Fig 1.
C. elegans granulins impair organismal fitness and resistance to ER stress.
(A) Wild-type (N2) and pgrn-1(-) animals with and without granulin expression were subjected to ER stress with tunicamycin (5 μg / ml). The fraction developing to L4 stage was quantified (n = 50, 3 biological replicates). (B) Wild-type (N2) and pgrn-1(-) animals with and without C. elegans progranulin over-expression (OE) were subjected to ER stress with tunicamycin (5 μg / ml). The fraction developing to L4 stage was quantified (n = 50, 3 biological replicates). (C) Total worm lysates from synchronized day 1 adult granulin-expressing animals were immunoblotted with an anti-HSP-4/BiP antibody (3 biological replicates). Anti-actin was used as a loading control. (D) Wild-type and pgrn-1(-) animals with and without granulin expression were staged as embryos. Animals were scored for development to L4 stage (n = 50, 12 biological replicates). (E) Measurement of body length at day 1 adulthood (n = 12). (F) Measurement of short-term associative learning (three biological replicates). The glutamate receptor mutant nmr-1(ak4) was used as a positive control. Throughout, error bars show mean ± SEM, one or two-way ANOVA with post-hoc Tukey multiple comparisons test. Comparisons are to wild-type unless otherwise indicated (*P<0.05, ***P<0.001, ****P<0.0001, ns = not significant, wt = wild-type).
Fig 2.
C. elegans progranulin and granulins localize to the endolysosomal compartment.
A translational PGRN-1::RFP reporter (red) co-localizes with (A) Golgi (mannosidase II::GFP) and (B) lysosomes (LMP-1::GFP) in the intestine of L1 stage larvae (anterior to the left). In scavenging coelomocytes, PGRN-1::RFP does not co-localize with (C) Golgi (mannosidase II::GFP), but does co-localize with (D) early, late and recycling endosomes (RME-8::GFP) and (E) lysosomes (LMP-1::GFP) (n = 8 animals per GFP marker). Dashed white lines mark the outline of each coelomocyte cell and white boxes show zoomed insets in Fig 2A-B, scale bar = 5 μm. Shown are representative images from confocal microscopy z-stack sections taken at 0.7 μm. (F-I) Subcellular fractionation of pgrn-1(-) animals (F), and pgrn-1(-) animals with expression of (G) granulin 1, (H) granulin 2, and (I) granulin 3. Whole lysate (WL), cytosol (C), lysosome (L) and endoplasmic reticulum (ER) fractions (10 μg total protein) were immunoblotted with anti-FLAG and anti-LMP-1 antibodies.
Fig 3.
Granulins are produced in an age- and stress-dependent manner and disrupt lysosomal morphology.
(A-B) Western blot of C. elegans PGRN-1::RFP lysates with (A) aging and (B) starvation. Immunoblotting was performed with an anti-granulin 3 antibody. NS = non-specific band. The most prominent cleavage product at ~30kDa was recognized by both granulin 3 and RFP antibodies (see S3E Fig). (C) Subcellular fractionation of pgrn-1(-); PGRN::RFP animals. Whole lysate (WL), cytosol (C), lysosome (L) and endoplasmic reticulum (ER) fractions from fed and starved (70 hours off-food) animals were immunoblotted with anti-granulin 3 and anti-LMP-1 antibodies. The same progranulin full-length and cleavage bands were also identified with an anti-RFP antibody (S3F Fig). Well-fed pgrn-1(-) animals are shown as a control for non-specific bands (NS). (D-G) Representative light and fluorescent confocal images of anterior coelomocyte cells expressing LMP-1::GFP in (D) wild-type, (E) pgrn-1(-), (F) pgrn-1(-); granulin 2(+) and (G) pgrn-1(-); granulin 3(+) animals. Animals were imaged at L4 stage. Scale bars are 10 μm in the wild-type panel and 5 μm in remaining panels. Dashed white lines mark the outline of each coelomocyte cell. Open white arrow heads indicate spherical lysosomes and closed white arrow heads indicate tubular extensions (number of animals with tubular extensions: wt: 2/22, pgrn-1(-): 10/21, pgrn-1(-); granulin 2(+): 7/25, pgrn-1(-); granulin 3(+); 13/24). (H) Lysosomal diameter measurements from anterior coelomocyte cells of L4 stage animals (n = 60). Error bars show mean ± SEM, one-way ANOVA with post-hoc Tukey multiple comparisons test (mean values (μm): wt: 1.57 ± 0.04, pgrn-1(-): 1.37 ± 0.05, pgrn-1(-); granulin 2(+): 1.47 ± 0.05, pgrn-1(-); granulin 3(+): 1.16 ± 0.05). Comparisons are to wild-type unless otherwise indicated (*P<0.05, **P<0.01, ****P<0.0001, wt = wild-type).
Fig 4.
Granulin peptides impair lysosomal protease expression and activity.
Total worm lysates from synchronized day 1 adult granulin-expressing animals were immunoblotted with antibodies recognizing (A) the aspartyl protease ASP-3/CTSD and (B) the cysteine protease CPL-1/CTSL. An anti-actin antibody was used as a loading control. Representative Western blots are shown and data were quantified from 3 independent biological repeats. Enzymatic activity measured in total worm lysates for (C) ASP-3/CTSD and (D) cysteine protease activity for CPR/CTSB and CPL-1/CTSL. CTSB/Li, cathepsin B and L inhibitor. Data were quantified from 3 independent biological repeats. Throughout, values shown are mean ± SEM, one-way ANOVA with Tukey multiple comparisons test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Animals lacking asp-3 continue to have significant protease activity, likely due to other endogenous aspartyl proteases that are inhibited by the pan-aspartyl protease inhibitor, Pepstatin A.
Fig 5.
Computational analysis of granulin sequence across species highlights their distinct electrostatic and hydrophobicity profiles.
(A) Amino acid multiple sequence alignment for C. elegans, Homo sapiens, Mus musculus and Danio rerio (PGRNb) granulin domains using the MAFFT (Multiple Alignment using Fast Fourier Transform) server. Residues matching the granulin consensus sequence are highlighted in cyan. The identity score (%) between all granulin domains and C. elegans granulins 1, 2 and 3 from pairwise sequence alignment using the EMBOSS Needle server is indicated on the right. The granulin domains of different species sharing the highest identity score to C. elegans granulins 1, 2 and 3 are highlighted in square boxes. (B) pH-dependent electrostatic charge scale calculated by propKa 3.1. A 3-color scale is used for the granulin domains, colored from red (negative) to blue (positive) using a percentile scale. (C) Kyte and Doolittle (K&D) hydrophobicity scores for granulin domains of C. elegans. The central regions of granulins 2 and 3 are highlighted with circles since most of these residues have positive K&D scores, which is suggestive of a potential hydrophobic patch.
Fig 6.
Granulins activate the lysosomal CLEAR response and induce nuclear localization of HLH-30/TFEB.
(A) Heat map showing the fold-changes of gene expression in comparisons of day 1 adult animals as indicated. Data from four independent biological replicates are shown (except for granulin 1 where one sample was excluded as a quality control outlier). Significance cut-off was a false discovery rate (FDR) of P<0.05 (up-regulated = red, down-regulated = green). Number of HLH-30/TFEB binding sites identified/total number of DEGs: pgrn-1(-) vs wt: 67/233, pgrn-1(-); granulin 1(+) vs wt: 18/179, pgrn-1(-); granulin 2(+) vs wt: 408/4050, pgrn-1(-); granulin 3(+) vs wt: 1136/2560. See S1–S5 Tables for the complete gene lists. (B) Representative images of wild-type, pgrn-1(-) and pgrn-1(-); granulin 3(+) animals expressing HLH-30::GFP (scale bar = 200 μm). (C) Percentage of animals with nuclear localized HLH-30::GFP (n = 120 animals from 3 biological replicates). Wild-type, pgrn-1(-) and granulin-expressing animals with and without hlh-30 expression were staged as embryos, and animals were scored for (D) development to gravid adult (n = 50, 3 biological replicates), and (E) the number of larvae arresting at L1 and L2 stage (n = 50, 3 biological replicates). Throughout, values shown are mean ± SEM, one-way ANOVA and Tukey multiple comparisons test. Comparisons are to wild-type unless otherwise indicated (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns = not significant). The ~20% larval arrest observed in the pgrn-1(-) strains with wild-type hlh-30 reach significance when compared pairwise with wildtype using a Student’s t-test (*P<0.05). (F) Genetic model for progranulin and granulin function in lysosomal function, protein homeostasis and stress resistance.