Spiral waves and intracellular calcium signalling.

Confocal imaging of intracellular Ca2+ brings a new level of resolution to the study of hormonal control of intracellular Ca2+ release. This approach has demonstrated the existence of pulsatile circular and spiral waves of Ca+ release induced by receptor activation. The data obtained by confocal imaging support a new framework for understanding intracellular Ca2+ signalling. The goal of this chapter is to review our data on the complexity of intracellular Ca2+ release in Xenopus oocytes, introduce the concept of Ca2+ excitability as a model for Ca2+ release and discuss the implications for encoding intracellular signal information.     

Local positive feedback by calcium in the propagation of intracellular calcium waves.

In many types of eukaryotic cells, the activation of surface receptors leads to the production of inositol 1,4,5-trisphosphate and calcium release from intracellular stores. Calcium release can occur in complex spatial patterns, including waves of release that traverse the cytoplasm. Fluorescence video microscopy was used to view calcium waves in single mouse neuroblastoma cells. The propagation of calcium waves was slowed by buffers that bind calcium quickly, such as BAPTA, but not by a buffer with slower on-rate, EGTA. This shows that a key feedback event in wave propagation is rapid diffusion of calcium occurring locally on a scale of < 1 micron. The length-speed product of wavefronts was used to determine that calcium acting in feedback diffuses at nearly the rate expected for free diffusion in aqueous solution. In cytoplasm, which contains immobile Ca2+ buffers, this rate of diffusion occurs only in the first 0.2 ms after release, within 0.4 micron of a Ca2+ release channel mouth. Calcium diffusion from an open channel to neighboring release sites is, therefore, a rate-determining regenerative step in calcium wave propagation. The theoretical limitations of the wave front analysis are discussed.     

Mechanically induced intracellular calcium waves in osteoblasts demonstrate calcium fingerprints in bone cell mechanotransduction

An early response to mechanical stimulation of bone cells in vitro is an increase in intracellular calcium concentration ([Ca ²⁺]_i). This study analyzed the [Ca ²⁺]_i wave area, magnitude, duration, rise time, fall time, and time to onset in individual osteoblasts for two identical bouts of mechanical stimulation separated by a 30-min rest period. The area under the [Ca ²⁺]_i wave increased in the second loading bout compared to the first. This suggests that rest periods may potentiate mechanically induced intracellular calcium signals. Furthermore, many of the [Ca ²⁺]_i wave parameters were strongly, positively correlated between the two bouts of mechanical stimulation. For example, in individual primary osteoblasts, if a cell had a large [Ca ²⁺]_i wave area in the first bout it was likely to have a large [Ca ²⁺]_i wave area in the second bout (r ² = 0.933). These findings support the idea that individual bone cells have “calcium fingerprints” (i.e., a unique [Ca ²⁺]_i wave profile that is reproducible for repeated exposure to a given stimulus).     

Stabilization of proteins in confined spaces

We present theory showing that confining a protein to a small inert space (a "cage") should stabilize the protein against reversible unfolding. Examples of such spaces might include the pores within chromatography columns, the Anfinsen cage in chaperonins, the interiors of ribosomes, or regions of steric occlusion inside cells. Confinement eliminates some expanded configurations of the unfolded chain, shifting the equilibrium from the unfolded state toward the native state. The partition coefficient for a protein in a confined space is predicted to decrease significantly when the solvent is changed from native to denaturing conditions. Small cages are predicted to increase the stability of the native state by as much as 15 kcal/mol. Confinement may also increase the rates of protein or RNA folding.     

Shapes of semiflexible polymers in confined spaces

Biological macromolecules, living in the confines of a cell, often adopt conformations that are unlikely to occur in free space. In this paper, we investigate the effects of confinement on the shape of a semiflexible chain. Results of Monte Carlo simulations show the existence of a shape transition when the persistence length of the polymer becomes comparable to the dimensions of the box. An order parameter is introduced to quantify this behavior. A simple model is constructed to study the effect of the shape transition on the effective persistence length of the polymer.     

A single-pool model for intracellular calcium oscillations and waves in the Xenopus laevis oocyte.

We construct a minimal model of cytosolic free Ca2+ oscillations based on Ca2+ release via the inositol 1,4,5-trisphosphate (IP3) receptor/Ca2+ channel (IP3R) of a single intracellular Ca2+ pool. The model relies on experimental evidence that the cytosolic free calcium concentration ([Ca2+]c) modulates the IP3R in a biphasic manner, with Ca2+ release inhibited by low and high [Ca2+]c and facilitated by intermediate [Ca2+]c, and that channel inactivation occurs on a slower time scale than activation. The model produces [Ca2+]c oscillations at constant [IP3] and reproduces a number of crucial experiments. The two-dimensional spatial model with IP3 dynamics, cytosolic diffusion of IP3 (Dp = 300 microns 2 s-1), and cytosolic diffusion of Ca2+ (Dc = 20 microns 2 s-1) produces circular, planar, and spiral waves of Ca2+ with speeds of 7-15 microns.s-1, which annihilate upon collision. Increasing extracellular [Ca2+] influx increases wave speed and baseline [Ca2+]c. A [Ca2+]c-dependent Ca2+ diffusion coefficient does not alter the qualitative behavior of the model. An important model prediction is that channel inactivation must occur on a slower time scale than activation in order for waves to propagate. The model serves to capture the essential macroscopic mechanisms that are involved in the production of intracellular Ca2+ oscillations and traveling waves in the Xenopus laevis oocyte.     

Hemoglobin crystals in fluid specimens from confined body spaces

Hemoglobin crystals phagocytized by polymorphonuclear leukocytes were seen in cytologic preparations of a cerebrospinal fluid and two pleural fluids. In the last two cases, the crystals were seen within erythrocytes and also free in the background. Intraerythrocytic crystallization of hemoglobin is the result of polymerization of the hemoglobin molecules; it occurs in peripheral blood in certain hemoglobinopathies, being more pronounced in hemoglobin C disease. In our three cases, in which the crystallization occurred not in peripheral blood but in fluids of confined body spaces, there was no clinical evidence of hemoglobinopathy and blood hemoglobin electrophoresis performed in one of the cases revealed normal hemoglobin. Under laboratory conditions, we produced intraerythrocytic crystallization of hemoglobin in hemorrhagic pleural fluid specimens by subjecting them to agents that induced decreased oxygen concentration and osmotic dehydration of the cells. We suggest that similar processes operative in fluid accumulated in confined body spaces produce crystallization of the hemoglobin of extravasated red blood cells in the absence of hemoglobinopathy.     

Molecular dynamics modelling of tethered organics in confined spaces

A computational method for constructing and evaluating the dynamic behaviour of functionalised hexagonal mesoporous silica (HMS) MCM-41 models is reported. HMS with three pore diameters (1.7, 2.2 and 2.9 nm) were prepared, and, from these, two series of derivative structures were constructed – one with 1,3-diphenylpropyl (DPP) tethers and the other with smaller dimethylsilyl (DMS) tethers attached to the mesopores' internal surfaces. Comparison with experimental data shows that simulation results correctly predict the maximum tether density that can be achieved for each tether and each pore diameter. For the smaller pore models, the extent of DPP functionalisation that can be achieved is limited by the available pore volume. However, for the larger pore model, the extent of functionalisation is limited by access to potentially reactive sites on the pore surface. The dynamic behaviour of the models was investigated over a range of temperatures (240–648 K). At lower temperatures ( < 400 K), the mobility of DPP tethers in the 2.9 nm model is actually less than that observed in either the 2.2 nm model or the 1.7 nm model due to the extensive non-bonded interactions that are able to develop between tethers and the silica surface at this diameter. At higher temperatures, the free ends of these tethers break away from the surface, extend further into the pore space and the DPP mobility in the 2.9 nm model is higher than in the smaller pore systems.     

Fast calcium waves

Calcium waves are propagated in five main speed ranges which cover a billion-fold range of speeds. We define the fast speed range as 3-30μm/s after correction to a standard temperature of 20°C. Only waves which are not fertilization waves are considered here. 181 such cases are listed here. These are through organisms in all major taxa from cyanobacteria through mammals including human beings except for those through other bacteria, higher plants and fungi. Nearly two-thirds of these speeds lie between 12 and 24μm/s. We argue that their common mechanism in eukaryotes is a reaction-diffusion one involving calcium-induced calcium release, in which calcium waves are propagated along the endoplasmic reticulum. We propose that the gliding movements of some cyanobacteria are driven by fast calcium waves which are propagated along their plasma membranes. Fast calcium waves may drive materials to one end of developing embryos by cellular peristalsis, help coordinate complex cell movements during development and underlie brain injury waves. Moreover, we continue to argue that such waves greatly increase the likelihood that chronic injuries will initiate tumors and cancers before genetic damage occurs. Finally we propose numerous further studies.     

Protein folding and binding in confined spaces and in crowded solutions

Simple theoretical models are presented to illustrate the effects of spatial confinement and macromolecular crowding on the equilibria and rates of protein folding and binding. Confinement is expected to significantly stabilize the folded state, but for crowding only a marginal effect on protein stability is expected. In confinement the unfolded chain is restricted to a cage but in crowding the unfolded chain may explore different interstitial voids. Because confinement and crowding eliminate the more expanded conformations of the unfolded state, folding from the compact unfolded state is expected to speed up. Crowding will shift the binding equilibrium of proteins toward the bound state. The significant slowing down in protein diffusion by crowding, perhaps beneficial for chaperonin action, could result in a decrease in protein binding rates.     

Role of mitochondria in spontaneous rhythmic activity and intracellular calcium waves in the guinea pig gallbladder smooth muscle

Mitochondrial Ca(2+) handling has been implicated in spontaneous rhythmic activity in smooth muscle and interstitial cells of Cajal. In this investigation we evaluated the effect of mitochondrial inhibitors on spontaneous action potentials (APs), Ca(2+) flashes, and Ca(2+) waves in gallbladder smooth muscle (GBSM). Disruption of the mitochondrial membrane potential with carbonyl cyanide 3-chlorophenylhydrazone, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, rotenone, and antimycin A significantly reduced or eliminated APs, Ca(2+) flashes, and Ca(2+) waves in GBSM. Blockade of ATP production with oligomycin did not alter APs or Ca(2+) flashes but significantly reduced Ca(2+) wave frequency. Inhibition of mitochondrial Ca(2+) uptake and Ca(2+) release with Ru360 and CGP-37157, respectively, reduced the frequency of Ca(2+) flashes and Ca(2+) waves in GBSM. Similar to oligomycin, cyclosporin A did not alter AP and Ca(2+) flash frequency but significantly reduced Ca(2+) wave activity. These data suggest that mitochondrial Ca(2+) handling is necessary for the generation of spontaneous electrical activity and may therefore play an important role in gallbladder tone and motility.     

Surfing on calcium waves

A key question in brain development is how migration of neuronal precursors is guided to establish the ordered laminar layers. In the April 20, 2007 issue of Cell, Guan et al. show that the leading process of migrating cerebellar granule neurons senses repulsive Slit molecules by generating a Ca(2+) wave that propagates to the soma to cause reversal of cell polarity and migration.     

Polarity in intracellular calcium signaling.

The concentration of free calcium ions (Ca(2+)) in the cytosol is precisely regulated and can be rapidly increased in response to various types of stimuli. Since Ca(2+) can be used to control different processes in the same cell, the spatial organization of cytosolic Ca(2+) signals is of considerable importance. Polarized cells have advantages for Ca(2+) studies since localized signals can be related to particular organelles. The pancreatic acinar cell is well-characterized with a clearly polarized structure and function. Since the discovery of the intracellular Ca(2+)-releasing function of inositol 1,4,5-trisphosphate (IP(3)) in the pancreas in the early 1980s, this cell has become a popular study object and is now one of the best-characterized with regard to Ca(2+) signaling properties. Stimulation of pancreatic acinar cells with the neurotransmitter acetylcholine or the hormone cholecystokinin evokes Ca(2+) signals that are either local or global, depending on the agonist concentration and the length of the stimulation period. The nature of the Ca(2+) transport events across the basal and apical plasma membranes as well as the involvement of the endoplasmic reticulum (ER), the nucleus, the mitochondria, and the secretory granules in Ca(2+) signal generation and termination have become much clearer in recent years.     

The latest waves in calcium signaling

Ca2+ is a universal second messenger that is a key component of myriad processes in all cell types. Perturbations in normal intracellular Ca2+ concentrations underlie many common pathological conditions, ranging from cardiac hypertrophy to ischemic death of neurons. A recent meeting addressed the contributions of Ca2+ and Ca2+ binding proteins to health and disease. Insights gleaned from mechanistic studies offered the potential for new therapeutic approaches to combat a variety of diseases resulting from alterations in Ca2+ homeostasis.     

Chemotaxis of cell populations through confined spaces at single-cell resolution

Cell migration is crucial for both physiological and pathological processes. Current in vitro cell motility assays suffer from various drawbacks, including insufficient temporal and/or optical resolution, or the failure to include a controlled chemotactic stimulus. Here, we address these limitations with a migration chamber that utilizes a self-sustaining chemotactic gradient to induce locomotion through confined environments that emulate physiological settings. Dynamic real-time analysis of both population-scale and single-cell movement are achieved at high resolution. Interior surfaces can be functionalized through adsorption of extracellular matrix components, and pharmacological agents can be administered to cells directly, or indirectly through the chemotactic reservoir. Direct comparison of multiple cell types can be achieved in a single enclosed system to compare inherent migratory potentials. Our novel microfluidic design is therefore a powerful tool for the study of cellular chemotaxis, and is suitable for a wide range of biological and biomedical applications.