The rab7 GTPase resides on a vesicular compartment connected to lysosomes

1995 ◽  
Vol 108 (11) ◽  
pp. 3349-3358 ◽  
Author(s):  
S. Meresse ◽  
J.P. Gorvel ◽  
P. Chavrier
Keyword(s):  
Mammalian Cells ◽  
Intracellular Localization ◽  
Confocal Laser ◽  
Cell Periphery ◽  
Rab Gtpases ◽  
Wild Type ◽  
Microtubule Network ◽  
Late Endosomes ◽  
Hela Cell Lines ◽  
Vesicular Compartment

Rab GTPases belong to the Ras GTPase superfamily and are key regulators of membrane traffic. Among them, rab7 has been localized on late endosomes of NRK cells but its function remains unknown. In order to investigate its role, we generated stable HeLa cell lines that express either wild type or a GTPase-defective mutant of rab7 in an inducible manner. A morphological analysis of the intracellular localization of these proteins was performed by confocal laser microscopy. Here we show that, in HeLa cells, rab7 is present on a vesicular compartment that extends from the perinuclear area to the cell periphery and shows only a partial colocalization with the cation-independent mannose 6-phosphate receptor, a marker for late endosomes. The topology of this compartment is dependent on the microtubule network since nocodazole treatment results in its scattering throughout the cytoplasm. In addition, we observed that, in contrast to the wild-type protein, a rab7 mutant with a reduced GTPase activity is in part associated with lysosomal membranes. This observation was confirmed by subcellular fractionation in a Percoll gradient. Our data implicate rab7 as the first GTPase functioning on terminal endocytic structures in mammalian cells.

scholarly journals Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability, Localization, and Subsequent Budding of Virus-Like Particles

2009 ◽  
Vol 84 (5) ◽  
pp. 2294-2303 ◽  
Author(s):  
Yuliang Liu ◽  
Luis Cocka ◽  
Atsushi Okumura ◽  
Yong-An Zhang ◽  
J. Oriol Sunyer ◽  
...  
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Ebola Virus ◽  
Intracellular Localization ◽  
Gel Filtration ◽  
Point Mutations ◽  
Marburg Virus ◽  
Structure Stability ◽  
Wild Type ◽  
Virus Like Particles

ABSTRACT The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the 96LPLGVA101 sequence of eVP40 and the corresponding 84LPLGIM89 sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the 96LPLGVA101 motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the 96LPLGVA101 motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.

scholarly journals MLN64 Is Involved in Actin-mediated Dynamics of Late Endocytic Organelles

2005 ◽  
Vol 16 (8) ◽  
pp. 3873-3886 ◽  
Author(s):  
Maarit Hölttä-Vuori ◽  
Fabien Alpy ◽  
Kimmo Tanhuanpää ◽  
Eija Jokitalo ◽  
Aino-Liisa Mutka ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Unknown Function ◽  
Cell Periphery ◽  
Dependent Manner ◽  
Protein Overexpression ◽  
Wild Type ◽  
Functional Connection ◽  
Late Endosomes ◽  
Endosomal Membranes ◽  
Cholesterol Binding

MLN64 is a late endosomal cholesterol-binding membrane protein of an unknown function. Here, we show that MLN64 depletion results in the dispersion of late endocytic organelles to the cell periphery similarly as upon pharmacological actin disruption. The dispersed organelles in MLN64 knockdown cells exhibited decreased association with actin and the Arp2/3 complex subunit p34-Arc. MLN64 depletion was accompanied by impaired fusion of late endocytic organelles and delayed cargo degradation. MLN64 overexpression increased the number of actin and p34-Arc-positive patches on late endosomes, enhanced the fusion of late endocytic organelles in an actin-dependent manner, and stimulated the deposition of sterol in late endosomes harboring the protein. Overexpression of wild-type MLN64 was capable of rescuing the endosome dispersion in MLN64-depleted cells, whereas mutants of MLN64 defective in cholesterol binding were not, suggesting a functional connection between MLN64-mediated sterol transfer and actin-dependent late endosome dynamics. We propose that local sterol enrichment by MLN64 in the late endosomal membranes facilitates their association with actin, thereby governing actin-dependent fusion and degradative activity of late endocytic organelles.

Expression of the plasmodial pfmdr1 gene in mammalian cells is associated with increased susceptibility to chloroquine

1994 ◽  
Vol 14 (4) ◽  
pp. 2419-2428
Author(s):  
H H van Es ◽  
S Karcz ◽  
F Chu ◽  
A F Cowman ◽  
S Vidal ◽  
...  
Keyword(s):  
Cho Cells ◽  
Mammalian Cells ◽  
Intracellular Localization ◽  
Plasmodium Falciparum Malaria ◽  
Drug Susceptibility ◽  
Wild Type ◽  
Malaria Parasites ◽  
Pfmdr1 Gene ◽  
Alpha Galactosidase

Chloroquine (CQ)-resistant (CQR) Plasmodium falciparum malaria parasites show a strong decrease in CQ accumulation in comparison with chloroquine-sensitive parasites. Controversy exists over the role of the plasmodial pfmdr1 gene in the CQR phenotype. pfmdr1 is a member of the superfamily of ATP-binding cassette transporters. Other members of this family are the mammalian multidrug resistance genes and the CFTR gene. We have expressed the pfmdr1-encoded protein, Pgh1, in CHO cells and Xenopus oocytes. CHO cells expressing the Pgh1 protein demonstrated an increased, verapamil-insensitive susceptibility to CQ. Conversely, no increase in drug susceptibility to primaquine, quinine, adriamycin, or colchicine was observed in Pgh1-expressing cells. CQ uptake experiments revealed an increased, ATP-dependent accumulation of CQ in Pgh1-expressing cells over the level in nonexpressing control cells. The increased CQ accumulation in Pgh1-expressing cells coincided with an enhanced in vivo inhibition of lysosomal alpha-galactosidase by CQ. CHO cells expressing Pgh1 carrying two of the CQR-associated Pgh1 amino acid changes (S1034C and N1042D) did not display an increased CQ sensitivity. Immunofluorescence experiments revealed an intracellular localization of both mutant and wild-type forms of Pgh1. We conclude from our results that wild-type Pgh1 protein can mediate an increased intracellular accumulation of CQ and that this function is impaired in CQR-associated mutant forms of the protein. We speculate that the Pgh1 protein plays an important role in CQ import in CQ-sensitive malaria parasites.

scholarly journals Construction and validation of EGFP-expressingStaphylococcus aureusclinical strains for adhesion and internalization assays on epithelial cells

2019 ◽  
Author(s):  
Sana Charaoui-Boukerzaza ◽  
M. Fedy Morgene ◽  
Josselin Rigaill ◽  
Estelle Audoux ◽  
Zhiguo He ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Epithelial Cells ◽  
Mammalian Cells ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Egfp Expression ◽  
Confocal Laser ◽  
Wild Type ◽  
Clinical Strains ◽  
One Step

ABSTRACTBackgroundStaphylococcus aureusis both a major pathogen and a commensal bacterium in humans. It is able to adhere at the surface of epithelial cells of the anterior nares and can trigger its internalization inside these non-professional phagocytic cells. To better understand the interactions of clinical isolates with keratinocytes in the anterior nares, we developed and validated a one-step protocol expressing enhanced green fluorescent protein (EGFP) inS. aureusclinical strains with the aim to study adhesion to and internalization into mammalian cells.MethodsTwentyS. aureusclinical isolates belonging to clonal complexes 5, 8, 30, 45, 398 were selected for one-step transformation protocol with the EGFP-encoding plasmid pBSU101. EGFP expression was analysed by flow cytometry and confocal microscopy. Wild type and isogenic EGFP-expressing strains were compared for adhesion and internalization levels by using the HaCaT cell model.ResultsTransformation was achieved in all theS. aureusstrains regardless of their genetic background. The flow cytometry analysis showed that the mean proportion of EGFP-expressing bacteria was 97.2% (± 2.1) after 4h of incubation. Adhesion and internalization levels were similar in wild-type and isogenic EGFP-expressingS. aureusstrains. Confocal laser scanning microscopy confirmed that EGFP-expressingS. aureusbacteria could be easily identified inside HaCaT keratinocytes.ConclusionThis study reports an efficient protocol for expressing EGFP inS. aureusclinical strains and demonstrates that these EGFP-expressing strains are suitable for adhesion and internalization assays using HaCaT cells, which allows to perform static and dynamicin vitrostudies ofS. aureuscolonization.

scholarly journals Expression of the plasmodial pfmdr1 gene in mammalian cells is associated with increased susceptibility to chloroquine.

1994 ◽  
Vol 14 (4) ◽  
pp. 2419-2428 ◽  
Author(s):  
H H van Es ◽  
S Karcz ◽  
F Chu ◽  
A F Cowman ◽  
S Vidal ◽  
...  
Keyword(s):  
Cho Cells ◽  
Mammalian Cells ◽  
Intracellular Localization ◽  
Plasmodium Falciparum Malaria ◽  
Drug Susceptibility ◽  
Wild Type ◽  
Malaria Parasites ◽  
Pfmdr1 Gene ◽  
Alpha Galactosidase

Chloroquine (CQ)-resistant (CQR) Plasmodium falciparum malaria parasites show a strong decrease in CQ accumulation in comparison with chloroquine-sensitive parasites. Controversy exists over the role of the plasmodial pfmdr1 gene in the CQR phenotype. pfmdr1 is a member of the superfamily of ATP-binding cassette transporters. Other members of this family are the mammalian multidrug resistance genes and the CFTR gene. We have expressed the pfmdr1-encoded protein, Pgh1, in CHO cells and Xenopus oocytes. CHO cells expressing the Pgh1 protein demonstrated an increased, verapamil-insensitive susceptibility to CQ. Conversely, no increase in drug susceptibility to primaquine, quinine, adriamycin, or colchicine was observed in Pgh1-expressing cells. CQ uptake experiments revealed an increased, ATP-dependent accumulation of CQ in Pgh1-expressing cells over the level in nonexpressing control cells. The increased CQ accumulation in Pgh1-expressing cells coincided with an enhanced in vivo inhibition of lysosomal alpha-galactosidase by CQ. CHO cells expressing Pgh1 carrying two of the CQR-associated Pgh1 amino acid changes (S1034C and N1042D) did not display an increased CQ sensitivity. Immunofluorescence experiments revealed an intracellular localization of both mutant and wild-type forms of Pgh1. We conclude from our results that wild-type Pgh1 protein can mediate an increased intracellular accumulation of CQ and that this function is impaired in CQR-associated mutant forms of the protein. We speculate that the Pgh1 protein plays an important role in CQ import in CQ-sensitive malaria parasites.

scholarly journals The Oxysterol-binding Protein Homologue ORP1L Interacts with Rab7 and Alters Functional Properties of Late Endocytic Compartments

2005 ◽  
Vol 16 (12) ◽  
pp. 5480-5492 ◽  
Author(s):  
Marie Johansson ◽  
Markku Lehto ◽  
Kimmo Tanhuanpää ◽  
Timothy L. Cover ◽  
Vesa M. Olkkonen
Keyword(s):  
Membrane Trafficking ◽  
Binding Protein ◽  
Pleckstrin Homology Domain ◽  
Rab Gtpases ◽  
Microtubule Network ◽  
Oxysterol Binding Protein ◽  
Motor Complex ◽  
Late Endosomes ◽  
Endocytic Compartments ◽  
Fluorescent Dextran

ORP1L is a member of the human oxysterol-binding protein (OSBP) family. ORP1L localizes to late endosomes (LEs)/lysosomes, colocalizing with the GTPases Rab7 and Rab9 and lysosome-associated membrane protein-1. We demonstrate that ORP1L interacts physically with Rab7, preferentially with its GTP-bound form, and provide evidence that ORP1L stabilizes GTP-bound Rab7 on LEs/lysosomes. The Rab7-binding determinant is mapped to the ankyrin repeat (ANK) region of ORP1L. The pleckstrin homology domain (PHD) of ORP1L binds phosphoinositides with low affinity and specificity. ORP1L ANK- and ANK+PHD fragments induce perinuclear clustering of LE/lysosomes. This is dependent on an intact microtubule network and a functional dynein/dynactin motor complex. The dominant inhibitory Rab7 mutant T22N reverses the LE clustering, suggesting that the effect is dependent on active Rab7. Transport of fluorescent dextran to LEs is inhibited by overexpression of ORP1L. Overexpression of ORP1L, and in particular the N-terminal fragments of ORP1L, inhibits vacuolation of LE caused by Helicobacter pylori toxin VacA, a process also involving Rab7. The present study demonstrates that ORP1L binds to Rab7, modifies its functional cycle, and can interfere with LE/lysosome organization and endocytic membrane trafficking. This is the first report of a direct connection between the OSBP-related protein family and the Rab GTPases.

scholarly journals The Salmonella Effector PipB2 Affects Late Endosome/Lysosome Distribution to Mediate Sif Extension

2005 ◽  
Vol 16 (9) ◽  
pp. 4108-4123 ◽  
Author(s):  
Leigh A. Knodler ◽  
Olivia Steele-Mortimer
Keyword(s):  
Host Cell ◽  
Mammalian Cells ◽  
Ectopic Expression ◽  
Bacterial Pathogen ◽  
Sequence Divergence ◽  
Cell Periphery ◽  
Host Cell Proteins ◽  
Late Endosomes ◽  
Membrane Bound ◽  
Endocytic Compartments

After internalization into mammalian cells, the bacterial pathogen Salmonella enterica resides within a membrane-bound compartment, the Salmonella-containing vacuole (SCV). During its maturation process, the SCV interacts extensively with host cell endocytic compartments, especially late endosomes/lysosomes (LE/Lys) at later stages. These interactions are mediated by the activities of multiple bacterial and host cell proteins. Here, we show that the Salmonella type III effector PipB2 reorganizes LE/Lys compartments in mammalian cells. This activity results in the centrifugal extension of lysosomal glycoprotein-rich membrane tubules, known as Salmonella-induced filaments, away from the SCV along microtubules. Salmonella overexpressing pipB2 induce the peripheral accumulation of LE/Lys compartments, reducing the frequency of LE/Lys tubulation. Furthermore, ectopic expression of pipB2 redistributes LE/Lys, but not other cellular organelles, to the cell periphery. In coexpression studies, PipB2 can overcome the effects of dominant-active Rab7 or Rab34 on LE/Lys positioning. Deletion of a C-terminal pentapeptide motif of PipB2, LFNEF, prevents its peripheral targeting and effect on organelle positioning. The PipB2 homologue PipB does not possess this motif or the same biological activity as PipB2. Therefore, it seems that a divergence in the biological functions of these two effectors can be accounted for by sequence divergence in their C termini.

scholarly journals Essential Role for the Legionella pneumophila Rep Helicase Homologue in Intracellular Infection of Mammalian Cells

2000 ◽  
Vol 68 (12) ◽  
pp. 6970-6978 ◽  
Author(s):  
Omar S. Harb ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Alveolar Epithelial Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wild Type ◽  
Intracellular Replication ◽  
Simultaneous Exposure ◽  
U937 Macrophage

ABSTRACT We have previously isolated 32 mutants of Legionella pneumophila that are defective in the infection of mammalian cells but not protozoa. The mutated loci have been designated macrophage-specific infectivity (mil) loci. In this study we characterized the mil mutant GK11. This mutant was incapable of growth within U937 macrophage-like cells and WI-26 alveolar epithelial cells. This defect in intracellular replication correlated with a defect in cytopathogenicity to these cells. Sequence analysis of the GK11 locus revealed it to be highly similar torep helicase genes of other bacteria. Since helicase mutants of Escherichia coli are hypersensitive to thymine starvation, we examined the sensitivity of GK11 to thymineless death (TLD). In the absence of thymine and thymidine, mutant GK11 did not undergo TLD but was defective for in vitro growth, and the defect was partially restored when these compounds were added to the growth medium. In addition, supplementation with thymidine or thymine partially restored the ability of GK11 to grow within and kill U937 macrophage-like cells. The data suggested that the low levels of thymine or thymidine in the L. pneumophila phagosome contributed to the defect of GK11 within macrophages. Using confocal laser scanning microscopy, we determined the effect of the mutation in the Rep helicase homologue on the intracellular trafficking of GK11 within macrophages. In contrast to the wild-type strain, phagosomes harboring GK11 colocalized with several late endosomal/lysosomal markers, including LAMP-1, LAMP-2, and cathepsin D. In addition, only 50% of the GK11 phagosomes colocalized with the endoplasmic reticulum marker BiP 4 h postinfection. Colocalization of BiP with GK11 phagosomes was absent 6 h postinfection, while 90% of the wild-type phagosomes colocalized with this marker at both time points. We propose that the low level of thymine within the L. pneumophila phagosome in combination with simultaneous exposure to multiple stress stimuli results in deleterious mutations that cannot be repaired in therep helicase homologue mutant, rendering it defective in intracellular replication.

scholarly journals Identification of a Novel Keratin 9 Missense Mutation in a Chinese Family with Epidermolytic Palmoplantar Keratoderma

2018 ◽  
Vol 46 (5) ◽  
pp. 1919-1929 ◽  
Author(s):  
Heng Xiao ◽  
Yi Guo ◽  
Junhui Yi ◽  
Hong Xia ◽  
Hongbo Xu ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Network Formation ◽  
Han Chinese ◽  
Dominant Negative ◽  
Confocal Laser ◽  
Chinese Family ◽  
Cell Periphery ◽  
Hacat Cells ◽  
Wild Type ◽  
Palmoplantar Keratoderma

Background/Aims: Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant genodermatosis. It is characterized by diffuse yellow keratoses on the palmoplantar epidermis, with an erythematous border. The keratin 9 gene (KRT9) and less frequently the keratin 1 gene (KRT1) are responsible for EPPK. This study aims to identify and analyse genetic defects responsible for EPPK in a Han Chinese pedigree. Methods: A four-generation Han Chinese pedigree containing five individuals affected with EPPK was recruited. Exome sequencing, Sanger sequencing, and bioinformatics tools were conducted to identify the mutation in this pedigree. HaCaT cells were transfected with either wild-type or mutated KRT9. Confocal laser immunofluorescence assay, imaging processing, and statistical analysis were performed to evaluate wild-type and mutant KRT9 groups. Results: A novel heterozygous c.1369C>T transition (p.Leu457Phe) in exon 6 of the KRT9 gene was identified in four patients. It co-segregated with the disorder in the family. Functional analysis showed that withdrawal of the filament network from the cell periphery and particle formation were present in about 10% of Leu457Phe-transfected HaCaT cells, while approximately 3% of cells transfected with wild-type KRT9 showed this phenotype. The particles in mutant group were larger than that in wild-type group (P-value < 0.05). Conclusion: The variant may be the disease-causing missense mutation and produce dominant negative effects by interrupting keratin network formation. This study indicates the pathogenic role of the KRT9 gene mutation in this pedigree with EPPK, and may be helpful in genetic counseling, prenatal diagnosis and gene-targeted therapies of EPPK.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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