Assembly and trafficking of caveolar domains in the cell

Using total internal reflection fluorescence microscopy (TIR-FM), fluorescence recovery after photobleaching (FRAP), and other light microscopy techniques, we analyzed the dynamics, the activation, and the assembly of caveolae labeled with fluorescently tagged caveolin-1 (Cav1). We found that when activated by simian virus 40 (SV40), a nonenveloped DNA virus that uses caveolae for cell entry, the fraction of mobile caveolae was dramatically enhanced both in the plasma membrane (PM) and in the caveosome, an intracellular organelle that functions as an intermediate station in caveolar endocytosis. Activation also resulted in increased microtubule (MT)-dependent, long-range movement of caveolar vesicles. We generated heterokaryons that contained GFP- and RFP-tagged caveolae by fusing cells expressing Cav1-GFP and -RFP, respectively, and showed that even when activated, individual caveolar domains underwent little exchange of Cav1. Only when the cells were subjected to transient cholesterol depletion, did the caveolae domain exchange Cav1. Thus, in contrast to clathrin-, or other types of coated transport vesicles, caveolae constitute stable, cholesterol-dependent membrane domains that can serve as fixed containers through vesicle traffic. Finally, we identified the Golgi complex as the site where newly assembled caveolar domains appeared first.

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Ganglioside GD1a Restores Infectibility to Mouse Cells Lacking Functional Receptors for Polyomavirus

Journal of Virology ◽

10.1128/jvi.79.1.615-618.2005 ◽

2005 ◽

Vol 79 (1) ◽

pp. 615-618 ◽

Cited By ~ 29

Author(s):

Joanna Gilbert ◽

Jean Dahl ◽

Cathy Riney ◽

John You ◽

Cunqi Cui ◽

...

Keyword(s):

Simian Virus 40 ◽

Simian Virus ◽

Cell Entry ◽

Heterologous Host ◽

Virus Binding ◽

Rat Glioma ◽

Caveolin 1 ◽

Ganglioside Biosynthesis ◽

A Cell ◽

Mouse Cells

ABSTRACT Recent investigations on the pathway of cell entry by polyomavirus (Py) and simian virus 40 (SV40) have defined specific gangliosides as functional receptors mediating virus binding and transport from the plasma membrane to the endoplasmic reticulum (B. Tsai et al., EMBO J. 22:4346-4355, 2003; Gilbert and Benjamin, in press). These studies were carried out with C6 rat glioma cells, a heterologous host chosen for its known deficiency in ganglioside biosynthesis. Here, a cell genetic approach was undertaken to identify components required for the early steps of infection using mouse cells as the natural host for Py. Receptor-negative (R−) mouse cells, screened based on resistance to Py infection, were shown to bind Py but failed to allow entry of the virus. R− cells were also found to be resistant to SV40. Infectibility was restored or enhanced by the addition of the same specific gangliosides found in earlier studies with C6 cells. In one R− line, overexpression of caveolin-1 also increased infectibility. These results support and extend findings on gangliosides in lipid rafts as functional receptors and mediators of internalization for Py and SV40.

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A Raft-derived, Pak1-regulated Entry Participates in α2β1 Integrin-dependent Sorting to Caveosomes

Molecular Biology of the Cell ◽

10.1091/mbc.e07-10-1094 ◽

2008 ◽

Vol 19 (7) ◽

pp. 2857-2869 ◽

Cited By ~ 71

Author(s):

Mikko Karjalainen ◽

Elina Kakkonen ◽

Paula Upla ◽

Heli Paloranta ◽

Pasi Kankaanpää ◽

...

Keyword(s):

Simian Virus 40 ◽

Fluid Phase ◽

Simian Virus ◽

Cell Entry ◽

Phosphatidylinositol 3 Kinase ◽

Caveolin 1 ◽

Entry Pathway ◽

Integrin Clustering ◽

P21 Activated Kinase ◽

Α2β1 Integrin

We have previously shown that a human picornavirus echovirus 1 (EV1) is transported to caveosomes during 2 h together with its receptor α2β1 integrin. Here, we show that the majority of early uptake does not occur through caveolae. α2β1 integrin, clustered by antibodies or by EV1 binding, is initially internalized from lipid rafts into tubulovesicular structures. These vesicles accumulate fluid-phase markers but do not initially colocalize with caveolin-1 or internalized simian virus 40 (SV40). Furthermore, the internalized endosomes do not contain glycosylphosphatidylinositol (GPI)-anchored proteins or flotillin 1, suggesting that clustered α2β1 integrin does not enter the GPI-anchored protein enriched endosomal compartment or flotillin pathways, respectively. Endosomes mature further into larger multivesicular bodies between 15 min to 2 h and concomitantly recruit caveolin-1 or SV40 inside. Cell entry is regulated by p21-activated kinase (Pak)1, Rac1, phosphatidylinositol 3-kinase, phospholipase C, and actin but not by dynamin 2 in SAOS-α2β1 cells. An amiloride analog, 5-(N-ethyl-N-isopropanyl) amiloride, blocks infection, causes integrin accumulation in early tubulovesicular structures, and prevents their structural maturation into multivesicular structures. Our results together suggest that α2β1 integrin clustering defines its own entry pathway that is Pak1 dependent but clathrin and caveolin independent and that is able to sort cargo to caveosomes.

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Clathrin- and caveolin-1–independent endocytosis

The Journal of Cell Biology ◽

10.1083/jcb.200407113 ◽

2005 ◽

Vol 168 (3) ◽

pp. 477-488 ◽

Cited By ~ 321

Author(s):

Eva-Maria Damm ◽

Lucas Pelkmans ◽

Jürgen Kartenbeck ◽

Anna Mezzacasa ◽

Teymuras Kurzchalia ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Knockout Mouse ◽

Simian Virus 40 ◽

Simian Virus ◽

Endocytic Pathway ◽

Host Cells ◽

Human Hepatoma ◽

Wild Type ◽

Embryonic Fibroblasts ◽

Caveolin 1

Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)–deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1–independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.

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Early Steps of Polyomavirus Entry into Cells

Journal of Virology ◽

10.1128/jvi.74.18.8582-8588.2000 ◽

2000 ◽

Vol 74 (18) ◽

pp. 8582-8588 ◽

Cited By ~ 54

Author(s):

Joanna M. Gilbert ◽

Thomas L. Benjamin

Keyword(s):

Jc Virus ◽

Simian Virus 40 ◽

Simian Virus ◽

Dominant Negative ◽

Independent Manner ◽

Caveolin 1 ◽

Dominant Negative Form ◽

Murine Polyomavirus ◽

Dynamin I ◽

Negative Form

ABSTRACT The mechanism by which murine polyomavirus penetrates cells and arrives at the nucleus, the site of viral replication, is not well understood. Simian virus 40 and JC virus, two closely related members of the polyomavirus subfamily, use caveola- and clathrin-mediated uptake pathways for entry, respectively. The data presented here indicate that compounds that block endocytosis of both caveola- and clathrin-derived vesicles have no effect on polyomavirus infectivity. Polyomavirus does not appear to colocalize with either clathrin light chain or caveolin-1 by immunofluorescence microscopy. Additionally, expression of a dominant-negative form of dynamin I has no effect on polyomavirus uptake and infectivity. Therefore, polyomavirus uptake occurs through a class of uncoated vesicles in a clathrin-, caveolin-1-, and dynamin I-independent manner.

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Bound simian virus 40 translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae.

Molecular Biology of the Cell ◽

10.1091/mbc.7.11.1825 ◽

1996 ◽

Vol 7 (11) ◽

pp. 1825-1834 ◽

Cited By ~ 276

Author(s):

H A Anderson ◽

Y Chen ◽

L C Norkin

Keyword(s):

Cell Surface ◽

Phorbol Ester ◽

Simian Virus 40 ◽

Simian Virus ◽

Coated Vesicles ◽

Membrane Domains ◽

Infectious Entry ◽

Triton X

Simian virus 40 (SV40) entry leading to infection occurred only after the virus was at the cell surface for 1.5 to 2 h. SV40 infectious entry was not sensitive to cytosol acidification, a treatment that blocks endocytosis via clathrin-coated vesicles. Instead, SV40 infectious entry was blocked by treating cells with the phorbol ester PMA or nystatin, which selectively disrupts caveolae. In control experiments, transferrin internalization was sensitive to cytosol acidification but was not sensitive to PMA or nystatin. Also, absorbed transferrin entered cells within minutes. Finally, bound SV40 translocated to caveolin-enriched membrane complexes isolated by a Triton X-100 insolubility protocol. Treatment with nystatin did not impair SV40 binding but did block the partitioning of virus into the caveolin-enriched complexes.

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Role for Influenza Virus Envelope Cholesterol in Virus Entry and Infection

Journal of Virology ◽

10.1128/jvi.77.23.12543-12551.2003 ◽

2003 ◽

Vol 77 (23) ◽

pp. 12543-12551 ◽

Cited By ~ 113

Author(s):

Xiangjie Sun ◽

Gary R. Whittaker

Keyword(s):

Influenza Virus ◽

Simian Virus 40 ◽

Critical Factor ◽

Simian Virus ◽

Host Cells ◽

Cholesterol Depletion ◽

Virus Infectivity ◽

Dependent Manner ◽

Virus Envelope ◽

Nonenveloped Virus

ABSTRACT Enveloped viruses are highly dependent on their lipid envelopes for entry into and infection of host cells. Here, we have examined the role of cholesterol in the virus envelope, using methyl-β-cyclodextrin depletion. Pretreatment of virions with methyl-β-cyclodextrin efficiently depleted envelope cholesterol from influenza virus and significantly reduced virus infectivity in a dose-dependent manner. A nonenveloped virus, simian virus 40, was not affected by methyl-β-cyclodextrin treatment. In the case of influenza virus, infectivity could be partially rescued by the addition of exogenous cholesterol. Influenza virus morphology, binding, and internalization were not affected by methyl-β-cyclodextrin depletion, whereas envelope cholesterol depletion markedly affected influenza virus fusion, as measured by a specific reduction in the infectivity of viruses induced to fuse at the cell surface and by fluorescence-dequenching assays. These data suggest that envelope cholesterol is a critical factor in the fusion process of influenza virus.

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Electron Microscopy of stained and unstained bovine papilloma virus: Comparison with polyoma virus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156079 ◽

1989 ◽

Vol 47 ◽

pp. 820-821

Author(s):

T.S. Baker ◽

N. H. Olson ◽

W. W. Newcomb ◽

J. C. Brown ◽

C. Olson

Keyword(s):

Electron Microscopy ◽

High Speed ◽

Simian Virus 40 ◽

Simian Virus ◽

Supernatant Fraction ◽

Epithelial Tissue ◽

Papilloma Virus ◽

Moderate Resolution ◽

Microscopy Techniques ◽

Bovine Papilloma Virus

Bovine papilloma virus type 1 (BPV-1) is a member of the papillomavirus genus, one of two in the Papovaviridae family. Papovaviruses are characterized by similarities in their capsid structure, genetics, biochemical composition and role in the formation of benign and cancerous tumors. The discoveries that the capsid structures of polyoma and simian virus 40 (SV40) (members of the genus polyomavirus) consist of 72 pentameric capsomeres raises the fundamental question of whether or not the capsids of the larger and more complex papilloma viruses have a similar, unexpected arrangement of the capsid subunits. The recent development of cryo-electron microscopy techniques, which facilitate direct visualization of the “native” morphology of biological specimens at moderate resolution (1-4 nm), provides an opportunity to critically examine the structure of BPV-1.The BPV-1 used in this study was originally isolated from a calf in 1965, passaged again in 1986, and stored in 50% glycerin and phosphate buffered saline. Virus was extracted and purified following the protocol of Cowsert et al. (1987): epithelial tissue rich in BPV-1 was mixed with an equal volume of buffer (1M NaCl, 20mM Tris, pH 7.5) and disrupted in a Waring blender. After high speed clarification, the supernatant fraction was mixed with an equal volume of Freon 113 and centrifuged at low speed. Virus from the aqueous phase was pelleted, resuspended in CsCl (ρ=l.33 g/cm3), density banded, pelleted and finally resuspended in 30 mM KCl and 6 mM Tris (pH 7.5) to a protein concentration of ˜3 mg/ml.

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Cholesterol-sensitive Modulation of Transcytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e06-08-0735 ◽

2007 ◽

Vol 18 (6) ◽

pp. 2057-2071 ◽

Cited By ~ 8

Author(s):

Julieta Leyt ◽

Naomi Melamed-Book ◽

Jean-Pierre Vaerman ◽

Shulamit Cohen ◽

Aryeh M. Weiss ◽

...

Keyword(s):

Plasma Membrane ◽

Lipid Rafts ◽

Receptor Internalization ◽

Cholesterol Depletion ◽

Compensatory Mechanism ◽

Membrane Domains ◽

Caveolin 1 ◽

Human Transferrin Receptor ◽

Detergent Resistant Membranes ◽

Tyrosine 14

Cholesterol-rich membrane domains (e.g., lipid rafts) are thought to act as molecular sorting machines, capable of coordinating the organization of signal transduction pathways within limited regions of the plasma membrane and organelles. The significance of these domains in polarized postendocytic sorting is currently not understood. We show that dimeric IgA stimulates the incorporation of its receptor into cholesterol-sensitive detergent-resistant membranes confined to the basolateral surface/basolateral endosomes. A fraction of human transferrin receptor was also found in basolateral detergent-resistant membranes. Disrupting these membrane domains by cholesterol depletion (using methyl-β-cyclodextrin) before ligand-receptor internalization caused depolarization of traffic from endosomes, suggesting that cholesterol in basolateral lipid rafts plays a role in polarized sorting after endocytosis. In contrast, cholesterol depletion performed after ligand internalization stimulated cargo transcytosis. It also stimulated caveolin-1 phosphorylation on tyrosine 14 and the appearance of the activated protein in dimeric IgA-containing apical organelles. We propose that cholesterol depletion stimulates the coupling of transcytotic and caveolin-1 signaling pathways, consequently prompting the membranes to shuttle from endosomes to the plasma membrane. This process may represent a unique compensatory mechanism required to maintain cholesterol balance on the cell surface of polarized epithelia.

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