Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

Podsumowanie

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.

Streszczenie

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 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'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.

Wprowadzenie

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 eIF2α (eukaryotic initiation factor 2 α). SGs are dynamic structures which exchange components with the polysomes and other granules like the P-bodies. They constitute a "triage center" 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 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 vitro^10-13. 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.

Protokół

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 ±1 °C, 55 ±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)

1. Autoclave thin forceps, as well as curved forceps and dissecting scissors. Store in a sterile container. Use sterile D-PBS (Dulbecco's Phosphate-Buffered Saline). Prepare complete MEFs medium: Dulbecco's modified Eagle medium (DMEM)/F12 supplemented with 10% fetal calf serum (FBS), 1 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acids, and 0.5 mM 2-mercaptoethanol and warm at 37 °C.
2. Euthanize a pregnant mouse (at 13.5 days post-coïtum (dpc)) by cervical dislocation. Remove the uterine horns from the mouse sanitized with 90% EtOH. Under a sterile and clean hood, place the horns with the embryos in 37 °C prewarmed D-PBS. Open the horns, place the embryos in a 100 mm sterile plastic Petri dish, clean them up from the umbilical cord material and wash them with warm D-PBS to remove blood excess. Under a binocular, remove the embryo’s limbs, the internal organs and the upper part of the head containing the brain.
3. In a new Petri dish, mince the embryos into very small pieces with a sterile surgical blade or scissors (at least for 1 min). In the case of embryos of different genotypes, be careful to put each embryo in individual Petri dishes and keep the tail or the upper head for genotyping.
4. Cover the embryo with 1x trypsin-EDTA (0.25% trypsin, 1 mM EDTA) . Incubate for 30 min to 1 hr in a 37 °C incubator. Add 10 ml of complete MEFs medium to stop trypsin reaction. Pipet up and down to remove all aggregates. Let sit the cell suspension into a 50 ml tube (filled with complete medium) for 10 min, and centrifuge the supernatant for 5 min at 300 x g at room temperature. Resuspend the pellet in complete medium (6 ml for 1 embryo). Viable nucleated cells can be counted using trypan blue (around 5 x 10⁶ cells can be expected from one embryo). Plate 3 ml per 60 mm Petri dish.
5. Change the medium the next day and allow the cells to grow until the dishes are confluent. Many cell types can be seen, but only fibroblasts will survive subculture.
6. Split the cells and allow them to grow in 35 mm dishes with glass bottom (important for time lapse experiment), until 50-70% confluency for the transfection. Test for Mycoplasma and mouse pathogens.

2. Culture Adaptations in the Case of Neurons

1. The day before the culture, coat 35 mm glass bottom Petri dishes with poly-L-lysine (200 µl of 0.1 mg/ml) under a sterile and clean hood, and leave overnight.
2. On the next morning, rinse with sterile pure water, twice for 5 min and once for 45 min to 1 hr. Replace with 2 ml of DMEM plus 10% FBS medium and keep in a 37 °C incubator.
3. Dissect embryos at 18.5 dpc. Under a sterile and clean hood, place the horns with the embryos in cold sterile HBSS (Hank's Balanced Salt Solution) in 100 mm Petri dish. Neonatal pups can also be used instead of embryos in order to preserve the life of the dam and enable it to produce more offspring, especially in the case of transgenic animals which can be difficult to obtain. In individual Petri dishes, take each embryo or newborn and cut the head with scissors. Hold the head by inserting curved forceps into the eyes, cut the skin and carefully open the soft skull from the back of the head until the eyes, on each side of the head. Cut the optic nerves and the brain stem, remove the brain, and put it in a new Petri dish containing HBSS. Under a stereoscope, remove all the meninges using two thin forceps. Separate the hippocampi, the cortex or any other part of the brain depending on the structure to study.
4. Immerse the dissected brain structure in 4.5 ml of cold HBSS prepared previously in 15 ml tubes and keep on ice until digestion with trypsin. Add 0.5 ml of 2.5% trypsin and incubate at 37 °C for 15 min to 20 min. Rinse the trypsin 3x with HBSS, being extremely careful to not discard the digested brain parts.
5. Resuspend in 500 µl (hippocampi) to 1 ml (cortex) DMEM plus 10% FBS and pipet up and down several times with a 1 ml micropipette equipped with a 1 ml tip, then equipped with 1 ml plus 200 µl tips, until there is no visible aggregate.
6. Distribute 100-200 µl of cell suspension to each 35 mm glass bottom dish containing DMEM plus 10% FBS and let the neurons adhere at 37 °C for at least 3 hr. Replace by prewarmed neuron complete medium (Neurobasal medium supplemented with 250 µM L-glutamine and NS21, prepared as described in Chen et al.¹⁹) and leave at 37 °C to allow neuronal growth. Transfect the neurons at 5 to 14 days in vitro (div) (the efficiency of transfection is higher after a couple of div but synaptic connections are better established from 7-10 div).

3. Transfection of EGFP-G3BP1 Construct

Transfect the cells with a vector containing the cDNA of your protein of interest (any component of SGs) fused to a fluorescent marker (GFP, YFP, etc.), using 3 µg of purified plasmid per 35 mm dish.

1. Transfect the MEFs using a commercial method, following the manufacturer's protocol (See Table of Materials/Reagents).
2. Transfect the neurons with a calcium phosphate method adapted from Xia et al.²⁰ Briefly:
   1. Prepare the solutions: DMEM-wash: DMEM containing 25 mM KCl; transfection solution: DMEM-wash containing 1x DMKY (HEPES 5 mM, MgCl₂ 10 mM, phenol red); and shock solution: HeBS 1x, DMKY 1x, and DMSO 2% (v/v); and keep them at 37 °C.
   2. Remove the media from the neurons, filtrate, and keep it at 37 °C. Wash with DMEM-wash then replace with transfection medium and keep at 37 °C during the preparation of the calcium phosphate-plasmid DNA precipitates.
   3. In a 1.5 ml microcentrifuge tube, add (in this order) Braun water (final volume 50 µl), 5 µl of CaCl₂ 2.5 M, and 3 µg of plasmid DNA. Drop this mix onto 50 µl of HeBS 2x already introduced in a round bottom polypropylene tube. Mix the tube by rotation along with the dropping. Let the precipitate form at room temperature during 30 min.
   4. Add the precipitate dropwise onto the neurons and leave at 37 °C during 30 to 50 min.
   5. Replace with shock solution for 1 min, then rinse with DMEM-wash and finally reintroduce the preconditioned medium. Keep the cells at 37 °C.

4. Visualization of G3BP Containing SGs Assembly and Dynamics

1. Replace the medium of the cells which contains phenol red by phenol red-free medium.
2. Use a confocal microscope equipped with fluorescence for the acquisitions. Turn on the microscope systems: mercury lamp, computer, and lasers. Warm up the chamber to 37 °C at least 20 min before the beginning of the acquisitions.
3. The over expression of G3BP induces the spontaneous formation of a type of SGs. The cells may thus be observed for the assembly of SGs 24 hr right after the transfection without stress induction. Alternatively, stress can be induced to further induce the assembly of SGs. Add 0.5 mM sodium arsenite and start the acquisition right after the addition of the compound (granules will be well formed within 1 hr).
4. Add oil to the immersion objective (use 40X or 63X objectives) and install the Petri dish over the objective in the adapted 35 mm microscope holder, stabilized on the stage holder. Check that the dish is flat otherwise the acquisitions will be altered. Turn on the fluorescence light according to the relevant fluorophore and visualize transfected cells. Turn off fluorescence once a cell of interest is in the field in order to minimize bleaching and cytotoxicity.
5. In the "acquire tab", select the lasers depending on the excitation wavelength of the tag fluorophore (488 nm in our protocol which uses EGFP-tagged G3BP) and select the adapted emission length for the PMT (photomultiplier tube). Adjust the laser power (usually not more than 10%) to minimize noise and oversaturation as well as toxicity, and set the gain and offset to modify the signal to noise ratio. Scan fast (1,000 Hz) in order to minimize the duration of laser exposition (line average 2, frame average 1). If desired, set the parameters for the Z stack to be able to reconstruct the image in 3D (a Z step of 1 µm is usually fine with cells). Determine the interval time between each acquisition: smaller intervals permit to obtain a more coherent movie but this may induce photobleaching and toxicity (an interval of 20 sec was used in the described experiments). Duration of total acquisition may vary depending on the stress type. Many G3BP-containing SGs are formed very rapidly under arsenite treatment and are well visible after 1 hr of treatment: in that case, choose rapidly the cells to film and start the acquisitions right after the stress, with a total duration of acquisitions of 15-20 min largely sufficient to observe the assembly of several SGs.
6. Save the pictures and reconstruct the stacks and movie using ImageJ software.

Wyniki

Stress granule 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

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
DMEM/F12	Gibco	21331	Prewarm at 37 °C
Sodium pyruvate	Gibco	11360
Nonessential amino acids	Gibco	11140
L-Glutamine	Gibco	25030
Trypsin	Gibco	15096
Glass bottom B-35	Greiner	627860/627861	Treated or not (treated: increases attachment of adherent cells)
Poly-L-lysine	Sigma-Aldrich	P2636
DMEM	Gibco	31966	Prewarm at 37 °C
HeBS	Sigma-Aldrich	51558
Neurobasal	Gibco	21103	Prewarm at 37 °C
2-mercaptoethanol	Gibco	31350
Forceps	Biotek	DU-110-A	Very thin, tips 0.1 mm (useful to remove meninges)
Curved forceps	Biotek	P-110-BUF	Very thin, tips 0.1 mm
Small scissors	Biotek	CM-85-BS	Can be useful to remove hippocampi
Polyplus transfection JetPEI reagent	Ozyme	101-10	MEFs transfection, follow the forward protocol
Inverted laser scanning confocal microscope	Leica	SP5