Skip to main content
NCBI home page
PLOS One logoLink to PLOS One
. 2024 Jul 9;19(7):e0305396. doi: 10.1371/journal.pone.0305396

Mutations in fibulin-1 and collagen IV suppress the short healthspan of mig-17/ADAMTS mutants in Caenorhabditis elegans

Yukimasa Shibata 1,*, Yijing Huang 1, Moeka Yoshida 1, Kiyoji Nishiwaki 1
Editor: Myeongwoo Lee2
PMCID: PMC11232982  PMID: 38980840

Abstract

The ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family metalloprotease MIG-17 plays a crucial role in the migration of gonadal distal tip cells (DTCs) in Caenorhabditis elegans. MIG-17 is secreted from the body wall muscle cells and localizes to the basement membranes (BMs) of various tissues including the gonadal BM where it regulates DTC migration through its catalytic activity. Missense mutations in the BM protein genes, let-2/collagen IV a2 and fbl-1/fibulin-1, have been identified as suppressors of the gonadal defects observed in mig-17 mutants. Genetic analyses indicate that LET-2 and FBL-1 act downstream of MIG-17 to regulate DTC migration. In addition to the control of DTC migration, MIG-17 also plays a role in healthspan, but not in lifespan. Here, we examined whether let-2 and fbl-1 alleles can suppress the age-related phenotypes of mig-17 mutants. let-2(k196) fully and fbl-1(k201) partly, but not let-2(k193) and fbl-1(k206), suppressed the senescence defects of mig-17. Interestingly, fbl-1(k206), but not fbl-1(k201) or let-2 alleles, exhibited an extended lifespan compared to the wild type when combined with mig-17. These results reveal allele specific interactions between let-2 or fbl-1 and mig-17 in age-related phenotypes, indicating that basement membrane physiology plays an important role in organismal aging.

Introduction

The BMs are complex structures composed of multiple types of molecules. To maintain their integrity, it is necessary to appropriately remove damaged components and incorporate new ones, ensuring proper turnover. However, as animals age, the metabolism of BMs slows down, leading to the accumulation of damage [1]. Collagen levels decrease with age, while spontaneous cross-links increase. Yet, the anti-aging effects of BMs remain poorly understood. In the skin, BM collagen is essential for stem cell maintenance through hemidesmosomes [2]. Loss of collagen is known to impair cell competition, thus contributing to aging. However, the relationship between BMs and the suppression of aging beyond skin stem cells remains unknown.

The ADAMTS family of zinc metalloproteases is a group of enzymes that play important roles in various biological processes by cleaving proteins in the extracellular matrix (ECM) and regulating the structure and function of tissues in the body [3]. Dysregulation of ADAMTS enzymes can contribute to various diseases and disorders, including arthritis, cardiovascular diseases, and cancer [4]. Although the functions of ADAMTS proteases in animal development and pathologies are well studied, their roles in organismal aging and lifespan remain mostly unsolved. In this study, we analyzed the functions of one of the C. elegans ADAMTS enzymes, MIG-17, in aging and lifespan.

MIG-17 is secreted from the body wall muscle cells as a proform and localizes to the BMs of various tissues, including gonad, intestine, body wall muscle, and hypodermis, where it is activated by auto-catalytic removal of its prodomain [5, 6] (Fig 1A and 1B). The protease activity of MIG-17 in the BM of DTCs is sufficient to regulate the directional migration of DTCs to generate the U-shaped gonad arms. In mig-17 mutants, the DTCs meander and stray, resulting in an abnormal gonadal shape [7]. Dominant gain-of-function (gf) mutations in the BM molecules collagen IV α2 chain and fibulin-1 suppress the gonadal defect of the mig-17 mutants [8–10] (Fig 1A). let-2(gf) mutations are amino-acid substitutions either in the triple helical domain or in the C-terminal non-collagenous 1 (NC1) domain (Fig 1A). fbl-1(gf) mutations are amino-acid substitutions found in the second EGF-like motif (Fig 1A). Genetic analyses revealed a regulatory pathway in which MIG-17 recruits and activates FBL-1C (C isoform), which then recruits NID-1 to the BM to control DTC migration. MIG-17 also activates collagen IV to induce nidogen-dependent and -independent pathways to control DTC migration [9]. In addition to its role in in gonadogenesis, MIG-17 also functions in regulating healthy aging. mig-17 mutants exhibit an earlier decline in periodic behaviors, including the defecation cycle and pumping rate, as well as a decrease in motility rate, and show earlier shortening of body length compared to the wild type. [11].

Fig 1. Schematic diagrams of MIG-17, FBL-1C, and LET-2A.

Fig 1

Open in a new tab

(A) The mutation sites of mig-17(k174), let-2(k193, k196), and fbl-1(k201, k206) are indicated. (B) A diagram showing the localization of MIG-17, LET-2, and FBL-1. These proteins are components of BMs.

In the present study, we examined the effects of suppressor mutations let-2 and fbl-1 on the age-dependent phenotypes of mig-17 mutants. We found that let-2 and fbl-1 mutations suppressed the mig-17 defects in an allele-specific manner.

Materials and methods

Strains and genetic analysis

Culture and handling of C. elegans were conducted as described [12]. Worms were cultured at 20°C. The following mutations and transgenes were used in this work: mig-17(k174), fbl-1(k201, k206), let-2 (k193, k196) [7–9].

Microscopy

Gonad migration phenotypes were scored using a DIC images of Nomarski microscope (Axioplan 2; Zeiss). Gonad migration phenotypes were scored using DIC images captured with a Zeiss Axioplan 2 microscope [13].

Analysis of growth rate

The growth rate was analyzed as described [14]. Briefly, newly hatched larvae were synchronized for one hour, grown at 20°C, and the growth rate was assessed based on the stages of vulval development at the L4 stage. A simple invagination occurs in early L4, followed by a Christmas tree-like invagination in mid-L4, and the invagination becomes mostly closed in late L4.

Analysis of body length

Body length was analyzed as previously described [11]. Briefly, young adult animals were collected as day 1 adults for synchronization. Body length was measured by ImageJ software. To measure body length, we traced a line along the body using ImageJ and then measured its length.

Behavioral analyses

Defecation cycle and pumping rate were analyzed as previously described [11]. Briefly, average of five defecation cycles were scored for individual animal. If the defecation cycle exceeded 3 minutes, further measurements were discontinued. Number of pumps during 30 seconds were counted and the average of three independent measurements was calculated for each individual.

Analysis of lifespan

Lifespan was analyzed as previously described [11]. Three independent experiments are performed. Synchronization was performed by collecting L4 animals. Animals were transferred to new OP50 seeded plates for every two days during reproduction period, and every three days during post-reproduction period.

Statistics

Growth rates were analyzed using Fisher’s exact test to compare adults and larvae.

One-way analysis of variance (ANOVA) was conducted using Excel to compare defecation cycle, pumping rate, and body length. If a significant difference was found by ANOVA, Tukey’s multiple comparison test was performed using Excel statistics [15].

The lifespan was analyzed using a log-rank test in R [11].

Results

fbl-1(k201) mutation suppresses growth retardation of mig-17 mutants

We previously reported that dominant gain-of-function mutations in let-2 and fbl-1could suppress the gonadal defects of mig-17 mutants (S1 Fig). We examined whether mig-17 and the suppressor mutations affect the larval growth rate. The growth of C. elegans from hatched larvae to adults involves four larval stages, namely larval stage 1 (L1) to L4, punctuated by molting. We first examined whether MIG-17 is required for normal growth rate. We synchronized the newly hatched larvae for one hour and grew them at 20°C. The growth rate was assessed by monitoring the stages of vulval development at the L4 stage, as described [14]. Our observations revealed that mig-17 mutants exhibited slower growth compared to wild-type animals (Fig 2A–2D). Since the gain-of-function mutations in collagen IV, let-2(k193) and let-2(k196), and fibulin-1, fbl-1(k201) and fbl-1(k206) suppress the gonadal defects of mig-17 mutants [8, 10], we further investigated whether they could also alleviate the growth retardation observed in mig-17 mutants. let-2(k193) and let-2(k196) mutants also displayed growth retardation (WT vs let-2(k196) p = 0.058). These let-2 mutations failed to suppress the slow growth observed in mig-17 mutants. Instead, let-2(k193) seemed to exacerbate the phenotype of mig-17 at 43 hours, as evidenced by the presence of 10% early L4 animals, a feature not observed in mig-17 alone (Fig 2A and 2B). The mutations fbl-1(k201) and fbl-1(k206) did not significantly impact the growth rate, and only fbl-1(k201) exhibited a weak suppression of mig-17 growth retardation at 43 hours (Fig 2C). These results indicate that although both the let-2 and fbl-1 alleles are strong suppressors of the gonadal defects of mig-17, they mostly fail to suppress the growth defect of mig-17.

Fig 2. Suppressors of the gonadal defects of mig-17 fail to suppress the growth defect.

Fig 2

Open in a new tab

Bar graphs represent developmental stages at 43 or 48 hours after hatch. The sample size is indicated at the bottom of each bar graph. Percentages of adult animals and those of L4 animals were compared in 43 and 48 hours, respectively. Black and red asterisks on the top of bar graph indicate p-values for Fisher’s exact test against WT and mig-17(k174), respectively: ***p < 0.005, *p < 0.05.

Suppression of short healthspan in mig-17 mutants

Aged decline in the defecation cycle and pumping rate are often used as indicators of senescence rate in C. elegans [11, 16, 17]. We examined the defecation cycle of wild type and mig-17 mutant animals. The cycle was around 50 seconds at the young adult stage (day 1) and extended to 90 seconds until day 5 in the wild type (Fig 3A–3F). Although mig-17 mutants showed a cycle comparable to the wild type at day 1, it became significantly longer after day 3 compared to the wild type, suggesting that the senescence rate of mig-17 mutants is faster than that of the wild type (Fig 3A–3F) [11]. We then investigated whether the let-2 and fbl-1 mutants could suppress the mig-17 defect. Both let-2 alleles showed a significantly longer defecation cycle compared to the wild type from day 1 to day 5. Interestingly, however, let-2(k196), but not let-2(k193), strongly suppressed the mig-17 defecation decline at day 3 and day 5 (Fig 3A–3C). Thus, mig-17(k174) and let-2(k196) can suppress each other for their defecation defects. fbl-1(k206) alleles showed significantly longer defecation cycles compared to the wild type after day 5 and fbl-1(k201) showed a trend toward suppression of the mig-17 defecation decline at day 5, although it was not statistically significant (t-test:p = 0.069) (Fig 3D–3F).

Fig 3. Suppression of slow defecation cycle in aged mig-17 mutants by let-2(k196).

Fig 3

Open in a new tab

Box and dot plots indicate the defecation cycle. The sample size is 30 animals. p-values against WT is indicate at the bottom of plots. p-values against mig-17(k174) are indicated by red asterisks. One-way ANOVA with Tukey’s multiple comparisons: *p≤ 0.05, **p≤ 0.01.

We examined pharyngeal pumping rates at day 1 and day 9 adults. The pumping rates decreased by 50% in day 9 adults. The pumping rates of mig-17 adults were much slower than those of wild type at day 1 and day 9, suggesting short healthspan of mig-17 animals (Fig 4C and 4D) [11]. Interestingly, let-2(k196), but not let-2(k193), showed a trend toward suppression of the mig-17 pumping rate decline at day 9 (Fig 4A and 4B), although it was not statistically significant. The pumping rate of mig-17(k174) (p = 0.0001) and fbl-1(k206) mig-17(k174) (p = 0.028), but not that of fbl-1(k201); mig-17(k174) (p = 0.105), showed significant differences from that of wild-type animals at day 9 adult (Fig 4C and 4D).

Fig 4. Suppression of slow pumping rate in aged mig-17 mutants by let-2(k196) and fbl-1(k201).

Fig 4

Open in a new tab

Box and dot plots indicate the number of pumping in 30 seconds. Sample size is 20 animals. p-values against WT is indicate at the bottom of plots. p-values against mig-17(k174) are indicated by red asterisks. One-way ANOVA with Tukey’s multiple comparisons: *p≤ 0.05, **p≤ 0.01.

We then examined age-dependent alteration of body length. In wild-type animals, the body length increased until day 3 to day 5 and gradually deceased after day 7 (Fig 5A and 5B). Although mig-17 animals showed body length similar to that of the wild type at day1, their body lengths at day 3 and day 5 were significantly shorter than those of the wild type (Fig 5A and 5B) [11]. The body length of day 1 adults was longer in mig-17; let-2(k196) and mig-17; let-2(k193) double mutants compared to the wild type or mig-17 mutants. At the day3 adult, the body length of mig-17(k174) and let-2(k193) mig-17(k174), but not that of let-2(k196) mig- 17(k174), showed significant differences from those of wild-type animals (Fig 5A). Both fbl-1(k201) and fbl-1(k206) suppressed the short body size of mig-17 at day 1 to day 3 (Fig 5B).

Fig 5. Suppression of body length defects in mig-17 mutants by let-2 and fbl-1.

Fig 5

Open in a new tab

Box and dot plots indicate the body length. Standard deviation is indicated. The sample size is 30 animals. p-values against WT are indicated at the bottom of the plots. Red and blue asterisks on the top of bar indicate p-values against mig-17(k174) and let-2 or fbl-1, respectively. One-way ANOVA with Tukey’s multiple comparisons: *p≤ 0.05, **p≤ 0.01.

The fbl-1(k206) mutation extends the lifespan of mig-17 mutants

We examined the lifespan of mig-17 mutants and analyzed the effects of let-2 and fbl-1 mutations on lifespan. The lifespan analysis was conducted from the young adult stage. The mig-17 mutants exhibited a lifespan comparable to the wild type (Fig 6A–6D) as we have reported [11]. However, the lifespans of let-2(k193) and let-2(k196) animals were significantly shorter than the wild type or mig-17 mutants. The let-2(k193); mig-17 and let-2(k196); mig-17 double mutants also showed a shortened lifespan (Fig 6A, 6B and 6E). In contrast, the lifespans of fbl-1(k201) and fbl-1(k206) single mutants, as well as fbl-1(k201); mig-17 double mutants, were comparable to those of the wild type or mig-17 mutants (Fig 6C–6E). Interestingly, however, fbl-1(k206); mig-17 double mutants exhibited a significantly longer lifespan compared to the wild type. Thus, as observed in the age-related phenotypes, let-2 and fbl-1 mutations differentially affected the lifespan of mig-17 mutants. The extension of lifespan in fbl-1(k206); mig-17 double mutants, but not in fbl-1(k206) single mutants, implies that fbl-1(k206) mutation can extend lifespan in the absence of the MIG-17 protease (since mig-17(k174) represents a null allele).

Fig 6. Lifespan extension by mig-22(k185gf).

Fig 6

Open in a new tab

(A-D) Survival curve shows the effect of let-2(k193, k196) or fbl-1(k201, k206) mutations in the mig-17(k174) background. The x axis represents lifespan in days of adulthood. The y axis shows the fraction of worms alive. (E) Table shows p-values for logrank test are indicated as: ***p < 0.005, **p < 0.01, *p < 0.05, NS Not significant. The sample size is 180 animals.

Discussion

In the present study, we analyzed the age-related phenotypes of the mig-17/ADAMTS mutant and its suppressor mutations for the gonadal defects in let-2/collagen IV and fbl-1/fibulin-1. The mig-17(k174) null mutant showed a normal lifespan but exhibited a slow growth rate and short healthspan, including earlier elongation of defecation cycle and a reduction of pharyngeal pumping rate. The mig-17 mutant also exhibited a defect in age-associated body length elongation. Despite all let-2 and fbl-1 alleles being robust suppressors of the mig-17 gonadal defects, they had differential effects on these mig-17 phenotypes.

As we observed in mig-17/ADAMTS mutants, growth retardation was also noted in Adamts1 as well as Adamts9/+; Adamts20 mutants in mice [18, 19]. Although the mig-17 mutants had no effect on lifespan, they exhibited phenotypes associated with short healthspan. While much of the research on ADAMTS proteases has focused on their roles in development and disease, some studies suggest that they may also have functions relevant to aging. For example, several ADAMTS proteases are induced in intervertebral disc degeneration during aging [20, 21]. Reduction of the reelin level by ADAMTS-4 and ADAMTS-5-dependent degradation is associated with hyperphosphorylated tau protein, forming neurofibrillary tangles leading to Alzheimer’s disease [22]. However, these represent deleterious effects of ADAMTS on aging. In contrast, MIG-17 has a function to promote healthy aging, suggesting that MIG-17-dependent remodeling of the basement membrane is crucial for delaying the senescence process. We previously demonstrated that chondroitin proteoglycan acts downstream of MIG-17 to mediate healthy aging [11].

Both let-2 mutations shortened lifespan. Thus, both of these type IV collagen mutations have a negative impact on lifespan, although it is not clear whether these mutations directly affect lifespan. However, let-2(k196) suppressed the early prolongation of mig-17 defecation cycle associated with aging (Table 1). let-2(k196) also suppressed mig-17 for its insufficient increase in body length with aging. In contrast, let-2(k193) did not suppress any of these aging defects in mig-17. Type IV collagen molecules self-assemble into a complex mesh-like network within basement membranes. This network contributes to the mechanical integrity and resilience of basement membranes, allowing them to withstand mechanical stresses [23]. In addition to these structural functions, type IV collagen is also known to be involved in TGF-β signaling. For example, mutations in COL4A1 and COL4A2 impair triple helical assembly into protomers and their secretion, leading to intracellular accumulation and extracellular deficiency. Accordingly, semi-dominant COL4A1 and COL4A2 mutations cause Gould syndrome characterized primary by cerebrovascular manifestation in which TGF-β signaling is overactivated [24]. Increased TGF-β1 and TGF-β-dependent SMAD3 signaling are associated with age-related aortic valve calcification in klotho knockout mice [25]. Interestingly, DAF-7/TGF-β regulates longevity through DAF-2/insulin signaling in C. elegans [26]. It may be possible that the let-2(k196) mutation alters the TGF-β signaling to correct the short healthspan of mig-17 mutants. Consistently, in humans, ADAMTS10, whose mutation causes Weill-Marchesani syndrome characterized by short stature and thickened skin, increases TGF-β activation in a dose-dependent manner [27, 28]. let-2(k196) is a missense mutation in the triple helical domain, whereas let-2(k193) is a missense mutation in the C-terminal NC1 domain of type IV collagen [9]. It might be possible that the triple helical domain may play a more important role than the NC1 domain in the regulation of aging.

Table 1.

Phenotype Developmental phenotype Aging
Gonad development Growth rate Defecation cycle Pumping rate Body length Lifespan
mig-17(k174) Mig Slow Slow Slow Short
mig-17(k174) let-2(k196) Slow Short
mig-17(k174) let-2(k193) Slow Slow Short Short
let-2(k196) Slow Slow Short
let-2(k193) Slow Short Short
mig-17(k174) fbl-1(k201)
mig-17(k174) fbl-1(k206) Slow Slow Slow Short Long
fbl-1(k201)
fbl-1(k206) Slow Short Short
Open in a new tab

fbl-1(k201), but not fbl-1(k206), suppressed the growth retardation and early decline in pumping rate of mig-17. Furthermore, both fbl-1 alleles suppressed the insufficient growth of body length associated with aging in mig-17. fbl-1(k201) had no effect on lifespan, but fbl-1(k206) had a slightly shorter lifespan than the wild type. However, surprisingly, the mig-17(k174); fbl-1(k206) double mutant lived significantly longer than the wild type animals. Interestingly, fbl-1(k201) and fbl-1(k206) mutations are amino acid substitutions in the second Calcium binding EGF-like motif (G249E and H251Y, respectively). Thus, they are only separated by one amino acid within the same EGF-like motif. It is surprising that only fbl-1(k206), but not fbl-1(k201), interacts with mig-17 to prolong lifespan. Fibulin-1 is an evolutionarily conserved basement membrane protein that provides structural support to cells and tissues [29]. Fibulin-1 is also known to regulate EGF signaling. Fibulin-1C and D isoforms bind to the EGF receptor, possibly through its EGF-like motifs, and inhibits its activation, localization and function of lung cancer cells [30]. In C. elegans, the LIN-3/EGF and LET-23/EGF receptor signaling promotes healthy aging and longevity [31]. Thus, it might be possible that FBL-1 regulates lifespan by modulating EGF signaling in C. elegans. Among two splicing isoforms, FBL-1C and FBL-1D, FBL-1C is essential for the suppression of mig-17 gonadal defects [8]. It is unknown which isoform acts in the suppression of age-related phenotypes of mig-17. It would be interesting to determine which isoform, or both, is required for the suppression of age-related phenotypes of mig-17 and whether they act in signaling pathways involving EGF.

Supporting information

S1 Fig. Suppression of the gonad migration defect of mig-17 by let-2 or fbl-1.

Percentage of DTC migration defects. Blue and orange bars represent defects in anterior and posterior gonad arms, respectively. Black and red asterisks indicate p-values for Fisher’s exact test against WT and mig-17(k174), respectively p-values for Fisher’s exact test are indicated: ***p < 0.005, *p < 0.05.

(TIF)

pone.0305396.s001.tif (140.2KB, tif)

Acknowledgments

We thank Noriko Nakagawa, Nami Okahashi, and Chizu Yoshikata for technical assistance. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

Grant-in-Aid for Research Activity Start-up by the Ministry of Education, Culture, Sports, Science and Technology to YS(22K20658) and by the Naito Grant for the advancement of natural science to KN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Ewald CY. The Matrisome during Aging and Longevity: A Systems-Level Approach toward Defining Matreotypes Promoting Healthy Aging. Gerontology. 2020;66(3):266–74. Epub 20191213. doi: 10.1159/000504295 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Liu N, Matsumura H, Kato T, Ichinose S, Takada A, Namiki T, et al. Stem cell competition orchestrates skin homeostasis and ageing. Nature. 2019;568(7752):344–50. Epub 20190403. doi: 10.1038/s41586-019-1085-7 . [DOI] [PubMed] [Google Scholar]
  • 3.Kelwick R, Desanlis I, Wheeler GN, Edwards DR. The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family. Genome Biol. 2015;16:113. doi: 10.1186/s13059-015-0676-3 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Mead TJ, Apte SS. ADAMTS proteins in human disorders. Matrix Biol. 2018;71–72:225–39. doi: 10.1016/j.matbio.2018.06.002 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Ihara S, Nishiwaki K. Prodomain-dependent tissue targeting of an ADAMTS protease controls cell migration in Caenorhabditis elegans. EMBO J. 2007;26(11):2607–20. doi: 10.1038/sj.emboj.7601718 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Ihara S, Nishiwaki K. Stage-specific activation of MIG-17/ADAMTS controls cell migration in Caenorhabditis elegans. FEBS J. 2008;275(17):4296–305. doi: 10.1111/j.1742-4658.2008.06573.x . [DOI] [PubMed] [Google Scholar]
  • 7.Nishiwaki K, Hisamoto N, Matsumoto K. A metalloprotease disintegrin that controls cell migration in Caenorhabditis elegans. Science. 2000;288(5474):2205–8. doi: 10.1126/science.288.5474.2205 . [DOI] [PubMed] [Google Scholar]
  • 8.Kubota Y, Kuroki R, Nishiwaki K. A fibulin-1 homolog interacts with an ADAM protease that controls cell migration in C. elegans. Curr Biol. 2004;14(22):2011–8. doi: 10.1016/j.cub.2004.10.047 . [DOI] [PubMed] [Google Scholar]
  • 9.Kubota Y, Ohkura K, Tamai KK, Nagata K, Nishiwaki K. MIG-17/ADAMTS controls cell migration by recruiting nidogen to the basement membrane in C. elegans. Proc Natl Acad Sci U S A. 2008;105(52):20804–9. doi: 10.1073/pnas.0804055106 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Imanishi A, Aoki Y, Kakehi M, Mori S, Takano T, Kubota Y, et al. Genetic interactions among ADAMTS metalloproteases and basement membrane molecules in cell migration in Caenorhabditis elegans. PLoS One. 2020;15(12):e0240571. doi: 10.1371/journal.pone.0240571 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Shibata Y, Tanaka Y, Sasakura H, Morioka Y, Sassa T, Fujii S, et al. Endogenous chondroitin extends the lifespan and healthspan in C. elegans. Sci Rep. 2024;14(1):4813. Epub 20240227. doi: 10.1038/s41598-024-55417-7 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77(1):71–94. doi: 10.1093/genetics/77.1.71 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Nishiwaki K. Mutations affecting symmetrical migration of distal tip cells in Caenorhabditis elegans. Genetics. 1999;152(3):985–97. doi: 10.1093/genetics/152.3.985 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.MacNeil LT, Watson E, Arda HE, Zhu LJ, Walhout AJ. Diet-induced developmental acceleration independent of TOR and insulin in C. elegans. Cell. 2013;153(1):240–52. doi: 10.1016/j.cell.2013.02.049 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Choi W, Ryu SE, Cheon Y, Park YJ, Kim S, Kim E, et al. A single chemosensory GPCR is required for a concentration-dependent behavioral switching in C. elegans. Curr Biol. 2022;32(2):398–411 e4. Epub 20211213. doi: 10.1016/j.cub.2021.11.035 . [DOI] [PubMed] [Google Scholar]
  • 16.Russell JC, Burnaevskiy N, Ma B, Mailig MA, Faust F, Crane M, et al. Electrophysiological Measures of Aging Pharynx Function in C. elegans Reveal Enhanced Organ Functionality in Older, Long-lived Mutants. J Gerontol A Biol Sci Med Sci. 2019;74(8):1173–9. doi: 10.1093/gerona/glx230 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Bolanowski MA, Russell RL, Jacobson LA. Quantitative measures of aging in the nematode Caenorhabditis elegans. I. Population and longitudinal studies of two behavioral parameters. Mech Ageing Dev. 1981;15(3):279–95. doi: 10.1016/0047-6374(81)90136-6 . [DOI] [PubMed] [Google Scholar]
  • 18.Shindo T, Kurihara H, Kuno K, Yokoyama H, Wada T, Kurihara Y, et al. ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function. J Clin Invest. 2000;105(10):1345–52. doi: 10.1172/JCI8635 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Enomoto H, Nelson CM, Somerville RP, Mielke K, Dixon LJ, Powell K, et al. Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation. Development. 2010;137(23):4029–38. doi: 10.1242/dev.050591 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Wang X, Wu H, Zhang Q, Liu F, Wang J, Wei C, et al. NFKB2 inhibits NRG1 transcription to affect nucleus pulposus cell degeneration and in fl ammation in intervertebral disc degeneration. Mech Ageing Dev. 2021;197:111511. doi: 10.1016/j.mad.2021.111511 . [DOI] [PubMed] [Google Scholar]
  • 21.Li X, Zhang J, Wang B, Chen C, Zhang E, Lv Z, et al. USP24-dependent stabilization of Runx2 recruits a p300/NCOA3 complex to transactivate ADAMTS genes and promote degeneration of intervertebral disc in chronic inflammation mice. Biol Direct. 2023;18(1):37. doi: 10.1186/s13062-023-00395-5 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Gurses MS, Ural MN, Gulec MA, Akyol O, Akyol S. Pathophysiological Function of ADAMTS Enzymes on Molecular Mechanism of Alzheimer’s Disease. Aging Dis. 2016;7(4):479–90. doi: 10.14336/AD.2016.0111 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Sundaramoorthy M, Meiyappan M, Todd P, Hudson BG. Crystal structure of NC1 domains. Structural basis for type IV collagen assembly in basement membranes. J Biol Chem. 2002;277(34):31142–53. doi: 10.1074/jbc.M201740200 . [DOI] [PubMed] [Google Scholar]
  • 24.Branyan K, Labelle-Dumais C, Wang X, Hayashi G, Lee B, Peltz Z, et al. Elevated TGFbeta signaling contributes to cerebral small vessel disease in mouse models of Gould syndrome. Matrix Biol. 2023;115:48–70. doi: 10.1016/j.matbio.2022.11.007 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Chakrabarti M, Bhattacharya A, Gebere MG, Johnson J, Ayub ZA, Chatzistamou I, et al. Increased TGFbeta1 and SMAD3 Contribute to Age-Related Aortic Valve Calcification. Front Cardiovasc Med. 2022;9:770065. doi: 10.3389/fcvm.2022.770065 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Shaw WM, Luo S, Landis J, Ashraf J, Murphy CT. The C. elegans TGF-beta Dauer pathway regulates longevity via insulin signaling. Curr Biol. 2007;17(19):1635–45. doi: 10.1016/j.cub.2007.08.058 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Cain SA, Woods S, Singh M, Kimber SJ, Baldock C. ADAMTS6 cleaves the large latent TGFbeta complex and increases the mechanotension of cells to activate TGFbeta. Matrix Biol. 2022;114:18–34. Epub 20221108. doi: 10.1016/j.matbio.2022.11.001 . [DOI] [PubMed] [Google Scholar]
  • 28.Mularczyk EJ, Singh M, Godwin ARF, Galli F, Humphreys N, Adamson AD, et al. ADAMTS10-mediated tissue disruption in Weill-Marchesani syndrome. Hum Mol Genet. 2018;27(21):3675–87. doi: 10.1093/hmg/ddy276 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Timpl R, Sasaki T, Kostka G, Chu ML. Fibulins: a versatile family of extracellular matrix proteins. Nat Rev Mol Cell Biol. 2003;4(6):479–89. doi: 10.1038/nrm1130 . [DOI] [PubMed] [Google Scholar]
  • 30.Harikrishnan K, Joshi O, Madangirikar S, Balasubramanian N. Cell Derived Matrix Fibulin-1 Associates With Epidermal Growth Factor Receptor to Inhibit Its Activation, Localization and Function in Lung Cancer Calu-1 Cells. Front Cell Dev Biol. 2020;8:522. doi: 10.3389/fcell.2020.00522 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Iwasa H, Yu S, Xue J, Driscoll M. Novel EGF pathway regulators modulate C. elegans healthspan and lifespan via EGF receptor, PLC-gamma, and IP3R activation. Aging Cell. 2010;9(4):490–505. doi: 10.1111/j.1474-9726.2010.00575.x . [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Myeongwoo Lee

9 Apr 2024

PONE-D-24-09277Mutations in fibulin-1 and collagen IV suppress the short healthspan of mig-17/ADAMTS mutants in Caenorhabditis elegansPLOS ONE

Dear Dr. Shibata,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. While reviewers think the work is interesting, both felt that the manuscript needs improvements. Please refer to the reviewer's comments. In the revised manuscript, attach a letter addressing reviewer's critiques point-by-point when submitting the revised manusript. 

Please submit your revised manuscript by May 24 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Myeongwoo Lee, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following financial disclosure: 

"Grant-in-Aid for Research Activity Start-up by the Ministry of Education, Culture, Sports, Science and Technology to YS(22K20658) and by the Naito Grant for the advancement of natural science to KN. "

Please state what role the funders took in the study.  If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" 

If this statement is not correct you must amend it as needed. 

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

"We thank Noriko Nakagawa, Nami Okahashi, and Chizu Yoshikata for technical assistance. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources. This work was supported by a Grant-in-Aid for Research Activity Start-up by the Ministry of Education, Culture, Sports, Science and Technology to YS(22K20658) and by the Naito Grant for the advancement of natural science to KN."

We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

"Grant-in-Aid for Research Activity Start-up by the Ministry of Education, Culture, Sports, Science and Technology to YS(22K20658) and by the Naito Grant for the advancement of natural science to KN. "

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This paper from a leader in the C. elegans organogenesis field describes how matrix proteins contribute to organismal aging. In addition to its role in DTC migration, the metalloprotease MIG-17/ADAMTS is important for healthy aging in the worm. Two other matrix proteins, LET-2/collagen and FBL-1/fibulin that interact genetically with MIG-17 in DTC migration also suppress age-related phenotypes in the mig-17 mutants in an allele-specific manner. I think this will be of interest to those interested in matrix and aging.

Recommendations:

Make it more clear in the abstract that let-2 and fbl-1 differentially affect the lifespan of mig-17 mutants, and that mig-17 shows age related phenotypes (say what these are) but not short lifespan per se. It might be more correct to say that mig-17 shows altered healthspan, not altered lifespan.

Provide a bit more information in the introduction about the roles of basement membrane in aging. What is known about this? (e.g. skin aging and collagen). This will make the article more accessible to a broader audience. Also provide a brief general description of the mig-17 age-dependent phenotypes.

Methods: Please provide more detail about the imaging (DIC, I assume?).

Statistics: Please also include a section describing the statistical methods. Each figure legend should more clearly describe the statistical methods used (just saying t-test is not sufficiently descriptive). Multiple comparisons corrections should be applied.

Include the supplementary diagram showing the position of the alleles on the proteins in the main text. Include MIG-17 too, and show that the allele studied is a null. This should be figure 1. Also a diagram of the worm and/or a photo showing where these proteins are localized might help.

Fig. 4 show all data as in Fig. 3. Not clear why there are asterisks above and below the columns. Effects seem very minor – check for multiple comparisons correction in statistics.

Fig. S1 % abnormal animals – abnormal in what way? Gonad morphogenesis defects would be more descriptive.

Minor:

p.5 Change ‘In addition to its plays a role in gonadogenesis, MIG-17 also function in regulating healthy aging’ to ‘In addition to its role in in gonadogenesis, MIG-17 also functions in regulating healthy aging’.

p. 6 ‘young adult animals were collected as day 1 adults’ and ‘number of pumps during 30 seconds’ ‘Average of three independent measurements were calculated for each individual’

p. 7 Add a period after ‘punctuated by molting’.

p.9 ‘Additionally we investigated changes in body length during aging in this study’. This sentence seems out of place. Recommend delete and just merge the previous sentence in with the next paragraph.

p. 9 last sentence Delete ‘were observed to’. It is fine to just say ‘The pumping rates decreased in day 9 adults’. Better would be to provide the quantification in the text. E.g decreased by 20% or whatever it is.

p.15 ‘Fibulin-1 IS also known to regulate…’

Fig. 1 Change ‘hatch’ to ‘hatching’ . T-tests not the appropriate stat for comparing percentages. Recommend using Chi-squared or Fisher’s exact.

All figures: X axis fonts are very tiny. Turning these to an angle might help.

Fig. 3 Y axis: Number of pumps in 30 sec

Fig. 5 fonts way too tiny

Fig. 6 should be Table 1.

Reviewer #2: This manuscript describes the genetic interaction between mig-17 and fbl-1 or let-2 viable alleles. The authors argue that allele-specific suppression of age-related phenotypes of the mig-17 null allele is present. This paper is an extension of their work published previously. This manuscript only displays behavioral analyses without cell biological data. However, their studies can provide important insight. The remodeling of the matrix may affect an animal's age-related markers.

While it contains exciting studies, the manuscript requires improvements to be published in PLOS One.

1. Introduction. The author should summarize how cell-matrix interaction links to age-related phenotypes, including examples from other systems.

2. Materials and Methods. The authors must include details of procedures (p.6). ‘Body length was measured using ImageJ software’; I suggest adding more detail. “ … the growth was assessed on the stage of vulva development …” also needs more detail.

3. Age-related phenotype suppression (Figure 5, difficult to read). I noticed that the median life span of mig-17 in panels A, B, and C ranged from 18 to 21 days, but the median of mig-17 in panel D appears to be around 16 days. The authors need to clarify the differences. Or, the experiment must be repeated.

4. Lifespan phenotypes of mutants. The authors described the reduced lifespan. In general, any mutations can reduce lifespan. Although the genetic study is interesting, it is difficult to convince that such mutations directly affect lifespan.

5. Minor comments.

a. Some typos need to be corrected. Ex) 'let-2(196)' and 'TGFβ'.

b. Graphs are difficult to understand. Figure 5 graph must be drawn using thicker lines.

c. Overall, the manuscript needs to be revised to a cohesive format.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Erin J. Cram

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2024 Jul 9;19(7):e0305396. doi: 10.1371/journal.pone.0305396.r002

Author response to Decision Letter 0

3 May 2024

Followings are responses to editor’s and reviewer’s comment.

Comments to the Author

Reviewer #1: This paper from a leader in the C. elegans organogenesis field describes how matrix proteins contribute to organismal aging. In addition to its role in DTC migration, the metalloprotease MIG-17/ADAMTS is important for healthy aging in the worm. Two other matrix proteins, LET-2/collagen and FBL-1/fibulin that interact genetically with MIG-17 in DTC migration also suppress age-related phenotypes in the mig-17 mutants in an allele-specific manner. I think this will be of interest to those interested in matrix and aging.

Recommendations:

1. Make it more clear in the abstract that let-2 and fbl-1 differentially affect the lifespan of mig-17 mutants, and that mig-17 shows age related phenotypes (say what these are) but not short lifespan per se. It might be more correct to say that mig-17 shows altered healthspan, not altered lifespan.

We added underlined phrase in the abstract.

Page 3, line 11

“Interestingly, fbl-1(k206), but not fbl-1(k201) or let-2 alleles, exhibited an extended lifespan compared to the wild type when combined with mig-17.”

Page 3, line 14

“In addition to the control of DTC migration, MIG-17 also plays a role in healthspan, but not in lifespan”

2. Provide a bit more information in the introduction about the roles of basement membrane in aging. What is known about this? (e.g. skin aging and collagen). This will make the article more accessible to a broader audience. Also provide a brief general description of the mig-17 age-dependent phenotypes.

We added following sentences to describe the roles of basement membrane in aging.

Page 4, line2

“The BMs are complex structures composed of multiple types of molecules. To maintain their integrity, it is necessary to appropriately remove damaged components and incorporate new ones, ensuring proper turnover. However, as animals age, the metabolism of BMs slows down, leading to the accumulation of damage [1]. Collagen levels decrease with age, while spontaneous cross-links increase. Yet, the anti-aging effects of BMs remain poorly understood. In the skin, BM collagen is essential for stem cell maintenance through hemidesmosomes [2]. Loss of collagen is known to impair cell competition, thus contributing to aging. However, the relationship between BMs and the suppression of aging beyond skin stem cells remains unknown.”

Accordingly, we added two references

1. Ewald CY. The Matrisome during Aging and Longevity: A Systems-Level Approach toward Defining Matreotypes Promoting Healthy Aging. Gerontology. 2020;66(3):266-74. Epub 20191213. doi: 10.1159/000504295. PubMed PMID: 31838471; PubMed Central PMCID: PMCPMC7214094.

2. Liu N, Matsumura H, Kato T, Ichinose S, Takada A, Namiki T, et al. Stem cell competition orchestrates skin homeostasis and ageing. Nature. 2019;568(7752):344-50. Epub 20190403. doi: 10.1038/s41586-019-1085-7. PubMed PMID: 30944469.

To describe the mig-17 age-dependent phenotypes, we modified the description as follows.

Page 5, 3rd line from the bottom

“mig-17 mutants exhibit an earlier decline in periodic behavior including the defecation cycle and pumping rate, and motility rate, as well as earlier shortening of body length compared to the wild type”

3. Methods: Please provide more detail about the imaging (DIC, I assume?).

We added underlined phrase.

Page 6, 5th line from the bottom

“Gonad migration phenotypes were scored using a DIC images of Nomarski microscope (Axioplan 2; Zeiss).”

4. Statistics: Please also include a section describing the statistical methods. Each figure legend should more clearly describe the statistical methods used (just saying t-test is not sufficiently descriptive). Multiple comparisons corrections should be applied.

Thank you for your suggestion. We performed multiple comparisons corrections in Fig. 3, 4, 5 using one-way ANOVA and Tukey’s multiple comparisons.

Accordingly, we changed figure legends as follows.

Legend of figure 3-5

We removed “for t-test” from following sentence “p-values for t-test against WT is indicate at the bottom of plots.”

We replaced“: *** p<0.005, ** p<0.01, *p<0.05.” to “One-way ANOVA with Tukey’s multiple comparisons:, *p≤ 0.05, **p≤ 0.01.”

We added statistics section in Materials and Methods.

Page 8, line 6

“Statistics

Growth rates were analyzed using Fisher's exact test to compare adults and larvae.

One-way analysis of variance (ANOVA) was conducted using Excel to compare defecation cycle, pumping rate, and body length. If a significant difference was found by ANOVA, Tukey’s multiple comparison test was performed using Excel statistics.

The lifespan was analyzed using a log-rank test in R.”

Accordingly, we changed result and discussion as follows.

Page 10, 4th line from the bottom

“fbl-1(k206) alleles showed significantly longer defecation cycles compared to the wild type after day 5 and fbl-1(k201) showed a trend toward suppression of the mig-17 defecation decline at day 5, although it was not statistically significant (t-test: p=0.069)”

Page 11, line 4

“The pumping rates of mig-17 adults were much slower than those of wild type at day 1 and day 9, suggesting short healthspan of mig-17 animals (Fig. 4C, D) [11]. Interestingly, let-2(k196), but not let-2(k193), showed a trend toward suppression of the mig-17 pumping rate decline at day 9 (Fig. 4A, B), although it was not statistically significant. The pumping rate of mig-17(k174) and fbl-1(k206); mig-17(k174), but not that of fbl-1(k201); mig- 17(k174), showed significant differences from that of wild-type animals at day 9 adult. (Fig. 4C, D).”

Page 11, 6th line from the bottom

“The body length of day 1 adults was longer in mig-17; let-2(k196) and mig-17; let-2(k193) double mutants compared to the wild type or mig-17 mutants. At the day3 adult, the body length of mig-17(k174) and let-2(k193) mig-17(k174), but not that of let-2(k196) mig- 17(k174), showed significant differences from those of wild-type animals”

Page 14, line 8

“However, let-2(k196) suppressed the early prolongation of mig-17 defecation cycle associated with aging.”

We also changed Table1 as follows.

Defecation cycle: mig-17(k174) fbl-1(k201) and fbl-1(k201) became blank.

Pumping rate: mig-17(k174) let-2(k193), let-2(k196), and let-2(k193) became blank.

Body length (represent day3 data): mig-17(k174) fbl-1(k206) and fbl-1(k206) became short.

5. Include the supplementary diagram showing the position of the alleles on the proteins in the main text. Include MIG-17 too, and show that the allele studied is a null. This should be figure 1. Also a diagram of the worm and/or a photo showing where these proteins are localized might help.

We added Figure 1, which includes a diagram showing the position of the alleles on the MIG-17, LET-2, and FBL-1 proteins. Additionally, we included a diagram indicating the localization of these proteins on BMs.

Accordingly, we added legend of Figure 1, as follows.

“Figure 1: Schematic diagrams of MIG-17, FBL-1C, and LET-2A.

(A) The mutation sites of mig-17(k174), let-2(k193, k196), and fbl-1(k201, k206) are indicated. (B) A diagram showing the localization of MIG-17, LET-2, and FBL-1. These proteins are components of BMs.

”

And we removed the description of alleles of FBL-1 and LET-2 from Sup Figure1.

Accordingly, we changed Fig. 1-5 to Fig. 2-6 and removed Sup Figure1A.

6. Fig. 4 show all data as in Fig. 3. Not clear why there are asterisks above and below the columns. Effects seem very minor – check for multiple comparisons correction in statistics.

Thank you for your suggestion. We represented Fig. 5(ex-Fig. 4) as box and dot plot.

Accordingly, we changed first sentence of legend as follows.

“Box and dot plots indicate the body length.”

7. Fig. S1 % abnormal animals – abnormal in what way? Gonad morphogenesis defects would be more descriptive.

We changed description of y axis to “% animals w gonad morphogenesis defects”

Minor:

p.5 Change ‘In addition to its plays a role in gonadogenesis, MIG-17 also function in regulating healthy aging’ to ‘In addition to its role in in gonadogenesis, MIG-17 also functions in regulating healthy aging’.

We followed reviewer’s suggestion.

p. 6 ‘young adult animals were collected as day 1 adults’ and ‘number of pumps during 30 seconds’ ‘Average of three independent measurements were calculated for each individual’

We followed reviewer’s suggestion.

p. 7 Add a period after ‘punctuated by molting’.

We followed reviewer’s suggestion.

p.9 ‘Additionally we investigated changes in body length during aging in this study’. This sentence seems out of place. Recommend delete and just merge the previous sentence in with the next paragraph.

We followed reviewer’s suggestion.

p. 9 last sentence Delete ‘were observed to’. It is fine to just say ‘The pumping rates decreased in day 9 adults’. Better would be to provide the quantification in the text. E.g decreased by 20% or whatever it is.

We changed to “The pumping rates decreased by 50% in day 9 adults.” (Page 10, 1st line)

p.15 ‘Fibulin-1 is also known to regulate…’

We followed reviewer’s suggestion.

Fig. 1 Change ‘hatch’ to ‘hatching’ . T-tests not the appropriate stat for comparing percentages. Recommend using Chi-squared or Fisher’s exact.

We followed reviewer’s suggestion.

Accordingly, we changed figure legend as follows.

“Black and red asterisks on the top of bar graph indicate p-values for Fisher’s exact test against WT and mig-17(k174), respectively”

In addition, we performed Fisher’s exact test to Supplementary data that analyzed suppression of the gonad migration defect of mig-17.

All figures: X axis fonts are very tiny. Turning these to an angle might help.

We followed reviewer’s suggestion.

Fig. 3 Y axis: Number of pumps in 30 sec

We followed reviewer’s suggestion.

Fig. 5 fonts way too tiny

We followed reviewer’s suggestion.

Fig. 6 should be Table 1.

We followed reviewer’s suggestion.

Reviewer #2: This manuscript describes the genetic interaction between mig-17 and fbl-1 or let-2 viable alleles. The authors argue that allele-specific suppression of age-related phenotypes of the mig-17 null allele is present. This paper is an extension of their work published previously. This manuscript only displays behavioral analyses without cell biological data. However, their studies can provide important insight. The remodeling of the matrix may affect an animal's age-related markers.

While it contains exciting studies, the manuscript requires improvements to be published in PLOS One.

1. Introduction. The author should summarize how cell-matrix interaction links to age-related phenotypes, including examples from other systems.

Please see response to reviwer1 point 2.

2. Materials and Methods. The authors must include details of procedures (p.6). ‘Body length was measured using ImageJ software’; I suggest adding more detail. “ … the growth was assessed on the stage of vulva development …” also needs more detail.

We added following sentences to explain details of procedures.

Page 7, line 8

“To measure body length, we traced a line along the body using ImageJ and then measured its length.”

Page 7, line 2

“A simple invagination occurs in early L4, followed by a Christmas tree-like invagination in mid-L4, and the invagination becomes mostly closed in late L4.”

3. Age-related phenotype suppression (Figure 5, difficult to read). I noticed that the median life span of mig-17 in panels A, B, and C ranged from 18 to 21 days, but the median of mig-17 in panel D appears to be around 16 days. The authors need to clarify the differences. Or, the experiment must be repeated.

C. elegans lifespans vary from experiment to experiment, both for wild type and mutants. This variability is believed to be influenced by multiple factors, including the degree of dryness of the plate and the amount of E. coli used as food. Given the difficulty in controlling these conditions, when measuring the lifespan of C. elegans, we compare the control and sample using plates seeded with E. coli prepared simultaneously. Discrepancies in lifespan may arise if plates are created at different times. The variance in the lifespan of mig-17 between experiments falls within the normal range. Moreover, in both instances, the results are reproducible, as there are no significant differences observed between the wild type and mig-17 mutants.

4. Lifespan phenotypes of mutants. The authors described the reduced lifespan. In general, any mutations can reduce lifespan. Although the genetic study is interesting, it is difficult to convince that such mutations directly affect lifespan.

We agree with reviewer’s suggestion. Therefore, we added underlined phrase in discussion part.

Page 14, line 6

“Thus, both of these type IV collagen mutations have a negative impact on lifespan, although it is not clear whether these mutations directly affect lifespan.”

5. Minor comments.

a. Some typos need to be corrected. Ex) 'let-2(196)' and 'TGFβ'.

We corrected these typo.

b. Graphs are difficult to understand. Figure 5 graph must be drawn using thicker lines.

We followed reviewer’s suggestion.

c. Overall, the manuscript needs to be revised to a cohesive format.

We followed reviewer’s suggestion.

Attachment

Submitted filename: Response to the reviewer 1st.docx

pone.0305396.s002.docx (27.9KB, docx)

Decision Letter 1

Myeongwoo Lee

20 May 2024

PONE-D-24-09277R1Mutations in fibulin-1 and collagen IV suppress the short healthspan of mig-17/ADAMTS mutants in Caenorhabditis elegansPLOS ONE

Dear Dr. Shibata,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Reviewers only require minor modifications and calrifications, which can be completed easily.

Please submit your revised manuscript by Jul 04 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Myeongwoo Lee, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Change the Microscopy section in the materials and methods to:

Gonad migration phenotypes were scored using DIC images captured with a Zeiss Axioplan 2

microscope.

Under ‘Behavioral Analysis’, recommend edits to make complete sentences: ‘Number of pumps

during 30 seconds were counted and the average of three independent measurements was calculated for each

individual.

Reviewer #2: 1. Please clarify the purpose of Figure 1. The domain structures were not mentioned in the Introduction. Specifically, Figure 1B should have been mentioned in the text. I suggest considering its deletion.

2. Materials and methods. References are needed for the statistical methods used in the study.

3. Page 9, line 9. “the gain of function mutations in collagen IV..” needs a reference.

4. Page 11, line 7. “.. showed significant differences ..” needs a p-value in parenthesis.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2024 Jul 9;19(7):e0305396. doi: 10.1371/journal.pone.0305396.r004

Author response to Decision Letter 1

22 May 2024

Comments to the Author

Reviewer #1: Change the Microscopy section in the materials and methods to:

Gonad migration phenotypes were scored using DIC images captured with a Zeiss Axioplan 2 microscope.

Thank you for your suggestion. We followed reviewer’s comment.

Under ‘Behavioral Analysis’, recommend edits to make complete sentences: ‘Number of pumps during 30 seconds were counted and the average of three independent measurements was calculated for each individual.

We followed reviewer’s comment.

Reviewer #2: 1. Please clarify the purpose of Figure 1. The domain structures were not mentioned in the Introduction. Specifically, Figure 1B should have been mentioned in the text. I suggest considering its deletion.

We cited Figure 1A, and 1B in Introduction as follows.

Page 5 line2

“MIG-17 is secreted from the body wall muscle cells as a proform and localizes to the BMs of various tissues, including gonad, intestine, body wall muscle, and hypodermis, where it is activated by auto-catalytic removal of its prodomain [5, 6] (Fig. 1A, B).”

Page 5 line7

“Dominant gain-of-function (gf) mutations in the BM molecules collagen IV �2 chain and fibulin-1 suppress the gonadal defect of the mig-17 mutants [8-10] (Fig. 1A). let-2(gf) mutations are amino-acid substitutions either in the triple helical domain or in the C-terminal non-collagenous 1 (NC1) domain (Fig. 1A). fbl-1(gf) mutations are amino-acid substitutions found in the second EGF-like motif (Fig. 1A).”

2. Materials and methods. References are needed for the statistical methods used in the study.

Thank you for your suggestion. We followed reviewer’s comment.

Followings are references.

Shibata Y, Tanaka Y, Sasakura H, Morioka Y, Sassa T, Fujii S, et al. Endogenous chondroitin extends the lifespan and healthspan in C. elegans. Sci Rep. 2024;14(1):4813. Epub 20240227. doi: 10.1038/s41598-024-55417-7. PubMed PMID: 38413743; PubMed Central PMCID: PMCPMC10899230.

Choi W, Ryu SE, Cheon Y, Park YJ, Kim S, Kim E, et al. A single chemosensory GPCR is required for a concentration-dependent behavioral switching in C. elegans. Curr Biol. 2022;32(2):398-411 e4. Epub 20211213. doi: 10.1016/j.cub.2021.11.035. PubMed PMID: 34906353.

3. Page 9, line 9. “the gain of function mutations in collagen IV..” needs a reference.

We followed reviewer’s comment.

Followings are references.

Kubota Y, Kuroki R, Nishiwaki K. A fibulin-1 homolog interacts with an ADAM protease that controls cell migration in C. elegans. Curr Biol. 2004;14(22):2011-8. doi: 10.1016/j.cub.2004.10.047. PubMed PMID: 15556863.

Imanishi A, Aoki Y, Kakehi M, Mori S, Takano T, Kubota Y, et al. Genetic interactions among ADAMTS metalloproteases and basement membrane molecules in cell migration in Caenorhabditis elegans. PLoS One. 2020;15(12):e0240571. doi: 10.1371/journal.pone.0240571. PubMed PMID: 33264296; PubMed Central PMCID: PMCPMC7710118.

4. Page 11, line 7. “.. showed significant differences ..” needs a p-value in parenthesis.

We added p-value as follows.

Page 11, line 7

“The pumping rate of mig-17(k174) (p=0.0001) and fbl-1(k206) mig-17(k174) (p=0.028), but not that of fbl-1(k201); mig-17(k174) (p=0.105), showed significant differences from that of wild-type animals at day 9 adult (Fig. 4C, D).”

Attachment

Submitted filename: Response to the reviewer 2nd.docx

pone.0305396.s003.docx (16.5KB, docx)

Decision Letter 2

Myeongwoo Lee

30 May 2024

Mutations in fibulin-1 and collagen IV suppress the short healthspan of mig-17/ADAMTS mutants in Caenorhabditis elegans

PONE-D-24-09277R2

Dear Dr. Shibata,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Myeongwoo Lee, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have addressed all of my concerns. I have no further comments on the paper, which is a nice addition to the cell migration literature.

Reviewer #2: No further comments, except for Figure 1B. I still don't understand why the authors need Figure 1B. It does not add anything to understand their model.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

Acceptance letter

Myeongwoo Lee

4 Jun 2024

PONE-D-24-09277R2

PLOS ONE

Dear Dr. Shibata,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Myeongwoo Lee

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Suppression of the gonad migration defect of mig-17 by let-2 or fbl-1.

    Percentage of DTC migration defects. Blue and orange bars represent defects in anterior and posterior gonad arms, respectively. Black and red asterisks indicate p-values for Fisher’s exact test against WT and mig-17(k174), respectively p-values for Fisher’s exact test are indicated: ***p < 0.005, *p < 0.05.

    (TIF)

    pone.0305396.s001.tif (140.2KB, tif)
    Attachment

    Submitted filename: Response to the reviewer 1st.docx

    pone.0305396.s002.docx (27.9KB, docx)
    Attachment

    Submitted filename: Response to the reviewer 2nd.docx

    pone.0305396.s003.docx (16.5KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting information files.

    Articles from PLOS ONE are provided here courtesy of PLOS

    RESOURCES