SparkMaster: automated calcium spark analysis with ImageJ

Ca sparks are elementary Ca-release events from intracellular Ca stores that are observed in virtually all types of muscle. Typically, Ca sparks are measured in the line-scan mode with confocal laser-scanning microscopes, yielding two-dimensional images (distance vs. time). The manual analysis of these images is time consuming and prone to errors as well as investigator bias. Therefore, we developed SparkMaster, an automated analysis program that allows rapid and reliable spark analysis. The underlying analysis algorithm is adapted from the threshold-based standard method of spark analysis developed by Cheng et al. (Biophys J 76: 606–617, 1999) and is implemented here in the freely available image-processing software ImageJ. SparkMaster offers a graphical user interface through which all analysis parameters and output options are selected. The analysis includes general image parameters (number of detected sparks, spark frequency) and individual spark parameters (amplitude, full width at half-maximum amplitude, full duration at half-maximum amplitude, full width, full duration, time to peak, maximum steepness of spark upstroke, time constant of spark decay). We validated the algorithm using images with synthetic sparks embedded into backgrounds with different signal-to-noise ratios to determine an analysis criteria at which a high sensitivity is combined with a low frequency of false-positive detections. Finally, we applied SparkMaster to analyze experimental data of sparks measured in intact and permeabilized ventricular cardiomyocytes, permeabilized mammalian skeletal muscle, and intact smooth muscle cells. We found that SparkMaster provides a reliable, easy to use, and fast way of analyzing Ca sparks in a wide variety of experimental conditions. myocytes; sarcoplasmic reticulum; confocal microscopy     

Ca2+ entry-independent effects of L-type Ca2+ channel modulators on Ca2+ sparks in ventricular myocytes

During the cardiac action potential, Ca²⁺ entry through dyhidropyridine receptor L-type Ca²⁺ channels (DHPRs) activates ryanodine receptors (RyRs) Ca²⁺-release channels, resulting in massive Ca²⁺ mobilization from the sarcoplasmic reticulum (SR). This global Ca²⁺ release arises from spatiotemporal summation of many localized elementary Ca²⁺-release events, Ca²⁺ sparks. We tested whether DHPRs modulate Ca²⁺sparks in a Ca²⁺ entry-independent manner. Negative modulation by DHPR of RyRs via physical interactions is accepted in resting skeletal muscle but remains controversial in the heart. Ca²⁺ sparks were studied in cat cardiac myocytes permeabilized with saponin or internally perfused via a patch pipette. Bathing and pipette solutions contained low Ca²⁺ (100 nM). Under these conditions, Ca²⁺ sparks were detected with a stable frequency of 3–5 sparks·s^–1·100 µm^–1. The DHPR blockers nifedipine, nimodipine, FS-2, and calciseptine decreased spark frequency, whereas the DHPR agonists Bay-K8644 and FPL-64176 increased it. None of these agents altered the spatiotemporal characteristics of Ca²⁺ sparks. The DHPR modulators were also without effect on SR Ca²⁺ load (caffeine-induced Ca²⁺ transients) or sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) activity (Ca²⁺ loading rates of isolated SR microsomes) and did not change cardiac RyR channel gating (planar lipid bilayer experiments). In summary, DHPR modulators affected spark frequency in the absence of DHPR-mediated Ca²⁺ entry. This action could not be attributed to a direct action of DHPR modulators on SERCA or RyRs. Our results suggest that the activity of RyR Ca²⁺-release units in ventricular myocytes is modulated by Ca²⁺ entry-independent conformational changes in neighboring DHPRs. exitation-contraction coupling; ryanodine receptor; sarco(endo)plasmic reticulum Ca²⁺-ATPase; dihydropyridine receptor; sarcoplasmic reticulum     

Role of glycolytically generated ATP for CaMKII-mediated regulation of intracellular Ca2+ signaling in bovine vascular endothelial cells

The role of glycolytically generated ATP in Ca²⁺/calmodulin-dependent kinase II (CaMKII)-mediated regulation of intracellular Ca²⁺ signaling was examined in cultured calf pulmonary artery endothelial (CPAE) cells. Exposure of cells (extracellular Ca²⁺ concentration = 2 mM) to glycolytic inhibitors 2-deoxy-D-glucose (2-DG), pyruvate (pyr) + -hydroxybutyrate (-HB), or iodoacetic acid (IAA) caused an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i). CaMKII inhibitors (KN-93, W-7) triggered a similar increase of [Ca²⁺]_i. The rise of [Ca²⁺]_i was characterized by a transient spike followed by a small sustained plateau of elevated [Ca²⁺]_i. In the absence of extracellular Ca²⁺ 2-DG caused an increase in [Ca²⁺]_i, suggesting that inhibition of glycolysis directly triggered release of Ca²⁺ from intracellular endoplasmic reticulum (ER) Ca²⁺ stores. The inositol-1,4,5-trisphosphate receptor (IP₃R) inhibitor 2-aminoethoxydiphenyl borate abolished the KN-93- and 2-DG-induced Ca²⁺ response. Ca²⁺ release was initiated in peripheral cytoplasmic processes from which activation propagated as a [Ca²⁺]_i wave toward the central region of the cell. Focal application of 2-DG resulted in spatially confined elevations of [Ca²⁺]_i. Propagating [Ca²⁺]_i waves were preceded by [Ca²⁺]_i oscillations and small, highly localized elevations of [Ca²⁺]_i (Ca²⁺ puffs). Inhibition of glycolysis with 2-DG reduced the KN-93-induced Ca²⁺ response, and vice versa during inhibition of CaMKII 2-DG-induced Ca²⁺ release was attenuated. Similar results were obtained with pyr + -HB and W-7. Furthermore, 2-DG and IAA caused a rapid increase of intracellular Mg²⁺ concentration, indicating a concomitant drop of cellular ATP levels. In conclusion, CaMKII exerts a profound inhibition of ER Ca²⁺ release in CPAE cells, which is mediated by glycolytically generated ATP, possibly through ATP-dependent phosphorylation of the IP₃R. Ca²⁺/calmodulin-dependent kinase II; glycolysis; calcium regulation     

Pharmacological inhibition of PARP-1 reduces alpha-synuclein- and MPP+-induced cytotoxicity in Parkinson's disease in vitro models

Treatments based on pharmacological inhibition of poly(ADP-ribose) polymerase-1 (PARP-1) have been suggested for a broad variety of human disorders, including Parkinson's disease (PD). The neuroprotective effects underlying the efficacy of PARP-1 inhibitors in PD models suggest a role for PARP-1 in neurodegeneration. In this study, we assessed the efficacy of PARP-1 inhibition in two distinct PD models. First, we tested a panel of small molecule PARP-1 inhibitors in alpha-synuclein (aSyn) cytotoxicity assay, where we observed compound-dependent ameliorating effects. Next, we tested the same panel in primary ventral mesencephalic neuronal cultures, treated with MPP(+). Dopaminergic neurons, the primary cells affected in PD, were selected and subjected to analysis. A significant ameliorating effect was achieved only with a highly potent PARP-1 inhibitor. Our data implicates aberrant PARP-1 function in different pathways of neurodegeneration. Further, our results suggest a rationale for the development of highly potent, bio-available, brain-penetrable PARP-1 inhibitors to provide therapeutic benefits for Parkinson's patients.     

Evolutionarily conserved E(y)2/Sus1 protein is essential for the barrier activity of Su(Hw)-dependent insulators in Drosophila

Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers for spreading of repressive chromatin. The Su(Hw) protein is responsible for activity of the best-studied Drosophila insulators. Here we demonstrate that an evolutionarily conserved protein, E(y)2/Sus1, is recruited to the Su(Hw) insulators via binding to the zinc-finger domain of Su(Hw). Partial inactivation of E(y)2 in a weak mutation, e(y)2(u1), impairs only the barrier, but not the enhancer-blocking, activity of the Su(Hw) insulators. Whereas neither su(Hw)(-) nor e(y)2(u1) affects fly viability, their combination proves lethal, testifying to functional interaction between Su(Hw) and E(y)2 in vivo. Apparently, different domains of Su(Hw) recruit proteins responsible for enhancer-blocking and for the barrier activity.     

Role of the PB-loop in ApcE and phycobilisome core function in cyanobacterium Synechocystis sp. PCC 6803

The phycobilisome (PBS) is a giant highly-structured pigment-protein antenna of cyanobacteria and red algae. PBS is composed of the phycobiliproteins and several linker polypeptides. The large core-membrane linker protein (L_CM or ApcE) influences many features and functions of PBS and consists of several domains including the chromophorylated PB-domain. Being homologous to the phycobiliprotein α-subunits this domain includes a so-called PB-loop insertion whose functions are still unknown. We have created the photoautotrophic mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 with lacking PB-loop. Using various spectral techniques we have demonstrated that this mutation does not destroy the PBS integrity and the internal PBS excitation energy transfer pathways. At the same time, the deletion of the PB-loop leads to the decrease of connectivity between the PBS and thylakoid membrane and to the compensatory increase of the relative photosystem II content. Mutation provokes the violation of the thylakoid membranes arrangement, the inability to perform state transitions, and diminishing of the OCP-dependent non-photochemical PBS quenching. In essence, even such a minute mutation of the PBS polypeptide component, like the PB-loop deletion, becomes important for the concerted function of the photosynthetic apparatus.     

Gly25-Ser26 Amyloid β-Protein Structural Isomorphs Produce Distinct Aβ42 Conformational Dynamics and Assembly Characteristics

One of the earliest events in amyloid β-protein (Aβ) self-association is nucleation of Aβ monomer folding through formation of a turn at Gly25-Lys28. We report here the effects of structural changes at the center of the turn, Gly25-Ser26, on Aβ42 conformational dynamics and assembly. We used “click peptide” chemistry to quasi-synchronously create Aβ42 from 26-O-acyliso-Aβ42 (iAβ42) through a pH jump from 3 to 7.4. We also synthesized N_α-acetyl-Ser26-iAβ42 (Ac-iAβ42), which cannot undergo O → N acyl chemistry, to study the behavior of this ester form of Aβ42 itself at neutral pH. Data from experiments monitoring increases in β-sheet formation (thioflavin T, CD), hydrodynamic radius (R_H), scattering intensity (quasielastic light scattering spectroscopy), and extent of oligomerization (ion mobility spectroscopy–mass spectrometry) were quite consistent. A rank order of Ac-iAβ42 > iAβ42 > Aβ42 was observed. Photochemically cross-linked iAβ42 displayed an oligomer distribution with a prominent dimer band that was not present with Aβ42. These dimers also were observed selectively in iAβ42 in ion mobility spectrometry experiments. The distinct biophysical behaviors of iAβ42 and Aβ42 appear to be due to the conversion of iAβ42 into “pure” Aβ42 monomer, a nascent form of Aβ42 that does not comprise the variety of oligomeric and aggregated states present in pre-existent Aβ42. These results emphasize the importance of the Gly25-Ser26 dipeptide in organizing Aβ42 monomer structure and thus suggest that drugs altering the interactions of this dipeptide with neighboring side-chain atoms or with the peptide backbone could be useful in therapeutic strategies targeting formation of Aβ oligomers and higher-order assemblies.     

Unexpected population genetic structure of European roe deer in Poland: an invasion of the mtDNA genome from Siberian roe deer

Introgressive hybridization is a widespread evolutionary phenomenon which may lead to increased allelic variation at selective neutral loci and to transfer of fitness‐related traits to introgressed lineages. We inferred the population genetic structure of the European roe deer (Capreolus capreolus) in Poland from mitochondrial (CR and cyt b) and sex‐linked markers (ZFX, SRY, DBY4 and DBY8). Analyses of CR mtDNA sequences from 452 individuals indicated widespread introgression of Siberian roe deer (C. pygargus) mtDNA in the European roe deer genome, 2000 km from the current distribution range of C. pygargus. Introgressed individuals constituted 16.6% of the deer studied. Nearly 75% of them possessed haplotypes belonging to the group which arose 23 kyr ago and have not been detected within the natural range of Siberian roe deer, indicating that majority of present introgression has ancient origin. Unlike the mtDNA results, sex‐specific markers did not show signs of introgression. Species distribution modelling analyses suggested that C. pygargus could have extended its range as far west as Central Europe after last glacial maximum. The main hybridization event was probably associated with range expansion of the most abundant European roe deer lineage from western refugia and took place in Central Europe after the Younger Dryas (10.8–10.0 ka BP). Initially, introgressed mtDNA variants could have spread out on the wave of expansion through the mechanism of gene surfing, reaching high frequencies in European roe deer populations and leading to observed asymmetrical gene flow. Human‐mediated introductions of C. pygargus had minimal effect on the extent of mtDNA introgression.     

Engineering of the Saccharomyces cerevisiae yeast strain with multiple chromosome-integrated genes of human alpha-fetoprotein and its high-yield secretory production, purification, structural and functional characterization

Alpha-fetoprotein (AFP) is a biological drug candidate of high medicinal potential in the treatment of autoimmune diseases, cancer, and regenerative medicine. Large-scale production of recombinant human alpha-fetoprotein (rhAFP) is desirable for structural and functional studies and applied research. In this study we cloned and expressed in the secreted form wild-type glycosylated human rhAFP and non-glycosylated mutant rhAFP(0) (N233S) in the yeast strain Saccharomyces cerevisiae with multiple chromosome-integrated synthetic human AFP genes. RhAFP and rhAFP(0) were successfully produced and purified from the culture liquids active naturally folded proteins. Elimination of the glycosylation by mutation reduced rhAFP(0) secretion about threefold as compared to the wild-type protein showing critical role of the N-linked glycan for heterologous protein folding and secretion. Structural similarity of rhAFP and rhAFP(0) with natural embryonic eAFP was confirmed by circular dichroism technique. Functional tests demonstrated similar type of tumor suppressive and immunosuppressive activity for both recombinant species rhAFP and rhAFP(0) as compared to natural eAFP. It was documented that both types of biological activities attributed to rhAFP and rhAFP(0) are due to the fast induction of apoptosis in tumor cells and mitogen-activated lymphocytes. Despite the fact that rhAFP and rhAFP(0) demonstrated slightly less effective tumor suppressive activity as compared to eAFP but rhAFP(0) had produced statistically notable increase in its ability to induce inhibition of in vitro lymphocyte proliferation as compared to the glycosylated rhAFP and eAFP. We conclude that N-linked glycosylation of rhAFP is required for efficient folding and secretion. However the presence of N-linked sugar moiety was shown to be unimportant for tumor suppressive activity but was critically important for its immunoregulative activity which demonstrates that different molecular mechanisms are involved in these two types of biological functional activities attributed to AFP.     

The role of integrin alpha D beta2 (CD11d/CD18) in monocyte/macrophage migration

Integrin α_Dβ₂ (CD11d/CD18) is a multiligand macrophage receptor with recognition specificity identical to that of the major myeloid cell-specific integrin α_Mβ₂ (CD11b/CD18, Mac-1). Despite its prominent upregulation on inflammatory macrophages, the role of α_Dβ₂ in monocyte and macrophage migration is unknown. In this study, we have generated model and natural cell lines expressing different densities of α_Dβ₂ and examined their migration to various extracellular matrix proteins. When expressed at a low density, α_Dβ₂ on the surface of recombinant HEK293 cells and murine IC-21 macrophages cooperates with β₁/β₃ integrins to support cell migration. However, its increased expression on the α_Dβ₂-expressing HEK293 cells and its upregulation by PMA on the IC-21 macrophages result in increased cell adhesiveness and inhibition of cell migration. Furthermore, ligation of α_Dβ₂ with anti-α_D blocking antibodies restores β₁/β₃-driven cell migration by removing the excess α_Dβ₂-mediated adhesive bonds. Consistent with in vitro data, increased numbers of inflammatory macrophages were recovered from the inflamed peritoneum of mice after the administration of anti-α_D antibody. These results demonstrate that the density of α_Dβ₂ is critically involved in modulating macrophage adhesiveness and their migration, and suggest that low levels of α_Dβ₂ contribute to monocyte migration while α_Dβ₂ upregulation on differentiated macrophages may facilitate their retention at sites of inflammation.