1. Cell Biology

Chronic optogenetic induction of stress granules is cytotoxic and reveals the evolution of ALS-FTD pathology

  1. Peipei Zhang
  2. Baochang Fan
  3. Peiguo Yang
  4. Jamshid Temirov
  5. James Messing
  6. Hong Joo Kim
  7. J Paul Taylor  Is a corresponding author
  1. St. Jude Children’s Research Hospital, United States
  2. Howard Hughes Medical Institute, United States
https://doi.org/10.7554/eLife.39578
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Cite this article
  1. Peipei Zhang
  2. Baochang Fan
  3. Peiguo Yang
  4. Jamshid Temirov
  5. James Messing
  6. Hong Joo Kim
  7. J Paul Taylor
(2019)
Chronic optogenetic induction of stress granules is cytotoxic and reveals the evolution of ALS-FTD pathology
eLife 8:e39578.
https://doi.org/10.7554/eLife.39578
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  3. Download .RIS
4 figures, 5 videos, 1 table and 1 additional file

Figures

Figure 1 with 4 supplements
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OptoGranules are light-inducible dynamic stress granules.

(a) Design of Opto-G3BP1 and Opto-Control constructs. (b) U2OS cells stably expressing Opto-Control or Opto-G3BP1 were stimulated with a single 5-msec pulse of 488 nm blue light (power density ~2.5 …

https://doi.org/10.7554/eLife.39578.002
Figure 1—figure supplement 1
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Opto-G3BP1 assembles light-dependent cytoplasmic clusters.

(a) Top: Immunoblot showing expression levels of mCherry (red) and G3BP1 (green) in U2OS cells stably expressing Opto-Control or Opto-G3BP1 with or without blue light stimulation. Bottom: …

https://doi.org/10.7554/eLife.39578.003
Figure 1—figure supplement 2
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OptoGranules are light-inducible stress granules.

(a-g) U2OS cells stably expressing Opto-G3BP1 with or without continuous blue light (~2 mW/cm2) using custom-made LED arrays for 5 hr were immunostained with antibodies against TIA1 (a), TIAR (b), …

https://doi.org/10.7554/eLife.39578.004
Figure 1—figure supplement 3
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OptoGranules rescue stress granule formation in G3BP1/2 double KO cells.

(a-h,) (a) WT U2OS cells (top) and G3BP1/2 double KO U2OS cells (bottom) with or without sodium arsenite (0.5 mM NaAsO2, 45 min) treatment were immunostained for G3BP1 as well as stress granule …

https://doi.org/10.7554/eLife.39578.005
Figure 1—figure supplement 4
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OptoDroplets are not stress granules.

(a) Schematic diagram of Opto-FUS (left) and Opto-TDP-43 (right) constructs. (b) U2OS cells were transiently co-transfected with stress granule marker G3BP1-GFP together with either Opto-FUS-IDR or …

https://doi.org/10.7554/eLife.39578.006
Figure 2 with 1 supplement
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OptoGranule formation is dependent on the local concentration of activated G3BP1 and dependent on polysome disassembly, but independent of eIF2α phosphorylation.

(a) U2OS cells stably expressing Opto-G3BP1 were intermittently exposed to a blue-light laser (488 nm) for activation followed by image acquisition with a 561 nm channel. Blue light intensity was …

https://doi.org/10.7554/eLife.39578.010
Figure 2—figure supplement 1
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OptoGranule formation is independent of eIF2α phosphorylation.

Immunoblot showing phosphorylated eIF2α and actin levels in cells treated with sodium arsenite (0.5 mM NaAsO2) for 45 min, exposed to 42°C heat shock for 1 hr, or exposed to continuous blue light …

https://doi.org/10.7554/eLife.39578.011
Figure 3 with 1 supplement
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Persistent OptoGranules are cytotoxic and evolve to pathological inclusions.

(a,b) U2OS cells stably expressing Opto-Control or Opto-G3BP1 were stimulated with continuous blue light (~2 mW/cm2) for indicated times using custom-made LED arrays and viability was assessed by …

https://doi.org/10.7554/eLife.39578.012
Figure 3—figure supplement 1
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OptoGranules evolve to pathological inclusions.

(a) U2OS cells stably expressing Opto-G3BP1 were exposed to chronic persistent blue light stimulation (same data as shown in Figure 3c) or chronic intermittent blue light stimulation (same data as …

https://doi.org/10.7554/eLife.39578.013
Figure 4 with 2 supplements
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Persistent OptoGranules are cytotoxic and evolve to pathological inclusions in human iPSC-derived neurons.

(a) Schematic illustrating generation of iPSC-derived neurons stably expressing Opto-G3BP1. (b) iPSC-derived neurons expressing Opto-Control (mRuby) or Opto-G3BP1 (mRuby) were intermittently exposed …

https://doi.org/10.7554/eLife.39578.014
Figure 4—figure supplement 1
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iPSC-derived neurons form OptoGranules.

(ab) Quantification of mRNA levels in iPSC-derived neurons before or after 1 and 2 weeks of Ngn2 lentiviral infection. Levels are normalized for RPP30 mRNA levels as an internal control. Panel b is …

https://doi.org/10.7554/eLife.39578.015
Figure 4—figure supplement 2
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OptoGranules evolve to pathological inclusions in iPSC-derived neurons.

(a) Schematic illustrating generation of iPSC-derived neurons in which doxycycline induces both differentiation and Opto-G3BP1(mCherry) expression. (b) iPSC-derived neurons expressing …

https://doi.org/10.7554/eLife.39578.016

Videos

Video 1
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Opto-Control fails to assemble light-dependent cytoplasmic clusters.

U2OS cells stably expressing Opto-Control were stimulated with a single 5-msec pulse of 488 nm blue light (power density ~2.5 MW/cm2) in a defined ROI. See Video 2 for corresponding Opto-G3BP1 …

https://doi.org/10.7554/eLife.39578.007
Video 2
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Opto-G3BP1 assembles light-dependent cytoplasmic clusters.

U2OS cells stably expressing Opto-G3BP1 were stimulated with a single 5-msec pulse of 488 nm blue light (power density ~2.5 MW/cm2) in a defined ROI. Opto-G3BP1 assembles highly dynamic, liquid-like …

https://doi.org/10.7554/eLife.39578.008
Video 3
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Dynamic recruitment of the stress granule marker GFP-TIA1 into light-induced Opto-G3BP1 granules.

U2OS cells transiently transfected with Opto-G3BP1 and the stress granule marker GFP-TIA1 were stimulated with a blue-light laser (power density ~2.5 MW/cm2) for 5 msec. Cells were sequentially …

https://doi.org/10.7554/eLife.39578.009
Video 4
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Blue light activation fails to induce the assembly of OptoGranules in iPSC-derived neurons expressing Opto-Control.

iPSC-derived neurons expressing Opto-Control (mRuby) were intermittently exposed to a 488 nm blue-light laser (95% laser power, power density 6.5 W/cm2) followed by image acquisition with a 561 nm ch…

https://doi.org/10.7554/eLife.39578.017
Video 5
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Blue light activation induces the assembly of OptoGranules in iPSC-derived neurons expressing Opto-G3BP1.

iPSC-derived neurons expressing Opto-G3BP1 (mRuby) were intermittently exposed to a 488 nm blue-light laser (95% laser power, power density 6.5 W/cm2) followed by image acquisition with a 561 nm chan…

https://doi.org/10.7554/eLife.39578.018

Tables

Key resources table
Reagent type
(species) or
resource		DesignationSource or
reference		IdentifiersAdditional information
Cell line (Human)U-2 OSATCCHTB-96; RRID:CVCL_0042
Cell line (Human)Lenti-X 293T(293LE)Clontech632180; RRID: CVCL_4401
Cell line (Human)iPSCsBuilding a KidneyBJFF6; RRID: CVCL_VU02
Recombinant DNA reagentpCRY2PHR-mCherryN1Addgene26866; RRID:Addgene_26866
Recombinant DNA reagentpCMV-CRY2-mCherryAddgene58368;
RRID:Addgene_58368
Recombinant DNA reagentphND2-N174Addgene31822;
RRID:Addgene_31822
Recombinant DNA reagentpKanCMV-mClover3-mRuby3Addgene74252;
RRID:Addgene_74252
Recombinant DNA reagentpTight-hND2-N106Addgene31875; RRID:Addgene_31875
Recombinant DNA reagentpsPAX2Addgene12260;
RRID:Addgene_12260
Recombinant DNA reagentCRY2olig-mCherryAddgene60032; RRID:Addgene_60032
Recombinant DNA reagentpMD2.GAddgene12259;
RRID:Addgene_12259
Recombinant DNA reagentlinear hygromycin markerClontech631625; RRID:Addgene_60032
Antibodygoat polyclonal
anti-β-actin		Santa Cruz
Biotechnology		sc-1616; RRID: AB_630836(1:1000)
Antibodymouse monoclonal anti-eIF2αSanta Cruz
Biotechnology		sc-133132; RRID: AB_1562699(1:1000)
Antibodyrabbit polyclonal anti-β-actinCell Signaling3597S; RRID: AB_390740(1:1000)
Antibodyrabbit polyclonal anti-mCherryAbcam167453; RRID: AB_2571870(1:1000)
Antibodymouse monoclonal anti-G3BP1BD Biosciences611126; RRID: AB_398437(1:1000)
Antibodyrabbit polyclonal anti-PABPAbcamab21060; RRID: AB_777008(1:400)
Antibodyrabbit polyclonal anti-eIF4GSanta Cruz
Biotechnology		sc-11373;
RRID: AB_2095750		(1:400)
Antibodyrabbit polyclonal anti-TDP-43Proteintech12892–1-AP; RRID: AB_2200505(1:400)
Antibodymouse monoclonal
anti-phospho-TDP-43 (M01)		Cosmo Bio COTIP-PTD-MO1; RRID: AB_1961900(1:1000)
Antibodyrabbit polyclonal anti-phospho-TDP-43 (P01)Cosmo Bio COTIP-PTD-PO1; RRID: AB_1961899(1:400)
Antibodymouse monoclonal anti-VCPBD Biosciences612183; RRID: AB_399554(1:100)
Antibodyrabbit polyclonal anti-amyloid-oligomer A11Thermo Fisher
Scientific		AHB0052; RRID: AB_2536236(1:100)
Antibodyrabbit polyclonal anti-UbiquitinDakoZ0458;
RRID: AB_2315524		(1:100)
Antibodymouse monoclonal anti-p62Abcamab56416;
RRID: AB_945626		(1:400)
Antibodymouse monoclonal anti-MAP2SigmaM9942;
RRID: AB_477256		(1:400)
Antibodygoat polyclonal
anti-TIA1		Santa Cruz Biotechnologysc-1751; RRID: AB_2201433(1:400)
Antibodymouse monoclonal anti-TIARBD Biosciences610352; RRID: AB_397742(1:400)
Antibodyrabbit polyclonal anti-ataxin 2Proteintech21776–1-AP; RRID: AB_10858483(1:400)
Antibodygoat polyclonal
anti-eIF3η		Santa Cruz
Biotechnology		sc-16377;
RRID: AB_671941		(1:400)
Antibodyrabbit polyclonal anti-
GLE1		Abcamab96007; RRID: AB_10678755(1:400)
Antibodyrabbit polyclonal anti-
FUS		Bethyl
Laboratories		A300-302A; RRID: AB_309445(1:400)
Antibodyrabbit polyclonal anti-
OPTN		Proteintech10837–1-AP; RRID: AB_2156665(1:400)
Commercial
assay or kit		FuGENE 6PromegaE2691
Commercial assay or kitNEBuilder HiFi
DNA Assembly
Master Mix kit		NEBE2621
Commercial assay or kitQ5 site-directed
mutagenesis		NEBE0054
Commercial assay or kitRNA 3′ End
Biotinylation Kit		Pierce20160
Commercial assay or kitCellTiter-Glo 2.0 assay kitPromegaG9242
Chemical compound, drugISRIBSigmaSML0843200 nM
Chemical compound, drugcycloheximideSigmaC4859100 µg/ml
Chemical compound, drugsodium arseniteSigma35000–1 L-R0.5 mM
Chemical compound, drughygromycin BThermo Fisher
Scientific		10687010200 μg/ml
Chemical compound, drugdoxycycline hyclateSigma-AldrichD98911 µg/ml
Chemical compound, drugpuromycinThermo Fisher
Scientific		A11138031 µg/ml
Software, algorithmImageJNIHhttps://imagej.nih.gov/ij/, RRID:SCR_003073
Software, algorithmGraphPad PrismGraphPad
Software Inc		http://www.graphpad.com/
scientific-software/prism/
RRID:SCR_002798
Software, algorithmSlideBook 6Intelligent Imaging Innovationshttps://www.intelligent-imaging.com/slidebook.php
RRID:SCR_014300
Software, algorithmImage StudioLI-CORhttps://www.licor.com/bio/products/software/image_studio_lite/?utm_source=BIO+Blog&utm_medium=28Aug13post&utm_content=ISLite1&utm_campaign=ISLite, RRID: SCR_014211
RRID:SCR_015795
Software, algorithmLAS X SoftwareLeicahttps://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp8/
RRID:SCR_013673

Additional files

Transparent reporting form
https://doi.org/10.7554/eLife.39578.019

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