Nancy K. Dwyer

The Niemann-Pick C1 Protein Resides in a Vesicular Compartment Linked to Retrograde Transport of Multiple Lysosomal Cargo

Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking. Immunocytochemical studies in fibroblasts reveal that theNPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate receptor(−) vesicles that can be distinguished from cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21 °C, consistent with vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles may transiently interact with lysosomes to facilitate sterol relocation. Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein. We conclude that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol.

MLN64 Mediates Mobilization of Lysosomal Cholesterol to Steroidogenic Mitochondria

This study demonstrates that the steroidogenic acute regulatory protein-related lipid transfer (START) domain-containing protein, MLN64, participates in intracellular cholesterol trafficking. Analysis of the intracellular itinerary of MLN64 and MLN64 mutants tagged with green fluorescent protein showed that the N-terminal transmembrane domains mediate endocytosis of MLN64 from the plasma membrane to late endocytic compartments. MLN64 constitutively traffics via dynamic NPC1-containing late endosomal tubules in normal cells; this dynamic movement was inhibited in cholesterol-loaded cells, and MLN64 is trapped at the periphery of cholesterol-laden lysosomes. The MLN64 START domain stimulated free cholesterol transfer from donor to acceptor mitochondrial membranes and enhanced steroidogenesis by placental mitochondria. Expression of a truncated form of MLN64 (ΔSTART-MLN64), which contains N-terminal transmembrane domains but lacks the START domain, caused free cholesterol accumulation in lysosomes and inhibited late endocytic dynamics. The ΔSTART-MLN64 dominant negative protein was located at the surface of the cholesterol-laden lysosomes. This dominant negative mutant suppressed steroidogenesis in COS cells expressing the mitochondrial cholesterol side chain cleavage system. We conclude that MLN64 participates in mobilization and utilization of lysosomal cholesterol by virtue of the START domains role in cholesterol transport.

Cessation of rapid late endosomal tubulovesicular trafficking in Niemann–Pick type C1 disease

Niemann–Pick type C1 (NPC1) disease results from a defect in the NPC1 protein and is characterized by a pathological accumulation of cholesterol and glycolipids in endocytic organelles. We followed the biosynthesis and trafficking of NPC1 with the use of a functional green fluorescent protein-fused NPC1. Newly synthesized NPC1 is exported from the endoplasmic reticulum and requires transit through the Golgi before it is targeted to late endosomes. NPC1-containing late endosomes then move by a dynamic process involving tubulation and fission, followed by rapid retrograde and anterograde migration along microtubules. Cell fusion studies with normal and mutant NPC1 cells show that exchange of contents between late endosomes and lysosomes depends upon ongoing tubulovesicular late endocytic trafficking. In turn, rapid endosomal tubular movement requires an intact NPC1 sterol-sensing domain and is retarded by an elevated endosomal cholesterol content. We conclude that the neuropathology and cellular lysosomal lipid accumulation in NPC1 disease results, at least in part, from striking defects in late endosomal tubulovesicular trafficking.

Mutations in the Leucine Zipper Motif and Sterol-sensing Domain Inactivate the Niemann-Pick C1 Glycoprotein

Niemann-Pick type C (NPC) disease, characterized by accumulation of low density lipoprotein-derived free cholesterol in lysosomes, is caused by mutations in the NPC1 gene. We examined the ability of wild-type NPC1 and NPC1 mutants to correct the NPC sterol trafficking defect and their subcellular localization in CT60 cells. Cells transfected with wild-type NPC1 expressed 170- and 190-kDa proteins. Tunicamycin treatment resulted in a 140-kDa protein, the deduced size of NPC1, suggesting that NPC1 isN-glycosylated. Mutation of all four asparagines in potential N-terminal N-glycosylation sites to glutamines resulted in a 20-kDa reduction of the expressed protein. Proteins with a single N-glycosylation site mutation localized to late endosome/lysosomal compartments, as did wild-type NPC1, and each corrected the cholesterol trafficking defect. However, mutation of all four potential N-glycosylation sites reduced ability to correct the NPC phenotype commensurate with reduced expression of the protein. Mutations in the putative sterol-sensing domain resulted in inactive proteins targeted to lysosomal membranes encircling cholesterol-laden cores. N-terminal leucine zipper motif mutants could not correct the NPC defect, although they accumulated in lysosomal membranes. We conclude that NPC1 is a glycoprotein that must have an intact sterol-sensing domain and leucine zipper motif for cholesterol-mobilizing activity.

Immunocytochemical Evidence that GLUT4 Resides in a Specialized Translocation Post-endosomal VAMP2-positive Compartment in Rat Adipose Cells in the Absence of Insulin

Insulin stimulates glucose transport in rat adipose cells through the translocation of GLUT4 from a poorly defined intracellular compartment to the cell surface. We employed confocal microscopy to determine the in situ localization of GLUT4 relative to vesicle, Golgi, and endosomal proteins in these physiological insulin target cells. Three-dimensional analyses of GLUT4 immunostaining in basal cells revealed an intracellular punctate, patchy distribution both in the perinuclear region and scattered throughout the cytoplasm. VAMP2 closely associates with GLUT4 in many punctate vesicle-like structures. A small fraction of GLUT4 overlaps with TGN38-mannosidase ll, γ-adaptin, and mannose-6-phosphate receptors in the perinuclear region, presumably corresponding to late endosome and trans-Golgi network structures. GLUT4 does not co-localize with transferrin receptors, clathrin, and lgp-120. After insulin treatment, GLUT4 partially redistributes to the cell surface and decreases in the perinuclear area. However, GLUT4 remains co-localized with TGN38-mannosidase ll and γ-adaptin. Therefore, the basal compartment from which GLUT4 is translocated in response to insulin comprises specialized post-endosomal VAMP2-positive vesicles, distinct from the constitutively recycling endosomes. These results are consistent with a kinetic model in which GLUT4 is sequestered through two or more intracellular pools in series. (J Histochem Cytochem 45:1083–1096, 1997)

Differential trafficking of the Niemann-Pick C1 and 2 proteins highlights distinct roles in late endocytic lipid trafficking

The cellular location of Niemann‐Pick C2 protein (NPC2) in cultured human fibroblasts and Chinese hamster ovary cells was examined immunocytochemically and in living cells by expression of a functional red fluorescent protein chimeric analogue. Results: NPC2 is present in the lysosomes of both cholesterol‐depleted and ‐replenished cells, unlike Niemann‐Pick C1 protein (NPC1) which is recruited to late endosomes only upon uptake of low‐density lipoprotein. With mobilization of cholesterol from lysosomes, immunocytochemical detection of NPC2 in lysosomes is greatly diminished, whereas NPC1 remains in the late endosomal compartment. We found a partial overlap in the trafficking and organellar sites of accumulation of NPC2 and NPC1. In living cells, NPC2 traffics with NPC1 in late endosomal tubules. However, in contrast to NPC1, which remains either in late endosomal vesicles and tubules or at the peripheries of cholesterol‐laden lysosomes, NPC2 moves into the central core of lysosomes. Glycolipid analysis reveals that, in contrast to null mutant NPC1 cells, which accumulate GM2 ganglioside only at the plasma membrane, with no endocytic storage, absence of NPC2 protein in null mutant NPC2 cells does not block internalization of GM2 into endocytic vesicles. This difference in the cellular distribution of GM2 in NPC1 and NPC2 null mutants is the first report of a variation in the phenotypic expression of these genotypically distinct lesions.

Cytochemical studies of lipid metabolism: immunogold probes for lipoprotein lipase and cholesterol

In this article, cytochemical methods are presented for the study of lipid metabolism both in normal cells and in mutant cells with genetic disorders characterized by abnormal lipid metabolism. The benefit of using an immunocytochemical approach to the study of lipase in tissues is discussed, and a review is presented of the results on immunolocalization of lipoprotein lipase in cardiac tissue of normal mice. Immunocytochemical techniques are applied to the study of lysosomal proliferation in hepatocytes from liver of mutant mice with a genetic defect responsible for the lack of hepatic lipase and lipoprotein lipase activity in these animals. Localization of lipids in tissues with structural techniques has been an area of great interest to our laboratory for many years. Attention is called to the development of a technique for the visualization of fatty acids as a function of their ionization state and the production of fatty-acid myelin figures in membranes. Results on the use of filipin to detect unesterified cholesterol in membranes are reviewed. Filipin produces fluorescent filipin-cholesterol complexes but also perturbs cell membranes. Application of this cytochemical probe, in combination with immunocytochemistry of lysosomes, produced useful information on defects in low-density lipoprotein-derived cholesterol translocation in mutant human fibroblasts. Initial results on the application of immunological techniques to the study of cholesterol in lipid model systems indicate a novel approach, which may be applicable to specialized cell systems. Recent advances in cryoultramicrotomy and development of immunoprobes present valuable opportunities for the structural assessment of lipids and lipases in cell organelles and cell membranes.

INCORPORATION OF MOUSE ZONA PELLUCIDA PROTEINS INTO THE ENVELOPE OF XENOPUS LAEVIS OOCYTES

Abstract All vertebrate eggs have extracellular matrices, referred to as the zona pellucida in Mus musculus and the vitelline envelope in Xenopuslaevis. The mouse zona, composed of three sulfated glycoproteins (ZP1, ZP2, ZP3), is critical for fertilization and early development, and mice lacking a zona pellucida produce no live offspring. The primary structures of mouse ZP1 (623 amino acids), ZP2 (713 amino acids) and ZP3 (424 amino acids) have been deduced from full-length cDNAs, but posttranslational modifications result in mature zona proteins with molecular masses of 200–180 kDa, 140–120 kDa, and 83 kDa, respectively. The vitelline envelope forms a similar structure around Xenopus eggs and contains three glycoproteins that are structurally related (39–48% amino acid similarity) to the three mouse zona proteins. To investigate whether the structural semblances are sufficient to allow incorporation of the mouse zona proteins into the Xenopus vitelline envelope, capped synthetic mRNAs encoding ZP1, ZP2, and ZP3 proteins were injected into the cytoplasm of stage VI Xenopus oocytes. After 20 h of incubation the oocytes were harvested, and posttranslationally modified zona proteins were detected with monoclonal antibodies specific to mouse ZP1, ZP2, and ZP3. The oocytes were imaged with confocal microscopy to detect individual zona proteins in the extracellular matrix of the oocytes, and this localization was confirmed biochemically. Thus the mouse zona proteins appear to have been sufficiently conserved through 350 million years of evolution to be incorporated into the extracellular envelope surrounding Xenopus eggs.

Microtubules are not required for glucocorticoid receptor mediated gene induction

Steroid-free glucocorticoid receptors are generally considered to reside in the cytoplasm of cells. After the binding of steroids, the receptors translocate into the nucleus in a manner that has been proposed to involve microtubules. However, some results with inhibitors of microtubule assembly argue to the contrary. In all of these studies, only the whole cell localization of receptors has been examined; the biological activity of these receptors has not been determined. We now report that steroid-induced gene expression is maintained in the absence of intact microtubules. This argues that microtubules are not required for either the nuclear translocation or biological activity of glucocorticoid receptors.

Type C Niemann-Pick disease: dimethyl sulfoxide moderates abnormal LDL-cholesterol processing in mutant fibroblasts

Biochemical and cytochemical studies have revealed that abnormal processing of low-density-lipoprotein (LDL) cholesterol can be reversed in mutant Niemann-Pick C (NP-C) fibroblasts when 2% dimethyl sulfoxide (DMSO) is added to the culture medium. Both the excessive lysosomal accumulation of LDL cholesterol and the delayed induction of cellular homeostatic responses associated with the uptake of LDL by the mutant cells were substantially reversed by DMSO. DMSO appears to accelerate the intracellular mobilization of LDL-derived cholesterol through effects that may reflect enhanced membrane permeability or cholesterol solubilization.