Functional organization of calcium stores in polarized secretory cells and transcellular calcium transport

In this short review, we will first discuss localized cytoplasmic calcium signals in pancreatic acinar cells. In the second part of the review, we will describe recently discovered polarized calcium efflux and calcium propagation through the lumen of the endoplasmic reticulum — ER (a phenomenon we have termed “calcium tunnelling”). Finally, we will present a hypothesis concerning the roles that these mechanisms could play in transcellular calcium flux.     

An intracellular Ca2+ subsystem as a biologically plausible source of intrinsic conditional bistability in a network model of working memory

We have developed a firing rate network model for working memory that combines Mexican-hat-like synaptic coupling with intrinsic or cellular dynamics that are conditionally bistable. While our approach is in the spirit of Camperi and Wang (1998) we include a specific and plausible mechanism for the cellular bistability. Modulatory neurotransmitters are known to activate second messenger signaling systems, and our model includes an intracellular Ca²⁺ handling subsystem whose dynamics depend upon the level of the second messenger inositol 1,4,5 trisphosphate (IP3). This Ca²⁺ subsystem endows individual units with conditional intrinsic bistability for a range of IP3. The full “hybrid” network sustains IP3-dependent persistent (“bump”) activity in response to a brief transient stimulus. The bump response in our hybrid model, like that of Camperi-Wang, is resistant to noise – its position does not drift with time. Action Editor: Upinder Bhalla     

Insect octopamine receptors: a new classification scheme based on studies of cloned Drosophila G-protein coupled receptors

Summary Insect octopamine receptors are G-protein coupled receptors. They can be coupled to second messenger pathways to mediate either increases or decreases in intracellular cyclic AMP levels or the generation of intracellular calcium signals. Insect octopamine receptors were originally classified on the basis of second messenger changes induced in a variety of intact tissue preparations. Such a classification system is problematic if more than one receptor subtype is present in the same tissue preparation. Recent progress on the cloning and characterization in heterologous cell systems of octopamine receptors from Drosophila and other insects is reviewed. A new classification system for insect octopamine receptors into “α-adrenergic-like octopamine receptors (OctαRs)”, “β-adrenergic-like octopamine receptors (OctβRs)” and “octopamine/tyramine (or tyraminergic) receptors” is proposed based on their similarities in structure and in signalling properties with vertebrate adrenergic receptors. In future studies on the molecular basis of octopamine signalling in individual tissues it will be essential to identify the relative expression levels of the different classes of octopamine receptor present. In addition, it will be essential to identify if co-expression of such receptors in the same cells results in the formation of oligomeric receptors with specific emergent pharmacological and signalling properties.     

Calcium dependency and the effect of calcium antagonists on molluscan proboscis smooth muscles from the whelk,Buccinum undatum

In all four proboscis muscles of the whelk Buccinum undatum, the potassium-induced depolarization response was acutely dependent upon extracellular calcium, being eliminated in calcium-free conditions. The responses to acetylcholine were found to be partly dependent upon intracellular calcium. Responses to the peptides phenylalanine-methionine-arginine-phenylalanine-NH₂ and phenylalanine-leucine-arginine-phenylalanine-NH₂ were much more resistant to calcium-free conditions and appeared to engage the excitation-contraction coupling mechanism by mobilizing stored intracellular calcium. Sucrose-gap studies of radular retractor muscles showed that the organic calcium “antagonist” nifedipine enhanced potassium-induced depolarization responses, initiating spike-like action potentials and associated fast twitch activity. The inorganic calcium antagonist gadolinium exerted concentration-dependent inhibitory actions on these muscles. Basal tonus and fast twitch activity in response to potassium-induced depolarization were eliminated as were the spike-like action potentials of the membrane electrical response. The inorganic calcium “antagonist” cadmium greatly enhanced potassium-induced contractures in all four muscles, and on its own it induced tonic force and fast twitches in all the muscles. It seems likely that cadmium may have displaced stored intracellular calcium to induce myofilament activation. While these molluscan smooth muscles appear to possess calcium channels with fast and slow characteristics, their behaviour and pharmacological manipulation is very different from their more well known mammalian transient and long-lasting channel counterparts.     

Basic mechanisms determining the characteristics of calcium signals in nerve cells

The paper summarizes recent data about the mechanisms that determine the kinetics and amplitude of transient elevations in the intracellular level of free calcium (calcium signals) in excitable cells. The relative role of various types of voltage-operated calcium channels, fast cytosolic buffering, active accumulation in intracellular stores, and extrusion of ions from the cell are discussed. New technical approaches enabling resolution of these questions are described.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 5–8, January–February, 1994.     

Calcium regulates scallop muscle by changing myosin flexibility

Muscle myosins are molecular motors that convert the chemical free energy available from ATP hydrolysis into mechanical displacement of actin filaments, bringing about muscle contraction. Myosin cross-bridges exert force on actin filaments during a cycle of attached and detached states that are coupled to each round of ATP hydrolysis. Contraction and ATPase activity of the striated adductor muscle of scallop is controlled by calcium ion binding to myosin. This mechanism of the so-called “thick filament regulation” is quite different to vertebrate striated muscle which is switched on and off via “thin filament regulation” whereby calcium ions bind to regulatory proteins associated with the actin filaments. We have used an optically based single molecule technique to measure the angular disposition adopted by the two myosin heads whilst bound to actin in the presence and absence of calcium ions. This has allowed us to directly observe the movement of individual myosin heads in aqueous solution at room temperature in real time. We address the issue of how scallop striated muscle myosin might be regulated by calcium and have interpreted our results in terms of the structures of smooth muscle myosin that also exhibit thick filament regulation. This paper is not being submitted elsewhere and the authors have no competing financial interests     

Generic physical mechanisms of morphogenesis and pattern formation as determinants in the evolution of multicellular organization

Early embryos of metazoan species are subject to the same set of physical forces and interactions as any small parcels of semi-solid material, living or nonliving. It is proposed that such “generic” properties of embryonic tissues have played a major role in the evolution of biological form and pattern by providing an array of morphological templates, during the early stages of metazoan phylogeny, upon which natural selection could act. The generic physical mechanisms considered include sedimentation, diffusion, and reaction-diffusion coupling, all of which can give rise to chemical nonuniformities (including periodic patterns) in eggs and small multicellular aggregates, and differential adhesion, which can lead to the formation of boundaries of non-mixing between adjacent cell populations. Generic mechanisms that produce chemical patterns, acting in concern with the capacity of cells to modulate their adhesivity (presumed to be a primitive, defining property of metazoa), could lead to multilayered gastrulae of various types, segmental organization, and many of the other distinguishing characteristics of extant and extinct metazoan body plans. Similar generic mechanisms, acting on small tissue primordia during and subsequent to the establishment of the major body plans, could have given rise to the forms of organs, such as the vertebrate limbs. Generic physical processes acting on a single system of cells and cell products can often produce a widely divergent set of morphological phenotypes, and these are proposed to be the raw material of the evolution of form. The establishment of any ecologically successful form by these mechanisms will be followed, under this hypothesis, by a period of genetic evolution, in which the recruitment of gene products to produce the “generically templated” morphologies by redundant pathways would be favoured by intense selection, leading to extensive genetic change with little impact on the fossil record. In this view, the stabilizing and reinforcing functions of natural selection are more important than its ability to effect incremental change in morphology. Aspects of evolution which are problematic from the standard neo-Darwinian viewpoint, or not considered within that framework, but which follow in a straightforward fashion from the view presented here, include the beginnings of an understanding of why organisms have the structure and appearance they’ do, why homoplasy (the recurrent evolution of certain forms) is so prevalent, why evolution has the tempo and mode it does (“punctuated equilibrium”), and why a “rapid” burst of morphological evolution occurred so soon after the origin of the metazoa.     

Intracellular calcium redistribution during mating in Chlamydomonas reinhardii

Gametes of the unicellular green alga Chlamydomonas reinhardii recognize and adhere to cells of the opposite mating type by flagellar contact. Adhesion between these specialized organelles signals a rapid series of mating events which result in gamete fusion. The sequence of morphological changes (flagellar tip activation, cell wall loss, and mating structure elongation), which occur as a consequence of the sexual signalling, have been characterized. The signalling mechanisms have, however, not been defined. Calcium is known to be involved during fertilization of animal species. Increased intracellular free calcium, which can be achieved either by calcium influx or by mobilization of ions from intracellular stores, has been observed during activation of both eggs and sperm. A recent report by Bloodgood & Levin that gametes of C. reinhardii preloaded with 45Ca showed a transient increase in Ca efflux following mating, suggests that intracellular Ca redistribution may also accompany mating in this algal species. We have used X-ray microanalysis to analyze the subcellular distribution of bound calcium during mating in Chlamydomonas reinhardii. X-ray maps reveal that calcium is sequestered in discrete granules within the gamete cell body prior to mating and that during activation and cell fusion, calcium is diffuse throughout the cell. This suggests the possibility that calcium serves as a second messenger in this species.     

Distant interactions in bacteria

Exchange of information between bacteria via physical signals, referred to as “distant interactions” (DI), is the subject of this review. All cases of DI reported to date are discussed, as well as the history of these studies and the place of DI in bacterial communication. Bacterial DI are a particular case of DI occurring in nature (in plants, animals, and fungi). Along with the chemical signals of intracellular communications, DI play a significant role in the life of microorganisms, especially during critical and transitional periods.     

Rapid Endocytosis and Vesicle Recycling in Neuroendocrine Cells

Endocytosis is a crucial process for neuroendocrine cells that ensures membrane homeostasis, vesicle recycling, and hormone release reliability. Different endocytic mechanisms have been described in chromaffin cells, such as clathrin-dependent slow endocytosis and clathrin-independent rapid endocytosis. Rapid endocytosis, classically measured in terms of a fast decrease in membrane capacitance, exhibits two different forms, “rapid compensatory endocytosis” and “excess retrieval.” While excess retrieval seems to be associated with formation of long-lasting endosomes, rapid compensatory endocytosis is well correlated with exocytotic activity, and it is regarded as a mechanism associated to rapid vesicle recycling during normal secretory activity. It has been suggested that rapid compensatory endocytosis may be related to the prevalence of a transient fusion mode of exo-endocytosis. In the latter mode, the fusion pore, a nanometric-sized channel formed at the onset of exocytosis, remains open for a few hundred milliseconds and later abruptly closes, releasing a small amount of transmitters. By this mechanism, endocrine cell selectively releases low molecular weight transmitters, and rapidly recycles the secretory vesicles. In this article, we discuss the cellular and molecular mechanisms that define the different forms of exocytosis and endocytosis and their impact on vesicle recycling pathways.     

How environmental factors regulate mutagenesis and gene transfer in microorganisms

This review is focused on the physiological and evolutionary strategies of the processes occurring during the entry of microbial cells into stationary phase and the subsequent period of stasis. The molecular mechanisms adapting microorganisms from exponential growth to a static state involve activation and complex regulation of the stationary factor Sigma-S, which directs RNA polymerase to the specific promoters. As a result the static cells acquire general resistance (simultaneous tolerances) to different environmental stresses. In parallel with the physiological adaptation to stasis, diverse genetical processes are aimed towards resuming the growth of the static cells. Different types of mutagenesis occur: (i) in cells entering stasis and (ii) in static cells (adaptive mutagenesis). Cessation of growth induces the transient hypermutator state resulting in the accumulation of random mutations in the subpopulation of the static cells. If by chance, one or a few of such mutations lead to resumption of division, the growing cell will return to a normal mechanism of spontaneous mutagenesis. Another mechanism for generating genetical variability in stressed cells involves transposons and conjugative plasmids. Stresses can stimulate the excision of some transposons, which, in turn, can generate chromosomal mutations and activate intracellular mechanisms of mutagenesis. Under stress some conjugative plasmids activate genes encoding antirestriction proteins that repress restriction-modification systems of the recipient cells. Moreover, under stress special cellular mechanisms decrease (alleviate) the activity of restriction-modification systems which, in turn, enhance the probability of gene transfer into the stressed cells. Under stress, the efficiency of inter-species genetical barriers also decreases. This, stimulates inter-species gene transfer and may lead to a burst of incipient speciation in the population of non-growing cells. After resumption of growth the genetical barriers leading to isolation will be restored. In general, the cessation of growth “switches on”, and resumption of growth “switches off”, a set of special processes that are responsible for generating bursts of genetical variability in populations of microorganisms. This article is dedicated to the memory of Nikolai V Timofeev-Ressovsky (1900–1981).     

Transient alterations in cellular permeability in cultured human proximal tubule cells: Implications for transport studies

Summary Primary cultures of human proximal tubule (HPT) cells possess the characteristics of a tight epithelium and retain the characteristics of in vivo renal function. HPT cells from confluent monolayers when grown on collagen-coated polycarbonate inserts in a hormonally defined serum-free medium. However, initial studies of transepithelial transport observed large bidirectional fluxes of the paracellular probe inulin. The present studies were designed to assess the transformation of HPT cell tight junctions to a “leaky” state and subsequent recovery. The apparent transepithelial electrical resistance of HPT cells at confluence was 952.0±70.0 ohms*cm², suggesting a well-developed tight junction-mediated paracellular pathway in this epithelium. However, replacement of the growth media produced an immediate 90% drop in the initial resistance, which was paralleld by an increased flux of inulin and of phenol red. This transient abolition of barrier function spontaneously reestablished over 1–2 h by a process that was dependent on the ionic composition of the added media. Complete recovery of cellular resistance was paralleled by markedly decreased fluxes of inulin and of phenol red. The recovery of cellular barrier function was inhibited by cytochalasin B suggesting an intracellular action, not a physical disruption of the monolayer. These results suggest that the tight junctions in these cells appear to transiently produce a leaky state during removal of the media, but rearrange to a “tight conformation” when incubated in the appropriate media.     

Pioneer neurons: A basis or limiting factor of lophotrochozoa nervous system diversity?

It has been demonstrated by us and other authors that first nervous cells in developing larvae from various trochozoan groups differentiate at the periphery. These pioneer neurons are distinguished by the set of characters. They are located outside the forming central ganglia; outgrowing fibers of central neurons use their processes as a “scaffolding” transmitter expression in these neurons is transient. On the one hand, pioneer neurons mark the “frame” of the adult nervous system and thus play a limiting role. On the other hand, pioneering navigation provides possible mechanisms for evolutional plasticity of the nervous system in adults. In addition, pioneer neurons can underlie functional adaptation of trochophore animals, which minimizes fitness decrease during the transition from the larval to the adult form during metamorphosis.     

BioMetals: a historical and personal perspective

Understanding of BioMetals developed basically from a starting point about 60 years ago to current mechanistic understanding of the biological behavior of many metal ions from protein structural and functional studies. Figure 1 shows a Biochemical Periodic Table, element by element, with requirements, roles and biochemistry of the specific ions indicated. With few exceptions, the biology is of the ions formed and not of the elemental state of each. Early BioMetals efforts defined nutritional growth needs for animals, plants and microbes for inorganic “macro-nutrients” such as magnesium, calcium, potassium, sodium, and phosphate and of “micronutrients” such as copper, iron, manganese and zinc. Surprises came early with regard to microbes, for example the finding that Escherichia coli (then and now the standard microbial model) grows happily in the apparent total absence of calcium, sodium, and chloride, which are certainly major animal nutrients. Some elements such as mercury and arsenic are never required by living cells, but are always toxic, often at very low levels. Therefore, the division into nutrient elements and toxic elements came soon. For most inorganic nutrients, excessive amounts can be toxic as well, for example for copper and iron.     

Biophysics of nerve excitation

Experimental studies testifying to the presence of an interrelation between the physiological functions of the organism and physical and chemical processes in nerves are discussed. Changes in some physical and chemical parameters observed both upon elicited rhythmic excitation of nerves and during the spontaneous rhythmic activity of neurons are analyzed. Upon rhythmic excitation, a complex of physical and chemical processes is triggered, and reversible structural and metabolic rearrangements at the subcellular and molecular levels occur that do not take place during the generation of a single action potential. Thus, only in conditions of rhythmic excitation of a nerve, it is possible to reveal those processes that provide excitation of nerves in the organism. The future possibilities of the investigations combining the biophysical and physiological approaches are substantiated. Characteristic changes in physicochemical parameters are observed in nerves during the generation of a series of action potentials of different frequency and duration (“frequency dependence”) under normal physiological conditions, as well as in extreme situations and in nerve pathology. The structural and metabolic rearrangements are directly related to the mode of rhythmic excitation and proceed both in the course of rhythmic excitation and after its termination. Shown also is participation of the basic components of the nervous trunk (axon, Schwann cell, myelin, subcellular organelles) in the realization of rhythmic excitation. In the coordination of all processes involved in rhythmic excitation, the main role is played by the systems of redistribution and transport of intercellular and intracellular calcium. The idea is put forward that myelin of nerve fibers is not only an insulator, but also an “intercellular depot” of calcium and participates in the redistribution of different ions. Thus, the rhythmic excitation is of great importance in the realization of some physiological functions, the adaptation to changing conditions, the liquidation of consequences of paralogical processes, the formation of mechanisms of “memory,” etc.     

Analyzing folding and degradation of metabolically labelled polypeptides by conventional and diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Efficient protein folding and quality control are essential for unperturbed cell viability. Defects in these processes may lead to production of aberrant polypeptides that are either degraded leading to “loss-of-function” phenotypes, or deposited in or outside cells leading to “gain-of-toxic-function” phenotypes. Elucidation of molecular mechanisms regulating folding and quality control of newly synthesized polypeptides is therefore of greatest interest. Here we describe protocols for metabolic labelling of transfected/infected mammalian cells with [³⁵S]-methionine and [³⁵S]-cysteine, for immunoisolation from detergent extracts of the selected model proteins and for the investigation of the model polypeptide’s intracellular fate in response to chaperone-deletions or to cell exposure to folding or degradation inhibitors.     

Inflammasomes in infection and inflammation

Two of the main challenges that multicellular organisms faced during evolution were to cope with invading microorganisms and eliminate and replace dying cells. Our innate immune system evolved to handle both tasks. Key aspects of innate immunity are the detection of invaders or tissue injury and the activation of inflammation that alarms the system through the action of cytokine and chemokine cascades. While inflammation is essential for host resistance to infections, it is detrimental when produced chronically or in excess and is linked to various diseases, most notably auto-immune diseases, auto-inflammatory disorders, cancer and septic shock. Essential regulators of inflammation are enzymes termed “the inflammatory caspases”. They are activated by cellular sensors of danger signals, the inflammasomes, and subsequently convert pro-inflammatory cytokines into their mature active forms. In addition, they regulate non-conventional protein secretion of alarmins and cytokines, glycolysis and lipid biogenesis, and the execution of an inflammatory form of cell death termed “pyroptosis”. By acting as key regulators of inflammation, energy metabolism and cell death, inflammatory caspases and inflammasomes exert profound influences on innate immunity and infectious and non-infectious inflammatory diseases. Christian R. McIntire and Garabet Yeretssian have contributed equally to this review.     

Brain-type creatine kinase BB-CK interacts with the Golgi Matrix Protein GM130 in early prophase

Creatine kinase (CK) isoenzymes are essential for storing, buffering and intracellular transport of “energy-rich” phosphate compounds in tissues with fluctuating high energy demand such as muscle, brain and other tissues and cells where CK is expressed. In brain and many non-muscle cells, ubiquitous cytosolic “brain-type” BB-CK and ubiquitous mitochondrial CK (uMtCK) act as components of a phosphocreatine shuttle to maintain cellular energy pools and distribute energy flux. To date, still relatively little is known about direct coupling of functional dimeric BB-CK with other partner proteins or enzymes that are important for cell function. Using a global yeast two-hybrid (Y2H) screen with monomeric B-CK as bait and a representative brain cDNA library to search for interaction partners of B-CK with proteins of the brain, we repeatedly identified the cis-Golgi Matrix protein (GM130) as recurrent interacting partner of B-CK. Since HeLa cells also express both BB-CK and GM130, we subsequently used this cellular model system to verify and characterize the BB-CK-GM130 complex by GST-pulldown experiments, as well as by in vivo co-localization studies with confocal microscopy. Using dividing HeLa cells, we report here for the first time that GM130 and BB-CK co-localize specifically in a transient fashion during early prophase of mitosis, when GM130 plays an important role in Golgi fragmentation that starts also at early prophase. These data may shed new light on BB-CK function for energy provision for Golgi-fragmentation that is initiated by cell signalling cascades in the early phases of mitosis.     

Synaptic transmission in sympathetic vasoconstrictor pathways and its modification after injuries

The discharge of vasoconstrictor pathways arising in the CNS is largely unmodified as it passes through the sympathetic ganglia to the vasculature. The underlying synaptic events have been revealed by intracellular recordings from sympathetic paravertebral ganglion cells in the course of ongoing and reflex activity in anesthetized animals, first made in Skok’s Laboratory in Kyiv (Ukraine). Each preganglionic neuron diverges to contact a number of post-ganglionic neurons, on each of which several pre-ganglionic inputs converge. However, only suprathreshold “strong,” or “dominant” synapses are effective in transmitting the CNS signals. Strong synapses differ from the other subthreshold “weak,” or “accessory” inputs: (a) excitatory synaptic currents are >1 nA in their amplitude, (b) 3 to ≈>30 times more quanta of acetylcholine are released, (c) pre-synaptic Ca²⁺ entry through channels resistant to all-known antagonists triggers acetylcholine release, and (d) post-synaptic Ca²⁺ entry boosts and prolongs the nicotinic current. While the majority of postganglionic neurons have only one strong input, a proportion receives two or, rarely, three such inputs. In cells with multiple strong inputs, an equivalent number of discrete Ca²⁺ currents can be evoked at distinct foci electrically distant from the soma, suggesting that each strong input has a unique dendritic association with a cluster of Ca²⁺ channels. When strong preganglionic inputs are destroyed, residual weak synapses sprout and rapidly restore the suprathreshold connections. While much remains to be discovered about how strong synapses are established, their high safety factor ensures the wide and secure distribution of vasoconstrictor command signals from the CNS. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 294–301, July–October, 2007.