Supporting Online Material for
PROTEASOMAL REGULATION OF THE HYPOXIC RESPONSE MODULATES AGING IN C. ELEGANS
Ranjana Mehta, Katherine A. Steinkraus, George L. Sutphin, Fresnida J. Ramos, Lara S. Shamieh, Alexander Huh, Christina Davis, Devon Chandler-Brown, Matt Kaeberlein*
*Corresponding author: Matt Kaeberlein Ph: (206) 543−4849 Fax:(206) 543−3644 kaeber/at/u.washington.edu
This file includes
Materials and Methods
Figs. S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12
Tables S1, S2, S3, S4, S5, S6
Materials and Methods
Strains and growth conditions. Standard procedures for C. elegans strain maintenance and manipulation were used, as previously described (1−3). Except for RNAi experiments, all experimental procedures were performed on animals fed UV-killed E. coli OP50 from hatching. Experimental animals were maintained on solid Nematode Growth Medium (NGM) supplemented with 50 μg/ml ampicillin. All experiments were conducted on animals maintained throughout life at 20°C except when being examined by microscopy, unless otherwise noted in the text. Nematode strains used in this study are described in Table S3. For RNAi experiments, animals were maintained on RNAi feeding strains. RNAi plates consisted of NGM supplemented with 1mM β-D-isothiogalactopyranoside (IPTG) and 25 μg/ml carbenicillin. Unless otherwise indicated, worms were raised on RNAi bacteria from hatching. RNAi clones were verified by sequencing the region of the RNAi plasmid expressing the double-stranded RNA after purification from the corresponding bacterial clone and by phenotypic analysis of animals maintained on the RNAi bacteria. Sequence verified RNAi clones used in this study are listed in Table S4. DNA sequencing results for hif-1 and vhl-1 RNAi clone inserts are provided in Table S5. For hypoxic growth, animals were kept in a hypoxia chamber at 0.5% oxygen. Oxygen levels were held constant by addition of nitrogen, as needed, using a Biospherix (Lacona, NY) ProOx sensor/controller.
Quantification of life span and proteotoxicity-induced paralysis. . Life span analyses and bacterial deprivation (BD) experiments were carried out as previously described (1−3). Statistical analysis and replication of life span experiments is provided in Table S6. The paralysis of worms expressing polyglutamine (Q35YFP) or amyloid beta (Aβ) was determined by visual analysis as described (4). Worms were scored as paralyzed if they were unable to make forward progress on the NGM surface in response to both plate-tapping and tail-prodding. If paralyzed, worms were scored as alive if nose-tapping resulted in nose movement. If nose movement was not observed, worms were scored as dead. No animals were censored from life span experiments. Animals that foraged off the surface of the plate during the course of the experiment were not considered.
Fluorescence microscopy was performed using a Zeiss SteREO Lumar.V12 microscope as previously described (4
). Briefly, the worms were paralyzed with 25mM sodium azide and placed on a Teflon printed 8-well glass slide with 6mm well diameter. The DAPI filter (excitation filter centered at 365 nm and 445/50 nm emission band-pass filter) was used for imaging of age-pigments. The Image J 1.341.5.0_07 software (available from NIH website http://rsb.info.nih.gov/ij/java
) was employed for calculating the integrated density of the auto-fluorescent age-pigments in worms.
Quantification of DAF-16::GFP puncta: Eggs were prepared from gravid DAF-16::GFP adult worms and placed on NGM supplemented with OP50, EV, or RNAi bacteria. Animals were grown at 20°C for 3 days and then imaged using a Zeiss SteREO Lumar.V12 microscope as previously described (4). For the transient heat shock experiment, animals were exposed to 37°C for 90 minutes immediately prior to imaging. Fluorescence microscopy was performed. Animals were paralyzed with 25mM sodium azide and placed on a teflon printed 8-well glass slide with 6mm well diameter (Electron Microscopy Sciences). The GFP filter (470/40 excitation band-pass filter and 525/50 emission band pass filter) was employed for imaging the DAF-16::GFP worms at 150X magnification with exposure time of 40 ms for bright field and 500 ms for GFP filter. The Image J 1.341.5.0_07 software was employed for inverting the images and converting them to 32-bit format and then manually counting the puncta for all the worms.
Quantification of LGG1-GFP puncta. Adult LGG-1::GFP animals were allowed to lay eggs on RNAi or EV bacteria then removed. Hatchlings were allowed to develop until L3 then imaged using an Axiocam HRm camera on an Axiovert 200m inverted fluorescence microscope at 1000x. The image area was processed using Adobe Photoshop and Axiovision Rel. 4.6 software and foci were counted manually.
Determination of brood size. Age synchronized animals were obtained from a 2 hour timed egg laying and allowed to develop at 20°C. At L4, individual animals were transferred onto NGM plates (one worm per plate) and allowed to lay eggs overnight. On subsequent days each adult was transferred onto a fresh NGM plate and allowed to lay eggs overnight until no eggs were produced. The number of viable progeny produced each day was scored by counting hatched larvae after 48 hours.
Quantification of pharyngeal pumping. Age synchronized animals were obtained from a 2 hour timed egg laying and allowed to develop at 20°C. Video recordings of pharyngeal pumping rate for individual animals were obtained by attaching a Canon Power shot S3IS digital camera to the eye-piece of Zeiss SteREO Lumar.V12 microscope and recording pharyngeal pumping at 150X magnification for 1 minute. To quantify pumping rate, the videos were viewed at slow speed in Windows Media Player.
Statistical analysis. A Wilcoxon Rank-Sum test (MATLAB ‘ranksum’ function) was used to generate p-values to determine statistical significance for life span and paralysis assays. The mean life spans and paralysis, number of animals, number of replicate experiments, and p-values are provided in Tables S1 and S6. A 2-tailed Student's T-test was performed using the TTEST function in Microsoft Excel to calculate p-values for brood size, age-pigment accumulation, LGG-1::GFP foci, DAF-16::GFP foci, and pharyngeal pumping rate assays.
Figure S1. Model for regulation of longevity and proteotoxicity by VHL-1 and HF-1. Reduced vhl-1 activity leads to increased life span and enhanced resistance to proteotoxicity due to reduced degradation of HIF-1 and increased activity of HIF-1 target genes, including hypoxic response genes. VHL-1 and HIF-1 act to modulate aging and protein homeostasis in a pathway that is distinct from both dietary restriction and insulin/IGF-1-like signaling, but which may share a core set of similar or shared downstream target genes.
Figure S2. Proteasome inhibition reduces resistance to polyglutamine toxicity in C. elegans. Polyglutamine-induced paralysis is accelerated in Q35YFP animals upon treatment with the proteasome inhibitors lactacystin (p=0.02) or MG 132 (p<1×10−5).
Figure S3. Cullin complex core components promote resistance to polyglutamine toxicity. (A) Cullin E3 ubiquitin ligases generally consist of a cullin protein (e.g. cul-1, cul-2), an adaptor protein (e.g. skr-1), a RING protein (e.g. rbx-1) and a substrate recognition subunit (SRS). This complex promotes the transfer of ubiquitin (Ub) from the E2 to the substrate target protein. RNAi knock-down of (B) cul-1(p=1.0×10−4), cul-2 (p=2.5×10−3), (C) rbx-1 (p<1×10−5), or skr-1 (p<1×10−5) significantly accelerates polyglutamine-induced paralysis in Q35YFP animals.
Figure S4. Regulation of hypoxic response genes by VHL-1 and EGL-9. VHL-1 and EGL-9 repress HIF-1 under normoxic conditions by promoting ubiquitination and degradation of HIF-1. Under hypoxic conditions ubiquitination of HIF-1 is repressed, leading to persistence of HIF-1 and increased expression of HIF-1 regulated hypoxic response genes.
Figure S5. Mutation of vhl-1 reduces accumulation of auto-fluorescent age-pigments. Relative to N2, vhl-1(ok161) mutants accumulate significantly less auto-fluorescent age pigments. This phenotype is dependent on hif-1 . Five representative 15 day old animals are shown for each genotype.
Figure S6. Hypoxia reduces fecundity in C. elegans. (A) N2 animals were maintained under either normoxic or hypoxic (0.5% oxygen) conditions beginning at L4, and the number of progeny produced per animal was quantified each day. (B) Mean brood size per animal was significantly reduced by hypoxia. At least 5 animals were examined for each condition in three replicate experiments. Brood size was reduced in each replicate (T-test p-value < 1× 10−5 in each case). Pooled data is shown. Pooled t-test p-value < 1× 10−5.
Figure S7. Exposure to hypoxia reduces fecundity in reproductive adults. N2 animals were allowed to hatch and reach reproductive maturity under normoxic conditions. After the first day of reproductive maturity, animals were transferred to fresh plates that were maintained under either normoxic or hypoxic (0.5% oxygen) conditions and the number of progeny produced per animal was quantified. (A) Fecundity was significantly reduced by hypoxia on the 2nd and 3rd day of adulthood (1st and 2nd day following transfer). (B) Total fecundity from day 2−5 of adulthood was also reduced by hypoxia. T-test p-value was less than 1× 10−5 for each comparison. 6 animals were examined for each condition. Error bars are standard error.
Figure S8. Exposure to hypoxia increases life span. N2 animals were allowed to hatch and reach L4 under normoxic conditions. At the L4 stage, animals were transferred to fresh plates that were maintained under either normoxic or hypoxic (0.5% oxygen) conditions. Animals subjected to transient hypoxia were returned to normoxic conditions after 24 hours (blue triangles). Adult hypoxia refers to animals maintained under hypoxic conditions until death. Transient (p=0.001) or continuous (p=0.0002) hypoxia resulted in a significant increase in life span, relative to animals kept in normoxia.
Figure S9. Knock-down of vhl-1 or hypoxic growth do not induced autophagy. Autophagy can be monitored by the presence of LGG-1::GFP puncta, which are known to increase in response to DR. No change in LGG-1::GFP puncta was observed for vhl-1(RNAi) or hypoxic growth (hyp, 0.5% oxygen). Representative images are shown.
Figure S10. Knock-down of vhl-1 does not cause a significant relocalization of DAF-16 to the nucleus. Representative images of animals expressing a DAF-16 GFP fusion protein. Relative to animals maintained on empty vector (EV) bacterial, vhl-1(RNAi) failed to increase the number of nuclear DAF-16 foci. Daf-2(RNAi) or transient heat shock induced significant relocalization of DAF-16 to the nucleus (arrows).
Figure S11. Knock-down of dafl6 does not suppress the reduced auto-fluorescence associated with deletion of vhl-1. Deletion of vhl-1 significantly reduces auto-fluorescence in 3 day old adult animals, relative to N2 controls fed empty vector (EV) bacteria or daf-16(RNAi). RNAi knock-down of hif-1 fully suppresses the reduced auto-fluorescence of vhl-1(ok161) animals. Representative images of individual animals are shown.
Figure S12. VHL-1 promotes longevity by a mechanism distinct from inuslin/IGF-1-like signaling. (A) RNAi knock-down of daf-16 does not block life span extension from deletion of vhl-1 (see ), but is sufficient to prevent life span extension from mutation of daf-2. (B) RNAi knock-down of vhl-1 significantly increases life span of daf-16(mu86) animals (p=5.2 × 10−4).
Table S1. E3 ligase-related RNAi clones tested for their effect on resistance to polyglutamine stress. Q35YFP animals were treated with the following RNAi clones beginning at the L4 stage of development and paralysis was monitored as a function of age. Each row represents a single experiment. The genomic RNAi library from which the clone was derived is noted next to the gene name as either “V” (Vidal library) or “A” (Ahringer library). The day of adulthood at which 50% of the animals became paralyzed is shown for the RNAi-treated animals (p50 RNAi) and for experiment-matched animals grown on empty vector bacteria (p50 Control). The number of animals in each group is shown in parentheses (N). If less than 50% of the animals were paralyzed at the last day of analysis, p50 is noted as >14.
Table S2. Dauer formation at 20°C and 25°C. Gravid adult animals were allowed to lay eggs at the designated temperature then removed. Following hatching, animals were maintained at the designated temperature. At least 5 plates with >100 larval animals per plate were observed for each genotype at each temperature.
Table S3. C. elegans strains used in this study.
Table S4. Verified RNAi clones used in life span and paralysis assays. RNAi clones used to knock-down gene expression for life span and paralysis experiments were from the ORF RNAi libraries generated by the Ahringer (5) and Vidal (6) labs, except for the daf-2 RNAi clone, which was a gift from J. McElwee and has been previously described (4). The presence of the appropriate gene sequence in each plasmid was independently verified.
Table S5. DNA sequence of the RNAi clone inserts used to target hif-1 and vhl-1 in this study.
Table S6. Life span data for replicate experiments in this manuscript. Individual experiments are shown. Mean life spans ± standard error are shown for treatment and experiment-matched controls. Number of animals in each group is shown in parentheses (N). The p-value is the result of a Wilcoxon Rank-Sum test. *Life span was performed in the RNAi sensitive rrf-3(pk1426) background. Otherwise, control life span refers to N2.
Supporting References and Notes
S1. T. L. Kaeberlein et al., Aging Cell 5, 487 (Dec, 2006).
S2. E. D. Smith et al., BMC Dev Biol 8, 49 (May 5, 2008).
S3. G. L. Sutphin, M. Kaeberlein, Exp Gerontol 43, 130 (Mar, 2008).
S4. K. A. Steinkraus et al., Aging Cell, (Feb 20, 2008).
S5. R. S. Kamath et al., Nature 421, 231 (Jan 16, 2003).
S6. J. F. Rual et al., Genome research 14, 2162 (Oct, 2004).