Electrical coupling and dye transfer between axon segments in the medium giant axon of the earthworm

Electrical coupling between axon segments has been studied in the medium giant axon, which had been partially isolated from the ventral nerve cord of the earthworm. Evidence has been obtained that in addition to the coupling structures in the septum there are diffusion- and current-pathways through the pseudo-myelin which seem to be more permeable to fluorescein than the channels in the septum. These findings offer an explanation for the discrepancy of the experimentally determined specific resistance of the septum and the expected values based on nexus density and channel size in the septum.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Propagation of Action Potentials and the Structure of the Nexus in Cardiac Muscle

The hypothesis that the nexus is a specialized structure allowing current flow between cell interiors is corroborated by concomitant structural changes of the nexus and changes of electrical coupling between cells due to soaking in solutions of abnormal tonicity. Fusiform frog atrial fibers are interconnected by nexuses. The nexuses, desmosomes, and regions of myofibrillar attachment of this muscle are not associated in a manner similar to intercalated discs of guinea pig cardiac muscle. Indeed, nexuses occur wherever cell membranes are closely apposed. Action potentials of frog atrial bundles detected extracellularly across a sucrose gap change from monophasic to diphasic when the gap is shunted by a resistor. This indicates that action potentials are transmitted across the gap when sufficient excitatory current is allowed to flow across the gap. When the sucrose solution in the gap is made hypertonic, propagation past the gap is blocked and the resistance between the cells in the gap increases. Electron micrographs demonstrate that the nexuses of frog atrium and guinea pig ventricle are ruptured by hypertonic solutions.     

Nexal membrane permeability to anions

The permeability of the septa of the earthworm in the median axon has been calculated for the anions fluorescein and its halogen derivatives. The values ranged from 5.4 X 10(-5) to 4 X 10(-6) cm/s. Previously, the septa had been shown to contain nexuses. By using freeze-fracture material, the surface area of nexus on the septal membranes was determined to be 4.5%, very similar to the percentage of nexus in the intercalated disk of mammalian myocardium. Plasma membrane permeability to these dyes was also calculated and shown to be much less than that of the septal membranes. In addition, an estimate of cytoplasmic binding for each dye was made, and most dyes showed little or no binding with the exception of aminofluorescein.     

The Structure of Mytilus Smooth Muscle and the Electrical Constants of the Resting Muscle

The individual muscle fibers of the anterior byssus retractor muscle (ABRM) of Mytilus edulis L. are uninucleate, 1.2–1.8 mm in length, 5 µm in diameter, and organized into bundles 100–200 µm in diameter, surrounded by connective tissue. Some bundles run the length of the whole muscle. Adjacent muscle cell membranes are interconnected by nexuses at frequent intervals. Specialized attachments exist between muscle fibers and connective tissue. Electrical constants of the resting muscle membrane were measured with intracellular recording electrodes and both extracellular and intracellular current-passing electrodes. With an intracellular current-passing electrode, the time constant τ, was 4.3 ± 1.5 ms. With current delivered via an extracellular electrode τ was 68.3 ± 15 ms. The space constant, λ, was 1.8 mm ± 0.4. The membrane input resistance, R_eff, ranged from 23 to 51 MΩ. The observations that values of τ depend on the method of passing current, and that the value of λ is large relative to fiber length and diameter are considered evidence that the individual muscle fibers are electrically interconnected within bundles in a three-dimensional network. Estimations are made of the membrane resistance, R_m, to compare the values to fast and slow striated muscle fibers and mammalian smooth muscles. The implications of this study in reinterpreting previous mechanical and electrical studies are discussed.     

Freeze-fracture studies of the sinoatrial and atrioventricular nodes of the caprine heart,with special reference to the nexus

Summary The caprine sinoatrial node (SAN) and atrioventricular node (AVN) were studied by freeze-fracture techniques, and their nexus or gap junction structure were compared with that of ordinary atrial and ventricular muscle cells. The general features of the nexus in both the SAN and AVN were essentially identical. Approximately two-thirds of the nexuses observed in the nodal cells consisted of typical macular arrangements of nexal particles, and the remaining third, of atypical configurations of either circular arrangements or linear arrays of particles in continuity with the macular nexuses. Such atypical nexuses were never observed in the ordinary adult myocardial cells. Quantitative analysis revealed that all of the nexuses in the nodal cells measured, were less than 0.1 m², whereas the majority of the nexuses in ordinary myocardial cells (64% in the atrium and 76% in the ventricle) were larger than 0.1 m². No significant differences in diameter and center-to-center distance of nexal particle were found between the nodal cells and ordinary myocardial cells.     

Could Public Restrooms Be an Environment for Bacterial Resistomes?

Antibiotic resistance in bacteria remains a major problem and environments that help to maintain such resistance, represent a significant problem to infection control in the community. Restrooms have always been regarded as potential sources of infectious diseases and we suggest they have the potential to sustain bacterial “resistomes”. Recent studies have demonstrated the wide range of different bacterial phyla that can be found in non-healthcare restrooms. In our study we focused on the Staphylococci. These species are often skin contaminants on man and have been reported as common restroom isolates in recent molecular studies. We collected samples from 18 toilets sited in 4 different public buildings. Using MALDI-TOF-MS and other techniques, we identified a wide range of antibiotic resistant Staphylococci and other bacteria from our samples. We identified 19 different Staphylococcal species within our isolates and 37.8% of the isolates were drug resistant. We also identified different Staphylococcal species with the same antibiograms inhabiting the same restrooms. Bacterial “resistomes” are communities of bacteria often localised in specific areas and within these environments drug resistance determinants may be freely transferred. Our study shows that non-healthcare restrooms are a source of antibiotic resistant bacteria where a collection of antibiotic resistance genes in pathogenic and non-pathogenic bacteria could form a resistome containing a “nexus of genetic diversity”     

Molecular Diagnostics of Fine Needle Aspiration for the Presurgical Screening of Thyroid Nodules

“The incidence of thyroid cancer, the most common endocrine malignancy, is rising. The two most common types of thyroid cancer are papillary and follicular” thyroid carcinomas. “Fine-needle aspiration (FNA) of thyroid nodules” can permit to detect many genetic mutations and other molecular alterations, including RAS and BRAF point mutations, PAX8/peroxisome proliferator-activated receptor (PPAR)γ and “RET/PTC rearrangements, occurring in thyroid papillary and follicular carcinomas” (more than 70% of cases), which can be used successfully to improve the diagnosis “and the management of patients with thyroid nodules”. The most extensive experience has been accumulated with “the diagnostic use of BRAF mutation”, which is highly specific for malignancy. “Testing FNA samples for a panel of mutations” that typically includes RAS, BRAF, PAX8/PPARγ and RET/PTC could permit to achieve the biggest diagnostic impact. “The accuracy of cancer diagnosis in thyroid nodules could be improved significantly using these and other emerging molecular markers”.     

Cytoplasmic Continuity Revisited: Closure of Septa of the Filamentous Fungus Schizophyllum commune in Response to Environmental Conditions

Background

Mycelia of higher fungi consist of interconnected hyphae that are compartmentalized by septa. These septa contain large pores that allow streaming of cytoplasm and even organelles. The cytoplasm of such mycelia is therefore considered to be continuous.

Methodology/Principal Findings

Here, we show by laser dissection that septa of Schizophyllum commune can be closed depending on the environmental conditions. The most apical septum of growing hyphae was open when this basidiomycete was grown in minimal medium with glucose as a carbon source. In contrast, the second and the third septum were closed in more than 50% and 90% of the cases, respectively. Interestingly, only 24 and 37% of these septa were closed when hyphae were growing in the absence of glucose. Whether a septum was open or closed also depended on physical conditions of the environment or the presence of toxic agents. The first septum closed when hyphae were exposed to high temperature, to hypertonic conditions, or to the antibiotic nourseothricin. In the case of high temperature, septa opened again when the mycelium was placed back to the normal growth temperature.

Conclusions/Significance

Taken together, it is concluded that the septal pores of S. commune are dynamic structures that open or close depending on the environmental conditions. Our findings imply that the cytoplasm in the mycelium of a higher fungus is not continuous perse.     

Circulating CD138 enhances disease progression by augmenting autoreactive antibody production in a mouse model of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a progressive autoimmune disease characterized by high levels of antibodies directed against nuclear antigens. Elevated serum CD138, a heparan sulfate–bearing proteoglycan, correlates with increased disease activity in patients with SLE, but the contribution of CD138 to lupus disease is not known. Corroborating patient data, we detected an increase in serum CD138 in MRL/MpJ-Fas^lpr/J (MRL/Lpr) mice (a model for SLE disease) parallel to disease activity. Although T-cell receptor β (TCRβ)+CD138+ T cells typically expand in MRL/Lpr mice as the disease progresses, surprisingly, TCRβ+CD138− cells were the primary source of circulating CD138, as the transfer of TCRβ+CD138− cells, but not TCRβ+CD138+ cells, to young MRL/Lpr mice resulted in higher serum CD138 in the recipients. We found that trypsin was able to cleave CD138 from TCRβ+CD138+ cells, and that trypsin was highly expressed in TCRβ+CD138− cells. Moreover, trypsin inhibitors, the “defined trypsin inhibitor” and leupeptin, increased CD138 expression on TCRβ+CD138− cells, suggesting a contribution of cleaved CD138 to the increase in blood CD138 levels. Furthermore, soluble CD138 was able to bind “a proliferation-inducing ligand” (APRIL) and enhance APRIL-mediated plasma cell generation and autoreactive antibody production through the phosphorylation of extracellular signal–regulated kinase in B cells. The APRIL receptor “transmembrane activator, calcium modulator, and cyclophilin ligand interactor” was involved in the enhancement of APRIL activity by CD138, as the synergistic effect of APRIL and CD138 was ablated in transmembrane activator, calcium modulator, and cyclophilin ligand interactor–deficient B cells. These findings indicate a regulatory role for soluble CD138 in B-cell differentiation and autoreactive antibody production in SLE disease.     

Phase Plane Trajectories of the Muscle Spike Potential

To facilitate a study of the transmembrane action current, the striated muscle spike potential was recorded against its first time derivative. The specialized recording methods are described, as well as several mathematical transformations between a coordinate system in V, t, and the present coordinate system in V, dV/dt. The particular properties of the present recording method permitted an estimation for the “sodium” potential in muscle fibers at +39 mv. The maximum membrane conductance during spike production is in the order of 150 to 200 mmhos/cm². The changes in the shape of the recorded response indicate that the ionic currents and membrane conductances are reduced by Tris buffer or hypertonic Ringer's fluid. However, no marked changes in the properties of active membrane were observed when chloride ion was replaced by sulfate.     

Defining Extracellular Integrin α-Chain Sites That Affect Cell Adhesion and Adhesion Strengthening without Altering Soluble Ligand Binding

It was previously shown that mutations of integrin α4 chain sites, within putative EF-hand-type divalent cation-binding domains, each caused a marked reduction in α4β1-dependent cell adhesion. Some reports have suggested that α-chain “EF-hand” sites may interact directly with ligands. However, we show here that mutations of three different α4 “EF-hand” sites each had no effect on binding of soluble monovalent or bivalent vascular cell adhesion molecule 1 whether measured indirectly or directly. Furthermore, these mutations had minimal effect on α4β1-dependent cell tethering to vascular cell adhesion molecule 1 under shear. However, EF-hand mutants did show severe impairments in cellular resistance to detachment under shear flow. Thus, mutation of integrin α4 “EF-hand-like” sites may impair 1) static cell adhesion and 2) adhesion strengthening under shear flow by a mechanism that does not involve alterations of initial ligand binding.     

A novel membrane-dependent on/off switch mechanism of talin FERM domain at sites of cell adhesion

The activation of heterodimeric (α/β) integrin transmembrane receptors by cytosolic protein talin is crucial for regulating diverse cell-adhesion-dependent processes, including blood coagulation, tissue remodeling, and cancer metastasis. This process is triggered by the coincident binding of N-terminal FERM (four-point-one-protein/ezrin/radixin/moesin) domain of talin (talin-FERM) to the inner membrane surface and integrin β cytoplasmic tail, but how these binding events are spatiotemporally regulated remains obscure. Here we report the crystal structure of a dormant talin, revealing how a C-terminal talin rod segment (talin-RS) self-masks a key integrin-binding site on talin-FERM via a large interface. Unexpectedly, the structure also reveals a distinct negatively charged surface on talin-RS that electrostatically hinders the talin-FERM binding to the membrane. Such a dual inhibitory topology for talin is consistent with the biochemical and functional data, but differs significantly from a previous model. We show that upon enrichment with phosphotidylinositol-4,5-bisphosphate (PIP2) – a known talin activator, membrane strongly attracts a positively charged surface on talin-FERM and simultaneously repels the negatively charged surface on talin-RS. Such an electrostatic “pull-push” process promotes the relief of the dual inhibition of talin-FERM, which differs from the classic “steric clash” model for conventional PIP2-induced FERM domain activation. These data therefore unravel a new type of membrane-dependent FERM domain regulation and illustrate how it mediates the talin on/off switches to regulate integrin transmembrane signaling and cell adhesion.     

Mitochondrial biogenesis in epithelial cancer cells promotes breast cancer tumor growth and confers autophagy resistance

Here, we set out to test the novel hypothesis that increased mitochondrial biogenesis in epithelial cancer cells would “fuel” enhanced tumor growth. For this purpose, we generated MDA-MB-231 cells (a triple-negative human breast cancer cell line) overexpressing PGC-1α and MitoNEET, which are established molecules that drive mitochondrial biogenesis and increased mitochondrial oxidative phosphorylation (OXPHOS). Interestingly, both PGC-1α and MitoNEET increased the abundance of OXPHOS protein complexes, conferred autophagy resistance under conditions of starvation and increased tumor growth by up to ~3-fold. However, this increase in tumor growth was independent of neo-angiogenesis, as assessed by immunostaining and quantitation of vessel density using CD31 antibodies. Quantitatively similar increases in tumor growth were also observed by overexpression of PGC-1β and POLRMT in MDA-MB-231 cells, which are also responsible for mediating increased mitochondrial biogenesis. Thus, we propose that increased mitochondrial “power” in epithelial cancer cells oncogenically promotes tumor growth by conferring autophagy resistance. As such, PGC-1α, PGC-1β, mitoNEET and POLRMT should all be considered as tumor promoters or “metabolic oncogenes.” Our results are consistent with numerous previous clinical studies showing that metformin (a weak mitochondrial “poison”) prevents the onset of nearly all types of human cancers in diabetic patients. Therefore, metformin (a complex I inhibitor) and other mitochondrial inhibitors should be developed as novel anticancer therapies, targeting mitochondrial metabolism in cancer cells.     

A STUDY OF THE STRUCTURE AND DISTRIBUTION OF THE NEXUS

Nexuses, that is, fusions of plasma membranes of adjacent cells, are described in mammalian smooth and cardiac muscle, median giant axon of earthworm, frog skin, and rat submandibular gland. In smooth muscle they usually occur where a process from one cell either meets a process of, or projects into a neighboring cell. On the other hand, in mammalian heart muscle and in earthworm giant axon the nexuses occur along the intercalated disc and intercellular segmental septa, respectively. Their occurrence between these excitable cells is correlated with propagation of action potentials by an electrical rather than chemical mechanism. Since the nexuses may offer pathways for electric current between cell interiors, it seems possible that they constitute a link in the structural basis for electrical transmission in these systems. In epithelia, nexuses usually appear as part of a terminal bar complex. This is true in the rat salivary gland studied here. In the epidermis of frog skin, nexuses are less numerous between the basilar columnar cells than between the subjacent squamous cells. The nexuses which occur in epithelia in frog skin and rat salivary gland are distributed as though to provide seals against electrochemical backleaks and sites of chemical exchange between cell interiors.     

Extracellular cell wall β(1,3)glucan is required to couple septation to actomyosin ring contraction

Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall β(1,3)glucan. We show that Bgs4-derived β(1,3)glucan is required for correct and stable actomyosin ring positioning in the cell middle, before the start of septum formation and anchorage to the cell wall. Consequently, β(1,3)glucan loss generated ring sliding, oblique positioned rings and septa, misdirected septum synthesis indicative of relaxed rings, and uncoupling between a fast ring and membrane ingression and slow septum synthesis, suggesting that cytokinesis can progress with defective septum pushing and/or ring pulling forces. Moreover, Bgs4-derived β(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular β(1,3)glucan is required for cytokinesis to connect the cell wall with the plasma membrane and for contractile ring function, as proposed for the equivalent extracellular matrix in animal cells.     

Structural requirements for the binding of phenylglycosides to the surface of protoplasts

A variety of phenylglycosides have been synthesized and tested for binding to the surface of protoplasts from suspension-cultured cells of “Paul's Scarlet” rose (Rosa sp.). Multivalent phenylglycosides in the form of Yariv antigens (1,3,5,-tri-[p-glycosyloxyphenylazo]-2,4,6,-trihydroxybenzene) agglutinated the protoplasts. Fluorescence-labeled derivatives of other monovalent and polyvalent phenyl-β-glycosides did not bind to the protoplast surface. Agglutination was induced by Yariv antigens only if these probes contained β-anomeric, O-glycosidic linkages. Yariv antigens containing α-anomeric or thio-glycosidic linkages did not agglutinate protoplasts. These same structural features of Yariv antigens were also required for the precipitation of gum arabic-Yariv antigen complexes. The results suggest that plasma membranes of “Paul's Scarlet” rose protoplasts contain arabinogalactan-proteins that interact with phenyl-β-glycosides. The results further show that binding at these plasma membrane sites is not solely dependent upon the carbohydrate portion of single phenylglycosides, but may also require specific spatial orientations of adjacent phenylglycosides.     

Both Transmembrane Domains of BK β1 Subunits Are Essential to Confer the Normal Phenotype of β1-Containing BK Channels

Voltage/Ca²⁺ _i-gated, large conductance K⁺ (BK) channels result from tetrameric association of α (slo1) subunits. In most tissues, BK protein complexes include regulatory β subunits that contain two transmembrane domains (TM1, TM2), an extracellular loop, and two short intracellular termini. Four BK β types have been identified, each presenting a rather selective tissue-specific expression profile. Thus, BK β modifies current phenotype to suit physiology in a tissue-specific manner. The smooth muscle-abundant BK β1 drastically increases the channel''s apparent Ca²⁺ _i sensitivity. The resulting phenotype is critical for BK channel activity to increase in response to Ca²⁺ levels reached near the channel during depolarization-induced Ca²⁺ influx and myocyte contraction. The eventual BK channel activation generates outward K⁺ currents that drive the membrane potential in the negative direction and eventually counteract depolarization-induced Ca²⁺ influx. The BK β1 regions responsible for the characteristic phenotype of β1-containing BK channels remain to be identified. We used patch-clamp electrophysiology on channels resulting from the combination of smooth muscle slo1 (cbv1) subunits with smooth muscle-abundant β1, neuron-abundant β4, or chimeras constructed by swapping β1 and β4 regions, and determined the contribution of specific β1 regions to the BK phenotype. At Ca²⁺ levels found near the channel during myocyte contraction (10 µM), channel complexes that included chimeras having both TMs from β1 and the remaining regions (“background”) from β4 showed a phenotype (V_half, τ_act, τ_deact) identical to that of complexes containing wt β1. This phenotype could not be evoked by complexes that included chimeras combining either β1 TM1 or β1 TM2 with a β4 background. Likewise, β “halves” (each including β1 TM1 or β1 TM2) resulting from interrupting the continuity of the EC loop failed to render the normal phenotype, indicating that physical connection between β1 TMs via the EC loop is also necessary for proper channel function.     

α-Proteobacterial Symbionts of Marine Bryozoans in the Genus Watersipora

Bacterial symbionts that resembled mollicutes were discovered in the marine bryozoan Watersipora arcuata in the 1980s. In this study, we used PCR and sequencing of 16S rRNA genes, specific fluorescence in situ hybridization, and phylogenetic analysis to determine that the bacterial symbionts of “W. subtorquata” and “W. arcuata” from several locations along the California coast are actually closely related α-Proteobacteria, not mollicutes. We propose the names “Candidatus Endowatersipora palomitas” and “Candidatus Endowatersipora rubus” for the symbionts of “W. subtorquata” and “W. arcuata,” respectively.     

The Integrin β1 Subunit Regulates Paracellular Permeability of Kidney Proximal Tubule Cells

Epithelial cells lining the gastrointestinal tract and kidney have different abilities to facilitate paracellular and transcellular transport of water and solutes. In the kidney, the proximal tubule allows both transcellular and paracellular transport, while the collecting duct primarily facilitates transcellular transport. The claudins and E-cadherin are major structural and functional components regulating paracellular transport. In this study we present the novel finding that the transmembrane matrix receptors, integrins, play a role in regulating paracellular transport of renal proximal tubule cells. Deleting the integrin β1 subunit in these cells converts them from a “loose” epithelium, characterized by low expression of E-cadherin and claudin-7 and high expression of claudin-2, to a “tight” epithelium with increased E-cadherin and claudin-7 expression and decreased claudin-2 expression. This effect is mediated by the integrin β1 cytoplasmic tail and does not entail β1 heterodimerization with an α-subunit or its localization to the cell surface. In addition, we demonstrate that deleting the β1 subunit in the proximal tubule of the kidney results in a major urine-concentrating defect. Thus, the integrin β1 tail plays a key role in regulating the composition and function of tight and adherens junctions that define paracellular transport properties of terminally differentiated renal proximal tubule epithelial cells.     

Molecular insights into the primary nucleation of polymorphic amyloid β dimers in DOPC lipid bilayer membrane

Alzheimer''s disease (AD) pathology is characterized by loss of memory cognitive and behavioral deterioration. One of the hallmarks of AD is amyloid β (Aβ) plaques in the brain that consists of Aβ oligomers and fibrils. It is accepted that oligomers, particularly dimers, are toxic species that are produced extracellularly and intracellularly in membranes. It is believed that the disruption of membranes by polymorphic Aβ oligomers is the key for the pathology of AD. This is a first study that investigate the effect of polymorphic “α‐helix/random coil” and “fibril‐like” Aβ dimers on 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) membrane. It has been found that the DOPC membrane promotes Aβ_1–42 “fibril‐like” dimers and impedes Aβ_1–42 “α‐helix/random coil” dimers. The N‐termini domains within Aβ_1–42 dimers play a role in Aβ aggregation in membrane milieus. In addition, the aromatic π–π interactions (involving residues F19 and F20 in Aβ_1–42) are the driving forces for the hydrophobic interactions that initiate the primary nucleation of polymorphic Aβ_1–42 dimers within DOPC membrane. Finally, the DOPC bilayer membrane thickness is locally decreased, and it is disrupted by an embedded distinct Aβ_1–42 dimer, due to relatively large contacts between Aβ_1–42 monomers and the DOPC membrane. This study reveals insights into the molecular mechanisms by which polymorphic early‐stage Aβ_1–42 dimers have distinct impacts on DOPC membrane.