ArfGAP1 Activity and COPI Vesicle Biogenesis

Golgi-derived coat protein I (COPI) vesicles mediate transport in the early secretory pathway. The minimal machinery required for COPI vesicle formation from Golgi membranes in vitro consists of (i) the hetero-heptameric protein complex coatomer, (ii) the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) and (iii) transmembrane proteins that function as coat receptors, such as p24 proteins. Various and opposing reports exist on a role of ArfGAP1 in COPI vesicle biogenesis. In this study, we show that, in contrast to data in the literature, ArfGAP1 is not required for COPI vesicle formation. To investigate roles of ArfGAP1 in vesicle formation, we titrated the enzyme into a defined reconstitution assay to form and purify COPI vesicles. We find that catalytic amounts of Arf1GAP1 significantly reduce the yield of purified COPI vesicles and that Arf1 rather than ArfGAP1 constitutes a stoichiometric component of the COPI coat. Combining the controversial reports with the results presented in this study, we suggest a novel role for ArfGAP1 in membrane trafficking.     

Intramembrane Charge Movement Associated with Endogenous K+ Channel Activity in HEK-293 Cells

1. The use of molecular biology in combination with electrophysiology in the HEK-293 cell line has given fascinating insights into neuronal ion channel function. Nevertheless, to fully understand the properties of channels exogenously expressed in these cells, a detailed evaluation of endogenous channels is indispensable. 2. Previous studies have shown the expression of endogenous voltage-gated K+, Ca2+, and Cl- channels and this predicts that changes in membrane potential will cause intramembrane charge movement, though this gating charge translocation remain undefined. Here, we confirm this prediction by performing patch-clamp experiments to record ionic and gating currents. Our data show that HEK-293 cells express at least two types of K+-selective endogenous channels which sustain the majority of the ionic current, and exclude a significant contribution from Ca2+ and Cl- channels to the whole-cell current. 3. Gating currents were unambiguously resolved after ionic current blockade enabling this first report of intramembrane charge movement in HEK-293 cells arising entirely from endogenous K+ channel activity, and providing valuable information concerning the activation mechanism of voltage-gated K+ channels in these cells.     

Molecular regulation of cardiac ryanodine receptor ion channel

The release of Ca(2+) ions from intracellular stores is a key step in a wide variety of cellular functions. In striated muscle, the release of Ca(2+) from the sarcoplasmic reticulum (SR) leads to muscle contraction. Ca(2+) release occurs through large, high-conductance Ca(2+) release channels, also known as ryanodine receptors (RyRs) because they bind the plant alkaloid ryanodine with high affinity and specificity. The RyRs are isolated as 30S protein complexes comprised of four 560 kDa RyR2 subunits and four 12 kDa FK506 binding protein (FKBP12) subunits. Multiple endogenous effector molecules and posttranslational modifications regulate the RyRs. This review focuses on current research toward understanding the control of the isolated cardiac Ca(2+) release channel/ryanodine receptor (RyR2) by Ca(2+), calmodulin, thiol oxidation/reduction and nitrosylation, and protein phosphorylation.     

Deep Proteomic Evaluation of Primary and Cell Line Motoneuron Disease Models Delineates Major Differences in Neuronal Characteristics

The fatal neurodegenerative disorders amyotrophic lateral sclerosis and spinal muscular atrophy are, respectively, the most common motoneuron disease and genetic cause of infant death. Various in vitro model systems have been established to investigate motoneuron disease mechanisms, in particular immortalized cell lines and primary neurons. Using quantitative mass-spectrometry-based proteomics, we compared the proteomes of primary motoneurons to motoneuron-like cell lines NSC-34 and N2a, as well as to non-neuronal control cells, at a depth of 10,000 proteins. We used this resource to evaluate the suitability of murine in vitro model systems for cell biological and biochemical analysis of motoneuron disease mechanisms. Individual protein and pathway analysis indicated substantial differences between motoneuron-like cell lines and primary motoneurons, especially for proteins involved in differentiation, cytoskeleton, and receptor signaling, whereas common metabolic pathways were more similar. The proteins associated with amyotrophic lateral sclerosis also showed distinct differences between cell lines and primary motoneurons, providing a molecular basis for understanding fundamental alterations between cell lines and neurons with respect to neuronal pathways with relevance for disease mechanisms. Our study provides a proteomics resource for motoneuron research and presents a paradigm of how mass-spectrometry-based proteomics can be used to evaluate disease model systems.Motoneurons are extremely extended neurons that mediate the control of all muscle types by the central nervous system. Therefore, diseases involving progressive motoneuron degeneration such as amyotrophic lateral sclerosis (ALS)¹ (OMIM: 105400) or spinal muscle atrophy (OMIM: 253300) are particularly devastating and generally fatal disorders. Today, ALS is believed to form a phenotypic continuum with the disease entity frontotemporal lobe degeneration (OMIM: 600274) (1, 2). About 10% of ALS cases are known to be inherited, but the vast majority are considered sporadic. The number of inherited cases might be underestimated because of incomplete family histories, non-paternity, early death of family members, or incomplete penetrance (3).Mutations in several genes have been reported for the familial form, including in Sod1 (4), Als2 (5), Setx (6), Vapb (7), Tardbp (8, 9), Fus/Tls (10, 11), Vcp (12), Pfn1 (13), and several others (reviewed in Ref. 14). The most frequent genetic cause of inherited ALS was recently shown to be a hexanucleotide repeat expansion in an intron of a gene of unknown function called C9orf72 (15–17). Based on the spectrum of known mutations, several disease mechanisms for ALS have been proposed, including dysfunction of protein folding, axonal transport, RNA splicing, and metabolism (reviewed in Refs. 14, 18, and 19). Despite intensive research, it is still unclear whether a main common molecular pathway or mechanism underlies motoneuron degeneration in ALS and frontotemporal lobe degeneration. Spinal muscle atrophy is caused by homozygous mutations or deletions in the survival of motor neuron gene (Smn1) that presumably impair the RNA metabolism through diminished functionality of the Smn1 gene product (20). Over recent decades several model systems have been established to investigate ALS (21). These include transgenic animal models such as mouse (22), drosophila (23), and zebrafish (24). In cell-based studies, primary motoneurons cultured from rodent embryos (25) or motoneuron-like cell lines are employed. Primary cells are considered to more closely mimic the in vivo situation, but they are more challenging to establish and maintain. In contrast, the degree of functional relevance of cell lines can be difficult to establish, but they can be propagated without limitation and are well suited for high-throughput analysis. In particular, the spinal cord neuron–neuroblastoma hybrid cell line NSC-34 (26) and the mouse neuroblastoma cell line N2a (27) are widely used not only to assess motoneuron function, but also to study disease mechanisms in motoneurons (28, 29).As proteins are the functional actors in cells, proteomics should be able to make important contributions to the characterization and evaluation of cellular models. In particular, by identifying and quantifying the expressed proteins and bioinformatically interpreting the results, one can obtain enough information to infer functional differences. Our laboratory has previously shown proof of concept of such an approach by comparing the expression levels of about 4,000 proteins between primary hepatocytes and a hepatoma cell line (30). Very recently, mass-spectrometry-based proteomics has achieved sufficient depth and accuracy to quantify almost the entire proteome of mammalian cell lines (31–33). Furthermore, new instrumentation and algorithms now make it possible to perform label-free quantification between multiple cellular systems and with an accuracy previously associated only with stable isotope labeling techniques (34, 35).To evaluate the suitability of motoneuron-like cell lines as cellular model systems for research on ALS and related disorders, we characterized the proteomes of two widely used cell lines, NSC-34 and N2a, and compared them with the proteomes of mouse primary motoneurons and non-neuronal control cell lines. To generate primary motoneurons, we employed a recently described culturing system that makes it possible to isolate highly enriched motoneuron populations in less than 8 h (25). We identified more than 10,000 proteins and investigated differences in quantitative levels of individual neuron-associated proteins and pathways related to motoneuron function and disease mechanisms.     

Ancestral and neo-sex chromosomes contribute to population divergence in a dioecious plant

Empirical evidence from several animal groups suggests sex chromosomes disproportionately contribute to reproductive isolation. This effect may be enhanced when sex chromosomes are associated with turnover of sex determination systems resulting from structural rearrangements to the chromosomes. We investigated these predictions in the dioecious plant Rumex hastatulus, which is composed of populations of two different sex chromosome cytotypes caused by an X-autosome fusion. Using population genomic analyses, we investigated the demographic history of R. hastatulus and explored the contributions of ancestral and neo-sex chromosomes to population genetic divergence. Our study revealed that the cytotypes represent genetically divergent populations with evidence for historical but not contemporary gene flow between them. In agreement with classical predictions, we found that the ancestral X chromosome was disproportionately divergent compared with the rest of the genome. Excess differentiation was also observed on the Y chromosome, even when we used measures of differentiation that control for differences in effective population size. Our estimates of the timing of the origin of neo-sex chromosomes in R. hastatulus are coincident with cessation of gene flow, suggesting that the chromosomal fusion event that gave rise to the origin of the XYY cytotype may have also contributed to reproductive isolation.     

Incubation Temperature and Substrate Quality Modulate Sporulation by Aquatic Hyphomycetes

Frequency and amplitude of temperature oscillations can profoundly affect structure and function of ecosystems. Unless the rate of a biological process changes linearly within the range of these fluctuations, the cumulative effect of temperature differs from the effect measured at the average temperature (Jensen's inequality). Here, we measured numbers and types of spores released by aquatic hyphomycetes from oak and alder leaves that had been exposed in a Portuguese stream for between 7 and 94 days. Recovered leaves were incubated at four temperatures between 5 and 20 °C. Over this range, the sporulation response to temperature was decelerating, with an estimated optimum around 12.5 °C. Assuming a linear response, therefore, overestimates spore release from decaying leaves. The calculated discrepancy was more pronounced with recalcitrant oak leaves (greater toughness, phenolics concentration, lower N and P concentration than alder), and reached 26.6 % when temperature was assumed to oscillate between 1 and 9 °C, rather than remaining constant at 5 °C. The maximum fluctuation of water temperature over 48 h during the field experiment was approximately 3 °C, which would result in a discrepancy of up to 6 %. The composition of the fungal community (assessed by species identification of released spores) was significantly influenced by the state of decomposition, but not by leaf species or temperature. When quantifying the potential impact of global change on aquatic fungal communities, the average increase as well as fluctuations of the temperature have to be considered.     

A Symbolic Model for the Regulation by Bone Metabolism of the Blood Calcium Level in Rats

The control by bone metabolism of the blood calcium level in young rats may be described in terms of a regulator-type system. The model presented here comprises a feedback loop involving only a proportional control in thyroparathyroidectomized, and a combination of proportional and integral controls in normal animals. It accounts for the variations observed when the system was subjected to a variety of experimental constraints. The implications, limitations, and possible extensions of the model are discussed.