Microinjection of Xenopus Laevis Oocytes

Cohen, Sarah; Au, Shelly; Panté, Nelly

doi:10.3791/1106

Cited by 13 publications

(6 citation statements)

References 4 publications

(6 reference statements)

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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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“…This process is also time efficient compared to stable cell line generation. Moreover, several protocols of oocyte preparation and RNA microinjections have already been described in particular for studying cell cycle or nucleocytoplasmic transport 14,15 . Regarding the limits of such protocols to study translation, a particular attention should be taken regarding the concentration of mRNA.…”

Section: Discussionmentioning

confidence: 99%

<em>Xenopus laevis</em> as a Model to Identify Translation Impairment

Broucker

Semaille

Cailliau

et al. 2015

JoVE

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Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

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“…In plants cells, snRNP biogenesis is thought to proceed via similar pathways as described in mammalian cells ( Lorković and Barta, 2004 ; Shaw and Brown, 2012 ). However, experimental evidence pertaining to snRNP biogenesis processes in plant cells is limited, partly due to the absence of a suitable experimental system in which to examine the assembly and translocation of snRNAs and related proteins in plants, analogous to the Xenopus oocyte injection system ( Cohen et al, 2009 ). In humans, spliceosome disorders have been linked to severe inherited diseases, such as spinal muscular atrophy, which is caused by reduced levels of SMN proteins ( Matera and Wang, 2014 ; Lanfranco et al, 2017 ).…”

Section: Current Model Of Spliceosomal Snrnp Assembly Based On Mammalmentioning

confidence: 99%

Plant snRNP Biogenesis: A Perspective from the Nucleolus and Cajal Bodies

Ohtani

2018

Front. Plant Sci.

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Small nuclear ribonucleoproteins (snRNPs) are protein–RNA complexes composed of specific snRNP-associated proteins along with small nuclear RNAs (snRNAs), which are non-coding RNA molecules abundant in the nucleus. snRNPs mainly function as core components of the spliceosome, the molecular machinery for pre-mRNA splicing. Thus, snRNP biogenesis is a critical issue for plants, essential for the determination of a cell’s activity through the regulation of gene expression. The complex process of snRNP biogenesis is initiated by transcription of the snRNA in the nucleus, continues in the cytoplasm, and terminates back in the nucleus. Critical steps of snRNP biogenesis, such as chemical modification of the snRNA and snRNP maturation, occur in the nucleolus and its related sub-nuclear structures, Cajal bodies. In this review, I discuss roles for the nucleolus and Cajal bodies in snRNP biogenesis, and a possible linkage between the regulation of snRNP biogenesis and plant development and environmental responses.

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Microinjection of Xenopus Laevis Oocytes

Cited by 13 publications

References 4 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

<em>Xenopus laevis</em> as a Model to Identify Translation Impairment

Plant snRNP Biogenesis: A Perspective from the Nucleolus and Cajal Bodies

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