Name: visualization of g3bp stress granules dynamics in live primary cells
Uploaded: 2014-05-21T13:00:00
Description: Watch this Scientific Journal Video about Visualization of G3BP Stress Granules Dynamics in Live Primary Cells at JoVE.com

The authors would like to acknowledge the Montpellier Rio Imaging (MRI) platform where the acquisitions were performed. They thank Isabel Cristina Lopez Mejia, Alexandra Metz, Irina Lassot, Solange Desagher, Fabien Loustalot, and Virginie Georget for their help in different parts of the protocol. This work was supported by the Fondation pour la Recherche M&#233;dicale (FRM) (Equipe FRM 2011 -n&#176;DEQ20111223745).

All the animal procedures in this protocol are in strict adherence with the guidelines of the European Community Council Directive of 24 November 1986 (86-609/EEC). House the mice in group, allowing food and water ad libitum. Maintain them in a controlled environment (22 &#177;1 &#176;C, 55 &#177;5% humidity) with a 12 hr:12 hr light:dark cycle (light on at 7:00 am).
1. Culture of Murine Primary Cells: Mouse Embryonic Fibroblasts (MEFs)
<ol>
	<li>Autoclave thin forceps, as well as c...

<ol>
	<li>Thomas, M. G., Loschi, M., Desbats, M. A., Boccaccio, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+granules:+The+good,+the+bad+and+the+ugly.">RNA granules: The good, the bad and the ugly.</a> Cellular Signalling. 23 (2), 324-334 (2011).</li><li>Kedersha, N., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granules:+sites+of+mRNA+triage+that+regulate+mRNA+stability+and+translatability.">Stress granules: sites of mRNA triage that regulate mRNA stability and translatability.</a> Biochemical Society transactions. 30 (6), 963-969 (2002).</li><li>Anderson, P., Kedersha, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granules:+the+Tao+of+RNA+triage.">Stress granules: the Tao of RNA triage.</a> Trends in Biochemical Sciences. 33 (3), 141-150 (2008).</li><li>Gilks, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granule+assembly+is+mediated+by+prion-like+aggregation+of+TIA-1.">Stress granule assembly is mediated by prion-like aggregation of TIA-1.</a> Molecular biology of the cell. 15 (12), 5383-5398 (2004).</li><li>Tourri&#232;re, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RasGAP-associated+endoribonuclease+G3BP+assembles+stress+granules.">The RasGAP-associated endoribonuclease G3BP assembles stress granules.</a> The Journal of Cell Biology. 160 (6), 823-831 (2003).</li><li>Parker, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Ras-GTPase-activating+protein+SH3-domain-binding+protein.">A Ras-GTPase-activating protein SH3-domain-binding protein.</a> Molecular and Cellular Biology. 16 (6), 2561-2569 (1996).</li><li>Annibaldi, A., Dousse, A., Martin, S., Tazi, J., Widmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+G3BP1+as+a+RasGAP+binding+protein:+sensitization+of+tumor+cells+to+chemotherapy+by+the+RasGAP+317-326+sequence+does+not+involve+G3BP1.">Revisiting G3BP1 as a RasGAP binding protein: sensitization of tumor cells to chemotherapy by the RasGAP 317-326 sequence does not involve G3BP1.</a> PLOS ONE. 6 (12), (2011).</li><li>Kennedy, D., French, J., Guitard, E., Ru, K., Tocque, B., Mattick, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+G3BPs:+tissue+specific+expression,+chromosomal+localisation+and+rasGAP(120)+binding+studies.">Characterization of G3BPs: tissue specific expression, chromosomal localisation and rasGAP(120) binding studies.</a> Journal of Cellular Biochemistry. 84 (1), 173-187 (2001).</li><li>Kobayashi, T., Winslow, S., Sunesson, L., Hellman, U., Larsson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKC&#945;+Binds+G3BP2+and+Regulates+Stress+Granule+Formation+Following+Cellular+Stress.">PKC&#945; Binds G3BP2 and Regulates Stress Granule Formation Following Cellular Stress.</a> PLOS ONE. 7 (4), (2012).</li><li>Tourri&#232;re, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RasGAP-associated+endoribonuclease+G3Bp:+selective+RNA+degradation+and+phosphorylation-dependent+localization.">RasGAP-associated endoribonuclease G3Bp: selective RNA degradation and phosphorylation-dependent localization.</a> Molecular and Cellular Biology. 21 (22), 7747-7760 (2001).</li><li>Costa, M., Ochem, A., Staub, A., Falaschi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+DNA+helicase+VIII:+a+DNA+and+RNA+helicase+corresponding+to+the+G3BP+protein,+an+element+of+the+ras+transduction+pathway.">Human DNA helicase VIII: a DNA and RNA helicase corresponding to the G3BP protein, an element of the ras transduction pathway.</a> Nucleic acids research. 27 (3), 817-821 (1999).</li><li>Solomon, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+structural+features+of+caprin-1+mediate+its+interaction+with+G3BP-1+and+its+induction+of+phosphorylation+of+eukaryotic+translation+initiation+factor+2alpha,+entry+to+cytoplasmic+stress+granules,+and+selective+interaction+with+a+subset+of+mRNAs.">Distinct structural features of caprin-1 mediate its interaction with G3BP-1 and its induction of phosphorylation of eukaryotic translation initiation factor 2alpha, entry to cytoplasmic stress granules, and selective interaction with a subset of mRNAs.</a> Molecular and Cellular Biology. 27 (6), 2324-2342 (2007).</li><li>Soncini, C., Berdo, I., Draetta, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ras-GAP+SH3+domain+binding+protein+(G3BP)+is+a+modulator+of+USP10,+a+novel+human+ubiquitin+specific+protease.">Ras-GAP SH3 domain binding protein (G3BP) is a modulator of USP10, a novel human ubiquitin specific protease.</a> Oncogene. 20 (29), 3869-3879 (2001).</li><li>Zekri, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+fetal+growth+and+neonatal+survival+by+the+RasGAP-associated+endoribonuclease+G3BP.">Control of fetal growth and neonatal survival by the RasGAP-associated endoribonuclease G3BP.</a> Molecular and Cellular Biology. 25 (19), 8703-8716 (2005).</li><li>Martin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+of+G3BP1,+the+stress+granules+assembly+factor,+results+in+abnormal+synaptic+plasticity+and+calcium+homeostasis+in+neurons.">Deficiency of G3BP1, the stress granules assembly factor, results in abnormal synaptic plasticity and calcium homeostasis in neurons.</a> Journal of neurochemistry. , (2013).</li><li>Mazroui, R., Huot, M. -. E., Tremblay, S., Filion, C., Labelle, Y., Khandjian, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+of+messenger+RNA+by+Fragile+X+Mental+Retardation+protein+into+cytoplasmic+granules+induces+translation+repression.">Trapping of messenger RNA by Fragile X Mental Retardation protein into cytoplasmic granules induces translation repression.</a> Human molecular genetics. 11 (24), 3007-3017 (2002).</li><li>Colombrita, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43+is+recruited+to+stress+granules+in+conditions+of+oxidative+insult.">TDP-43 is recruited to stress granules in conditions of oxidative insult.</a> Journal of neurochemistry. 111 (4), 1051-1061 (2009).</li><li>Thomas, M. G., Tosar, L. J. M., Desbats, M. A., Leishman, C. C., Boccaccio, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+Staufen+1+is+recruited+to+stress+granules+and+impairs+their+assembly.">Mammalian Staufen 1 is recruited to stress granules and impairs their assembly.</a> Journal of Cell Science. 122 (4), 563-573 (2009).</li><li>Chen, Y., Stevens, B., Chang, J., Milbrandt, J., Barres, B. A., Hell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NS21:+re-defined+and+modified+supplement+B27+for+neuronal+cultures.">NS21: re-defined and modified supplement B27 for neuronal cultures.</a> Journal of neuroscience. 171 (2), 239-247 (2008).</li><li>Xia, Z., Dudek, H., Miranti, C. K., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Influx+via+the+NMDA+Receptor+Induces+Immediate+Early+Gene+Transcription+by+a+MAP+Kinase/ERK-Dependent+Mechanism.">Calcium Influx via the NMDA Receptor Induces Immediate Early Gene Transcription by a MAP Kinase/ERK-Dependent Mechanism.</a> The Journal of Neuroscience. 16 (17), 5425-5436 (1996).</li><li>Dudek, H., Ghosh, A., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+phosphate+transfection+of+DNA+into+neurons+in+primary+culture.">Calcium phosphate transfection of DNA into neurons in primary culture.</a> Current protocols in neuroscience. 3, (2001).</li><li>Reineke, L. C., Dougherty, J. D., Pierre, P., Lloyd, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+G3BP-induced+granules+trigger+eIF2&#945;+phosphorylation.">Large G3BP-induced granules trigger eIF2&#945; phosphorylation.</a> Molecular biology of the cell. 23 (18), (2012).</li><li>Kedersha, N., Tisdale, S., Hickman, T., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+and+quantitative+imaging+of+mammalian+stress+granules+and+processing+bodies.">Real-time and quantitative imaging of mammalian stress granules and processing bodies.</a> Methods in enzymology. 448, 521-552 (2008).</li></ol>

The most critical steps in the protocol concern the transfection and more particularly the time lapse acquisitions, which have to be carefully monitored in order to lower down the cytotoxicity.
Culture of primary cells is not a difficult part, as long as sterile conditions are maintained and caution is taken in order to prevent damage during the dissection and cell dissociation steps. MEFs can be kept frozen at early passages. Neuron transfection with calcium phosphate has been shown to work w...

Stress granules (SGs) are non-membranous cytoplasmic foci formed as a cellular protective response to environmental stress, such as elevated temperature, oxidative stress, hypoxia, osmotic shock, UV irradiation, glucose deprivation, or viral infection1. They can be induced chemically by treatment with compounds like sodium arsenite, which triggers oxidative stress. SGs accumulate stalled translation arrested messenger ribonucleoprotein (mRNPs) complexes2, sequestering mRNAs from the translational machinery, and their assembly can be triggered by the phosphorylation of eIF2&#945; (eukaryotic initiation factor 2 &#945;). SGs are dynamic structures which exchange components with the polysomes and other granules like the P-bodies. They constitute a &#34;triage center&#34; where mRNAs are sorted and processed for either translation, reinitiation, degradation, or packaging into stable non-polysomal mRNPs3. The assembly of SGs is fast but it is a gradual process with initial numerous small aggregates which coalesce into larger granules. The use of chemical inhibitors that disrupt or stabilize microtubules shows that the microtubule network is required for SG dynamics including assembly, coalescence and disassembly processes.
The dynamic assembly of SGs is also promoted by aggregation of specific RNA-binding proteins (RNA-BP) like TIA-1 (T-cell internal antigen-1) and TIAR (TIA-1-related protein), which are able to dimerize and promote polysome disassembly and the routing of mRNAs into SGs4. G3BP (RasGAP SH3 domain binding protein) is such an RNA-BP that localizes to SGs when cells are stressed with arsenite or high temperature, and overexpression of dephosphorylated G3BP can induce SGs assembly5.
G3BP is an evolutionarily conserved RNA-BP that was initially characterized through its interaction with a Ras-GTPase activating protein (RasGAP p120 6); but this interaction was recently revisited7. The G3BP family includes two members in mammals, G3BP1 (referred to as G3BP) and G3BP2&#160;8. Both proteins colocalize in SGs, when cells are subjected to stress9. G3BPs comprise an N-terminal NTF2 domain suggested to influence their localization and oligomerization, followed by proline rich (PxxP) motifs, then C-terminal motifs associated with RNA binding: the canonical RNA-Recognition Motif (RRM) with conserved RNP1 and RNP2 motifs, followed by an arginine-glycine rich (RGG) box. Interestingly, the analysis of different parts of the protein by constructing different domains fused to EGFP showed that the NTF2-like domain and the RNA-binding domain were the most efficiently recruited to SGs, suggesting the importance of the properties of dimerization and RNA binding in the assembly of SGs. Diverse models have revealed different functions of G3BP proteins in vitro10-13. Disruption of G3BP in mice have shown the importance of this protein in developmental growth and survival14, as well as an important role for G3BP in the Central Nervous System (CNS), characterized by ataxia and defects in spatial working memory15. G3BP deficiency leads to altered neuronal plasticity and calcium homeostasis, establishing a direct link between SG formation and neurodegenerative diseases15. It is thus important to be able to study the dynamics of SGs in primary cells like neurons.
This protocol provides a simple way to observe the assembly of G3BP1-containing SGs in primary cells under arsenite treatment. It can be used to study SGs assembly under different conditions, for example different kinds of stresses. It can also be adapted to other granules or other constituents of SGs. Indeed, this protocol focuses on G3BP1, but there are other stress granules markers like TIA-1/R, TTP (tristetraprolin)2, FMRP (Fragile X Mental Retardation Syndrome protein)16, TDP-43 (transactive response DNA binding protein 43)17 or Staufen18. In particular, proteins like TIA-1/R are, like G3BP, nucleating RNA binding proteins that can induce SGs assembly when overexpressed, even if the different formed SGs can differ in function, regulation and associated transcripts. Transfection of fluorophore-tagged version of any of these key components or nucleators of SGs can be performed to image particular SGs assembly and dynamics.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Name of Material/ Equipment</td>
			<td style="width:153px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:312px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">DMEM/F12</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">21331</td>
			<td>Pre-warm at 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sodium Pyruvate</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">11360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Non essential amino acids</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">11140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">L-Glutamine</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">25030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Trypsin</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">15096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Glass bottom B-35</td>
			<td style="width:153px;">Greiner</td>
			<td style="width:119px;">627860/627861</td>
			<td>Treated or not (treated: increases attachment of adherent cells)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Poly-L-lysine</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">DMEM</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">31966</td>
			<td>Pre-warm at 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">HeBS</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">51558</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Neurobasal</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">21103</td>
			<td>Pre-warm at 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">2-mercaptoethanol</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">31350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Forceps</td>
			<td style="width:153px;">Biotek</td>
			<td style="width:119px;">DU-110-A</td>
			<td>Very thin, tips 0,1 mm (useful to remove meninges)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Curved forceps</td>
			<td style="width:153px;">Biotek</td>
			<td style="width:119px;">P-110-BUF</td>
			<td>Very thin, tips 0,1 mm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Small scissors</td>
			<td style="width:153px;">Biotek</td>
			<td style="width:119px;">CM-85-BS</td>
			<td>Can be useful to remove hippocampi</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyplus transfection JetPEI reagent&#160;</td>
			<td style="width:153px;">Ozyme</td>
			<td style="width:119px;">101-10</td>
			<td>MEFs transfection, follow the forward protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted laser scanning confocal microscope</td>
			<td style="width:153px;">Leica&#160;</td>
			<td style="width:119px;">SP5</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of g3bp stress granules dynamics in live primary cells

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice&#160;and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer&#39;s disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.

Stress granule&#160;formation is important in the response of cells to stress, permitting a cellular adaptation with stalled translation, until the stress has cleared, associated to prevention of apoptosis. This assay permits to study the SGs in primary cells, by following the localization of key SGs protein components (Figure 1). G3BP, a key factor of SGs assembly, is present rather diffusively in the cytoplasm of cultured MEFs or neurons (Figure 2A a and c</str...

Watch this Scientific Journal Video about Visualization of G3BP Stress Granules Dynamics in Live Primary Cells at JoVE.com

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...

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