scholarly journals Low-Dose Fluvoxamine Modulates Endocytic Trafficking of SARS-CoV-2 Spike Protein: A Potential Mechanism for Anti-COVID-19 Protection by Antidepressants

2021 ◽  
Vol 12 ◽  
Author(s):  
Oleg O. Glebov
Keyword(s):  
Membrane Trafficking ◽  
Low Dose ◽  
Protein A ◽  
Antidepressant Drug ◽  
Fluid Phase ◽  
Spike Protein ◽  
Endosomal Trafficking ◽  
Therapeutic Concentration ◽  
Early Endosomes ◽  
Fluid Phase Endocytosis

Commonly prescribed antidepressants may be associated with protection against severe COVID-19. The mechanism of their action in this context, however, remains unknown. Here, I investigated the effect of an antidepressant drug fluvoxamine on membrane trafficking of the SARS-CoV-2 spike protein and its cell host receptor ACE2 in HEK293T cells. A sub-therapeutic concentration (80 nM) of fluvoxamine rapidly upregulated fluid-phase endocytosis, resulting in enhanced accumulation of the spike-ACE2 complex in enlarged early endosomes. Diversion of endosomal trafficking provides a simple cell biological mechanism consistent with the protective effect of antidepressants against COVID-19, highlighting their therapeutic and prophylactic potential.

scholarly journals Low-dose fluvoxamine modulates endocytic trafficking of SARS-CoV-2 spike protein: a potential mechanism for anti-COVID-19 protection by antidepressants

2021 ◽  
Author(s):  
Oleg O Glebov
Keyword(s):  
Membrane Trafficking ◽  
Low Dose ◽  
Protein A ◽  
Fluid Phase ◽  
Spike Protein ◽  
Endosomal Trafficking ◽  
Therapeutic Concentration ◽  
Phase 3 ◽  
Early Endosomes ◽  
Fluid Phase Endocytosis

Commonly prescribed antidepressants may be associated with protection against severe COVID-19, with one drug (fluvoxamine) currently undergoing a Phase 3 clinical trial. The mechanism of their action, however, remains unknown. Here, I investigated the effect of fluvoxamine on membrane trafficking of the SARS-CoV-2 spike protein and its cell host receptor ACE2 in HEK293T cells. A sub-therapeutic concentration (80 nM) of fluvoxamine rapidly upregulated fluid-phase endocytosis, resulting in enhanced accumulation of the spike-ACE2 complex in enlarged early endosomes. Diversion of endosomal trafficking provides a simple cell biological mechanism consistent with the protective effect of antidepressants against COVID-19, highlighting their therapeutic and prophylactic potential.

scholarly journals Fluid phase endocytosis and galactosyl receptor-mediated endocytosis employ different early endosomes

1999 ◽  
Vol 1421 (2) ◽  
pp. 317-328 ◽  
Author(s):  
Marianne Synnes ◽  
Kristian Prydz ◽  
Torunn Løvdal ◽  
Andreas Brech ◽  
Trond Berg
Keyword(s):  
Fluid Phase ◽  
Receptor Mediated Endocytosis ◽  
Early Endosomes ◽  
Fluid Phase Endocytosis

scholarly journals Neonatal Fc Receptor Mediates Internalization of Fc in Transfected Human Endothelial Cells

2008 ◽  
Vol 19 (12) ◽  
pp. 5490-5505 ◽  
Author(s):  
Nancy A. Goebl ◽  
Clifford M. Babbey ◽  
Amita Datta-Mannan ◽  
Derrick R. Witcher ◽  
Victor J. Wroblewski ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Fluid Phase ◽  
Surface Expression ◽  
Fc Receptor ◽  
Expression Level ◽  
Surface Binding ◽  
Neonatal Fc Receptor ◽  
Binding Forms ◽  
Early Endosomes ◽  
Fluid Phase Endocytosis

The neonatal Fc receptor, FcRn mediates an endocytic salvage pathway that prevents degradation of IgG, thus contributing to the homeostasis of circulating IgG. Based on the low affinity of IgG for FcRn at neutral pH, internalization of IgG by endothelial cells is generally believed to occur via fluid-phase endocytosis. To investigate the role of FcRn in IgG internalization, we used quantitative confocal microscopy to characterize internalization of fluorescent Fc molecules by HULEC-5A lung microvascular endothelia transfected with GFP fusion proteins of human or mouse FcRn. In these studies, cells transfected with FcRn accumulated significantly more intracellular Fc than untransfected cells. Internalization of FcRn-binding forms of Fc was proportional to FcRn expression level, was enriched relative to dextran internalization in proportion to FcRn expression level, and was blocked by incubation with excess unlabeled Fc. Because we were unable to detect either surface expression of FcRn or surface binding of Fc, these results suggest that FcRn-dependent internalization of Fc may occur through sequestration of Fc by FcRn in early endosomes. These studies indicate that FcRn-dependent internalization of IgG may be important not only in cells taking up IgG from an extracellular acidic space, but also in endothelial cells participating in homeostatic regulation of circulating IgG levels.

Phorbol ester promotes endocytosis by activating a factor involved in endosome fusion

1999 ◽  
Vol 112 (15) ◽  
pp. 2549-2557 ◽  
Author(s):  
A. Aballay ◽  
P.D. Stahl ◽  
L.S. Mayorga
Keyword(s):  
Phorbol Ester ◽  
Fluid Phase ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Early Endosomes ◽  
Zinc Depletion ◽  
Fluid Phase Endocytosis ◽  
Endocytic Vesicles

Previous studies indicate that a zinc- and phorbol ester-binding factor is necessary for in vitro endosome fusion and for the effect of Rab5 on endosome fusion. Rab5 is a small GTPase that regulates membrane fusion between early endosomes derived from either receptor-mediated endocytosis or fluid-phase endocytosis. In its GTP-bound form, Rab5 promotes endocytosis and enhances fusion among early endosomes. To determine if PMA stimulates endocytosis by activating a factor required for endosome fusion, we overexpressed wild-type Rab5, a dominant negative mutant (Rab5:S34N), and a GTPase deficient mutant (Rab5:Q79L) in BHK-21 cells. The phorbol ester PMA stimulates endocytosis and increases the number and the size of endocytic vesicles, even in the presence of Rab5:S34N. Zinc depletion with N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and addition of calphostin C (CPC), an inhibitor of PKC that interacts with zinc and phorbol ester binding motifs, inhibited both basal and Rab5-stimulated fluid phase endocytosis. These two reagents also inhibited the size and number of endocytic vesicles promoted by Rab5. These results suggest that PMA stimulates endocytosis by regulating the dynamics of the early endosome compartment.

scholarly journals Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

2015 ◽  
Vol 89 (16) ◽  
pp. 8365-8382 ◽  
Author(s):  
Jye-Chian Hsiao ◽  
Li-Wei Chu ◽  
Yung-Tsun Lo ◽  
Sue-Ping Lee ◽  
Tzu-Jung Chen ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Membrane Fusion ◽  
Hela Cells ◽  
Fluid Phase ◽  
Content Type ◽  
Virus Core ◽  
Early Endosomes ◽  
Fluid Phase Endocytosis ◽  
Mature Virus ◽  
In Cells

ABSTRACTVaccinia virus, the prototype of theOrthopoxvirusgenus in the familyPoxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm.IMPORTANCEVaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm.

scholarly journals Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells.

1991 ◽  
Vol 115 (6) ◽  
pp. 1561-1572 ◽  
Author(s):  
T Ludwig ◽  
G Griffiths ◽  
B Hoflack
Keyword(s):  
Lysosomal Enzymes ◽  
Rat Kidney ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Early Endosomes ◽  
Fluid Phase Endocytosis ◽  
Normal Rat ◽  
Endocytic Compartments ◽  
Endocytic Pathways

We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.

Establishment and analysis of conditional Rab1 and Rab5 knockout cells by using the auxin-inducible degron system

2021 ◽  
Author(s):  
Yuki Hatoyama ◽  
Yuta Homma ◽  
Shu Hiragi ◽  
Mitsunori Fukuda
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Fluid Phase ◽  
Small Gtpases ◽  
Golgi Stack ◽  
Protein Depletion ◽  
Intracellular Signals ◽  
Early Endosomes ◽  
Inducible Protein ◽  
The Impact

Two small GTPases, Rab1 and Rab5, are key membrane trafficking regulators that are conserved in all eukaryotes. Since they have recently been shown to be essential for cell survival and/or growth in cultured mammalian cells, thereby precluding the establishment of Rab1-knockout (KO) and Rab5-KO cells, it has been extremely difficult to assess the impact of complete Rab1 or Rab5 protein depletion on cellular functions. Here, we generated and analyzed conditional KO (CKO) cells for Rab1 (Rab1A/1B) and Rab5 (Rab5A/5B/5C) by using the auxin-inducible protein degradation system. Rab1-CKO and Rab5-CKO led to eventual cell death >18 h and >48 h, respectively, after auxin exposure. After acute Rab1 protein depletion, the Golgi stack and ribbon structures were completely disrupted, and ER-to-Golgi trafficking was severely inhibited. Moreover, we discovered a novel Rab1-depletion phenotype: perinuclear clustering of early endosomes and delayed transferrin recycling. In contrast, acute Rab5 protein depletion resulted in loss of early endosomes and late endosomes, but lysosomes appeared to be normal. We also observed a dramatic reduction in the intracellular signals of endocytic cargos via receptor-mediated or fluid-phase endocytosis in Rab5-depleted cells.

scholarly journals Inactivation of lmpA, Encoding a LIMPII-related Endosomal Protein, Suppresses the Internalization and Endosomal Trafficking Defects in Profilin-null Mutants

2000 ◽  
Vol 11 (6) ◽  
pp. 2019-2031 ◽  
Author(s):  
Lesly Temesvari ◽  
Linyi Zhang ◽  
Brent Fodera ◽  
Klaus-Peter Janssen ◽  
Michael Schleicher ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Fluid Phase ◽  
Actin Binding ◽  
Endosomal Trafficking ◽  
Wild Type ◽  
Gene Encoding ◽  
Biochemical Assays ◽  
Endosomal Pathway ◽  
Trafficking Defects

Profilin is a key phosphoinositide and actin-binding protein connecting and coordinating changes in signal transduction pathways with alterations in the actin cytoskeleton. Using biochemical assays and microscopic approaches, we demonstrate that profilin-null cells are defective in macropinocytosis, fluid phase efflux, and secretion of lysosomal enzymes but are unexpectedly more efficient in phagocytosis than wild-type cells. Disruption of the lmpA gene encoding a protein (DdLIMP) belonging to the CD36/LIMPII family suppressed, to different degrees, most of the profilin-minus defects, including the increase in F-actin, but did not rescue the secretion defect. Immunofluorescence microscopy indicated that DdLIMP, which is also capable of binding phosphoinositides, was associated with macropinosomes but was not detected in the plasma membrane. Also, inactivation of the lmpA gene in wild-type strains resulted in defects in macropinocytosis and fluid phase efflux but not in phagocytosis. These results suggest an important role for profilin in regulating the internalization of fluid and particles and the movement of material along the endosomal pathway; they also demonstrate a functional interaction between profilin and DdLIMP that may connect phosphoinositide-based signaling through the actin cytoskeleton with endolysosomal membrane trafficking events.

scholarly journals Novel Proteins Linking the Actin Cytoskeleton to the Endocytic Machinery in Saccharomyces cerevisiae

2002 ◽  
Vol 13 (10) ◽  
pp. 3646-3661 ◽  
Author(s):  
H. Dewar ◽  
D. T. Warren ◽  
F. C. Gardiner ◽  
C. G. Gourlay ◽  
N. Satish ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Elevated Temperatures ◽  
Adaptor Protein ◽  
Fluid Phase ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Fluid Phase Endocytosis ◽  
Endocytic Machinery

The importance of coupling the process of endocytosis to factors regulating actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified and characterized. Here we demonstrate the importance of two additional cortical components, Ysc84p and Lsb5p, which together are essential for the organization of the actin cytoskeleton and for fluid phase endocytosis. Both Ysc84p and Lsb5p were identified through two-hybrid screens with different domains of the adaptor protein Sla1p. Ysc84p colocalizes with cortical actin and requires the presence of an intact actin cytoskeleton for its cortical localization. Ycl034w/Lsb5p localizes to the cell cortex but does not colocalize with actin. The Lsb5 protein contains putative VHS and GAT domains as well as an NPF motif, which are all domains characteristic of proteins involved in membrane trafficking. Deletion of either gene alone does not confer any dramatic phenotype on cells. However, deletion of both genes is lethal at elevated temperatures. Furthermore, at all temperatures this double mutant has depolarized actin and an almost undetectable level of fluid phase endocytosis. Our data demonstrate that Ysc84p and Lsb5p are important components of complexes involved in overlapping pathways coupling endocytosis with the actin cytoskeleton in yeast.

Inhibition of phosphatidylinositol 3-kinase does not alter forskolin-stimulated Cl− secretion by T84 cells

2000 ◽  
Vol 278 (5) ◽  
pp. C865-C872 ◽  
Author(s):  
Jeffrey L. Dickson ◽  
Tracy D. Conner ◽  
Tom W. Ecay
Keyword(s):  
Membrane Trafficking ◽  
Short Circuit ◽  
Fluid Phase ◽  
Short Circuit Current ◽  
Transmembrane Conductance Regulator ◽  
Phosphatidylinositol 3 Kinase ◽  
T84 Cells ◽  
Fluid Phase Endocytosis ◽  
Membrane Compartment ◽  
Phosphatidylinositol 3

Wortmannin is a potent inhibitor of phosphatidylinositol 3-kinase (PI3K) and membrane trafficking in many cells. To test the hypothesis that cystic fibrosis transmembrane conductance regulator (CFTR) traffics into and out of the plasma membrane during cAMP-stimulated epithelial Cl−secretion, we have studied the effects of wortmannin on forskolin-stimulated Cl− secretion by the human colonic cell line T84. At the PI3K inhibitory concentration of 100 nM, wortmannin did not affect significantly forskolin-stimulated Cl− secretion measured as short-circuit current ( I SC). However, 500 nM wortmannin significantly inhibited forskolin-stimulated I SC. cAMP activation of apical membrane CFTR Cl− channels in α-toxin-permeabilized monolayers was not reduced by 500 nM wortmannin, suggesting that inhibition of other transporters accounts for the observed reduction in T84 Cl− secretion. Forskolin inhibits apical endocytosis of horseradish peroxidase (HRP), but wortmannin did not alter forskolin inhibition of apical HRP endocytosis. In the absence of forskolin, wortmannin stimulated HRP endocytosis significantly. We conclude that, in T84 cells, apical fluid phase endocytosis is not dependent on PI3K activity and that CFTR does not recycle through a PI3K-dependent and wortmannin-sensitive membrane compartment.

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