Supplemental Figure 1
1a. A morphological biomarker of aging: vacuolar structures accumulate in the body with increasing age.
Animals of indicated age (day 0 is the day the egg is laid) were photographed using Nomarski DIC optics; lateral mid-body view is presented, anterior to left, dorsal up. Arrows highlight prominent vacuolar structures that superficially resemble neurons undergoing necrosis, but may actually represent yolk/lipid inclusions.
1b.
Age-associated behavioral decline occurs with variable onset within a population.
Animals were scored at days indicated as A (green boxes, no fill), demonstrable spontaneous movement and significant sinsusoidal locomotion in response to prodding; B (yellow), little spontaneous locomotion and modest nonsinusoidal movement in response to prodding; C (red), immobile with only head movement or twitch in response to prodding; D (blue) dead
—unresponsive to prodding, no head or tail movement, no evidence of pharyngeal pumping. We reevaluated individuals every 1-3 days and reclassified into A, B and C categories repeatedly until all animals had died. Ratio is calculated as the remaining number of animals in a given class (A, B, or C) counted on the day indicated over the total number of animals present at the start of the experiment (n). This definition of ratio also pertains to the count of dead worms; thus, the dead worm count on a given day

is not inclusive of counts of dead worms from previous days. Similar results were obtained in 5 independent trials.
1c. Statistical analysis of life expectancy predicted by behavioral class. In order to determine whether behavioral class is a predictor of life expectancy, independent of chronological age, data was analyzed from representative experiments containing large sample numbers. To hold chronological age constant, life expectancies were determined for Class A, Class B and Class C animals from single day time points.
Mean life expectancy values and standard deviations were obtained for each class.
Data sets from different classes were compared using unpaired t-tests; p-values are indicated. In most cases when all three classes are present, life expectancies were significantly different between the classes. In later stages of life, when the majority of animals are Class B or Class C, the statistical differences between Class A and
Class B animals diminish. Our results indicate that behavioral class is a better predictor of life expectancy than chronological age.
Supplemental Figure 2
2a. High resolution fluorescence microscopy examination (1000x) revealed a striking change in the distribution of the nuclear-localized GFP fluorescence beginning in mid-life.
Montages were prepared with 20 randomly-selected images of bodywall muscle cell nuclei from nematodes transgenic for a nuclear-localized
GFP reporter, p myo-3
GFP/NLS. Starting at day 7, we detected small, dark patches appearing within many muscle nuclei, and we detected a loss of the circular shape

of the nuclei and nucleoli. Also, we observe an increase in nucleolar size relative to nuclear size. The degree of mottling progressively increases over the lifespan, so that later in life GFP fluorescence appears even more dispersed, finally even acquiring a completely fragmented appearance at some of the latest time points.
2b. A correlation of degree of nuclear patching with locomotory class (A, B, C).
We prepared montages of 20 randomly selected photographs of nuclei from Class A,
B, or C animals and asked naive subjects to order the three montages according to increasing severity of patching in the collection. 16/20 test subjects placed the montages in A, B, C order and 20/20 picked the Class A montage as first, confirming our impression of progressive decline. This suggests the nuclear status could influence locomotory ability and may reflect health of the muscle. Although the trend is clear, it is important to note that at most of the mid-life and late-life time points
(days 9-20), we find that GFP-reported nuclear changes in different muscle cells from individual animals exhibited variations in morphology ranging from the nonmottled to a severely mottled phenotype, indicating that stochastic factors influence age-related nuclear changes on a cell-by cell basis .
2c. age-1(hx546) mutant nematodes exhibit a delay in the progressive mottling of muscle cell nuclei. We prepared montages of 20 randomly selected GFP-labeled nuclei from age-1(hx546) mutant nematodes. While age-1(hx546) nuclei do exhibit similar age-related changes in GFP-labeled nuclear morphologies, there is a delay in this progression at mid-life time points (see Suppl. Fig. 2d).

2d.
Results of a blind survey comparing age-1(hx546) muscle cell nuclear morphology to wild type muscle cell nuclear morphology.
Individuals were asked to match unlabeled montages for each time point from age-1(hx546) mutants to a series of montages from wild type worms that included all 6 timepoints (Day 7,
9, 11, 14, 16, 18), randomly placed. 60 different surveys were performed.
Montages from age-1(hx546) days 9, 11 and 14 were consistently matched to earlier wild type time point montages. For later time points after day 14, age-1(hx546) montages were matched mostly with the identical wild type time point. At the latest time points (day 18), the extensive and variable mottling of nuclear morphologies yielded a wider variation in survey time point matches. Each bar graph represents a single time point for age-1(hx546) .
Supplemental Figure 3
A GFP-reporter for the C. elegans integrin beta1 subunit reveals no changes in muscle dense body morphology and positioning with age. The transgenic nematode line rhIs2 expresses a bPAT-3 integrin protein fused to a GFP reporter
(Plenefisch et al., 2000. Development 127, 1197-1207). This construct highlights integrin attachment sites, including dense bodies and M-lines. No changes are observed in dense bodies from young (Day 5) and old (Day 18) nematodes.
Supplemental Figure 4

a, b, Hypodermal nuclei as visualized by a nuclearly-localized p col-12
NLS-GFP reporter fusion. Arrows indicate representative nuclei ( a , day 5; b , day 16, Class C). Note prominent nucleolus and roughened appearance in day 16 animal. c, d, Lipid inclusions in intestinal cells of ( c ) young (day 4) and ( d ) old (day 18, Class A) animals. Lipids indicated by arrows; V, microvilli; Y, extracellular yolk protein
(confirmed by immuno-EM); *, a detached muscle fragment surrounded by yolk.
Scale bars indicate 5 microns.
Supplemental Figure 5
5a. Enhanced autophagy evident in intestinal cell of aged nematode.
Electron micrograph of an intestinal cell exhibiting few intact organelles, a loss of chromatin, an enlarged nucleus (N) and nucleolus (Nu) and multiple autophagic vacuoles containing internalized, membrane bound structures (
*
). Note the still intact intestinal lumen (Lu). Scale bar indicates 1 micron.
5b. Early stages of necrotic cell death evident in intestinal cell of aged nematode.
Electron micrograph showing the edge of an intestinal filled with multiple lipid inclusions (L). Black arrows indicate localized breaks in the plasma membrane.
White arrows indicate regions of increased electron density, indicative of areas of protein degradation, progressively moving inward from the plasma membrane breakpoints. Scale bar indicates 1 micron.

5c. More advanced stages of necrotic cell death are evident in a hypodermal cell of an aged nematode. Electron micrograph showing breakage of the plasma membrane (indicated by black arrow) of a hypodermal cell. Pseudocoelomic fluid outside cell (P) is in continuity with cytoplasm through the break. A clearing of the black, electron dense regions of protein degradation is occurring. Voids created by the clearing indicated by asterisks. Organelles no longer can be detected, while only lipid inclusions (L) remain. A neighboring excretory canal cell (excr) is healthy and unaffected. Remnants of granulated hypodermal cytoplasm remain at upper right. Scale bar indicates 1 micron.
5d.
Necrotic lysis of a hypodermal cell of an aged nematode. Electron micrograph of a severe break of the plasma membrane of a hypodermal cell (black arrow).
Extracellular space (ECS) and intracellular space (ICS) are in continuity through the break. Electron dense regions of protein degradation are completely cleared leaving a large void area, almost indistinguishable from the extracellular space. Large lipid inclusions (L) are seen bordering the region, within the hypodermal cell. Scale bar indicates 1 micron.
5e. Degrading nucleus of a hypodermal cell of an aged nematode exhibits hallmarks of apoptotic cell death. Within a misshapen, shrunken nucleus (N) and alongside a misshapen and enlarged nucleolus (Nu), chromatin clumping (cc) can be observed. In the condensed cytoplasmic region (cyt), the endoplasmic reticulum

(ER) and mitochondria (M) still are present. The presence of such apoptotically dying cells is rare in aged worms. Scale bar indicates 1 micron.
5f. Infiltrate in pseudocoelomic space of aged nematode identified as yolk protein by immuno-electron microscopy. Antibodies against the yolk protein YP170 are labeled by immunogold particles (white arrows) in an electron micrograph of the electron dense material filling the body cavity of an aged worm. Such extracellular deposits are very extensive in aged worms. Background label over cytoplasm (lower portion of panel) is very low. Scale bar indicates 0.5 microns.