Eukaryotic stress granules are cleared by autophagy and Cdc48/VCP function

Abstract

Stress granules and P bodies are conserved cytoplasmic aggregates of nontranslating messenger ribonucleoprotein complexes (mRNPs) implicated in the regulation of mRNA translation and decay and are related to RNP granules in embryos, neurons, and pathological inclusions in some degenerative diseases. Using baker's yeast, 125 genes were identified in a genetic screen that affected the dynamics of P bodies and/or stress granules. Analyses of such mutants, including CDC48 alleles, provide evidence that stress granules can be targeted to the vacuole by autophagy, in a process termed granulophagy. Moreover, stress granule clearance in mammalian cells is reduced by inhibition of autophagy or by depletion or pathogenic mutations in valosin-containing protein (VCP), the human ortholog of CDC48. Because mutations in VCP predispose humans to amyotrophic lateral sclerosis, frontotemporal lobar degeneration, inclusion body myopathy, and multisystem proteinopathy, this work suggests that autophagic clearance of stress granule related and pathogenic RNP granules that arise in degenerative diseases may be important in reducing their pathology.

Copyright © 2013 Elsevier Inc. All rights reserved.

Figures

Figure 1. Overview of screen for genes affecting stress granule and P-body assembly

A.) Typical screen phenotypes (log. growth). Pab1-GFP and Edc3-mCh serve as stress granule and P-body markers respectively. Examples of P-body localized stress granules (rtc2Δ, cho2Δ, rlf2Δ), increased P-bodies (pgd1Δ) and decreased P-bodies (rp28bΔ) are shown. B.) 125 screen hits categorized by GO-terms. C.) Network analysis of known physical and genetic connections of screen hits. See also Figure S3 and Table S1.

Figure 2. Several autophagy and Cdc48-complex factors exhibit stress granule phenotypes

A.) Logarithmically growing strains lacking autophagy factors exhibit accumulation and/or mis-localization of stress granule and P-body proteins. B.) W303 strain background ts alleles of Cdc48, Ufd1 and Npl4 exhibit increased stress granules relative to WT at the non-permissive temperature. Numbers indicate the average number of stress granule foci/cell in each strain, based on 3 independent experiments. See also Figure S4 and Table S5.

Figure 3. Atg15-induced IVCs are decreased by upstream mutations in autophagy pathways and in Cdc48

A.) BY4741-background strains in early stationary phase. IVCs present in an atg15Δ strain are reduced by secondary mutations in autophagy factors acting at/prior to autophagosome-vacuolar membrane fusion. B.) Quantitation of data in A, mean values based on 3 independent experiments +/− SD. P-values (* = <0.05, ** = < 0.005) relative to atg15Δ indicated. C.) W303 strain background, examined in early stationary phase following 0, 1 or 2hrs of 34°C heatshock. Inactivation of Cdc48 results in decreased IVCs caused by atg15 deletion. D.) Quantitation of data in C, mean values based on a minimum of 3 replicate experiments +/− SD. P-values as above. See also Table S5.

Figure 4. Impairment of cytoplasmic mRNA decay increases accumulation of granule proteins in IVCs

A.) RP840-background strains in early stationary phase. Temperature shifted strains were subject to 1hr at 37°C (lower row). B.) Quantitation of data in A, mean values based on a minimum of 3 replicate experiments +/− SD. P-values (* = atg15Δ indicated. See also Table S5.

Figure 5. Mammalian stress granule clearance is impaired by defective autophagy

A.) atg7−/− MEFs showed a low level of cells with clearly visible stress granules even in the absence of exogenous stressors. B.) Heat shock for 1hr at 43°C resulted in robust stress granule formation in both WT and atg7−/− MEFs. C.) After shift from 43°C back to 37°C stress granules clear within minutes in wild type MEFs, but persist for the duration of the assay (2hrs) in atg7−/− MEFs. Scale bar equals 10μm. D) Quantitation of the data in A-C, mean values based on a minimum of 3 replicate experiments +/− SEM. E) Real time quantitative PCR verifies the expression levels of atg7 in wild type and atg7−/− MEFs. See also Figure S7.

Figure 6. VCP is essential for stress granule clearance

A.) HeLa cells examined by immunofluorescence for the stress granule marker G3BP. Cells treated with non-targeting siRNA show stress granule assembly upon heat shock at 43°C (2hrs) and rapid clearance of stress granules upon return to 37°C. Cells treated with VCP-targeting siRNA show normal stress granule assembly, but these stress granules fail to clear following return to 37°C (2hrs). Scale bar equals 10μm. B.) Quantification of data in A, mean values based on a minimum of 3 replicate experiments +/− SEM. C). and D.) Western blot and quantification of VCP protein levels in cells from A, mean values based on a minimum of 3 replicate experiments +/− SEM. E.) HeLa cells examined by immunofluorescence for the stress granule markers TIAR and eIF3b. Pre-treatment with chemical inhibitors of VCP impairs clearance of stress granules. Cells were treated with vehicle (DMSO) or specific inhibitors Eer1 (4hrs pre-treatment with 8μM Eeyarestatin I, per Wang et al, 2010); DBeQ (3hrs pre-treatment with 10μM DBeQ, per Chou et al, 2011); ML240 (2hrs pre-treatment with 2.5μM ML240, per Chou et al, 2013). Scale bar equal 10μm. F.) Quantification of the data in E, mean values based on a minimum of 3 replicate experiments +/− SEM.

Figure 7. VCP is recruited to stress granules and disease-causing mutations in VCP induce constitutive stress granules that contain the disease protein TDP-43

A.) HeLa cells stained for endogenous VCP and the stress granule marker eIF4G. VCP is recruited to stress granules induced by three distinct stimuli: heat shock at 43°C for 2hrs, incubation with 20μM clotrimazole for 1hr, or incubation with 0.6M sorbitol for 1hr. Scale bar equal 10μm. B.) HeLa cells transfected with plasmid expressing wild type or mutant (A232 and R155H) VCP-GFP and stained for eIF3b and TDP-43. Over-expression of mutant but not wild type VCP results in the formation of constitutive stress granules that contain the disease protein TDP-43. Scale bar equals 10μm.