Novel standards in the measurement of rat insulin granules combining electron microscopy, high-content image analysis and in silico modelling.

AIMS/HYPOTHESIS: Knowledge of number, size and content of insulin secretory granules is pivotal for understanding the physiology of pancreatic beta cells. Here we re-evaluated key structural features of rat beta cells, including insulin granule size, number and distribution as well as cell size. METHODS: Electron micrographs of rat beta cells fixed either chemically or by high-pressure freezing were compared using a high-content analysis approach. These data were used to develop three-dimensional in silico beta cell models, the slicing of which would reproduce the experimental datasets. RESULTS: As previously reported, chemically fixed insulin secretory granules appeared as hollow spheres with a mean diameter of ∼350 nm. Remarkably, most granules fixed by high-pressure freezing lacked the characteristic halo between the dense core and the limiting membrane and were smaller than their chemically fixed counterparts. Based on our analyses, we conclude that the mean diameter of rat insulin secretory granules is 243 nm, corresponding to a surface area of 0.19 µm(2). Rat beta cells have a mean volume of 763 µm(3) and contain 5,000-6,000 granules. CONCLUSIONS/INTERPRETATION: A major reason for the lower mean granule number/rat beta cell relative to previous accounts is a reduced estimation of the mean beta cell volume. These findings imply that each granule contains about twofold more insulin, while its exocytosis increases membrane capacitance about twofold less than assumed previously. Our integrated approach defines new standards for quantitative image analysis of beta cells and could be applied to other cellular systems.

Sorting of Golgi resident proteins into different subpopulations of COPI vesicles: a role for ArfGAP1.

We present evidence for two subpopulations of coatomer protein I vesicles, both containing high amounts of Golgi resident proteins but only minor amounts of anterograde cargo. Early Golgi proteins p24alpha2, beta1, delta1, and gamma3 are shown to be sorted together into vesicles that are distinct from those containing mannosidase II, a glycosidase of the medial Golgi stack, and GS28, a SNARE protein of the Golgi stack. Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride. Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes. We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.

Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins.

Addition of the weak base ammonium chloride (NH4Cl) or the proton pump inhibitor bafilomycin A1 to cultured HeLa and LS 174T cells effectively neutralized the pH gradient of the secretory pathway. This resulted in relocalization of the three studied glycosyltransferases, N-acetylgalactosaminyltransferase 2, beta1,2 N-acetylglucosaminyltransferase I, and beta1,4 galactosyltransferase 1, normally localized to the Golgi stack, the medial/trans-Golgi and the trans-Golgi/TGN, respectively. Indirect immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation of the tagged or native glycosyltransferases showed that NH4Cl caused a relocalization of the enzymes mainly to vesicles of endosomal type, whereas bafilomycin A1 gave mainly cell surface staining. The general morphology of the endoplasmic reticulum and Golgi apparatus was retained as judged from immunofluorescence and electron microscopy studies. When the O-glycans on the guanidinium chloride insoluble gel-forming mucins from the LS 174T cells were analyzed by gas chromatography-mass spectrometry after neutralization of the secretory pathway pH by NH4Cl over 10 days shorter O-glycans were observed. However, no decrease in the number of oligosaccharide chains was indicated. Together, the results suggest that pH is a contributing factor for proper steady-state distribution of glycosyltransferases over the Golgi apparatus and that altered pH may cause alterations in glycosylation possibly due to a relocalization of glycosyltransferases.

GTP hydrolysis by arf-1 mediates sorting and concentration of Golgi resident enzymes into functional COP I vesicles.

Upon addition of GTPgammaS to in vitro budding reactions, COP I vesicles form but retain their coat, making them easy to isolate and analyze. We have developed an in vitro budding assay that reconstitutes the formation of COP I-derived vesicles under conditions where GTP hydrolysis can occur. Once formed, vesicles are uncoated and appear functional as they fuse readily with acceptor membranes. Electron microscopy shows a homogeneous population of uncoated vesicles that contain the medial/trans Golgi enzyme alpha1, 2-mannosidase II. Biochemical quantitation of vesicles reveals that resident Golgi enzymes are up to 10-fold more concentrated than in donor membranes, but vesicles formed in the presence of GTPgammaS show an average density of resident Golgi enzymes similar to that seen in donor membranes. We show that the sorting process is mediated by the small GTPase arf-1 as addition of a dominant, hydrolysis-deficient arf-1 (Q)71(L) mutant produced results similar to that of GTPgammaS. Strikingly, the average density of the anterograde cargo protein, polymeric IgA receptor, in COP I-derived vesicles was similar to that found in starting membranes and was independent of GTP hydrolysis. We conclude that hydrolysis of GTP bound to arf-1 promotes selective segregation and concentration of Golgi resident enzymes into COP I vesicles.

Analysis of a naturally occurring mutation in sucrase-isomaltase: glutamine 1098 is not essential for transport to the surface of COS-1 cells.

A glutamine for proline substitution at position 1098 was previously shown to result in accumulation of brush-border sucrase-isomaltase in the Golgi apparatus. The substitution is present in a highly homologous region of the protein, and results in a comparable accumulation when introduced into the same region in lysosomal alpha-glucosidase. To study the importance of the glutamine-1098, we analyzed the transport compatibility of two mutants in which glutamine-1098 is substituted by lysine or alanine. Both mutants were transported to the cell surface and processed comparable to wild type. We concluded that glutamine-1098 is not essential for transport to the cell surface.

Routing and processing of lactase-phlorizin hydrolase in transfected Caco-2 cells.

Human lactase-phlorizin hydrolase (LPH) is a digestive enzyme that is expressed in the small intestinal brush-border membrane. After terminal glycosylation in the Golgi apparatus, the 230-kDa pro-LPH is cleaved into the 160-kDa brush-border LPHbeta and the 100-kDa profragment (LPHalpha). Since LPHbeta is not transport-competent when it is expressed separately from LPHalpha in COS-1 cells, it was suggested that LPHalpha functions as an intramolecular chaperone. What happens to LPHalpha after cleavage is still unclear. To analyze and localize LPHalpha in polarized epithelial cells, wild type and tagged LPH were stably expressed in Caco-2 cells. In tagged LPH, a vesicular stomatitis virus epitope tag was inserted into the LPHalpha region. Wild type and tagged proteins were processed at similar rates, and both cleaved LPHbeta forms were expressed at the apical cell surface. Pro-LPH was recognized by antibodies against LPH, a profragment epitope and the vesicular stomatitis virus tag. LPHalpha alone, however, could not be recovered by these antibodies. Our data suggest that LPHalpha is degraded immediately after cleavage.

A mutation in a highly conserved region in brush-border sucrase-isomaltase and lysosomal alpha-glucosidase results in Golgi retention.

A point mutation in the cDNA of human intestinal sucrase-isomaltase has been recently identified in phenotype II of congenital sucrase-isomaltase deficiency. The mutation results in a substitution of glutamine by proline at position 1098 (Q1098P) in the sucrase subunit. Expression of this mutant sucrase-isomaltase cDNA in COS-1 cells results in an accumulation of sucrase-isomaltase in the ER, intermediate compartment and the cis-Golgi cisternae similar to the accumulation in phenotype II intestinal cells. An interesting feature of the Q1098P substitution is its location in a region of the sucrase subunit that shares striking similarities with the isomaltase subunit and other functionally related enzymes, such as human lysosomal acid alpha-glucosidase and Schwanniomyces occidentalis glucoamylase. We speculated that the Q-->P substitution in these highly conserved regions may result in a comparable accumulation. Here we examined this hypothesis using lysosomal alpha-glucosidase as a reporter gene. Mutagenesis of the glutamine residue at position 244 in the homologous region of alpha-glucosidase to proline results in a protein that is neither transported to the lysosomes nor secreted extracellularly but accumulates in the ER, intermediate compartment and cis-Golgi as a mannose-rich polypeptide similar to mutant sucrase-isomaltase in phenotype II. We propose that the Q1098P and Q244P mutations (in sucrase-isomaltase and alpha-glucosidase, respectively) generate structural alterations that are recognized by a control mechanism, operating beyond the ER in the intermediate compartment or cis-Golgi.

Congenital sucrase-isomaltase deficiency. Identification of a glutamine to proline substitution that leads to a transport block of sucrase-isomaltase in a pre-Golgi compartment.

Congenital sucrase-isomaltase deficiency is an example of a disease in which mutant phenotypes generate transport-incompetent molecules. Here, we analyze at the molecular level a phenotype of congenital sucrase-isomaltase deficiency in which sucrase-isomaltase (SI) is not transported to the brush border membrane but accumulates as a mannose-rich precursor in the endoplasmic reticulum (ER), ER-Golgi intermediate compartment, and the cis-Golgi, where it is finally degraded. A 6-kb clone containing the full-length cDNA encoding SI was isolated from the patient's intestinal tissue and from normal controls. Sequencing of the cDNA revealed a single mutation, A/C at nucleotide 3298 in the coding region of the sucrase subunit of the enzyme complex. The mutation leads to a substitution of the glutamine residue by a proline at amino acid 1098 (Q1098P). The Q1098P mutation lies in a region that is highly conserved between sucrase and isomaltase from different species and several other structurally and functionally related proteins. This is the first report that characterizes a point mutation in the SI gene that is responsible for the transport incompetence of SI and for its retention between the ER and the Golgi.

A young female with a cholangiocarcinoma in the past: is pregnancy allowed?

BACKGROUND: A 28-year-old woman developed a cholangiocarcinoma after using oral contraceptives for eight years. The common bile duct and the gallbladder were removed and four hepaticojejunostomies were performed. RESULTS: Seven years after surgery no evidence of disease recurrence is detectable and the patient wants advice regarding her wish for pregnancy. CONCLUSION: The possible role of oestrogens in the development of cholangiocarcinoma makes it difficult to give an adequate advice. As in patients with breast cancer in the past, it seems reasonable to admit pregnancy in our patient, because of a disease-free interval of longer than five years.

PIP: Long-term oral contraceptive (OC) use has been implicated as a possible factor in the development of hepatocellular carcinoma and cholangiocarcinoma. Presented, in this paper, is the case of a 35-year-old Dutch woman who developed a cholangiocarcinoma at 28 years of age after 8 years of OC use. The common bile duct and gall bladder were removed and 4 hepaticojejunostomies were performed. Seven years after surgery, with no evidence of disease recurrence, this woman sought advice regarding the feasibility of pregnancy. The possible role of estrogens in cholangiocarcinomas makes this a difficult question. The only such case reported in the literature involved a woman with inoperable cholangiocarcinoma who became pregnant with no evidence that the pregnancy stimulated tumor growth. Most studies of the interactive effects of pregnancy and recurrent malignancies have involved breast cancer. Recommended, for breast cancer patients, is postponement of pregnancy until a disease-free interval of 3-5 years has been achieved. In the absence of specific guidelines for cholangiocarcinoma survivors, it seems advisable to adhere to this same principle.