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 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.

The role of calcium and other ions in sorting and delivery in the late endocytic pathway

2007 ◽  
Vol 35 (5) ◽  
pp. 1088-1091 ◽  
Author(s):  
J.P. Luzio ◽  
N.A. Bright ◽  
P.R. Pryor
Keyword(s):  
Mammalian Cells ◽  
Ionic Composition ◽  
Endocytic Pathway ◽  
Lysosomal Storage ◽  
Acid Hydrolases ◽  
Gene Encoding ◽  
Endosomal Membrane ◽  
Type Iv ◽  
Late Endosomes ◽  
The Many

The passage of endocytosed receptor-bound ligands and membrane proteins through the endocytic pathway of mammalian cells to lysosomes occurs via early and late endosomes. The latter contain many luminal vesicles and are often referred to as MVBs (multivesicular bodies). The overall morphology of endosomal compartments is, in major part, a consequence of the many fusion events occurring in the endocytic pathway. Kissing events and direct fusion between late endosomes and lysosomes provide a means of delivery to lysosomes. The luminal ionic composition of organelles in the endocytic pathway is of considerable importance both in the trafficking of endocytosed ligands and in the membrane fusion events. In particular, H+ ions play a role in sorting processes and providing an appropriate environment for the action of lysosomal acid hydrolases. Na+/H+ exchangers in the endosomal membrane have been implicated in the formation of MVBs and sorting into luminal vesicles. Ca2+ ions are required for fusion events and luminal content condensation in the lysosome. Consistent with an important role for luminal Ca2+ in traffic through the late endocytic pathway, mutations in the gene encoding mucolipin-1, a lysosomal non-specific cation channel, result in abnormalities in lipid traffic and are associated with the autosomal recessive lysosomal storage disease MLIV (mucolipidosis type IV).

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 Transcriptome analysis and molecular mechanism of linseed (Linum usitatissimum L.) drought tolerance under repeated drought using single-molecule long-read sequencing

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Wei Wang ◽  
Lei Wang ◽  
Ling Wang ◽  
Meilian Tan ◽  
Collins O. Ogutu ◽  
...  
Keyword(s):  
Dna Repair ◽  
Drought Tolerance ◽  
Single Molecule ◽  
Linum Usitatissimum ◽  
Expression Patterns ◽  
Transcriptional Response ◽  
Enrichment Analysis ◽  
Resistant Variety ◽  
Proline Biosynthesis ◽  
Interacting Protein

Abstract Background Oil flax (linseed, Linum usitatissimum L.) is one of the most important oil crops., However, the increases in drought resulting from climate change have dramatically reduces linseed yield and quality, but very little is known about how linseed coordinates the expression of drought resistance gene in response to different level of drought stress (DS) on the genome-wide level. Results To explore the linseed transcriptional response of DS and repeated drought (RD) stress, we determined the drought tolerance of different linseed varieties. Then we performed full-length transcriptome sequencing of drought-resistant variety (Z141) and drought-sensitive variety (NY-17) under DS and RD stress at the seedling stage using single-molecule real-time sequencing and RNA-sequencing. Gene Ontology (GO) and reduce and visualize GO (REVIGO) enrichment analysis showed that upregulated genes of Z141 were enriched in more functional pathways related to plant drought tolerance than those of NY-17 were under DS. In addition, 4436 linseed transcription factors were identified, and 1190 were responsive to stress treatments. Moreover, protein-protein interaction (PPI) network analysis showed that the proline biosynthesis pathway interacts with stress response genes through RAD50 (DNA repair protein 50) interacting protein 1 (RIN-1). Finally, proline biosynthesis and DNA repair structural gene expression patterns were verified by RT- PCR. Conclusions The drought tolerance of Z141 may be related to its upregulation of drought tolerance genes under DS. Proline may play an important role in linseed drought tolerance by maintaining cell osmotic and protecting DNA from ROS damage. In summary, this study provides a new perspective to understand the drought adaptability of linseed.

scholarly journals Relationship between invariant chain expression and major histocompatibility complex class II transport into early and late endocytic compartments.

1993 ◽  
Vol 177 (3) ◽  
pp. 583-596 ◽  
Author(s):  
P Romagnoli ◽  
C Layet ◽  
J Yewdell ◽  
O Bakke ◽  
R N Germain
Keyword(s):  
Major Histocompatibility Complex ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Invariant Chain ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endocytic Compartments

Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the characteristics of late endosomes/prelysosomes. The Ii chains in these late endocytic vesicles have undergone proteolytic cleavage in the lumenal region postulated to control MHC class II peptide binding. These data indicate that the association of class II with Ii results in initial movement to early endosomes. At high levels of Ii expression, egress to later endocytic compartments is delayed and class II-Ii complexes accumulate together with endocytosed material. At lower levels of Ii expression, class II-Ii complexes are found primarily in late endosomes/prelysosomes. These data provide evidence that the route of class II transport to the site of antigen processing and loading involves movement through early endosomes to late endosomes/prelysosomes. Our results also reveal an unexpected ability of intact Ii to modify the structure and function of the early endosomal compartment, which may play a role in regulating this processing pathway.

scholarly journals Complex II subunit SDHD is critical for cell growth and metabolism, which can be partially restored with a synthetic ubiquinone analog

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Aloka B. Bandara ◽  
Joshua C. Drake ◽  
David A. Brown
Keyword(s):  
Mammalian Cells ◽  
Krebs Cycle ◽  
Atp Synthesis ◽  
Hek293 Cells ◽  
Knockout Mutant ◽  
Complex Ii ◽  
Growth Defects ◽  
Transport Chain ◽  
Mutant Cells

Abstract Background Succinate dehydrogenase (Complex II) plays a dual role in respiration by catalyzing the oxidation of succinate to fumarate in the mitochondrial Krebs cycle and transferring electrons from succinate to ubiquinone in the mitochondrial electron transport chain (ETC). Mutations in Complex II are associated with a number of pathologies. SDHD, one of the four subunits of Complex II, serves by anchoring the complex to the inner-membrane and transferring electrons from the complex to ubiquinone. Thus, modeling SDHD dysfunction could be a valuable tool for understanding its importance in metabolism and developing novel therapeutics, however no suitable models exist. Results Via CRISPR/Cas9, we mutated SDHD in HEK293 cells and investigated the in vitro role of SDHD in metabolism. Compared to the parent HEK293, the knockout mutant HEK293ΔSDHD produced significantly less number of cells in culture. The mutant cells predictably had suppressed Complex II-mediated mitochondrial respiration, but also Complex I-mediated respiration. SDHD mutation also adversely affected glycolytic capacity and ATP synthesis. Mutant cells were more apoptotic and susceptible to necrosis. Treatment with the mitochondrial therapeutic idebenone partially improved oxygen consumption and growth of mutant cells. Conclusions Overall, our results suggest that SDHD is vital for growth and metabolism of mammalian cells, and that respiratory and growth defects can be partially restored with treatment of a ubiquinone analog. This is the first report to use CRISPR/Cas9 approach to construct a knockout SDHD cell line and evaluate the efficacy of an established mitochondrial therapeutic candidate to improve bioenergetic capacity.

Trafficking of interleukin 2 and transferrin in endosomal fractions of T lymphocytes

1994 ◽  
Vol 107 (5) ◽  
pp. 1289-1295 ◽  
Author(s):  
V. Duprez ◽  
M. Smoljanovic ◽  
M. Lieb ◽  
A. Dautry-Varsat
Keyword(s):  
Horseradish Peroxidase ◽  
Interleukin 2 ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Recycling Endosomes ◽  
High Affinity ◽  
Human T Cell ◽  
Late Endosomes ◽  
Discontinuous Sucrose Gradient ◽  
Acidic Organelles

The T lymphocyte growth factor interleukin 2 binds to surface high-affinity receptors and is rapidly internalized and degraded in acidic organelles. The alpha and beta chains of high-affinity interleukin 2 receptors are internalized together with interleukin 2. To identify the intracellular pathway followed by interleukin 2, we have compared the subcellular distribution of interleukin 2, transferrin and a fluid-phase marker, horseradish peroxidase, in the human T cell line IARC 301.5. Transferrin was used as a marker of early and recycling endosomes, and horseradish peroxidase to probe for the whole endocytic pathway. Fractionation of intracellular organelles on a discontinuous sucrose gradient showed that internalized interleukin 2 is initially mostly found in compartments with similar densities to transferrin, e.g. early and recycling endosomes. The kinetics of entry and exit of interleukin 2 from such organelles was much slower than that of transferrin. Later on, interleukin 2 is predominantly found in dense lysosome-containing fractions. Very little, if any, interleukin 2 was found in fractions corresponding to late endosomes containing horseradish peroxidase. These results suggest that, after endocytosis, interleukin 2 enters early or recycling endosomes before it reaches dense lysosomes.

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.

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