EEA1, a Tethering Protein of the Early Sorting Endosome, Shows a Polarized Distribution in Hippocampal Neurons, Epithelial Cells, and Fibroblasts

EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of “basolateral-type” endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.

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Myo6 Facilitates the Translocation of Endocytic Vesicles from Cell Peripheries

Molecular Biology of the Cell ◽

10.1091/mbc.e02-11-0767 ◽

2003 ◽

Vol 14 (7) ◽

pp. 2728-2743 ◽

Cited By ~ 134

Author(s):

Laura Aschenbrenner ◽

TinThu Lee ◽

Tama Hasson

Keyword(s):

Epithelial Cells ◽

Fluorescent Protein ◽

Early Endosome ◽

Sufficient Time ◽

Significant Delay ◽

Transfected Cells ◽

Early Endosomes ◽

Green Fluorescent ◽

Endocytic Vesicles ◽

Transferrin Uptake

Immunolocalization studies in epithelial cells revealed myo6 was associated with peripherally located vesicles that contained the transferrin receptor. Pulse-chase experiments after transferrin uptake showed that these vesicles were newly uncoated endocytic vesicles and that myo6 was recruited to these vesicles immediately after uncoating. GIPC, a putative myo6 tail binding protein, was also present. Myo6 was not present on early endosomes, suggesting that myo6 has a transient association with endocytic vesicles and is released upon early endosome fusion. Green fluorescent protein (GFP) fused to myo6 as well as the cargo-binding tail (M6tail) alone targeted to the nascent endocytic vesicles. Overexpression of GFP-M6tail had no effect on a variety of organelle markers; however, GFP-M6tail displaced the endogenous myo6 from nascent vesicles and resulted in a significant delay in transferrin uptake. Pulse-chase experiments revealed that transferrin accumulated in uncoated vesicles within the peripheries of transfected cells and that Rab5 was recruited to the surface of these vesicles. Given sufficient time, the transferrin did traffic to the perinuclear sorting endosome. These data suggest that myo6 is an accessory protein required for the efficient transportation of nascent endocytic vesicles from the actin-rich peripheries of epithelial cells, allowing for timely fusion of endocytic vesicles with the early endosome.

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Autocrine signals enable chondrocytes to survive in culture.

The Journal of Cell Biology ◽

10.1083/jcb.126.4.1069 ◽

1994 ◽

Vol 126 (4) ◽

pp. 1069-1077 ◽

Cited By ~ 88

Author(s):

Y Ishizaki ◽

J F Burne ◽

M C Raff

Keyword(s):

Epithelial Cells ◽

Cell Density ◽

Mammalian Cells ◽

Cell Types ◽

High Density ◽

Lens Epithelial Cells ◽

Low Density ◽

Cell Type ◽

Adult Rats ◽

And Cartilage

We recently proposed that most mammalian cells other than blastomeres may be programmed to kill themselves unless continuously signaled by other cells not to. Many observations indicate that some mammalian cells are programmed in this way, but is it the case for most mammalian cells? As it is impractical to test all of the hundreds of types of mammalian cells, we have focused on two tissues--lens and cartilage--which each contain only a single cell type: if there are cells that do not require signals from other cells to avoid programmed cell death (PCD), lens epithelial cells and cartilage cells (chondrocytes) might be expected to be among them. We have previously shown that rat lens epithelial cells can survive in serum-free culture without signals from other cell types but seem to require signals from other lens epithelial cells to survive: without such signals they undergo PCD. We show here that the same is true for rat (and chick) chondrocytes. They can survive for weeks in culture at high cell density in the absence of other cell types, serum, or exogenous proteins or signaling molecules, but they die with the morphological features of apoptosis in these conditions at low cell density. Medium from high density cultures, FCS, or a combination of known growth factors, all support prolonged chondrocyte survival in low density cultures, as long as antioxidants are also present. Moreover, medium from high density chondrocyte cultures promotes the survival of lens epithelial cells in low density cultures and vice versa. Chondrocytes isolated from adult rats behave similarly to those isolated from developing rats. These findings support the hypothesis that most mammalian cells require signals from other cells to avoid PCD, although the signals can sometimes be provided by cells of the same type, at least in tissues that contain only one cell type.

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The production and effect of interferon on influenza virus growth in chick embryo lung epithelial and fibroblast cell cultures

Canadian Journal of Microbiology ◽

10.1139/m69-050 ◽

1969 ◽

Vol 15 (3) ◽

pp. 273-277 ◽

Cited By ~ 2

Author(s):

Sunidhkumar S. Gandhi ◽

Robert B. Stewart

Keyword(s):

Epithelial Cells ◽

Chick Embryo ◽

Cell Cultures ◽

Fibroblast Cell ◽

Cell Types ◽

Fibroblastic Cell ◽

Virus Growth ◽

Lung Epithelial ◽

Fibroblastic Cells ◽

Virus Strains

Cultures of fibroblastic cells prepared from chick embryo lung infected with low multiplicities of influenza type A virus strains were found to produce more interferon than did cultures of epithelial cells prepared from the same organ. Fibroblastic cell cultures were also found to be more sensitive to the action of interferon than were epithelial cells with respect to the levels of infectious virus produced and the duration of interferon action. Cultures of the two cell types treated with interferon did not differ with respect to the number of cells involved in virus synthesis.

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Rab10 Regulates Membrane Transport through Early Endosomes of Polarized Madin-Darby Canine Kidney Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e05-08-0799 ◽

2006 ◽

Vol 17 (7) ◽

pp. 3156-3175 ◽

Cited By ~ 108

Author(s):

Clifford M. Babbey ◽

Nahid Ahktar ◽

Exing Wang ◽

Carlos Chih-Hsiung Chen ◽

Barth D. Grant ◽

...

Keyword(s):

Membrane Transport ◽

Mammalian Cells ◽

Mdck Cells ◽

Endocytic Pathway ◽

Rab Proteins ◽

Madin Darby Canine Kidney ◽

Early Endosomes ◽

Mutant Forms ◽

Darby Canine Kidney ◽

Canine Kidney

Rab10, a protein originally isolated from Madin-Darby Canine Kidney (MDCK) epithelial cells, belongs to a family of Rab proteins that includes Rab8 and Rab13. Although both Rab8 and Rab13 have been found to mediate polarized membrane transport, the function of Rab10 in mammalian cells has not yet been established. We have used quantitative confocal microscopy of polarized MDCK cells expressing GFP chimeras of wild-type and mutant forms of Rab10 to analyze the function of Rab10 in polarized cells. These studies demonstrate that Rab10 is specifically associated with the common endosomes of MDCK cells, accessible to endocytic probes internalized from either the apical or basolateral plasma membrane domains. Expression of mutant Rab10 defective for either GTP hydrolysis or GTP binding increased recycling from early compartments on the basolateral endocytic pathway without affecting recycling from later compartments or the apical recycling pathway. These results suggest that Rab10 mediates transport from basolateral sorting endosomes to common endosomes.

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Axonal and dendritic endocytic pathways in cultured neurons.

The Journal of Cell Biology ◽

10.1083/jcb.119.1.123 ◽

1992 ◽

Vol 119 (1) ◽

pp. 123-137 ◽

Cited By ~ 162

Author(s):

R G Parton ◽

K Simons ◽

C G Dotti

Keyword(s):

Cell Body ◽

Hippocampal Neurons ◽

Multivesicular Body ◽

Nerve Terminals ◽

Multivesicular Bodies ◽

Late Endosomes ◽

Distal Side ◽

Early Endosomes ◽

Fast Transport ◽

Endocytic Pathways

The endocytic pathways from the axonal and dendritic surfaces of cultured polarized hippocampal neurons were examined. The dendrites and cell body contained extensive networks of tubular early endosomes which received endocytosed markers from the somatodendritic domain. In axons early endosomes were confined to presynaptic terminals and to varicosities. The somatodendritic but not the presynaptic early endosomes were labeled by internalized transferrin. In contrast to early endosomes, late endosomes and lysosomes were shown to be predominantly located in the cell body. Video microscopy was used to follow the transport of internalized markers from the periphery of axons and dendrites back to the cell body. Labeled structures in both domains moved unidirectionally by retrograde fast transport. Axonally transported organelles were sectioned for EM after video microscopic observation and shown to be large multivesicular body-like structures. Similar structures accumulated at the distal side of an axonal lesion. Multivesicular bodies therefore appear to be the major structures mediating transport of endocytosed markers between the nerve terminals and the cell body. Late endocytic structures were also shown to be highly mobile and were observed moving within the cell body and proximal dendritic segments. The results show that the organization of the endosomes differs in the axons and dendrites of cultured rat hippocampal neurons and that the different compartments or stages of the endocytic pathways can be resolved spatially.

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FITC-dextran as a probe for endosome function and localization in kidney

AJP Cell Physiology ◽

10.1152/ajpcell.1990.258.2.c309 ◽

1990 ◽

Vol 258 (2) ◽

pp. C309-C317 ◽

Cited By ~ 75

Author(s):

W. I. Lencer ◽

P. Weyer ◽

A. S. Verkman ◽

D. A. Ausiello ◽

D. Brown

Keyword(s):

Epithelial Cells ◽

Water Channel ◽

Fluorescein Isothiocyanate ◽

Endocytic Pathway ◽

Water Permeability Coefficient ◽

Osmotic Water ◽

Endocytic Vesicles ◽

Excised Tissue ◽

Specific Membrane ◽

Endocytic Pathways

Fluorescein isothiocyanate (FITC)-labeled endosomes were localized in kidney epithelial cells after tissue fixation and sectioning, and specific membrane transport properties of isolated endocytic vesicles were measured using the same probe. Rats were infused intravenously with 10 kDa FITC-dextran, and kidneys were fixed with paraformaldehyde lysine periodate. FITC-labeled vesicles were visualized in semithin (1 micron) frozen sections of excised tissue by epifluorescent microscopy and by electron microscopy after a photoconversion reaction. Most FITC-labeled endosomes were apically located in epithelial cells lining the urinary tubules. By immunocytochemistry the anti-lysosomal glycoprotein LGP 120 was absent from most of the FITC-labeled vesicles, although some colocalization was noted. The limiting membrane of FITC-labeled endosomes contained a vacuolar proton pump (pHmin = 6.23 +/- 0.033) and a water channel (osmotic water permeability coefficient, Pf = 0.052 +/- 0.005 cm/s) and was highly permeable to ethylene glycol and urea but relatively impermeable to glucose. Methods allowing the attribution of specific membrane functions to vesicles that can be visualized in the apical endocytic pathway of epithelial cells should be of general use for the study of endocytic pathways in a variety of systems.

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Intracellular routing of wild-type and mutated polymeric immunoglobulin receptor in hippocampal neurons in culture.

The Journal of Cell Biology ◽

10.1083/jcb.130.6.1447 ◽

1995 ◽

Vol 130 (6) ◽

pp. 1447-1459 ◽

Cited By ~ 34

Author(s):

M de Hoop ◽

C von Poser ◽

C Lange ◽

E Ikonen ◽

W Hunziker ◽

...

Keyword(s):

Plasma Membrane ◽

Epithelial Cells ◽

Hippocampal Neurons ◽

Expression System ◽

Neuronal Cells ◽

Apical Plasma Membrane ◽

Polymeric Immunoglobulin Receptor ◽

Mutant Form ◽

Immunoglobulin Receptor ◽

Early Endosomes

Certain epithelial cells synthesize the polymeric immunoglobulin receptor (pIgR) to transport immunoglobulins (Igs) A and M into external secretions. In polarized epithelia, newly synthesized receptor is first delivered to the basolateral plasma membrane and is then, after binding the Ig, transcytosed to the apical plasma membrane, where the receptor-ligand complex is released by proteolytic cleavage. In a previous work (Ikonen et al., 1993), we implied the existence of a dendro-axonal transcytotic pathway for the rabbit pIgR expressed in hippocampal neurons via the Semliki Forest Virus (SFV) expression system. By labeling surface-exposed pIgR in live neuronal cells, we now show (a) internalization of the receptor from the dendritic plasma membrane to the dendritic early endosomes, (b) redistribution of the receptor from the dendritic to the axonal domain, (c) inhibition of this movement by brefeldin A (BFA) and (d) stimulation by the activation of protein kinase C (PKC) via phorbol myristate acetate (PMA). In addition, we show that a mutant form of the receptor lacking the epithelial basolateral sorting signal is directly delivered to the axonal domain of hippocampal neurons. Although this mutant is internalized into early endosomes, no transcytosis to the dendrites could be observed. In epithelial Madin-Darby Canine Kidney (MDCK) cells, the mutant receptor could also be internalized into basolaterally derived early endosomes. These results suggest the existence of a dendro-axonal transcytotic pathway in neuronal cells which shares similarities with the basolateral to apical transcytosis in epithelial cells and constitute the basis for the future analysis of its physiological role.

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Clathrin-independent pathways do not contribute significantly to endocytic flux

eLife ◽

10.7554/elife.03970 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 87

Author(s):

Vassilis Bitsikas ◽

Ivan R Corrêa ◽

Benjamin J Nichols

Keyword(s):

Mammalian Cells ◽

High Temporal Resolution ◽

Coated Pits ◽

Alternative Pathways ◽

Specific Proteins ◽

Clathrin Adaptor ◽

And Function ◽

Endocytic Vesicles ◽

Data Call ◽

Endocytic Pathways

Several different endocytic pathways have been proposed to function in mammalian cells. Clathrin-coated pits are well defined, but the identity, mechanism and function of alternative pathways have been controversial. Here we apply universal chemical labelling of plasma membrane proteins to define all primary endocytic vesicles, and labelling of specific proteins with a reducible SNAP-tag substrate. These approaches provide high temporal resolution and stringent discrimination between surface-connected and intracellular membranes. We find that at least 95% of the earliest detectable endocytic vesicles arise from clathrin-coated pits. GPI-anchored proteins, candidate cargoes for alternate pathways, are also found to enter the cell predominantly via coated pits. Experiments employing a mutated clathrin adaptor reveal distinct mechanisms for sorting into coated pits, and thereby explain differential effects on the uptake of transferrin and GPI-anchored proteins. These data call for a revision of models for the activity and diversity of endocytic pathways in mammalian cells.

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The Human Cytomegalovirus Trimer and Pentamer Promote Sequential Steps in Entry into Epithelial and Endothelial Cells at Cell Surfaces and Endosomes

Journal of Virology ◽

10.1128/jvi.01336-18 ◽

2018 ◽

Vol 92 (21) ◽

Cited By ~ 21

Author(s):

Jing Liu ◽

Ted S. Jardetzky ◽

Andrea L. Chin ◽

David C. Johnson ◽

Adam L. Vanarsdall

Keyword(s):

Endothelial Cells ◽

Epithelial Cells ◽

Cell Surface ◽

Human Cytomegalovirus ◽

Antiviral Agents ◽

Cell Types ◽

Surface Binding ◽

Surface Receptors ◽

Virus Particles ◽

Early Endosomes

ABSTRACTHuman cytomegalovirus (HCMV) infects a wide variety of human cell types by different entry pathways that involve distinct envelope glycoprotein complexes that include gH/gL, a trimer complex consisting of gHgL/gO, and a pentamer complex consisting of gH/gL/UL128/UL130/UL131. We characterized the effects of soluble forms of these proteins on HCMV entry. Soluble trimer and pentamer blocked entry of HCMV into epithelial and endothelial cells, whereas soluble gH/gL did not. Trimer inhibited HCMV entry into fibroblast cells, but pentamer and gH/gL did not. Both trimer and pentamer bound to the surfaces of fibroblasts and epithelial cells, whereas gH/gL did not bind to either cell type. Cell surface binding of trimer and pentamer did not involve heparin sulfate moieties. The ability of soluble trimer to block entry of HCMV into epithelial cells did not involve platelet-derived growth factor PDGFRα, which has been reported as a trimer receptor for fibroblasts. Soluble trimer reduced the amount of virus particles that could be adsorbed onto the surface of epithelial cells, whereas soluble pentamer had no effect on virus adsorption. However, soluble pentamer reduced the ability of virus particles to exit from early endosomes into the cytoplasm and then travel to the nucleus. These studies support a model in which both the trimer and pentamer are required for HCMV entry into epithelial and endothelial cells, with trimer interacting with cell surface receptors other than PDGFR and pentamer acting later in the entry pathway to promote egress from endosomes.IMPORTANCEHCMV infects nearly 80% of the world's population and causes significant morbidity and mortality. The current antiviral agents used to treat HCMV infections are prone to resistance and can be toxic to patients, and there is no current vaccine against HCMV available. The data in this report will lead to a better understanding of how essential HCMV envelope glycoproteins function during infection of biologically important cell types and will have significant implications for understanding HCMV pathogenesis for developing new therapeutics.

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PIWI-piRNA pathway-mediated transposable element repression in Hydra somatic stem cells

10.1101/731695 ◽

2019 ◽

Author(s):

Bryan B. Teefy ◽

Stefan Siebert ◽

Jack F. Cazet ◽

Haifan Lin ◽

Celina E. Juliano

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Epithelial Cells ◽

Mammalian Cells ◽

Cell Types ◽

Somatic Tissue ◽

Epithelial Stem Cells ◽

Conserved Sequence ◽

Rna Immunoprecipitation ◽

Pirna Pathway

AbstractTransposable elements (TEs) can damage genomes, thus organisms employ a variety of mechanisms to repress TE expression. However, these mechanisms often fail over time leading to de-repression of TEs in aging tissues. The PIWI-piRNA pathway is a small RNA pathway that represses TE expression in the germline of animals. Here we explore the function of the pathway in the epithelial stem cells of Hydra, a long-lived freshwater cnidarian. Hydra have three stem cell populations; endodermal and ectodermal epithelial stem cells are strictly somatic, whereas the interstitial stem cells retain germline competence. In our previous study, we found that the PIWI proteins are expressed in all three Hydra stem cell types. In this study, we focus on the ectodermal and endodermal epithelial stem cells to understand the somatic function of the pathway. We isolated piRNAs from Hydra that lack the interstitial lineage and found that these somatic piRNAs map predominantly to TE transcripts and display the conserved sequence signatures typical of germline piRNAs. Three lines of evidence suggest that the PIWI-piRNA pathway represses TEs in Hydra epithelial stem cells. First, epithelial knockdown of the Hydra PIWI protein hywi resulted in upregulation of TE expression. Second, degradome sequencing revealed evidence of PIWI-mediated cleavage of TE RNAs in epithelial cells using the ping-pong mechanism. Finally, we demonstrated a direct association between Hywi protein and TE transcripts in epithelial cells using RNA immunoprecipitation. Interestingly, we found that RNAi knockdown of hywi leads to an upregulation of genes involved in innate immunity, which may be in response to TE upregulation; this is consistent with recent studies on TE expression in mammalian cells. Altogether, this study suggests a function for the PIWI-piRNA pathway in maintaining the long-lived somatic cell lineages of Hydra and may point to a broader role for this pathway in protecting somatic tissue from TE-induced damage.

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