An algorithm for automated detection,localization and measurement of local calcium signals from camera-based imaging

Local Ca²⁺ transients such as puffs and sparks form the building blocks of cellular Ca²⁺ signaling in numerous cell types. They have traditionally been studied by linescan confocal microscopy, but advances in TIRF microscopy together with improved electron-multiplied CCD (EMCCD) cameras now enable rapid (>500 frames s⁻¹) imaging of subcellular Ca²⁺ signals with high spatial resolution in two dimensions. This approach yields vastly more information (ca. 1 Gb min⁻¹) than linescan imaging, rendering visual identification and analysis of local events imaged both laborious and subject to user bias. Here we describe a routine to rapidly automate identification and analysis of local Ca²⁺ events. This features an intuitive graphical user-interfaces and runs under Matlab and the open-source Python software. The underlying algorithm features spatial and temporal noise filtering to reliably detect even small events in the presence of noisy and fluctuating baselines; localizes sites of Ca²⁺ release with sub-pixel resolution; facilitates user review and editing of data; and outputs time-sequences of fluorescence ratio signals for identified event sites along with Excel-compatible tables listing amplitudes and kinetics of events.     

At the right distance: ER-mitochondria juxtaposition in cell life and death

The interface between mitochondria and the endoplasmic reticulum is emerging as a crucial hub for calcium signalling, apoptosis, autophagy and lipid biosynthesis, with far reaching implications in cell life and death and in the regulation of mitochondrial and endoplasmic reticulum function. Here we review our current knowledge on the structural and functional aspects of this interorganellar juxtaposition. This article is part of a Special Issue entitled: Calcium Signaling In Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

The plant mitochondrial protein import apparatus — The differences make it interesting

Background

Mitochondria play essential roles in the life and death of almost all eukaryotic cells, ranging from single-celled to multi-cellular organisms that display tissue and developmental differentiation. As mitochondria only arose once in evolution, much can be learned from studying single celled model systems such as yeast and applying this knowledge to other organisms. However, two billion years of evolution have also resulted in substantial divergence in mitochondrial function between eukaryotic organisms.

Scope of Review

Here we review our current understanding of the mechanisms of mitochondrial protein import between plants and yeast (Saccharomyces cerevisiae) and identify a high level of conservation for the essential subunits of plant mitochondrial import apparatus. Furthermore, we investigate examples whereby divergence and acquisition of functions have arisen and highlight the emerging examples of interactions between the import apparatus and components of the respiratory chain.

Major conclusions

After more than three decades of research into the components and mechanisms of mitochondrial protein import of plants and yeast, the differences between these systems are examined. Specifically, expansions of the small gene families that encode the mitochondrial protein import apparatus in plants are detailed, and their essential role in seed viability is revealed.

General significance

These findings point to the essential role of the inner mitochondrial protein translocases in Arabidopsis, establishing their necessity for seed viability and the crucial role of mitochondrial biogenesis during germination. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

Regulatory networks,genes and glycerophospholipid biosynthesis pathway in schistosomiasis: A systems biology view for pharmacological intervention

Understanding network topology through embracing the global dynamical regulation of genes in an active state space rather than traditional one-gene–one trait approach facilitates the rational drug development process. Schistosomiasis, a neglected tropical disease, has glycerophospholipids as abundant molecules present on its surface. Lack of effective clinical solutions to treat pathogens encourages us to carry out systems-level studies that could contribute to the development of an effective therapy. Development of a strategy for identifying drug targets by combined genome-scale metabolic network and essentiality analyses through in silico approaches provides tantalizing opportunity to investigate the role of protein/substrate metabolism. A genome-scale metabolic network model reconstruction represents choline–phosphate cytidyltransferase as the rate limiting enzyme and regulates the rate of phosphatidylcholine (PC) biosynthesis. The uptake of choline was regulated by choline concentration, promoting the regulation of phosphocholine synthesis. In Schistosoma, the change in developmental stage could result from the availability of choline, hampering its developmental cycle. There are no structural reports for this protein. In order to inhibit the activity of choline–phosphate cytidyltransferase (CCT), it was modeled by homology modeling using 1COZ as the template from Bacillus subtilis. The transition-state stabilization and catalytic residues were mapped as ‘HXGH’ and ‘RTEGISTT’ motif. CCT catalyzes the formation of CDP-choline from phosphocholine in which nucleotidyltransferase adds CTP to phosphocholine. The presence of phosphocholine permits the parasite to survive in an immunologically hostile environment. This feature endeavors development of an inhibitor specific for cytidyltransferase in Schistosoma. Flavonolignans were used to inhibit this activity in which hydnowightin showed the highest affinity as compared to miltefosine.     

重组人源SNX7蛋白在大肠杆菌中的表达、纯化及磷脂结合特异性分析

Sorting nexins(SNXs)是一类含有SNX-PX结构域,并在细胞内吞和内体分选运输过程中发挥重要调节作用的蛋白。SNX7是SNXs家族中的一员,含有PX结构域和BAR结构域,属于SNX-PX-BAR亚家族。斑马鱼实验表明,SNX7是在肝脏中大量表达的抗凋亡蛋白,并在胚胎肝脏的发育中发挥关键作用。为了从蛋白水平对SNX7进行研究,首先将编码人源PX-BARSNX7(SNX7的一个片段,包含PX和BAR结构域)的cDNA片段插入到原核表达载体p28a中,再将重组质粒转化到大肠杆菌Rosseta 2(DE3)中诱导表达,并用亲和层析、离子交换和分子筛层析对PX-BARSNX7进行了纯化。Western blotting结果表明,亲和层析、离子交换和分子筛层析纯化后获得了高纯度的PX-BARSNX7蛋白。动态光散射实验显示PX-BARSNX7蛋白均一性良好。磷脂结合实验表明,PX-BARSNX7具有较为广泛的磷脂酰肌醇结合能力,能够与PtdIns(5)P、PtdIns(4,5)P2和PtdIns(3,4,5)P3结合。     

The α-Helical Region in p24γ2 Subunit of p24 Protein Cargo Receptor Is Pivotal for the Recognition and Transport of Glycosylphosphatidylinositol-anchored Proteins

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are group of proteins that depend on p24 cargo receptors for their transport from the endoplasmic reticulum to the Golgi apparatus. The GPI anchor is expected to act as a sorting and transport signal, but so far little is known about the recognition mechanism. In the present study we investigate the GPI-AP transport in cell knockdown of p24γ, the most diverse p24 subfamily. Knockdown of p24γ₂ but not of other p24γ family members impaired the transport of a reporter GPI-AP. Restoration of the knockdown-induced phenotype using chimeric constructs between p24γ₂ and the related p24γ₁ further implied a role of the α-helical region of p24γ₂ but not its GOLD domain in the specific binding of GPI-APs. We conclude that motifs in the membrane-adjacent α-helical region of p24γ₂ are involved in recognition of GPI-APs and are consequently responsible for the incorporation of these proteins into coat protein complex II-coated transport vesicles.     

Two Dipolar α-Helices within Hormone-encoding Regions of Proglucagon Are Sorting Signals to the Regulated Secretory Pathway

Proglucagon is expressed in pancreatic α cells, intestinal L cells, and some hypothalamic and brainstem neurons. Tissue-specific processing of proglucagon yields three major peptide hormones as follows: glucagon in the α cells and glucagon-like peptides (GLP)-1 and -2 in the L cells and neurons. Efficient sorting and packaging into the secretory granules of the regulated secretory pathway in each cell type are required for nutrient-regulated secretion of these proglucagon-derived peptides. Our previous work suggested that proglucagon is directed into granules by intrinsic sorting signals after initial processing to glicentin and major proglucagon fragment (McGirr, R., Guizzetti, L., and Dhanvantari, S. (2013) J. Endocrinol. 217, 229–240), leading to the hypothesis that sorting signals may be present in multiple domains. In the present study, we show that the α-helices within glucagon and GLP-1, but not GLP-2, act as sorting signals by efficiently directing a heterologous secretory protein to the regulated secretory pathway. Biophysical characterization of these peptides revealed that glucagon and GLP-1 each encode a nonamphipathic, dipolar α-helix, whereas the helix in GLP-2 is not dipolar. Surprisingly, glicentin and major proglucagon fragment were sorted with different efficiencies, thus providing evidence that proglucagon is first sorted to granules prior to processing. In contrast to many other prohormones in which sorting is directed by ordered prodomains, the sorting determinants of proglucagon lie within the ordered hormone domains of glucagon and GLP-1, illustrating that each prohormone has its own sorting “signature.”     

ESCRT-0 Protein Hepatocyte Growth Factor-regulated Tyrosine Kinase Substrate (Hrs) Is Targeted to Endosomes Independently of Signal-transducing Adaptor Molecule (STAM) and the Complex Formation with STAM Promotes Its Endosomal Dissociation

The ESCRT-0 complex, consisting of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal-transducing adaptor molecule (STAM) proteins, recognizes ubiquitylated cargo during the initial step of endosomal sorting. The endosomal accumulation of overexpressed Hrs has been reported previously to be associated with endosome enlargement. In this study, we have found that co-expressing exogenous STAM1 in Hrs-overexpressing cells leads to a diffuse localization for a large part of the Hrs accumulated on endosomes and a recovery of the impaired cargo protein degradation process, thus suggesting that exogenous STAM abrogates the abnormalities of the Hrs-positive endosomes. A fluorescently labeled Hrs, introduced into the cells by membrane permeabilization, exhibited endosomal localization in the absence of STAM1 and gradually dissociated from the endosomes upon the sequential addition of recombinant STAM1. Furthermore, when microinjected into cells, the fluorescently labeled Hrs also showed endosomal accumulation; however, ESCRT-0 complexes formed prior to the microinjection did not. Analysis of the state of the complex in HeLa cells using blue-native PAGE revealed that the membrane-associated Hrs exists partly as a monomer and not only in the STAM1-bound form. Thus, our data suggest that the membrane binding and dissociation cycle of the ESCRT-0 proteins on the endosomal membrane is a critical step during the cargo sorting process.     

Accuracy and precision of gait events derived from motion capture in horses during walk and trot

This study aimed to create an evidence base for detection of stance-phase timings from motion capture in horses. The objective was to compare the accuracy (bias) and precision (SD) for five published algorithms for the detection of hoof-on and hoof-off using force plates as the reference standard.