ATPase-Modulated Stress Granules Contain a Diverse Proteome and Substructure

Abstract

Stress granules are mRNA-protein granules that form when translation initiation is limited, and they are related to pathological granules in various neurodegenerative diseases. Super-resolution microscopy reveals stable substructures, referred to as cores, within stress granules that can be purified. Proteomic analysis of stress granule cores reveals a dense network of protein-protein interactions and links between stress granules and human diseases and identifies ATP-dependent helicases and protein remodelers as conserved stress granule components. ATP is required for stress granule assembly and dynamics. Moreover, multiple ATP-driven machines affect stress granules differently, with the CCT complex inhibiting stress granule assembly, while the MCM and RVB complexes promote stress granule persistence. Our observations suggest that stress granules contain a stable core structure surrounded by a dynamic shell with assembly, disassembly, and transitions between the core and shell modulated by numerous protein and RNA remodeling complexes.

Figure 1. Stress Granules are Stable in Cell Lysates

(A) Pab1-GFP carrying yeast cells and cell lysates from cells ± treatment with NaN₃ and ± treatment with Cycloheximide (CYH) or vanillin. (B) GFP-G3BP U-2 OS cells and cell lysates ± treatment with NaAsO₂, or heat shock for 60min, ± treatment with CYH. All scale bars are 2μm. See also Figures S1, S3.

Figure 2. Super Resolution Microscopy of Mammalian Stress Granules

(A) Comparison of size between granules in vivo and GFP-G3BP foci in lysates. Middle panel shows zoomed inset. Third panel shows foci in lysate. Scale bars are 2μm. (B) STORM images of stress granules in vivo, showing IF for GFP-G3BP (Alexa647-αGFP) and FISH for poly(A+) RNA (Alexa647-oligo(dT). Intensity map represents relative grey scale intensity. Scale bars represent 2μm. (C) 3D STORM image of a stress granule in vivo, showing IF for GFP-G3BP (Alexa647-αGFP). Grey surface represents surface of a stress granule. Cores are shown in green. Scale bar represents 500nm. (D) 3D STORM image of a stress granule in vivo for poly(A+) RNA (Alexa647-oligo(dT). Grey surface represents surface of a stress granule. Cores are shown in yellow. Scale bar represents 500nm. See also Figure S2 and Movie S1.

Figure 3. Mass Spectrometric Analysis of Stress Granule Cores

(A) Dynabeads bound to immunopurified yeast stress granule cores are shown. Pie chart shows GFP-tagged proteins localized to stress granules by overlap with Ded1-mCherry (see Supplemental Experimental Procedures). (B) Stress granule core enriched fraction from GFP-G3BP U-2 OS cells incubated with αGFP antibody, immunopurified using Protein A Dynabeads. Picture shows isolated granule cores bound to Dynabeads. Pie chart shows result of testing the co-localization of 15 putative stress granule proteins with GFP-G3BP by IF. (A) and (B) Scale bars are 2μm. (C) Table showing various conserved ATPases in stress granules. Red box indicates protein present only in the yeast granule proteome, blue indicates protein present only in the mammalian granule proteome and yellow indicates protein is conserved in both proteomes. See also Figure S4 and Tables S1, S2, S3.

Figure 4. Key Findings from the Stress Granule Proteome

(A) Fluorescence microscopy of cells with Ded1-mCherry and Tys1-GFP or Rpb2-GFP after NaN₃ treatment. (B) IF in NaAsO₂ stressed GFP-G3BP U-2 OS cells using antibodies against YARS or FAM120A detected by a Cy5 labeled secondary antibody. Arrowheads within magnified areas indicate examples of overlap. Scale bars are 2μm. (C) Properties of the yeast and mammalian stress granule proteomes. (D) Yeast and mammalian stress granule ‘connectomes’ showing physical interactions amongst stress granule proteins. Red nodes represent known RNA binding proteins. See also Figure S5 and Tables S1, S2.

Figure 5. ATP is Required for Stress Granule Assembly and Dynamicity in vivo

(A) Different times at which 2DG and CCCP were added to these cells are indicated. Scale bars are 5μm. (B) Granules shown prior to, at 0s after and at 145s after photobleaching. Cells were either treated for 60min with NaAsO₂ or with NaAsO₂ with 2DG and CCCP added 30min after the addition of NaAsO₂. Graph shows recovery curves as an average of 15 granules ± standard deviation. Scale bars are 2μm. Bar graph shows average total percentage recovery from 15 granules ± standard deviation. *** p-value <0.0001. See also Figure S6 and Movies S2, S3.

Figure 6. Inhibition of Cct4, Mcm2 or Rvb2 ATPase Activity Affects Stress Granule Assembly/Disassembly

(A) WT or cct4-1 cells carrying Pab1-GFP imaged after shifting to 37°C (60min) either before NaN₃ stress, after NaN₃ treatment for 10min, after NaN₃ treatment for 20min or with 1M KCl for 30min. (B) WT or cct4-1 cells carrying Pab1-GFP imaged prior to stress, after 30min of NaN₃, after 60min of recovery at 37°C. Graph shows mean from 3 experiments ± standard deviation. (C) WT and mcm2-1 yeast cells with Pab1 GFP prior to stress, after stress (30min), and upon recovery from stress. Graph shows mean from 3 experiments ± standard deviation. (D) Same as (C) but for rvb2-1 and the corresponding WT yeast cells. * p-value < 0.05. ** p-value < 0.01. *** p-value < 0.0001. White scale bars indicate 2μm. See also Figure S7.

Figure 7. Model for Stress Granule Assembly and Dynamicity

Dashed lines between mRNPs represent weak physical interactions in the Phase-Separated Shell. Red wavy lines represent strong interactions between Prion-Like Domains. Possible sites of activity of the CCT, RVB and MCM complexes are also shown.