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First published online April 24, 2006
doi: 10.1242/10.1242/jcs.02906

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Abnormal sterols in cholesterol-deficiency diseases cause secretory granule malformation and decreased membrane curvature

Marjorie C. Gondré-Lewis^1,*, Horia I. Petrache², Christopher A. Wassif³, Daniel Harries², Adrian Parsegian², Forbes D. Porter³ and Y. Peng Loh^1,

¹ Section on Cellular Neurobiology, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA
² Laboratory of Physical and Structural Biology, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA
³ Heritable Disorders Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA

View larger version (23K): [in a new window]   Fig. 1. Sterols in cholesterol defective mice. (A) Structure of lanosterol, lathosterol, dehydrocholesterol, and cholesterol, depicting a difference of one double bond between cholesterol and lathosterol, and of the location of the double bond in cholesterol and lathosterol (C5-C6 versus C7-C8, respectively). Lanosterol is an early precursor requiring multiple enzymatic steps before its conversion to cholesterol. (B) Biochemical analysis of sterol content in Dhcr7-/- mouse pancreas. 7-DHC, lathosterol and 8-DHC levels were measured in wild-type, heterozygous and mutant pancreatic tissue from newborn litters. In control animals, (Dhcr7+/+, Dhcr7+/-), cholesterol is the major sterol component and constitutes 99% of total sterols. In Dhcr7-/- mice, 7-DHC is elevated to constitute 58% of all sterols, concomitant with significant elevations of 8-DHC and lathosterol. ***P<0.001. Values are mean ± s.e.m. of five animals per genotype. (C) Biochemical analysis of sterol content in Sc5d-/- mouse pancreas. In Sc5d-/- animals, lathosterol makes up 70% of sterols analyzed, and cholesterol levels are significantly diminished compared with levels in the control. ***P<0.001. Values are mean ± s.e.m. of nine animals per genotype.

View larger version (125K): [in a new window]   Fig. 2. Pancreas and secretory granule phenotype in Dhcr7-/- mice at P0. Light images of thick sections (A-B) of pancreas show markedly decreased presence of dense core granules in sections of mutant pancreatic tissue (B), compared with numerous granules in Dhcr7+/+ mice (A). Electron microscopic analysis of normal exocrine granules reveal membrane-bound vesicles 0.05-1.5 µm in diameter filled with electron-dense granular material (C). In Dhcr7-/- animals, abnormal profiles include vesicles with condensed material surrounded by a light halo bound by membrane (F, arrows), fused vesicles (D,F, arrowheads), or light granular material that failed to form vesicular shaped structures altogether (E). Bars, 10 µm (A-B); 1 µm (C-F).

View larger version (92K): [in a new window]   Fig. 3. Exocrine pancreas and secretory granule morphology in Sc5d-/- mice (lathosterolosis model) at E18.5. (A,B) Sc5d-/- animals consistently show a marked reduction in the number of dense core granules. In addition, many aberrant morphological profiles were evident: (C) large phagocytic structures with features of late endosomes or lysosomes engulfing cellular debris and granular material (arrows); (D) complete absence of dense core vesicles, and an abundance of ribosomal structures and rough ER; (E) enlarged ER (arrows). (F) A few areas have dense core vesicles similar to Sc5d+/+ (WT), interspersed with profiles of enlarged ER. (G) Quantitative analysis of granule number in lathosterolosis mouse model. Sc5d+/+ and Sc5d-/- groups contain four animals each. Ten different areas of each pancreas were imaged at low EM magnification (3150x) and counted. Bars, 2 µm (A-C,F); 1 µm (D-E).

View larger version (10K): [in a new window]   Fig. 4. Quantitative analysis of secretory granule morphology in pancreas. SLOS animals exhibit a 30% decrease in total granule number (A), a 60% decrease in the number of granules with mature morphology (B), and a 55% increase in granules with immature morphology (C). Mature is defined as round vesicles with a membrane bound dense core and no light granular halo. Immature is defined as all other morphologies including `empty' granules. 10 images were analyzed per animal, at 3150X magnification, representative of each area within a section of pancreas. Each group represents the mean ± s.e.m. of four or five animals. ***P<0.001 compared with counts in the wild-type animals.

View larger version (17K): [in a new window]   Fig. 5. Fate of proteins within the regulated secretory pathway in SLOS mice. (A) In vivo expression of -amylase was analyzed by western blot of protein from whole pancreas of newborn SLOS littermates. (B) Quantification of bands shows abundant presence of -amylase in both WT and SLOS mutants, with SLOS tissues exhibiting a 15% decrease in accumulation at steady state. (C) Plasma levels of -amylase activity in blood. Activity was significantly elevated by 25% (***P<0.001) in circulating blood serum of SLOS mice compared with the level in WT serum. Values are mean ± s.e.m. of five animals from each group.

View larger version (58K): [in a new window]   Fig. 6. Synthesis and secretion of -amylase in cultured exocrine cells, and rescue of RSP. (A) Cells were pulse labeled for 30 minutes with [35S]methionine. Cell lysates were harvested and immunoprecipitated with anti--amylase. Average synthesis in control animals was normalized to total CPM and defined as 100%. Values reported are the mean % CPM relative to control ±s.e.m. (B) Western blot analysis of -amylase secreted in cultured pancreatic cells. Primary exocrine cells were equilibrated in basal media, and then a 1 hour basal secretion was collected. They were then stimulated with secretagogues for four 1-hour periods. B, basal release. S1 and S2, secretion during the third and fourth hour of stimulation, respectively. No secretion was evident during the first and second hour. (C) WT (left) and SLOS (middle and right) cells were cultured for 5.5 days in delipidated serum (LPDS) or fetal calf serum (FCS) and processed for electron microscopy. In the absence of lipid-containing serum, tubular profiles of ER-like structures were evident in SLOS (arrowheads), however when grown in the presence of lipid-containing FCS, electron dense granular vesicles were detected (arrows). (D) WT (left) and SLOS (middle and right) cells were cultured as in C, and probed for immunoreactivity to antibodies against the regulated secretory granule protein marker, CgA (green) or the Golgi marker p115 (red). In LPDS-containing media, CgA was localized to tubular profiles (arrowheads) of SLOS cells whereas when grown in the presence of FCS, they exhibited punctate vesicular structures (arrows), consistent with CgA localization to the RSP in controls. (E) In WT cells (top panels), vesicular staining of CgA (green) was detected upon addition of either cholesterol alone (left) or 7-DHC alone (right) to LPDS medium. In SLOS (bottom panels), vesicular patterns of CgA staining (green) were evident only if cholesterol (left) but not 7-DHC alone (right) was added to LPDS-medium. In the presence of 7-DHC, cells had numerous tubular profiles. Red, anti-p115. Bars, 2 µm (C); 10 µm (D,E).

View larger version (138K): [in a new window]   Fig. 7. Dense core granule formation in endocrine tissues of Sc5d-/- mice. At E18.5, DCGs are plentiful in the anterior pituitary (A), adrenal medulla (C) and endocrine pancreas (E) of control mice, whereas mice homozygous for the Sc5d mutation show absent or severely reduced presence of electron-dense granules (arrows) in equivalent regions of tissues (B,D,F). n, nucleus. Bars, 1 µm (A-B); 2 µm (C-D); 500 nm (E-F).

View larger version (18K): [in a new window]   Fig. 8. Impact of abnormal sterol expression on membrane curvature. (A) Membrane bending rigidity KC obtained from measured interactions of multilamellar DMPC vesicles at 35°C containing 30 mol% sterols. The importance of double-bond location in the sterol ring is illustrated, with cholesterol the most efficient at enhancing membrane rigidity. (B) X-ray scattering of highly curved, inverse hexagonal phase DOPE/30mol% sterol mixtures. The shift of scattering peaks with sterol type corresponds to changes in the radius of curvature of bent DOPE monolayers with the smallest values measured for cholesterol and the largest for 7-DHC. Depletion of cholesterol or replacement with 7-DHC or lathosterol modifies the distribution of forces (bending stress) within biomembranes contributing to cellular morphological changes. (C) Distribution of granule sizes in the lathosterol model. 30 images from each of three animals were analyzed. Granule areas are distributed over a greater range compared with the normal WT range.

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