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. 2015 Aug 4;4:e06807. doi: 10.7554/eLife.06807

Figure 6. Stress granule assembly is redundant and highly adaptable.

(A) Using a genetically encoded particle to study dynamic interactions in living yeast cells. Left: a fragment of the viral capsid protein μNS comprising the 250 C-terminal amino acids forms self-assembling particles in yeast cells. The particles were visualized using an N-terminal sfGFP tag. Right: interaction assay. Fusion of a protein X to sfGFP-tagged μNS particles allows interaction studies with a mCherry-tagged protein Y. (B) μNS particles carrying mutant luciferase on the surface interact with endogenous mCherry-tagged Hsp104. Cells were observed before and after a 10-min heat shock at 46°C. White lines indicate the cell boundaries. Scale bars: 5 µm. Also see related Figure 6—figure supplements 1, 2. (C) Same as (B), except that cells were used in which mCherry-tagged Pub1 was expressed from the endogenous locus. Only one representative μNS particle is shown at high magnification. Note that Pub1 only interacts with luciferase in cells exposed to robust heat stress. Also see related Figure 6—figure supplements 3–5. (D) Same as (C), except that mCherry-tagged Nrp1 was mildly overexpressed from a plasmid carrying an ADH1 promoter. Note that Nrp1 interacts with luciferase already in unstressed cells, and that the amount of Nrp1 accumulating on the particle is strongly increased upon heat stress. (E) Same as (D), except that the prion-like domain (PLD) of Nrp1 (Nrp1PLD) or a deletion mutant lacking the PLD (Nrp1ΔPLD) was observed at 25˚C. (F) The cellular chaperone machinery prevents interactions between misfolded proteins and stress granule components. Cells expressing Nrp1-GFP from the endogenous locus and mCherry-tagged mutated luciferase from a plasmid were exposed to a 10-min heat shock at 42°C or 46°C. The cells in the bottom panel were exposed to 3 mM guanidinium hydrochloride (GdnHCl) to inhibit Hsp104. Also see related Figure 6—figure supplement 6. (G) Same as (C), except that the temperature was increased slowly from 25°C to 46°C (preconditioning). Note that preconditioning prevents co-assembly of stress granules and misfolded proteins. Also see related Figure 6—figure supplement 7. (H) PLDs mediate interactions only when present in high local concentrations. Yeast cells were transformed with plasmids for the expression of GFP-tagged wild-type Nrp1 or deletion mutants lacking the RNA-binding domain (RBD) (Nrp1PLD) or PLD domain (Nrp1ΔPLD). The resulting cells were exposed to heat shock. (I) Upon heat shock, stress granules form on µNS particles that present Nrp1 on the surface. Same conditions as (C) and (D). Also see related Figure 6—figure supplements 8–9. (J) Same as (I), except that mutants lacking the RBD (Nrp1PLD) or PLD domain (Nrp1ΔPLD) were presented on the particle. Note that both mutants are able to recruit full-length Nrp1 at 25˚C. Also see related Figure 6—figure supplement 10–12.

DOI: http://dx.doi.org/10.7554/eLife.06807.034

Figure 6.

Figure 6—figure supplement 1. µNS particles do not interact with Hsp104 or Ssa1.

Figure 6—figure supplement 1.

Cells expressing tdimer2-tagged Hsp104 and Ssa1 from the endogenous locus were observed before and after heat shock at 46°C. White lines indicate the cell boundaries. Scale bars: 5 µm.
Figure 6—figure supplement 2. sfGFP-µNS particles carrying mutant luciferase or Ubc9ts on the surface interact with Hsp104 and Ssa1.

Figure 6—figure supplement 2.

Cells expressing tdimer2-tagged Hsp104 and Ssa1 from the endogenous locus were observed before and after heat shock at 46°C. Note that Ubc9ts is already interacting with chaperones in unstressed cells, suggesting that a fraction of Ubc9ts is already misfolded under normal conditions. White lines indicate the cell boundaries. Scale bars: 5 µm.
Figure 6—figure supplement 3. Misfolded proteins can nucleate stress granule formation under robust heat shock conditions.

Figure 6—figure supplement 3.

Same as Figure 6—figure supplement 2, except that cells were used in which mCherry-tagged Pab1 was expressed from the endogenous locus. Note that Pab1 only interacts with luciferase in cells that are exposed to robust heat stress. For presentation purposes, only one representative μNS particles is shown at high magnification for each experiment.
Figure 6—figure supplement 4. Control experiment showing that µNS particles do not interact with Pub1 or Pab1.

Figure 6—figure supplement 4.

Same as Figure 6—figure supplement 1, except that strains were used that expressed mCherry-tagged Pub1 or Pab1 from the endogenous locus. For presentation purposes, only one representative μNS particles is shown at high magnification for each experiment.
Figure 6—figure supplement 5. Misfolded proteins do not nucleate stress granule formation in glucose-deprived cells.

Figure 6—figure supplement 5.

Same as Figure 6—figure supplement 2, except that strains were used that expressed mCherry-tagged Pub1 or Pab1 from the endogenous locus, and stress was induced by removing glucose from the medium for 1 hr. For presentation purposes, only one representative μNS particles is shown at high magnification for each experiment.
Figure 6—figure supplement 6. Chemical inhibition of Hsp104 leads to co-aggregation of misfolded proteins and stress granule components even under mild heat shock conditions.

Figure 6—figure supplement 6.

Yeast cells expressing Nrp1-GFP from the endogenous locus and mCherry-tagged mutated Ubc9ts from a plasmid were exposed to heat shock at 42°C or 46°C. The cells in the bottom panel were exposed to 3 mM GdnHCl to inhibit Hsp104. White lines indicate the cell boundaries. Scale bars: 5 µm.
Figure 6—figure supplement 7. Misfolded proteins and stress granule components do not co-aggregate in preconditioned cells.

Figure 6—figure supplement 7.

Same as Figure 6—figure supplement 3, except that the temperature was increased slowly from 25°C to 46°C (preconditioning). For presentation purposes, only one representative μNS particles is shown at high magnification for each experiment.
Figure 6—figure supplement 8. Control experiment showing that Nrp1-µNS particles do not interact with Hsp104.

Figure 6—figure supplement 8.

A strain expressing tdimer2-tagged Hsp104 from the endogenous promoter was transformed with a construct for the expression of Nrp1-sfGFP-μNS. Note that particle-presented Nrp1 is not recognized by Hsp104 at 25˚C, indicating that it is not misfolded on the particle surface. White lines indicate the cell boundaries. Scale bar: 5 µm.
Figure 6—figure supplement 9. Nrp1 can nucleate stress granules upon glucose starvation stress.

Figure 6—figure supplement 9.

Same as Figure 6—figure supplement 8, except that strains were used that expressed mCherry-tagged Nrp1 and Pub1 and cells were exposed to glucose starvation for 1 hr. For presentation purposes, only one representative μNS particles is shown at high magnification for each experiment.
Figure 6—figure supplement 10. Same as Figure 6—figure supplement 8 except that mutants lacking the RBD (Nrp1PLD) or PLD domain (Nrp1ΔPLD) were presented on the particle.

Figure 6—figure supplement 10.

Note that both deletion mutants are not recognized by Hsp104 at 25˚C, suggesting that they are folded and accessible on the particle surface. White lines indicate the cell boundaries. Scale bars: 5 µm.
Figure 6—figure supplement 11. The PLD as well as the RBD of Nrp1 can nucleate stress granule formation.

Figure 6—figure supplement 11.

Same as Figure 6—figure supplement 10, except that a strain was used in which mCherry-tagged Pub1 was expressed from the endogenous locus and the cells were exposed to a heat shock at 46°C. For presentation purposes, only one representative μNS particles is shown at high magnification for each experiment.
Figure 6—figure supplement 12. The PLD or RBD of Nrp1 can nucleate stress granule formation through heterotypic interactions.

Figure 6—figure supplement 12.

A Pub1-mCherry-expressing strain lacking Nrp1 (Δnrp1) was transformed with a construct for expression of GFP-µNS fused to full-length Nrp1, Nrp1PLD, or Nrp1RBD. The cells were exposed to a heat shock at 46°C. For presentation purposes, only one representative µNS particle is shown at high magnification.