Use of Intact Xenopus Oocytes in Nucleocytoplasmic Transport Studies

Panté, Nelly

doi:10.1007/978-1-59745-000-3_21

Cited by 17 publications

(21 citation statements)

References 25 publications

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“…For the phospholipase A2 (PLA2) inhibition experiment, MVM was incubated with 10 M manoalide (Alexis Biochemicals) for 1 h at room temperature with agitation prior to microinjection. Where indicated, a 50 mM concentration of the caspase inhibitor benzyloxycarbonylVal-Ala-Asp-fluoromethyl ketone (zVAD-fmk), zDEVD-fmk, or zVEID-fmk (Tocris Biosciences) was included in the MVM solution to be injected, resulting in an intracellular inhibitor concentration of approximately 200 M. Oocytes were then incubated at room temperature for 2 h, followed by fixation and preparation for thin-section electron microscopy (EM) as previously described (2,5,39). Quantification of the proportion of ONM damaged was performed using ImageJ software (National Institutes of Health).…”

Section: Methodsmentioning

confidence: 99%

Nuclear Envelope Disruption Involving Host Caspases Plays a Role in the Parvovirus Replication Cycle

Cohen

Marr

Garcin

et al. 2011

J Virol

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Parvoviruses are small, nonenveloped, single-stranded DNA viruses which replicate in the nucleus of the host cell. We have previously found that early during infection the parvovirus minute virus of mice (MVM) causes small, transient disruptions of the nuclear envelope (NE). We have now investigated the mechanism used by MVM to disrupt the NE. Here we show that the viral phospholipase A2, the only known enzymatic domain on the parvovirus capsid, is not involved in causing NE disruption. Instead, the virus utilizes host cell caspases, which are proteases involved in causing NE breakdown during apoptosis, to facilitate these nuclear membrane disruptions. Studies with pharmacological inhibitors indicate that caspase-3 in particular is involved. A caspase-3 inhibitor prevents nuclear lamin cleavage and NE disruption in MVM-infected mouse fibroblast cells and reduces nuclear entry of MVM capsids and viral gene expression. Caspase-3 is, however, not activated above basal levels in MVM-infected cells, and other aspects of apoptosis are not triggered during early MVM infection. Instead, basally active caspase-3 is relocalized to the nuclei of infected cells. We propose that NE disruption involving caspases plays a role in (i) parvovirus entry into the nucleus and (ii) alteration of the compartmentalization of host proteins in a way that is favorable for the virus.In order to replicate successfully, viruses must overcome various barriers in the cell. For viruses that replicate in the cell nucleus, the nuclear envelope (NE) is one such barrier. The NE consists of an inner nuclear membrane (INM) and an outer nuclear membrane (ONM). These membranes are supported by an underlying protein meshwork called the nuclear lamina, composed of the intermediate filament proteins nuclear lamins, which is associated with the nuclear face of the NE. Embedded in the NE are the nuclear pore complexes (NPCs), which are large protein complexes that mediate active transport of molecules up to 39 nm in diameter into and out of the nucleus (40). Because the sizes and structures of viruses vary enormously, viruses have developed surprisingly diverse strategies for delivering their genome and accessory proteins into the nuclei of infected cells (21,26,60,61). Aside from some retroviruses, which are thought to enter the nucleus while the NE is disassembled during mitosis (19), most of these strategies involve partial disassembly of the virion and nuclear transport through the NPC using the cellular nuclear import machinery (i.e., nuclear localization signals, importins, GTP, and Ran) (55). The viral component entering the nucleus may be an intact capsid (e.g., hepatitis B virus capsid, which crosses the NPC intact [40,42]), a naked viral genome (e.g., for herpes simplex virus type 1 which ejects its DNA from its NPC-docked capsid into the nucleus, leaving empty capsids at the NPC [51]), or a viral genome in association with viral proteins (e.g., influenza virus ribonucleoprotein complexes [11]).In general, more is known about the nuclear entry of ...

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Section: Methodsmentioning

confidence: 99%

Nuclear Envelope Disruption Involving Host Caspases Plays a Role in the Parvovirus Replication Cycle

Cohen

Marr

Garcin

et al. 2011

J Virol

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“…Electron Microscopy-Colloidal gold particles (9 nm in diameter) were prepared and conjugated with the importin ␤⅐IBB complexes as previously described (33,52). Xenopus oocytes (stage VI) were microinjected into their cytoplasm with 50 nl of gold-conjugated complexes, and the injected oocytes were incubated at room temperature for 1 h and processed for embedding/thin sectioning electron microscopy as previously described (33,52).…”

Section: Methodsmentioning

confidence: 99%

The Importin β Binding Domain Modulates the Avidity of Importin β for the Nuclear Pore Complex

Lott

Bhardwaj

Mitrousis

et al. 2010

Journal of Biological Chemistry

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Importin ␤ mediates active passage of cellular substrates through the nuclear pore complex (NPC). Adaptors such as importin ␣ and snurportin associate with importin ␤ via an importin ␤ binding (IBB) domain. The intrinsic structural flexibility of importin ␤ allows its concerted interactions with IBB domains, phenylalanine-glycine nucleoporins, and the GTPase Ran during transport. In this paper, we provide evidence that the nature of the IBB domain modulates the affinity of the import complex for the NPC. In permeabilized cells, importin ␤ imports a cargo fused to the snurportin IBB (sIBB) with ϳ70% reduced energy requirement as compared with the classical importin ␣ IBB. At the molecular level, this is explained by ϳ200-fold reduced affinity of importin ␤ for Nup62, when bound to the sIBB. Consistently, in vivo, the importin ␤⅐sIBB complex has greatly reduced persistence inside the central channel of the NPC. We propose that by controlling the degree of strain in the tertiary structure of importin ␤, the IBB domain modulates the affinity of the import complex for nucleoporins, thus dictating its persistence inside the NPC.

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“…This system has been used to map distinct steps of import through the NPC, for example interaction of a nuclear import substrate with structural components of the NPC such as the cytoplasmic filaments and nuclear basket (reviewed by Panté, 2006). It has also been used to study the nuclear import of nuclear-replicating viruses (Panté and Kann, 2002;Rabe et al, 2003;Cohen and Panté, 2005).…”

Section: Discussionmentioning

confidence: 99%

“…It is also important to note that when dealing with a transport substrate that is not directly visible under the electron microscope, it is often necessary to label the substrate with an electron-opaque particle such as colloidal gold (reviewed by Panté, 2006). …”

Section: Discussionmentioning

confidence: 99%

“…Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Panté, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus.…”

Section: Introductionmentioning

confidence: 99%

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Microinjection of Xenopus Laevis Oocytes

Cohen

Panté

2009

JoVE

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Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Panté, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus.Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.Protocol

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Use of Intact Xenopus Oocytes in Nucleocytoplasmic Transport Studies

Cited by 17 publications

References 25 publications

Nuclear Envelope Disruption Involving Host Caspases Plays a Role in the Parvovirus Replication Cycle

Nuclear Envelope Disruption Involving Host Caspases Plays a Role in the Parvovirus Replication Cycle

The Importin β Binding Domain Modulates the Avidity of Importin β for the Nuclear Pore Complex

Microinjection of Xenopus Laevis Oocytes

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