-associated endocytic adaptor proteins that help to sort and recycle synaptic vesicles during presynaptic activity. These adaptors also serve additional functions in the turnover of defective or aged synaptic components and in the retrograde axonal transport of important signaling molecules by regulating the formation or transport of autophagosomes. Endocytic adaptors thus play multiple roles in the maintenance of synaptic function. Defects in their expression or function can lead to neurodegenerative and neurological diseases.

Fine structure of the cardiac muscle cells in the orb-web spider Nephila clavata

The fine structural characteristics of cardiac muscle cells and its myofibril organization in the orb web spider N. clavata were examined by transmission electron microscopy. Although myofibril striations are not remarkable as those of skeletal muscles, muscle fibers contain multiple myofibrils, abundant mitochondria, extensive sarcoplasmic reticulum and transverse tubules (T-tubules). Myofibrils are divided into distinct sarcomeres defined by Z-lines with average length of 2.0 μm, but the distinction between the A-band and the I-bands is not clear due to uniform striations over the length of the sarcomeres. Dyadic junction which consisted of a single T-tubule paired with a terminal cisterna of the sarcoplasmic reticulum is found mainly at the A-I level of sarcomere. Each cell is arranged to form multiple connections with neighboring cells through the intercalated discs. These specialized junctions include three types of intercellular junctions: gap junctions, fascia adherens and desmosomes for heart function. Our transmission electron microscopy (TEM) observations clearly show that spider’s cardiac muscle contraction is controlled by neurogenic rather than myogenic mechanism since each cardiac muscle fiber is innervated by a branch of motor neuron through neuromuscular junctions.

Релацията „стратегически иновации – ключови компетентности“ в условията на динамична конкурентна среда

The focus is placed on the leading issue in mesoeconomics: strategic innovation and the competitive mobility of business. The strategic innovations of companies are analyzed systematically in terms of the creation and distribution of value (worth) that is different from other market values, while key competencies are discussed as “special junctions” of routines and technologies without which it is impossible to diversify the potential markets. The necessity for the objective contradiction between competencies and markets to be continually overcome through strategic decisions and how the key competency for competitive cooperation (Co-opetition) becomes a strategic necessity in the dynamic conditions of the Fourth Industrial Revolution are justified. The analysis of the joint creation of new value factors with the help of clusters and other strategic alliances is carried out based on the following criteria: the minimization of possible costs and the maximization of possible sources for gaining added value; balancing the potential risks of interaction in the value network related to the contribution of each company towards achieving the common goal.

Theoretical study of network junction models for totally asymmetric exclusion processes with interacting particles

Biological transport phenomena frequently exhibit complex network behavior when several molecular fluxes converge to special junctions from which another fluxes move out in different directions. Similar behavior is also observed in vehicular transport. Stimulated by these observations, we developed a theoretical framework to investigate network junction models of totally asymmetric simple exclusion processes with interacting particles. Utilizing a two-site cluster mean field framework that takes into account some correlations in the system, stationary dynamic properties, such as phase diagrams, density profiles and correlations profiles, are explicitly evaluated. It is found that the number of stationary phases strongly depend on the number of segments that come and leave the network junction. The inter-particle interactions also have a strong effect of dynamic properties of the system. Our method can be extended to the systems with several junctions. All theoretical predictions are in good agreement with extensive Monte Carlo computer simulations.

Lamina propria: The connective tissue of rat urinary bladder mucosa.

To describe and illustrate the structure of the propria, the bladder of adult rats was fixed in controlled conditions of distension and examined by light and electron microscopy. The lamina propria, ~50 µm thick in the distended bladder, consists of a superficial part (the cellular component), adjacent to the urothelium, rich in nerves, capillaries, fibroblasts and thin bundles of collagen, and a deep, thicker part (the fibrous component), adjacent to the detrusor, rich in large collagen fibres and with few fibroblasts. In the cellular part there is an extensive plexus of afferent nerve fibers and a dense capillary network (with numerous pericytes), lying close to the urothelium, that is unique to the bladder. The main resident cells are fibroblasts, adhering to each other at the end of laminar extensions without forming specialized junctions. The deep part of the lamina propria is made of thick collagen fibers, interwoven and crisscrossing each other, with a few fibroblasts in the interstitial spaces between them. In summary, the superficial part of the lamina propria has most of the bladder afferent nerves, contains many fibroblasts and has a network of suburothelial capillaries. The deep part as a whole forms an ovoid balloon of woven fibrous material that is acted upon by the detrusor musculature attached to its outer surface. The lamina propria is a strong fibrous barrier between urothelium and musculature. The abundance of collagen points to the main role for its fibroblasts, that is, the production of collagen fibrils, assisting the mechanical role of the lamina propria.

Compensatory Changes in Mauthner Neurons in Goldfish Induced by Sensory Stimulation and Application of β-Amyloid

Objectives. To study the structure and formation of Mauthner neuron dendrites in goldfish exposed to neurotoxic β-amyloid fragment 25–35 and prolonged sensory stimulation influencing the afferent inputs to these neurons. Materials and methods. Goldfish Mauthner neurons were studied by light and electron microscopy. Individual dendrites were identified, their volumes were determined, and synapse structure was assessed using virtual 3D images of Mauthner neurons obtained from serial sections of thickness 3 μm. The functional status of Mauthner neurons was evaluated indirectly from the motor lateralization of the fish. Results. Mauthner neurons responded to application of β-amyloid combined with subsequent prolonged sensory stimulation with decreases in the volume of the ventral dendrites, damage to their ultrastructure, and degeneration of a proportion of synapses. Degeneration of more active neurons was more significant than that of less active cells. Newly formed medial dendrites had greater volume and less damaged synapse ultrastructure than ventral dendrites. There were no differences in the sizes of specialized junctions between synapses of the same type on ventral and medial synapses. Conclusions. Medial dendrite formation is a compensatory reaction to dystrophy of the ventral dendrite due to the experimental treatment.

Pareto-Optimal Coupling Conditions for the Aw--Rascle--Zhang Traffic Flow Model at Junctions

This article deals with macroscopic traffic flow models on a road network. More precisely, we consider coupling conditions at junctions for the Aw--Rascle--Zhang second order model consisting of a hyperbolic system of two conservation laws. These coupling conditions conserve both the number of vehicles and the mixing of Lagrangian attributes of traffic through the junction. The proposed Riemann solver is based on assignment coefficients, multiobjective optimization of fluxes, and priority parameters. We prove that this Riemann solver is well-posed in the case of special junctions, including 1-to-2 diverge and 2-to-1 merge.

Getting Nervous: An Evolutionary Overhaul for Communication.

The evolution of a nervous system as a control system of the body's functions is a key innovation of animals. Its fundamental units are neurons, highly specialized cells dedicated to fast cell-cell communication. Neurons pass signals to other neurons, muscle cells, or gland cells at specialized junctions, the synapses, where transmitters are released from vesicles in a Ca2+-dependent fashion to activate receptors in the membrane of the target cell. Reconstructing the origins of neuronal communication out of a more simple process remains a central challenge in biology. Recent genomic comparisons have revealed that all animals, including the nerveless poriferans and placozoans, share a basic set of genes for neuronal communication. This suggests that the first animal, the Urmetazoan, was already endowed with neurosecretory cells that probably started to connect into neuronal networks soon afterward. Here, we discuss scenarios for this pivotal transition in animal evolution.

Regulation of Endoplasmic Reticulum-Mitochondria Ca2+ Transfer and Its Importance for Anti-Cancer Therapies.

Inter-organelle membrane contact sites are emerging as major sites for the regulation of intracellular Ca2+ concentration and distribution. Here, extracellular stimuli operate on a wide array of channels, pumps, and ion exchangers to redistribute intracellular Ca2+ among several compartments. The resulting highly defined spatial and temporal patterns of Ca2+ movement can be used to elicit specific cellular responses, including cell proliferation, migration, or death. Plasma membrane (PM) also can directly contact mitochondria and endoplasmic reticulum (ER) through caveolae, small invaginations of the PM that ensure inter-organelle contacts, and can contribute to the regulation of numerous cellular functions through scaffolding proteins such as caveolins. PM and ER organize specialized junctions. Here, many components of the receptor-dependent Ca2+ signals are clustered, including the ORAI1-stromal interaction molecule 1 complex. This complex constitutes a primary mechanism for Ca2+ entry into non-excitable cells, modulated by intracellular Ca2+. Several contact sites between the ER and mitochondria, termed mitochondria-associated membranes, show a very complex and specialized structure and host a wide number of proteins that regulate Ca2+ transfer. In this review, we summarize current knowledge of the particular action of several oncogenes and tumor suppressors at these specialized check points and analyze anti-cancer therapies that specifically target Ca2+ flow at the inter-organelle contacts to alter the metabolism and fate of the cancer cell.

Abstract 114: Myocardin-Related Transcription Factors Are Required for Compensatory Signaling in Maladaptive Cardiac Remodeling

Intercellular communication is essential for coordinating the contraction of cardiomyocytes. This synchrony is established by coupling electrical activity and force transmission at specialized junctions called intercalated discs (ICDs). We have recently found that myocardin-related transcription factor A (MRTF-A) and –B (MRTFs) accumulate at ICDs of healthy mouse and human hearts, but are redistributed in cardiac pathology. MRTFs are mechanosensitive transcriptional co-activators of serum response factor (SRF) that regulate the actin cytoskeleton; however, the role of MRTFs in cardiomyocyte homeostasis remains unclear. We conditionally deleted Mrtfb in cardiomyocytes of mice harboring a homozygous null allele of Mrtfa (called MRTF cmdKO ). MRTF cmdKO mice exhibit no changes in cardiac function for up to a year; however, MRTF depletion results in conspicuous gap junction remodeling at ICDs and reduced cardiomyocyte communication. This phenotype stems from a significant reduction in genes important for trafficking gap junction proteins, including RP/EB family member 1 ( Mapre1 ), which we show is a direct transcriptional target of SRF/MRTFs. The absence of a functional phenotype amid gap junction displacement prompted us to model heart failure and mechanical stress at ICDs by subjecting MRTF cmdKO mice to transaortic constriction (TAC) surgery. Unexpectedly, MRTF cmdKO mice display partially penetrant sudden cardiac death within 2 weeks post-TAC surgery. MRTF cmdKO survivors undergo accelerated heart failure with a reduced adaptive gene response compared to controls. Future investigations are underway to determine the mechanisms governing accentuated heart failure in MRTF cmdKO mice. Taken together, the subcellular localization of MRTFs to ICDs creates a paradigm in which potent transcription factors are anchored to regions of electromechanical stress, which may represent a signaling circuit for ICD maintenance and remodeling in disease.

SENP3 grants tight junction integrity and cytoskeleton architecture in mouse Sertoli cells.

Germ cells develop in a sophisticated immune privileged microenvironment provided by specialized junctions contiguous the basement membrane of the adjacent Sertoli cells that constituted the blood-testis barrier (BTB) in seminiferous epithelium of testis in mammals. Deciphering the molecular regulatory machinery of BTB activity is central to improve male fertility and the role of post-translational modification including SUMOylation pathway is one of the key factors. Herein, we unveiled the mystery of the SUMO-2/3 specific protease SENP3 (Sentrin-specific protease 3) in BTB dynamics regulation. SENP3 is predominantly expressed in the nucleus of Sertoli and spermatocyte cells in adult mouse testis, and knockdown of SENP3 compromises tight junction in Sertoli cells by destructing the permeability function with a concomitant decline in trans-epithelial electrical resistance in primary Sertoli cells, which could attribute to the conspicuous dysfunction of tight junction (TJ) proteins (e.g., ZO-1, occludin) at the cell-cell interface due to the inactivation of STAT3. Moreover, SENP3 knockdown disrupts F-actin architecture in Sertoli cells through intervening Rac1/CDC42-N-WASP-Arp2/3 signaling pathway and Profilin-1 abundance. Our study pinpoints SENP3 might be a novel determinant of multiple pathways governing BTB dynamics in testis to support germ cells development in mammals.

Nanochimneys: Topology and Thermal Conductance of 3D Nanotube–Graphene Cone Junctions

Pillared 3D carbon architectures, with the graphene layers and carbon nanotubes connected by topological junctions, have been produced and observed, as reported recently. However, the atomistic details of such junctions are hard to discern in microscopy and remain presently unclear. The simplest junction contains six heptagons in the transition region between the nanotube and graphene. Although these junctions make the pillared architectures possible, they are susceptible to failure when the whole structure undergoes mechanical or thermal stress. In this work we consider “nanochimneys”, a variety of special junctions with cones in between the nanotube and graphene parts. We explore the structures of the nanochimneys (NCs) and determine their underlying topological requirements. We also study the thermal conductance of these pillared architectures and show that NCs conduct heat better than regular simple junctions.

The Riemann Problem at a Junction for a Phase Transition Traffic Model

We extend the Phase Transition model for traffic proposed in [8], by Colombo, Marcellini, and Rascle to the network case. More precisely, we consider the Riemann problem for such a system at a general junction with $n$ incoming and $m$ outgoing roads. We propose a Riemann solver at the junction which conserves both the number of cars and the maximal speed of each vehicle, which is a key feature of the Phase Transition model. For special junctions, we prove that the Riemann solver is well defined.

MAGI-1 Interacts with Nephrin to Maintain Slit Diaphragm Structure through Enhanced Rap1 Activation in Podocytes

MAGI-1 is a multidomain cytosolic scaffolding protein that in the kidney is specifically located at the podocyte slit diaphragm, a specialized junction that is universally injured in proteinuric diseases. There it interacts with several essential molecules, including nephrin and neph1, which are required for slit diaphragm formation and as an intracellular signaling hub. Here, we show that diminished MAGI-1 expression in cultured podocytes reduced nephrin and neph1 membrane localization and weakened tight junction integrity. Global magi1 knock-out mice, however, demonstrated normal glomerular histology and function into adulthood. We hypothesized that a second mild but complementary genetic insult might induce glomerular disease susceptibility in these mice. To identify such a gene, we utilized the developing fly eye to test for functional complementation between MAGI and its binding partners. In this way, we identified diminished expression of fly Hibris (nephrin) or Roughest (neph1) as dramatically exacerbating the effects of MAGI depletion. Indeed, when these combinations were studied in mice, the addition of nephrin, but not neph1, heterozygosity to homozygous deletion of MAGI-1 resulted in spontaneous glomerulosclerosis. In cultured podocytes, MAGI-1 depletion reduced intercellular contact-induced Rap1 activation, a pathway critical for proper podocyte function. Similarly, magi1 knock-out mice showed diminished glomerular Rap1 activation, an effect dramatically enhanced by concomitant nephrin haploinsufficiency. Finally, combined overexpression of MAGI-1 and nephrin increased Rap1 activation, but not when substituting a mutant MAGI-1 that cannot bind nephrin. We conclude that the interaction between nephrin and MAGI-1 regulates Rap1 activation in podocytes to maintain long term slit diaphragm structure.