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

2008 ◽  
Vol 19 (7) ◽  
pp. 2857-2869 ◽  
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.

scholarly journals Assembly and trafficking of caveolar domains in the cell

2005 ◽  
Vol 170 (5) ◽  
pp. 769-779 ◽  
Author(s):  
Akiko Tagawa ◽  
Anna Mezzacasa ◽  
Arnold Hayer ◽  
Andrea Longatti ◽  
Lucas Pelkmans ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cell Entry ◽  
Cholesterol Depletion ◽  
Membrane Domains ◽  
Vesicle Traffic ◽  
Caveolin 1 ◽  
Transport Vesicles ◽  
Microscopy Techniques

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.

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

2005 ◽  
Vol 79 (1) ◽  
pp. 615-618 ◽  
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.

scholarly journals Clathrin- and caveolin-1–independent endocytosis

2005 ◽  
Vol 168 (3) ◽  
pp. 477-488 ◽  
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.

scholarly journals Early Steps of Polyomavirus Entry into Cells

2000 ◽  
Vol 74 (18) ◽  
pp. 8582-8588 ◽  
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.

scholarly journals Cyclodextrin-Based Nanostructure Efficiently Delivers siRNA to Glioblastoma Cells Preferentially via Macropinocytosis

2020 ◽  
Vol 21 (23) ◽  
pp. 9306
Author(s):  
Darío Manzanares ◽  
María Dolores Pérez-Carrión ◽  
José Luis Jiménez Blanco ◽  
Carmen Ortiz Mellet ◽  
José Manuel García Fernández ◽  
...  
Keyword(s):  
Transfection Efficiency ◽  
Endocytic Pathway ◽  
Cyclodextrin Derivatives ◽  
Sirna Transfection ◽  
Cell Infection ◽  
Caveolin 1 ◽  
Entry Pathway ◽  
P21 Activated Kinase ◽  
Very High ◽  
Endocytic Pathways

Small interfering ribonucleic acid (siRNA) has the potential to revolutionize therapeutics since it can knockdown very efficiently the target protein. It is starting to be widely used to interfere with cell infection by HIV. However, naked siRNAs are unable to get into the cell, requiring the use of carriers to protect them from degradation and transporting them across the cell membrane. There is no information about which is the most efficient endocytosis route for high siRNA transfection efficiency. One of the most promising carriers to efficiently deliver siRNA are cyclodextrin derivatives. We have used nanocomplexes composed of siRNA and a β-cyclodextrin derivative, AMC6, with a very high transfection efficiency to selectively knockdown clathrin heavy chain, caveolin 1, and p21 Activated Kinase 1 to specifically block clathrin-mediated, caveolin-mediated and macropinocytosis endocytic pathways. The main objective was to identify whether there is a preferential endocytic pathway associated with high siRNA transfection efficiency. We have found that macropinocytosis is the preferential entry pathway for the nanoparticle and its associated siRNA cargo. However, blockade of macropinocytosis does not affect AMC6-mediated transfection efficiency, suggesting that macropinocytosis blockade can be functionally compensated by an increase in clathrin- and caveolin-mediated endocytosis.

scholarly journals Differential Mitogenic Signaling in Insulin Receptor-Deficient Fetal Pancreatic β-Cells

Endocrinology ◽  
2006 ◽  
Vol 147 (4) ◽  
pp. 1959-1968 ◽  
Author(s):  
C. Guillen ◽  
P. Navarro ◽  
M. Robledo ◽  
A. M. Valverde ◽  
M. Benito
Keyword(s):  
Insulin Receptor ◽  
Cell Lines ◽  
Cell Mass ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Phosphatidylinositol 3 Kinase ◽  
Β Cells ◽  
Β Cell ◽  
Phosphatidylinositol 3

Insulin receptor (IR) may play an essential role in the development of β-cell mass in the mouse pancreas. To further define the function of this signaling system in β-cell development, we generated IR-deficient β-cell lines. Fetal pancreata were dissected from mice harboring a floxed allele of the insulin receptor (IRLoxP) and used to isolate islets. These islets were infected with a retrovirus to express simian virus 40 large T antigen, a strategy for establishing β-cell lines (β-IRLoxP). Subsequently, these cells were infected with adenovirus encoding cre recombinase to delete insulin receptor (β-IR−/−). β-Cells expressed insulin and Pdx-1 mRNA in response to glucose. In β-IRLoxP β-cells, p44/p42 MAPK and phosphatidylinositol 3 kinase pathways, mammalian target of rapamycin (mTOR), and p70S6K phosphorylation and β-cell proliferation were stimulated in response to insulin. Wortmannin or PD98059 had no effect on insulin-mediated mTOR/p70S6K signaling and the corresponding mitogenic response. However, the presence of both inhibitors totally impaired these signaling pathways and mitogenesis in response to insulin. Rapamycin completely blocked insulin-activated mTOR/p70S6K signaling and mitogenesis. Interestingly, in β-IR−/− β-cells, glucose failed to stimulate phosphatidylinositol 3 kinase activity but induced p44/p42 MAPKs and mTOR/p70S6K phosphorylation and β-cell mitogenesis. PD98059, but not wortmannin, inhibited glucose-induced mTOR/p70S6K signaling and mitogenesis in those cells. Finally, rapamycin blocked glucose-mediated mitogenesis of β-IR−/− cells. In conclusion, independently of glucose, insulin can mediate mitogenesis in fetal pancreatic β-cell lines. However, in the absence of the insulin receptor, glucose induces β-cell mitogenesis.

scholarly journals Calcium Bridge Triggers Capsid Disassembly in the Cell Entry Process of Simian Virus 40

2009 ◽  
Vol 284 (50) ◽  
pp. 34703-34712 ◽  
Author(s):  
Masa-aki Kawano ◽  
Li Xing ◽  
Hiroko Tsukamoto ◽  
Takamasa Inoue ◽  
Hiroshi Handa ◽  
...  
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Cell Entry ◽  
Capsid Disassembly

scholarly journals Simian virus 40 induces lamin A/C fluctuations and nuclear envelope deformation during cell entry

Nucleus ◽  
2011 ◽  
Vol 2 (4) ◽  
pp. 320-330 ◽  
Author(s):  
Veronika Butin-Israeli ◽  
Orly Ben-nun-Shaul ◽  
Idit Kopatz ◽  
Stephen A. Adam ◽  
Takeshi Shimi ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cell Entry ◽  
Lamin A

scholarly journals Association of the Caveola Vesicular System with Cellular Entry by Filoviruses

2002 ◽  
Vol 76 (10) ◽  
pp. 5266-5270 ◽  
Author(s):  
Cyril J. Empig ◽  
Mark A. Goldsmith
Keyword(s):  
Cell Biology ◽  
Phorbol Esters ◽  
Marburg Virus ◽  
Cell Entry ◽  
Target Cells ◽  
Protein Marker ◽  
Caveolin 1 ◽  
Entry Pathway ◽  
Vesicular Stomatitis ◽  
Endocytic Vesicles

ABSTRACT The filoviruses Ebola Zaire virus and Marburg virus are believed to infect target cells through endocytic vesicles, but the details of this pathway are unknown. We used a pseudotyping strategy to investigate the cell biology of filovirus entry. We observed that specific inhibitors of the caveola system, including cholesterol-sequestering drugs and phorbol esters, inhibited the entry of filovirus pseudotypes into human cells. We also measured slower cell entry kinetics for both filovirus pseudotypes than for pseudotypes of vesicular stomatitis virus (VSV), which has been recognized to exploit the clathrin-mediated entry pathway. Finally, visualization by immunofluorescence and confocal microscopy revealed that the filovirus pseudotypes colocalized with the caveola protein marker caveolin-1 but that VSV pseudotypes did not. Collectively, these results provide evidence suggesting that filoviruses use caveolae to gain entry into cells.

scholarly journals Caveolar Endocytosis of Simian Virus 40 Is Followed by Brefeldin A-Sensitive Transport to the Endoplasmic Reticulum, Where the Virus Disassembles

2002 ◽  
Vol 76 (10) ◽  
pp. 5156-5166 ◽  
Author(s):  
Leonard C. Norkin ◽  
Howard A. Anderson ◽  
Scott A. Wolfrom ◽  
Ariella Oppenheim
Keyword(s):  
Endoplasmic Reticulum ◽  
Brefeldin A ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Capsid Proteins ◽  
Lysosomal Compartment ◽  
Caveolin 1 ◽  
Cell Biol ◽  
Intermediate Compartment

ABSTRACT Simian virus 40 (SV40) enters cells by atypical endocytosis mediated by caveolae that transports the virus to the endoplasmic reticulum (ER) instead of to the endosomal-lysosomal compartment, which is the usual destination for viruses and other cargo that enter by endocytosis. We show here that SV4O is transported to the ER via an intermediate compartment that contains β-COP, which is best known as a component of the COPI coatamer complexes that are required for the retrograde retrieval pathway from the Golgi to the ER. Additionally, transport of SV40 to the ER, as well as infection, is sensitive to brefeldin A. This drug acts by specifically inhibiting the ARF1 GTPase, which is known to regulate assembly of COPI coat complexes on Golgi cisternae. Moreover, some β-COP colocalizes with intracellular caveolin-1, which was previously shown to be present on a new organelle (termed the caveosome) that is an intermediate in the transport of SV40 to the ER (L. Pelkmans, J. Kartenbeck, and A. Helenius, Nat. Cell Biol. 3:473-483, 2001). We also show that the internal SV40 capsid proteins VP2 and VP3 become accessible to immunostaining starting at about 5 h. Most of that immunostaining overlays the ER, with some appearing outside of the ER. In contrast, immunostaining with anti-SV40 antisera remains confined to the ER.

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