A structural framework for a near-minimal form of life: mass and compositional analysis of the helical mollicute Spiroplasma melliferum BC3.

Spiroplasma melliferum is a wall-less bacterium with dynamic helical geometry. This organism is geometrically well defined and internally well ordered, and has an exceedingly small genome. Individual cells are chemotactic, polar, and swim actively. Their dynamic helicity can be traced at the molecular level to a highly ordered linear motor (composed essentially of the proteins fib and MreB) that is positioned on a defined helical line along the internal face of the cell's membrane. Using an array of complementary, informationally overlapping approaches, we have taken advantage of this uniquely simple, near-minimal life-form and its helical geometry to analyze the copy numbers of Spiroplasma's essential parts, as well as to elucidate how these components are spatially organized to subserve the whole living cell. Scanning transmission electron microscopy (STEM) was used to measure the mass-per-length and mass-per-area of whole cells, membrane fractions, intact cytoskeletons and cytoskeletal components. These local data were fit into whole-cell geometric parameters determined by a variety of light microscopy modalities. Hydrodynamic data obtained by analytical ultracentrifugation allowed computation of the hydration state of whole living cells, for which the relative amounts of protein, lipid, carbohydrate, DNA, and RNA were also estimated analytically. Finally, ribosome and RNA content, genome size and gene expression were also estimated (using stereology, spectroscopy and 2D-gel analysis, respectively). Taken together, the results provide a general framework for a minimal inventory and arrangement of the major cellular components needed to support life.

The interactive roles of zinc and calcium in mitochondrial dysfunction and neurodegeneration.

Zinc has been implicated in neurodegeneration following ischemia. In analogy with calcium, zinc has been proposed to induce toxicity via mitochondrial dysfunction, but the relative role of each cation in mitochondrial damage remains unclear. Here, we report that under conditions mimicking ischemia in hippocampal neurons - normal (2 mM) calcium plus elevated (> 100 µM) exogenous zinc - mitochondrial dysfunction evoked by glutamate, kainate or direct depolarization is, despite significant zinc uptake, primarily governed by calcium. Thus, robust mitochondrial ion accumulation, swelling, depolarization, and reactive oxygen species generation were only observed after toxic stimulation in calcium-containing media. This contrasts with the lack of any mitochondrial response in zinc-containing but calcium-free medium, even though zinc uptake and toxicity were strong under these conditions. Indeed, abnormally high, ionophore-induced zinc uptake was necessary to elicit any mitochondrial depolarization. In calcium- and zinc-containing media, depolarization-induced zinc uptake facilitated cell death and enhanced accumulation of mitochondrial calcium, which localized to characteristic matrix precipitates. Some of these contained detectable amounts of zinc. Together these data indicate that zinc uptake is generally insufficient to trigger mitochondrial dysfunction, so that mechanism(s) of zinc toxicity must be different from that of calcium.

Measurement of total calcium in neurons by electron probe X-ray microanalysis.

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires "cryofixation" of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 °C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at ≤-160 °C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 °C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their "characteristic" peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

Comparative impact of voltage-gated calcium channels and NMDA receptors on mitochondria-mediated neuronal injury.

Glutamate excitotoxicity, a major component of many neurodegenerative disorders, is characterized by excessive calcium influx selectively through NMDARs. However, there is a substantial uncertainty concerning why other known routes of significant calcium entry, in particular, VGCCs, are not similarly toxic. Here, we report that in the majority of neurons in rat hippocampal and cortical cultures, maximal L-type VGCC activation induces much lower calcium loading than toxic NMDAR activation. Consequently, few depolarization-activated neurons exhibit calcium deregulation and cell death. Activation of alternative routes of calcium entry induced neuronal death in proportion to the degree of calcium loading. In a small subset of neurons, depolarization evoked stronger calcium elevations, approaching those induced by toxic NMDA. These neurons were characterized by elevated expression of VGCCs and enhanced voltage-gated calcium currents, mitochondrial dysfunction and cell death. Preventing VGCC-dependent mitochondrial calcium loading resulted in stronger cytoplasmic calcium elevations, whereas inhibiting mitochondrial calcium clearance accelerated mitochondrial depolarization. Both observations further implicate mitochondrial dysfunction in VGCC-mediated cell death. Results indicate that neuronal vulnerability tracks the extent of calcium loading but does not appear to depend explicitly on the route of calcium entry.

The Bcl-2 gene polymorphism rs956572AA increases inositol 1,4,5-trisphosphate receptor-mediated endoplasmic reticulum calcium release in subjects with bipolar disorder.

BACKGROUND: Bipolar disorder (BPD) is characterized by altered intracellular calcium (Ca(2+)) homeostasis. Underlying mechanisms involve dysfunctions in endoplasmic reticulum (ER) and mitochondrial Ca(2+) handling, potentially mediated by B-cell lymphoma 2 (Bcl-2), a key protein that regulates Ca(2+) signaling by interacting directly with these organelles, and which has been implicated in the pathophysiology of BPD. Here, we examined the effects of the Bcl-2 gene single nucleotide polymorphism (SNP) rs956572 on intracellular Ca(2+) dynamics in patients with BPD. METHODS: Live cell fluorescence imaging and electron probe microanalysis were used to measure intracellular and intra-organelle free and total calcium in lymphoblasts from 18 subjects with BPD carrying the AA, AG, or GG variants of the rs956572 SNP. Analyses were carried out under basal conditions and in the presence of agents that affect Ca(2+) dynamics. RESULTS: Compared with GG homozygotes, variant AA-which expresses significantly reduced Bcl-2 messenger RNA and protein-exhibited elevated basal cytosolic Ca(2+) and larger increases in inositol 1,4,5-trisphosphate receptor-mediated cytosolic Ca(2+) elevations, the latter in parallel with enhanced depletion of the ER Ca(2+) pool. The aberrant behavior of AA cells was reversed by chronic lithium treatment and mimicked in variant GG by a Bcl-2 inhibitor. In contrast, no differences between SNP variants were found in ER or mitochondrial total Ca(2+) content or in basal store-operated Ca(2+) entry. CONCLUSIONS: These results demonstrate that, in patients with BPD, abnormal Bcl-2 gene expression in the AA variant contributes to dysfunctional Ca(2+) homeostasis through a specific ER inositol 1,4,5-trisphosphate receptor-dependent mechanism.

Calcium-dependent mitochondrial function and dysfunction in neurons.

Calcium is an extraordinarily versatile signaling ion, encoding cellular responses to a wide variety of external stimuli. In neurons, mitochondria can accumulate enormous amounts of calcium, with the consequence that mitochondrial calcium uptake, sequestration and release play pivotal roles in orchestrating calcium-dependent responses as diverse as gene transcription and cell death. In this review, we consider the basic chemistry of calcium as a 'sticky' cation, which leads to extremely high bound/free ratios, and discuss areas of current interest or controversy. Topics addressed include methodologies for measuring local intracellular calcium, mitochondrial calcium buffering and loading capacity, mitochondrially directed spatial calcium gradients, and the role of calcium overload-dependent mitochondrial dysfunction in glutamate-evoked excitotoxic injury and neurodegeneration. Finally, we consider the relationship between delayed calcium de-regulation, the mitochondrial permeability transition and the generation of reactive oxygen species, and propose a unified view of the 'source specificity' and 'calcium overload' models of N-methyl-d-aspartate (NMDA) receptor-dependent excitotoxicity. Non-NMDA receptor mechanisms of excitotoxicity are discussed briefly.

Differential NMDA receptor-dependent calcium loading and mitochondrial dysfunction in CA1 vs. CA3 hippocampal neurons.

Hippocampal CA1 pyramidal neurons are selectively vulnerable to ischemia, while adjacent CA3 neurons are relatively resistant. Although glutamate receptor-mediated mitochondrial Ca(2+) overload and dysfunction is a major component of ischemia-induced neuronal death, no direct relationship between selective neuronal vulnerability and mitochondrial dysfunction has been demonstrated in intact brain preparations. Here, we show that in organotypic slice cultures NMDA induces much larger Ca(2+) elevations in vulnerable CA1 neurons than in resistant CA3. Consequently, CA1 mitochondria exhibit stronger calcium accumulation, more extensive swelling and damage, stronger depolarization of their membrane potential, and a significant increase in ROS generation. NMDA-induced Ca(2+) and ROS elevations were abolished in Ca(2+)-free medium or by NMDAR antagonists, but not by zinc chelation. We conclude that Ca(2)(+) overload-dependent mitochondrial dysfunction is a determining factor in the selective vulnerability of CA1 neurons.

Coupling diverse routes of calcium entry to mitochondrial dysfunction and glutamate excitotoxicity.

Overactivation of NMDA receptors (NMDARs) is a critical early step in glutamate-evoked excitotoxic injury of CNS neurons. Distinct NMDAR-coupled pathways specified by, for example, receptor location or subunit composition seem to govern glutamate-induced excitotoxic death, but there is much uncertainty concerning the underlying mechanisms of pathway selection. Here we ask whether, and if so how, route-specific vulnerability is coupled to Ca(2+) overload and mitochondrial dysfunction, which is also a known, central component of exitotoxic injury. In cultured hippocampal neurons, overactivation of only extrasynaptic NMDARs resulted in Ca(2+) entry strong enough to promote Ca(2+) overload, which subsequently leads to mitochondrial dysfunction and cell death. Receptor composition per se appears not to be a primary factor for specifying signal coupling, as NR2B inhibition abolished Ca(2+) loading and was protective only in predominantly NR2B-expressing young neurons. In older neurons expressing comparable levels of NR2A- and NR2B-containing NMDARs, amelioration of Ca(2+) overload required the inhibition of extrasynaptic receptors containing both NR2 subunits. Prosurvival synaptic stimuli also evoked Ca(2+) entry through both N2A- and NR2B-containing NMDARs, but, in contrast to excitotoxic activation of extrasynaptic NMDARs, produced only low-amplitude cytoplasmic Ca(2+) spikes and modest, nondamaging mitochondrial Ca(2+) accumulation. The results--showing that the various routes of excitotoxic Ca(2+) entry converge on a common pathway involving Ca(2+) overload-induced mitochondrial dysfunction--reconcile and unify many aspects of the "route-specific" and "calcium load-dependent" views of exitotoxic injury.

Structure of the cytoskeleton of Spiroplasma melliferum BC3 and its interactions with the cell membrane.

Spiroplasma melliferum is a wall-less bacterium with dynamic helical symmetry. Taking advantage of the simplicity of this primitive lifeform, we have used structural (electron tomography and freeze fracture of whole cells; cryoelectron tomography and diffraction analysis of isolated cytoskeletons) and proteomic approaches to elucidate the basic organizing principles of its minimal yet functional cytoskeleton. From among approximately 30 Spiroplasma proteins present in a highly purified cytoskeletal fraction, we identify three major putative structural proteins: Fib, MreB, and elongation factor Tu. Fib assembles into a single flattened ribbon that follows the shortest helical line just under the plasma membrane and acts as a linear motor, whereas MreB is present as a matching array of membrane-associated fibrils parallel and associated with the motor. We also identify a prominent previously unknown filamentous network that occupies much of the cytoplasm and appears to cross-link the ribosomes. The abundant potentially filament-forming protein elongation factor Tu may be a component of this network, but the tomography data are most consistent with DNA as the core component. The results provide new information on the minimal organization necessary to support the scaffolding and motile functions of a minimal cytoskeleton.

Reduced calcium-dependent mitochondrial damage underlies the reduced vulnerability of excitotoxicity-tolerant hippocampal neurons.

In central neurons, over-stimulation of NMDA receptors leads to excessive mitochondrial calcium accumulation and damage, which is a critical step in excitotoxic death. This raises the possibility that low susceptibility to calcium overload-induced mitochondrial damage might characterize excitotoxicity-resistant neurons. In this study, we have exploited two complementary models of preconditioning-induced excitotoxicity resistance to demonstrate reduced calcium-dependent mitochondrial damage in NMDA-tolerant hippocampal neurons. We have further identified adaptations in mitochondrial calcium handling that account for enhanced mitochondrial integrity. In both models, enhanced tolerance was associated with improved preservation of mitochondrial membrane potential and structure. In the first model, which exhibited modest neuroprotection, mitochondria-dependent calcium deregulation was delayed, even though cytosolic and mitochondrial calcium loads were quantitatively unchanged, indicating that enhanced mitochondrial calcium capacity accounts for reduced injury. In contrast, the second model, which exhibited strong neuroprotection, displayed further delayed calcium deregulation and reduced mitochondrial damage because downregulation of NMDA receptor surface expression depressed calcium loading. Reducing calcium entry also modified the chemical composition of the calcium-buffering precipitates that form in calcium-loaded mitochondria. It thus appears that reduced mitochondrial calcium loading is a major factor underlying the robust neuroprotection seen in highly tolerant cells.

Calcium-induced precipitate formation in brain mitochondria: composition, calcium capacity, and retention.

Both isolated brain mitochondria and mitochondria in intact neurons are capable of accumulating large amounts of calcium, which leads to formation in the matrix of calcium- and phosphorus-rich precipitates, the chemical composition of which is largely unknown. Here, we have used inhibitors of the mitochondrial permeability transition (MPT) to determine how the amount and rate of mitochondrial calcium uptake relate to mitochondrial morphology, precipitate composition, and precipitate retention. Using isolated rat brain (RBM) or liver mitochondria (RLM) Ca(2+)-loaded by continuous cation infusion, precipitate composition was measured in situ in parallel with Ca(2+) uptake and mitochondrial swelling. In RBM, the endogenous MPT inhibitors adenosine 5'-diphosphate (ADP) and adenosine 5'-triphosphate (ATP) increased mitochondrial Ca(2+) loading capacity and facilitated formation of precipitates. In the presence of ADP, the Ca/P ratio approached 1.5, while ATP or reduced infusion rates decreased this ratio towards 1.0, indicating that precipitate chemical form varies with the conditions of loading. In both RBM and RLM, the presence of cyclosporine A in addition to ADP increased the Ca(2+) capacity and precipitate Ca/P ratio. Following MPT and/or depolarization, the release of accumulated Ca(2+) is rapid but incomplete; significant residual calcium in the form of precipitates is retained in damaged mitochondria for prolonged periods.

Real-time measurement of free Ca2+ changes in CNS myelin by two-photon microscopy.

Here we describe a technique for measuring changes in Ca2+ in the cytosolic domain of mature compact myelin of live axons in the central nervous system (CNS). We label the myelin sheath of optic nerve and dorsal column axons by using the Ca2+ indicator X-rhod-1 coupled with DiOC6(3) to produce bright myelin counterstaining, thereby providing unambiguous identification of the myelin sheath for analysis of two-photon excited fluorescence. We present evidence for localization of the Ca2+ reporter to the cytosolic domain of myelin, obtained by using fluorescence lifetime, spectral measurements and Mn2+ quenching. Chemical ischemia increased myelinic X-rhod-1 fluorescence (approximately 50% after 30 min) in a manner dependent on extracellular Ca2+. Inhibiting Na+-dependent glutamate transporters (with TBOA) or glycine transporters (with sarcosine and ALX-1393) reduced the ischemia-induced increase in Ca2+. We show that myelinic N-methyl-D-aspartate (NMDA) receptors are activated by the two conventional coagonists glutamate and glycine, which are released by specific transporters under conditions of cellular Na+ loading and depolarization in injured white matter. This new technique facilitates detailed studies of living myelin, a vital component of the mammalian CNS.

Strong calcium entry activates mitochondrial superoxide generation, upregulating kinase signaling in hippocampal neurons.

Large increases in cytosolic free Ca2+ ([Ca2+]i) activate several kinases that are important for neuronal plasticity, including Ca2+/calmodulin-dependent kinase II (CaMKII), protein kinase A (PKA), and protein kinase C (PKC). Because it is also known, mainly in non-neuronal systems, that superoxide radicals (O2-) activate these (and other) kinases and because O2- generation by mitochondria is in part [Ca2+]i dependent, we examined in hippocampal neurons the relationship between Ca2+ entry, O2- production, and kinase activity. We found that, after large stimulus-induced [Ca2+]i increases, O2- selectively produced by mitochondria near plasmalemmal sites of Ca2+ entry acts as a modulator to upregulate the two kinases, namely, CaMKII and PKA, whose activities are directly or indirectly phosphorylation dependent. The common mechanism involves O2- inhibition of inactivating protein phosphatases. Conversely, because small [Ca2+]i increases do not promote mitochondrial respiration and O2- generation, weak stimuli favor enhanced phosphatase activity, which therefore leads to suppressed kinase activity. Enhanced O2- production also promoted PKC activity but by a phosphatase-independent pathway. These results suggest that Ca2+-dependent upregulation of mitochondrial O2- production may be a general mechanism for linking Ca2+ entry to enhanced kinase activity and therefore to synaptic plasticity. This mechanism also represents yet another way that mitochondria, acting as calcium sensors, can play a role in neuronal signal transduction.

Excitotoxic calcium overload in a subpopulation of mitochondria triggers delayed death in hippocampal neurons.

In neurons, excitotoxic stimulation induces mitochondrial calcium overload and the release of pro-apoptotic proteins, which triggers delayed cell death. The precise mechanisms of apoptogen release, however, remain controversial. To characterize the linkage between mitochondrial calcium load and cell vulnerability, and to test the hypothesis that only a subpopulation of mitochondria damaged by calcium overload releases apoptogens, we have measured directly the concentrations of total Ca (free plus bound) in individual mitochondria and monitored in parallel structural changes and the subcellular localization of pro-apoptotic cytochrome c after NMDA overstimulation in cultured hippocampal neurons. Beyond transient elevation of cytosolic calcium and perturbation of Na+/K+ homeostasis, NMDA stimulation induced dramatic, but mainly reversible, changes in mitochondria, including strong calcium elevation, membrane potential depolarization, and variable swelling. Elevation of matrix Ca in the approximately one-third of mitochondria that were strongly swollen, as well as the absence of swelling when Ca2+ entry was abolished, indicate an essential role for Ca overload. Shortly after NMDA exposure, cytochrome c, normally localized to mitochondria, became diffusely distributed in the cytoplasm, coincident with the appearance of severely swollen mitochondria with ruptured outer membranes; under these conditions, cytochrome c was retained in intact mitochondria, implying that it was released mainly from damaged mitochondria. Consistent with the role of mitochondrial Ca overload, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone decreased Ca accumulation, prevented cytochrome c release, and was neuroprotective. These results support a mechanism in which delayed excitotoxic death involves apoptogen release from a subpopulation of calcium-overloaded mitochondria, whereas other, undamaged mitochondria maintain normal function.

Calcium-dependent mitochondrial superoxide modulates nuclear CREB phosphorylation in hippocampal neurons.

We report evidence that mitochondrially produced superoxide (O(2)(-)) is involved in signaling in hippocampal neurons by examining the relationship between strong but physiological increases in cytosolic free Ca(2+), mitochondrial calcium accumulation, O(2)(-) production, and CREB phosphorylation. Strong depolarization-induced Ca(2+) entry through NMDA or L-type Ca(2+) channels evoked large Ca(2+) transients, a sustained increase in O(2)(-), and a large rise in nuclear CaM and pCREB. Under these conditions, inhibition of mitochondrial Ca(2+) uptake and consequent O(2)(-) production suppressed Ca(2+) entry-induced pCREB elevation, indicating that O(2)(-) produced by mitochondria supports CREB phosphorylation. Similarly, inhibiting mitochondrial respiration blocked O(2)(-) production and also depressed the elevation of pCREB. Blocking calcineurin reversed this depression. We conclude that strong Ca(2+) entry promotes mitochondrial calcium accumulation and the subsequent enhancement of mitochondrial O(2)(-) production, which in turn prolongs the lifetime of pCREB by suppressing calcineurin-dependent pCREB dephosphorylation.

Mass distribution and spatial organization of the linear bacterial motor of Spiroplasma citri R8A2.

In the simple, helical, wall-less bacterial genus Spiroplasma, chemotaxis and motility are effected by a linear, contractile motor arranged as a flat cytoskeletal ribbon attached to the inner side of the membrane along the shortest helical line. With scanning transmission electron microscopy and diffraction analysis, we determined the hierarchical and spatial organization of the cytoskeleton of Spiroplasma citri R8A2. The structural unit appears to be a fibril, approximately 5 nm wide, composed of dimers of a 59-kDa protein; each ribbon is assembled from seven fibril pairs. The functional unit of the intact ribbon is a pair of aligned fibrils, along which pairs of dimers form tetrameric ring-like repeats. On average, isolated and purified ribbons contain 14 fibrils or seven well-aligned fibril pairs, which are the same structures observed in the intact cell. Scanning transmission electron microscopy mass analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified cytoskeletons indicate that the 59-kDa protein is the only constituent of the ribbons.

Correlated calcium uptake and release by mitochondria and endoplasmic reticulum of CA3 hippocampal dendrites after afferent synaptic stimulation.

Mitochondria and endoplasmic reticulum (ER) are important modulators of intracellular calcium signaling pathways, but the role of these organelles in shaping synaptic calcium transients in dendrites of pyramidal neurons remains speculative. We have measured directly the concentrations of total Ca (bound plus free) within intracellular compartments of proximal dendrites of CA3 hippocampal neurons at times after synaptic stimulation corresponding to the peak of the cytoplasmic free Ca2+ transient (1 sec), to just after its decay (30 sec), and to well after its return to prestimulus levels (180 sec). Electron probe microanalysis of cryosections from rapidly frozen slice cultures has revealed that afferent mossy fiber stimulation evokes large, rapid elevations in the concentration of total mitochondrial Ca ([Ca](mito)) in depolarized dendrites. A single tetanus (50 Hz/1 sec) elevated [Ca](mito) more than fivefold above characteristically low basal levels within 1 sec of stimulation and >10-fold by 30 sec after stimulation. This strong Ca accumulation was reversible, because [Ca](mito) had recovered by 180 sec after the tetanus. Ca sequestered within mitochondria was localized to small inclusions that were distributed heterogeneously within, and probably among, individual mitochondria. By 30 sec after stimulation an active subpopulation of ER cisterns had accumulated more Ca than had mitochondria despite a approximately 1 sec delay before the onset of accumulation. Active ER cisterns retained their Ca load much longer (>3 min) than mitochondria. The complementary time courses of mitochondrial versus ER Ca2+ uptake and release suggest that these organelles participate in a choreographed interplay, each shaping dendritic Ca2+ signals within characteristic regimes of cytosolic Ca2+ concentration and time.