Transport of dietary cholesterol from endocytic organelles to the endoplasmic reticulum (ER) is essential for cholesterol homoeostasis, but the mechanism and regulation of this transport remains ...poorly defined. Membrane contact sites (MCS), microdomains of close membrane apposition, are gaining attention as important platforms for non-vesicular, inter-organellar communication. Here we investigate the impact of ER-endocytic organelle MCS on cholesterol transport. We report a role for Niemann-Pick type C protein 1 (NPC1) in tethering ER-endocytic organelle MCS where it interacts with the ER-localised sterol transport protein Gramd1b to regulate cholesterol egress. We show that artificially tethering MCS rescues the cholesterol accumulation that characterises NPC1-deficient cells, consistent with direct lysosome to ER cholesterol transport across MCS. Finally, we identify an expanded population of lysosome-mitochondria MCS in cells depleted of NPC1 or Gramd1b that is dependent on the late endosomal sterol-binding protein STARD3, likely underlying the mitochondrial cholesterol accumulation in NPC1-deficient cells.
New light on photoreceptor renewal Burgoyne, T.; Meschede, I. P.; Futter, C. E.
Cell cycle (Georgetown, Tex.),
06/2016, Volume:
15, Issue:
11
Journal Article
Peer reviewed
Open access
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Isotopic compositions of carbon-bound hydrogen in individual compounds from eight different organisms were measured using isotope-ratio-monitoring gas chromatography–mass spectrometry. This technique ...is capable of measuring D/H ratios at natural abundance in individual lipids yielding as little as 20 nmol of H
2, and is applicable to a wide range of compounds including hydrocarbons, sterols, and fatty acids. The hydrogen isotopic compositions of lipids are controlled by three factors: isotopic compositions of biosynthetic precursors, fractionation and exchange accompanying biosynthesis, and hydrogenation during biosynthesis.
δD values of lipids from the eight organisms examined here suggest that all three processes are important for controlling natural variations in isotopic abundance.
n-Alkyl lipids are depleted in D relative to growth water by 113–262‰, while polyisoprenoid lipids are depleted in D relative to growth water by 142–376‰. Isotopic variations within compound classes (e.g.,
n-alkanes) are usually less than ∼50‰, but variations as large as 150‰ are observed among isoprenoid lipids from a single organism. Phytol is consistently depleted in D by up to 50‰ relative to other isoprenoid lipids. Inferred isotopic fractionations between cellular water and lipids are greater than those indicated by previous studies.
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The H3 factor, K, is a parameter required in high-precision, mass spectrometric analyses of hydrogen isotopic abundances. When H2 is used as the sample gas, R* = R − Ki 2, where R* is the true HD/H2 ...ratio, R is the observed (mass 3)/(mass 2) ion-current ratio, and i 2 is the ion current at mass 2. Four different methods for the determination of K were defined and tested under conditions characteristic of isotope ratio monitoring systems. Three of these were peak-based. The fourth employed steady flows of H2 from a conventional inlet system. Results obtained using the latter method were more precise (standard deviation of K = 0.1 versus ∼0.6 ppm mV-1 for the peak-based methods). However, use of the resulting values of K for correction of isotope ratio monitoring GC/MS results led to systematic errors as large as 9‰, whereas use of the peak-based values led to no systematic errors. Values of K were only weakly dependent on the pressure of He, declining ∼5% for each 10-fold increase in P He. Small variations in partial pressures of H2O and CH4, potential contaminants under isotope ratio monitoring conditions, had no significant effect on values of K.
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Two fundamentally different approaches, termed “pointwise” and “peakwise,” are currently used to correct hydrogen isotope ratio monitoring data for the presence of H3 + ion contributions. ...Consideration of the underlying assumptions shows that the peakwise approach is valid only for peaks with the same functional shape and only when background signals do not vary. The pointwise correction is much more versatile and can be used even when peak shapes and sizes, as well as background signals, vary significantly. It is not exact and is limited in accuracy by (1) the signal-broadening effects of electronic time constants, (2) the analog-to-digital conversion frequency, and (3) the highest frequency of the sample signal. To minimize errors for typical gas chromatographic signals, time constants of <500 ms and analog-to-digital sampling intervals of ≤250 ms are needed. Errors are further minimized by matching sample and standard peaks in both amplitude and D/H ratio. Using the pointwise algorithm, we demonstrate that a series of 14 homologous n-alkanes varying in concentration over a 5-fold range can be analyzed with a mean precision of 2.3‰ and no systematic errors.
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Space charge effects, and the matrix interferences they cause, are problems in inductively coupled plasma mass spectrometry (ICPMS). It has previously been observed that these deleterious space ...charge effects are not significantly present in sector-field instruments, a fact that has been attributed, but not demonstrated, to the high accelerating potentials they commonly employ. To examine the significance of space charge in our plasma-source mass spectrograph (which operates at only moderate accelerating potentials) and in other sector instruments, a graphite disk was placed ∼7 cm behind the skimmer. An inductively coupled plasma was operated for 17 h while a 0.01 mM multielement solution was introduced. This disk was then analyzed by spatially resolved laser ablation ICP time-of-flight MS. Second vacuum-stage acceleration appears to be an important factor that governs the elemental distribution within the ion beam. The ion beam width at m/z 208 is one-third of its width at m/z 7 using an accelerating potential of 800 V; at an accelerating potential of 4000 V, the ion beam width does not vary with mass.
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