A functioning artificial secretory cell

We present an amperometric study of content release from individual vesicles in an artificial secretory cell designed with the minimal components required to carry out exocytosis. Here, the membranes of the cell and vesicles are substituted for protein-free giant and large unilamellar vesicles respectively. In replacement of the SNARE-complex, the cell model was equipped with an analog composed of complimentary DNA constructs. The DNA constructs hybridize in a zipper-like fashion to bring about docking of the artificial secretory vesicles and following the addition of Ca2+ artificial exocytosis was completed. Exocytotic events recorded from the artificial cell closely approximate exocytosis in live cells. The results together with simulations of vesicular release demonstrate that the molecular flux in this model is attenuated and we suggest that this is the result of restricted diffusion through a semi-stable fusion pore or a partitioning of the signalling molecule out of the fused vesicle membrane

Cholesterol Alters the Dynamics of Release in Protein Independent Cell Models for Exocytosis

Neurons communicate via an essential process called exocytosis. Cholesterol, an abundant lipid in both secretory vesicles and cell plasma membrane can affect this process. In this study, amperometric recordings of vesicular dopamine release from two different artificial cell models created from a giant unilamellar liposome and a bleb cell plasma membrane, show that with higher membrane cholesterol the kinetics for vesicular release are decelerated in a concentration dependent manner. This reduction in exocytotic speed was consistent for two observed modes of exocytosis, full and partial release. Partial release events, which only occurred in the bleb cell model due to the higher tension in the system, exhibited amperometric spikes with three distinct shapes. In addition to the classic transient, some spikes displayed a current ramp or plateau following the maximum peak current. These post spike features represent neurotransmitter release from a dilated pore before constriction and show that enhancing membrane rigidity via cholesterol adds resistance to a dilated pore to re-close. This implies that the cholesterol dependent biophysical properties of the membrane directly affect the exocytosis kinetics and that membrane tension along with membrane rigidity can influence the fusion pore dynamics and stabilization which is central to regulation of neurochemical release

Arteriovenous Blood Metabolomics: A Readout of Intra-Tissue Metabostasis.

The human circulatory system consists of arterial blood that delivers nutrients to tissues, and venous blood that removes the metabolic by-products. Although it is well established that arterial blood generally has higher concentrations of glucose and oxygen relative to venous blood, a comprehensive biochemical characterization of arteriovenous differences has not yet been reported. Here we apply cutting-edge, mass spectrometry-based metabolomic technologies to provide a global characterization of metabolites that vary in concentration between the arterial and venous blood of human patients. Global profiling of paired arterial and venous plasma from 20 healthy individuals, followed up by targeted analysis made it possible to measure subtle (<2 fold), yet highly statistically significant and physiologically important differences in water soluble human plasma metabolome. While we detected changes in lactic acid, alanine, glutamine, and glutamate as expected from skeletal muscle activity, a number of unanticipated metabolites were also determined to be significantly altered including Krebs cycle intermediates, amino acids that have not been previously implicated in transport, and a few oxidized fatty acids. This study provides the most comprehensive assessment of metabolic changes in the blood during circulation to date and suggests that such profiling approach may offer new insights into organ homeostasis and organ specific pathology

Mechanics of lipid bilayer junctions affecting the size of a connecting lipid nanotube

In this study we report a physical analysis of the membrane mechanics affecting the size of the highly curved region of a lipid nanotube (LNT) that is either connected between a lipid bilayer vesicle and the tip of a glass microinjection pipette (tube-only) or between a lipid bilayer vesicle and a vesicle that is attached to the tip of a glass microinjection pipette (two-vesicle). For the tube-only configuration (TOC), a micropipette is used to pull a LNT into the interior of a surface-immobilized vesicle, where the length of the tube L is determined by the distance of the micropipette to the vesicle wall. For the two-vesicle configuration (TVC), a small vesicle is inflated at the tip of the micropipette tip and the length of the tube L is in this case determined by the distance between the two interconnected vesicles. An electrochemical method monitoring diffusion of electroactive molecules through the nanotube has been used to determine the radius of the nanotube R as a function of nanotube length L for the two configurations. The data show that the LNT connected in the TVC constricts to a smaller radius in comparison to the tube-only mode and that tube radius shrinks at shorter tube lengths. To explain these electrochemical data, we developed a theoretical model taking into account the free energy of the membrane regions of the vesicles, the LNT and the high curvature junctions. In particular, this model allows us to estimate the surface tension coefficients from R(L) measurements

Correlative High-Resolution Imaging of Iron Uptake in Lung Macrophages

Detection of iron at the subcellular level in order to gain insights into its transport, storage, and therapeutic prospects to prevent cytotoxic effects of excessive iron accumulation is still a challenge. Nanoscale magnetic sector secondary ion mass spectrometry (SIMS) is an excellent candidate for subcellular mapping of elements in cells since it provides high secondary ion collection efficiency and transmission, coupled with high-lateral-resolution capabilities enabled by nanoscale primary ion beams. In this study, we developed correlative methodologies that implement SIMS high-resolution imaging technologies to study accumulation and determine subcellular localization of iron in alveolar macrophages. We employed transmission electron microscopy (TEM) and backscattered electron (BSE) microscopy to obtain structural information and high-resolution analytical tools, NanoSIMS and helium ion microscopy-SIMS (HIM-SIMS) to trace the chemical signature of iron. Chemical information from NanoSIMS was correlated with TEM data, while high-spatial-resolution ion maps from HIM-SIMS analysis were correlated with BSE structural information of the cell. NanoSIMS revealed that iron is accumulating within mitochondria, and both NanoSIMS and HIM-SIMS showed accumulation of iron in electrolucent compartments such as vacuoles, lysosomes, and lipid droplets. This study provides insights into iron metabolism at the subcellular level and has future potential in finding therapeutics to reduce the cytotoxic effects of excessive iron loading

Visualization of Partial Exocytotic Content Release and Chemical Transport into Nanovesicles in Cells

For decades, "all-or-none"and "kiss-and-run"were thought to be the only major exocytotic release modes in cell-to-cell communication, while the significance of partial release has not yet been widely recognized and accepted owing to the lack of direct evidence for exocytotic partial release. Correlative imaging with transmission electron microscopy and NanoSIMS imaging and a dual stable isotope labeling approach was used to study the cargo status of vesicles before and after exocytosis; demonstrating a measurable loss of transmitter in individual vesicles following stimulation due to partial release. Model secretory cells were incubated with 13C-labeled l-3,4-dihydroxyphenylalanine, resulting in the loading of 13C-labeled dopamine into their vesicles. A second label, di-N-desethylamiodarone, having the stable isotope 127I, was introduced during stimulation. A significant drop in the level of 13C-labeled dopamine and a reduction in vesicle size, with an increasing level of 127I-, was observed in vesicles of stimulated cells. Colocalization of 13C and 127I- in several vesicles was observed after stimulation. Thus, chemical visualization shows transient opening of vesicles to the exterior of the cell without full release the dopamine cargo. We present a direct calculation for the fraction of neurotransmitter release from combined imaging data. The average vesicular release is 60% of the total catecholamine. An important observation is that extracellular molecules can be introduced to cells during the partial exocytotic release process. This nonendocytic transport process appears to be a general route of entry that might be exploited pharmacologically

Inhibition of HMG CoA reductase reveals an unexpected role for cholesterol during PGC migration in the mouse

<p>Abstract</p> <p>Background</p> <p>Primordial germ cells (PGCs) are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors.</p> <p>Results</p> <p>We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR) resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally.</p> <p>Conclusion</p> <p>In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.</p

Metabolic drift in the aging brain.

Brain function is highly dependent upon controlled energy metabolism whose loss heralds cognitive impairments. This is particularly notable in the aged individuals and in age-related neurodegenerative diseases. However, how metabolic homeostasis is disrupted in the aging brain is still poorly understood. Here we performed global, metabolomic and proteomic analyses across different anatomical regions of mouse brain at different stages of its adult lifespan. Interestingly, while severe proteomic imbalance was absent, global-untargeted metabolomics revealed an energymetabolic drift or significant imbalance in core metabolite levels in aged mouse brains. Metabolic imbalance was characterized by compromised cellular energy status (NAD decline, increased AMP/ATP, purine/pyrimidine accumulation) and significantly altered oxidative phosphorylation and nucleotide biosynthesis and degradation. The central energy metabolic drift suggests a failure of the cellular machinery to restore metabostasis (metabolite homeostasis) in the aged brain and therefore an inability to respond properly to external stimuli, likely driving the alterations in signaling activity and thus in neuronal function and communication

Subcellular Mass Spectrometry Imaging and Absolute Quantitative Analysis across Organelles

Mass spectrometry imaging is a field that promises to become a mainstream bioanalysis technology by allowing the combination of single-cell imaging and subcellular quantitative analysis. The frontier of single-cell imaging has advanced to the point where it is now possible to compare the chemical contents of individual organelles in terms of raw or normalized ion signal. However, to realize the full potential of this technology, it is necessary to move beyond this concept of relative quantification. Here we present a nanoSIMS imaging method that directly measures the absolute concentration of an organelle-associated, isotopically labeled, pro-drug directly from a mass spectrometry image. This is validated with a recently developed nanoelectrochemistry method for single organelles. We establish a limit of detection based on the number of isotopic labels used and the volume of the organelle of interest, also offering this calculation as a web application. This approach allows subcellular quantification of drugs and metabolites, an overarching and previously unmet goal in cell science and pharmaceutical development

Composition Based Strategies for Controlling Radii in Lipid Nanotubes

Nature routinely carries out small-scale chemistry within lipid bound cells and organelles. Liposome–lipid nanotube networks are being developed by many researchers in attempt to imitate these membrane enclosed environments, with the goal to perform small-scale chemical studies. These systems are well characterized in terms of the diameter of the giant unilamellar vesicles they are constructed from and the length of the nanotubes connecting them. Here we evaluate two methods based on intrinsic curvature for adjusting the diameter of the nanotube, an aspect of the network that has not previously been controllable. This was done by altering the lipid composition of the network membrane with two different approaches. In the first, the composition of the membrane was altered via lipid incubation of exogenous lipids; either with the addition of the low intrinsic curvature lipid soy phosphatidylcholine (soy-PC) or the high intrinsic curvature lipid soy phosphatidylethanolamine (soy-PE). In the second approach, exogenous lipids were added to the total lipid composition during liposome formation. Here we show that for both lipid augmentation methods, we observed a decrease in nanotube diameter following soy-PE additions but no significant change in size following the addition of soy-PC. Our results demonstrate that the effect of soy-PE on nanotube diameter is independent of the method of addition and suggests that high curvature soy-PE molecules facilitate tube membrane curvature