scholarly journals Dynamic Movements of Organelles Containing Niemann-Pick C1 Protein: NPC1 Involvement in Late Endocytic Events

2001 ◽  
Vol 12 (3) ◽  
pp. 601-614 ◽  
Author(s):  
Dennis C. Ko ◽  
Michael D. Gordon ◽  
Janet Y. Jin ◽  
Matthew P. Scott
Keyword(s):  
High Speed ◽  
Fluorescent Protein ◽  
Transmembrane Protein ◽  
Density Lipoprotein ◽  
Endocytic Pathway ◽  
Cell Periphery ◽  
Late Endosomes ◽  
Npc1 Gene ◽  
Niemann Pick ◽  
Mutant Cells

People homozygous for mutations in the Niemann-Pick type C1 (NPC1) gene have physiological defects, including excess accumulation of intracellular cholesterol and other lipids, that lead to drastic neural and liver degeneration. The NPC1 multipass transmembrane protein is resident in late endosomes and lysosomes, but its functions are unknown. We find that organelles containing functional NPC1-fluorescent protein fusions undergo dramatic movements, some in association with extending strands of endoplasmic reticulum. InNPC1 mutant cells the NPC1-bearing organelles that normally move at high speed between perinuclear regions and the periphery of the cell are largely absent. Pulse-chase experiments with dialkylindocarbocyanine low-density lipoprotein showed that NPC1 organelles function late in the endocytic pathway; NPC1 protein may aid the partitioning of endocytic and lysosomal compartments. The close connection between NPC1 and the drug U18666A, which causes NPC1-like organelle defects, was established by rescuing drug-treated cells with overproduced NPC1. U18666A inhibits outward movements of NPC1 organelles, trapping membranes and cholesterol in perinuclear organelles similar to those in NPC1 mutant cells, even when cells are grown in lipoprotein-depleted serum. We conclude that NPC1 protein promotes the creation and/or movement of particular late endosomes, which rapidly transport materials to and from the cell periphery.

scholarly journals Annexin A6 modulates TBC1D15/Rab7/StARD3 axis to control endosomal cholesterol export in NPC1 cells

2019 ◽  
Vol 77 (14) ◽  
pp. 2839-2857 ◽  
Author(s):  
Elsa Meneses-Salas ◽  
Ana García-Melero ◽  
Kristiina Kanerva ◽  
Patricia Blanco-Muñoz ◽  
Frederic Morales-Paytuvi ◽  
...  
Keyword(s):  
Late Endosome ◽  
Dependent Manner ◽  
Lipid Transfer ◽  
Cholesterol Accumulation ◽  
Membrane Contact Sites ◽  
Late Endosomes ◽  
Membrane Contact ◽  
Domain 3 ◽  
Niemann Pick ◽  
Mutant Cells

Abstract Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.

scholarly journals Pathophysiological In Vitro Profile of Neuronal Differentiated Cells Derived from Niemann-Pick Disease Type C2 Patient-Specific iPSCs Carrying the NPC2 Mutations c.58G>T/c.140G>T

2021 ◽  
Vol 22 (8) ◽  
pp. 4009
Author(s):  
Maik Liedtke ◽  
Christin Völkner ◽  
Alexandra V. Jürs ◽  
Franziska Peter ◽  
Michael Rabenstein ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transmembrane Protein ◽  
Hereditary Disease ◽  
Cholesterol Homeostasis ◽  
Patient Specific ◽  
Differentiated Cells ◽  
Npc1 Gene ◽  
Niemann Pick

Niemann-Pick type C2 (NP-C2) disease is a rare hereditary disease caused by mutations in the NPC2 gene. NPC2 is a small, soluble protein consisting of 151 amino acids, primarily expressed in late endosomes and lysosomes (LE/LY). Together with NPC1, a transmembrane protein found in these organelles, NPC2 accomplishes the exclusion of cholesterol; thus, both proteins are essential to maintain cellular cholesterol homeostasis. Consequently, mutations in the NPC2 or NPC1 gene result in pathophysiological accumulation of cholesterol and sphingolipids in LE/LY. The vast majority of Niemann-Pick type C disease patients, 95%, suffer from a mutation of NPC1, and only 5% display a mutation of NPC2. The biochemical phenotype of NP-C1 and NP-C2 appears to be indistinguishable, and both diseases share several commonalities in the clinical manifestation. Studies of the pathological mechanisms underlying NP-C2 are mostly based on NP-C2 animal models and NP-C2 patient-derived fibroblasts. Recently, we established induced pluripotent stem cells (iPSCs), derived from a donor carrying the NPC2 mutations c.58G>T/c.140G>T. Here, we present a profile of pathophysiological in vitro features, shared by NP-C1 and NP-C2, of neural differentiated cells obtained from the patient specific iPSCs. Profiling comprised a determination of the NPC2 protein level, detection of cholesterol accumulation by filipin staining, analysis of oxidative stress, and determination of autophagy. As expected, the NPC2-deficient cells displayed a significantly reduced amount of NPC2 protein, and, accordingly, we observed a significantly increased amount of cholesterol. Most notably, NPC2-deficient cells displayed only a slight increase of reactive oxygen species (ROS), suggesting that they do not suffer from oxidative stress and express catalase at a high level. As a site note, comparable NPC1-deficient cells suffer from a lack of catalase and display an increased level of ROS. In summary, this cell line provides a valuable tool to gain deeper understanding, not only of the pathogenic mechanism of NP-C2, but also of NP-C1.

Rab22a affects the morphology and function of the endocytic pathway

2001 ◽  
Vol 114 (22) ◽  
pp. 4041-4049 ◽  
Author(s):  
Rosana Mesa ◽  
Cristina Salomón ◽  
Marcelo Roggero ◽  
Philip D. Stahl ◽  
Luis S. Mayorga
Keyword(s):  
Fluorescent Protein ◽  
Fluid Phase ◽  
Small Gtpases ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Rab Proteins ◽  
Rab Protein ◽  
Late Endosomes ◽  
And Function ◽  
Negative Mutant

Soon after endocytosis, internalized material is sorted along different pathways in a process that requires the coordinated activity of several Rab proteins. Although abundant information is available about the subcellular distribution and function of some of the endocytosis-specific Rabs (e.g. Rab5 and Rab4), very little is known about some other members of this family of proteins. To unveil some of the properties of Rab22a, one of the less studied endosome-associated small GTPases, we have expressed the protein tagged with the green fluorescent protein in CHO cells. The results indicate that Rab22a associates with early and late endosomes (labeled by a 5 minute rhodamine-transferrin uptake and the cation-independent mannose 6-phosphate receptor, respectively) but not with lysosomes (labeled by 1 hour rhodamine horseradish peroxidase uptake followed by 1 hour chase). Overexpression of the protein causes a prominent morphological enlargement of the early and late endosomes. Two mutants were generated by site-directed mutagenesis, a negative mutant (Rab22aS19N, with reduced affinity for GTP) and a constitutively active mutant (Rab22aQ64L, with reduced endogenous GTPase activity). The distribution of the negative mutant was mostly cytosolic, whereas the positive mutant associated with early and late endosomes and, interestingly also with lysosomes and autophagosomes (labeled with monodansylcadaverine). Cells expressing Rab22a wild type and Rab22aS19N displayed decreased endocytosis of a fluid phase marker. Conversely, overexpression of Rab22aQ64L, which strongly affects the morphology of endosomes, did not inhibit bulk endocytosis. Our results show that Rab22a has a unique distribution along the endocytic pathway that is not shared by any other Rab protein, and that it strongly affects the morphology and function of endosomes.

The endocytic pathway in animal cells

1990 ◽  
Vol 48 (3) ◽  
pp. 41-41
Author(s):  
Gareth Griffiths
Keyword(s):  
Early Endosome ◽  
Endocytic Pathway ◽  
Three Dimensions ◽  
Cell Periphery ◽  
Animal Cells ◽  
The Third ◽  
Negative Stain ◽  
Late Endosomes ◽  
Work Done

Our recent data have provided evidence that the endocytic pathway comprises four structurally and functionally distinct compartments in addition to the clathrin coated vesicles. The most proximal of these compartments, the early endosome, is structurally complex, consists of cisternal, tubular, as well as vesicular domains and is predominantly localized to the cell periphery. We have proposed that the vesicular domains bud off the early endosome to form the spherical endosome (carrier) vesicles that transport material from early to late endosomes, a process that requires microtubules. These endosome vesicles are spherical (0.2-0.4μm diameter) structures whose lumen is packed with thin tubules. The structure of the latter has recently become more evident in negative stain preparations. These vesicles can also be shown to bind microtubules in vitro (work done in collaboration with Jean Gruenberg's group, EMBL). The third compartment, the late endosome or prelysosomal compartment (PLC) is, like the early endosome, complex in three-dimensions.

scholarly journals Rab8-dependent Recycling Promotes Endosomal Cholesterol Removal in Normal and Sphingolipidosis Cells

2007 ◽  
Vol 18 (1) ◽  
pp. 47-56 ◽  
Author(s):  
Matts D. Linder ◽  
Riikka-Liisa Uronen ◽  
Maarit Hölttä-Vuori ◽  
Peter van der Sluijs ◽  
Johan Peränen ◽  
...  
Keyword(s):  
Cholesterol Efflux ◽  
Ldl Cholesterol ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Receptor Activation ◽  
Cholesterol Esterification ◽  
Rab Gtpase ◽  
Cell Periphery ◽  
Regulatory Machinery ◽  
Niemann Pick

The mechanisms by which low-density lipoprotein (LDL)-cholesterol exits the endocytic circuits are not well understood. The process is defective in Niemann–Pick type C (NPC) disease in which cholesterol and sphingolipids accumulate in late endosomal compartments. This is accompanied by defective cholesterol esterification in the endoplasmic reticulum and impaired ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. We show here that overexpression of the recycling/exocytic Rab GTPase Rab8 rescued the late endosomal cholesterol deposition and sphingolipid mistrafficking in NPC fibroblasts. Rab8 redistributed cholesterol from late endosomes to the cell periphery and stimulated cholesterol efflux to the ABCA1-ligand apolipoprotein A-I (apoA-I) without increasing cholesterol esterification. Depletion of Rab8 from wild-type fibroblasts resulted in cholesterol deposition within late endosomal compartments. This cholesterol accumulation was accompanied by impaired clearance of LDL-cholesterol from endocytic circuits to apoA-I and could not be bypassed by liver X receptor activation. Our findings establish Rab8 as a key component of the regulatory machinery that leads to ABCA1-dependent removal of cholesterol from endocytic circuits.

scholarly journals Phagocytic and pinocytic uptake of cholesterol in Tetrahymena thermophila impact differently on gene regulation for sterol homeostasis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Josefina Hernández ◽  
Matías Gabrielli ◽  
Joaquín Costa ◽  
Antonio D. Uttaro
Keyword(s):  
Mammalian Cells ◽  
Tetrahymena Thermophila ◽  
Expression Patterns ◽  
Transcriptional Response ◽  
Endocytic Pathway ◽  
Sterol Uptake ◽  
Partial Conservation ◽  
Late Endosomes ◽  
Cholesterol Intake ◽  
Mutant Cells

AbstractThe ciliate Tetrahymena thermophila can either synthesize tetrahymanol or when available, assimilate and modify sterols from its diet. This metabolic shift is mainly driven by transcriptional regulation of genes for tetrahymanol synthesis (TS) and sterol bioconversion (SB). The mechanistic details of sterol uptake, intracellular trafficking and the associated gene expression changes are unknown. By following cholesterol incorporation over time in a conditional phagocytosis-deficient mutant, we found that although phagocytosis is the main sterol intake route, a secondary endocytic pathway exists. Different expression patterns for TS and SB genes were associated with these entry mechanisms. Squalene synthase was down-regulated by a massive cholesterol intake only attainable by phagocytosis-proficient cells, whereas C22-sterol desaturase required ten times less cholesterol and was up-regulated in both wild-type and mutant cells. These patterns are suggestive of at least two different signaling pathways. Sterol trafficking beyond phagosomes and esterification was impaired by the NPC1 inhibitor U18666A. NPC1 is a protein that mediates cholesterol export from late endosomes/lysosomes in mammalian cells. U18666A also produced a delay in the transcriptional response to cholesterol, suggesting that the regulatory signals are triggered between lysosomes and the endoplasmic reticulum. These findings could hint at partial conservation of sterol homeostasis between eukaryote lineages.

End13p/Vps4p is required for efficient transport from early to late endosomes in Saccharomyces cerevisiae

2001 ◽  
Vol 114 (10) ◽  
pp. 1935-1947 ◽  
Author(s):  
R. Zahn ◽  
B.J. Stevenson ◽  
S. Schroder-Kohne ◽  
B. Zanolari ◽  
H. Riezman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Dyes ◽  
Membrane Receptors ◽  
Endocytic Pathway ◽  
Aaa Atpase ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Plasma Membrane Receptors ◽  
Vacuolar Protein ◽  
Mutant Cells

end13-1 was isolated in a screen for endocytosis mutants and has been shown to have a post-internalisation defect in endocytic transport as well as a defect in vacuolar protein sorting (Vps(-) phenotype), leading to secretion of newly synthesised vacuolar proteins. Here we demonstrate that END13 is identical to VPS4, encoding an AAA (ATPase associated with a variety of cellular activities)-family ATPase. We also report that the end13-1 mutation is a serine 335 to phenylalanine substitution in the AAA-ATPase domain of End13p/Vps4p. It has been reported that mutant cells lacking End13p/Vps4p (end13(vps4)((Dgr;)) accumulate endocytosed marker dyes, plasma membrane receptors and newly synthesised vacuolar hydrolase precursors in an endosomal compartment adjacent to the vacuole (prevacuolar compartment, or PVC). We find, however, that the end13 mutants have defects in transport of endocytosed fluorescent dyes, plasma membrane receptors and ligands from small peripherally located early endosomes to larger late endosomes, which are often located adjacent to the vacuole. Our results indicate that End13p/Vps4p may play an important role in multiple steps of membrane traffic through the endocytic pathway.

scholarly journals Annexin A6 and NPC1 regulate LDL-inducible cell migration and distribution of focal adhesions

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Jaimy Jose ◽  
Monira Hoque ◽  
Johanna Engel ◽  
Syed S. Beevi ◽  
Mohamed Wahba ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Motility ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Migration And Invasion ◽  
3 Dimensional ◽  
Late Endosomes ◽  
Niemann Pick ◽  
Mutant Cells

AbstractCholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.

scholarly journals Visualization of TGN to Endosome Trafficking through Fluorescently Labeled MPR and AP-1 in Living Cells

2003 ◽  
Vol 14 (1) ◽  
pp. 142-155 ◽  
Author(s):  
Satoshi Waguri ◽  
Frédérique Dewitte ◽  
Roland Le Borgne ◽  
Yves Rouillé ◽  
Yasuo Uchiyama ◽  
...  
Keyword(s):  
Hela Cells ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Time Lapse ◽  
Living Cells ◽  
Endocytic Pathway ◽  
Cell Periphery ◽  
Endosome Trafficking ◽  
Early Endosomes ◽  
Green Fluorescent

We have stably expressed in HeLa cells a chimeric protein made of the green fluorescent protein (GFP) fused to the transmembrane and cytoplasmic domains of the mannose 6-phosphate/insulin like growth factor II receptor in order to study its dynamics in living cells. At steady state, the bulk of this chimeric protein (GFP-CI-MPR) localizes to the trans-Golgi network (TGN), but significant amounts are also detected in peripheral, tubulo-vesicular structures and early endosomes as well as at the plasma membrane. Time-lapse videomicroscopy shows that the GFP-CI-MPR is ubiquitously detected in tubular elements that detach from the TGN and move toward the cell periphery, sometimes breaking into smaller tubular fragments. The formation of the TGN-derived tubules is temperature dependent, requires the presence of intact microtubule and actin networks, and is regulated by the ARF-1 GTPase. The TGN-derived tubules fuse with peripheral, tubulo-vesicular structures also containing the GFP-CI-MPR. These structures are highly dynamic, fusing with each other as well as with early endosomes. Time-lapse videomicroscopy performed on HeLa cells coexpressing the CFP-CI-MPR and the AP-1 complex whose γ-subunit was fused to YFP shows that AP-1 is present not only on the TGN and peripheral CFP-CI-MPR containing structures but also on TGN-derived tubules containing the CFP-CI-MPR. The data support the notion that tubular elements can mediate MPR transport from the TGN to a peripheral, tubulo-vesicular network dynamically connected with the endocytic pathway and that the AP-1 coat may facilitate MPR sorting in the TGN and endosomes.

scholarly journals Cholesterol Overload: Contact Sites to the Rescue!

Contact ◽  
2019 ◽  
Vol 2 ◽  
pp. 251525641989350 ◽  
Author(s):  
Carlos Enrich ◽  
Carles Rentero ◽  
Thomas Grewal ◽  
Clare E. Futter ◽  
Emily R. Eden
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Cholesterol Homeostasis ◽  
Low Density ◽  
Cholesterol Accumulation ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Late Endosomes ◽  
Membrane Contact ◽  
Niemann Pick

Delivery of low-density lipoprotein-derived cholesterol to the endoplasmic reticulum (ER) is essential for cholesterol homeostasis, yet the mechanism of this transport has largely remained elusive. Two recent reports shed some light on this process, uncovering a role for Niemann Pick type-C1 protein (NPC1) in the formation of membrane contact sites (MCS) between late endosomes (LE)/lysosomes (Lys) and the ER. Both studies identified a loss of MCS in cells lacking functional NPC1, where cholesterol accumulates in late endocytic organelles. Remarkably, and taking different approaches, both studies have made a striking observation that expansion of LE/Lys-ER MCS can rescue the cholesterol accumulation phenotype in NPC1 mutant or deficient cells. In both cases, the cholesterol was shown to be transported to the ER, demonstrating the importance of ER-LE/Lys contact sites in the direct transport of low-density lipoprotein-derived cholesterol to the ER.

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