Intermediate filament network perturbation in the C. elegans intestine causes systemic dysfunctions
Curation statements for this article:-
Curated by eLife
eLife Assessment:
Mutations in a variety of intermediate filament proteins and their regulators lead to abnormal development, reduced lifetime, and increased stress sensitivity. This manuscript rigorously demonstrates that such defects result from inappropriate assembly of intermediate filament networks, as mutations in a central intermediate filament protein prevent assembly of both the normal network and these inappropriate assemblages and largely rescue most of the defects. This has important implications for our understanding of the assembly of intermediate filament structures and for understanding and potentially treating diseases resulting from mutations in intermediate filament protein genes.
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Abstract
Intermediate filaments (IFs) are major components of the metazoan cytoskeleton. A long-standing debate concerns the question whether IF network organization only reflects or also determines cell and tissue function. Using Caenorhabditis elegans , we have recently described mutants of the mitogen-activated protein kinase (MAPK) SMA-5 which perturb the organization of the intestinal IF cytoskeleton resulting in luminal widening and cytoplasmic invaginations. Besides these structural phenotypes, systemic dysfunctions were also observed. We now identify the IF polypeptide IFB-2 as a highly efficient suppressor of both the structural and functional deficiencies of mutant sma-5 animals by removing the aberrant IF network. Mechanistically, perturbed IF network morphogenesis is linked to hyperphosphorylation of multiple sites throughout the entire IFB-2 molecule. The rescuing capability is IF isotype-specific and not restricted to sma-5 mutants but extends to mutants that disrupt the function of the cytoskeletal linker IFO-1 and the IF-associated protein BBLN-1. The findings provide strong evidence for adverse consequences of the deranged IF networks with implications for diseases that are characterized by altered IF network organization.
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Author Response
Reviewer #2 (Public Review):
The authors describe in the nematode C. elegans the effects of perturbed organization of Intermediate filaments (IFs), which form the cytoskeleton of animal cells together with actin filaments. They focus on a previously identified mutant of the kinase SMA-5, which when mutated leads to disorganized IF structure in intestinal cells of C. elegans. The authors found that the phenotypes caused by the mutated SMA-5 kinase concerning gut morphology and animal health can be reversed by removing IF network components such as the protein IFB-2. This finding is extended to other components of the IF network, which also display a certain degree of sma-5 phenotype alleviation when depleted.
Strength:
The finding that suppressing the intestinal phenotypes caused in sma-5 mutants can be suppressed …
Author Response
Reviewer #2 (Public Review):
The authors describe in the nematode C. elegans the effects of perturbed organization of Intermediate filaments (IFs), which form the cytoskeleton of animal cells together with actin filaments. They focus on a previously identified mutant of the kinase SMA-5, which when mutated leads to disorganized IF structure in intestinal cells of C. elegans. The authors found that the phenotypes caused by the mutated SMA-5 kinase concerning gut morphology and animal health can be reversed by removing IF network components such as the protein IFB-2. This finding is extended to other components of the IF network, which also display a certain degree of sma-5 phenotype alleviation when depleted.
Strength:
The finding that suppressing the intestinal phenotypes caused in sma-5 mutants can be suppressed by removing functional IF components is an interesting observation. It confirms a previous study showing that bbln-1 mutation-caused IF phenotypes can be suppressed by depleting IFB-2.
Weakness:
- The finding of suppressing the intestinal phenotypes caused in sma-5 mutants can be considered a minor conceptual advancement. However, the study comes short of providing insight into the molecular processes of how deranged IF networks and its consequence can be rescued/suppressed by removing e.g. the IFB-2 filaments. Many statements concerning the relationship between SMA-5 and the IFs are based on assumptions. The study requires protein biochemical analysis to show whether SMA-5 phosphorylates the IF proteins - mainly the IFB-2 polypeptide. The relationship between SMA-5 / IFB-2 is a central aspect of this study but the main conclusions are based on the notion that IFB-2 and other IF proteins may be phosphorylated by SMA-5. Mutating putative phosphorylation sites of IFB-2 without having shown any proof that the modification occurs by SMA-5 is futile. This important open question needs to be addressed. And will allow statements whether the ifb-2(kc20) mutant allele-encoded shorter IFB-2 protein lacks phosphorylation or not.
We have addressed the major concern of the Reviewer by performing phosphorylation analyses of IFB-2 showing that loss of SMA-5 induces phosphorylation of multiple sites throughout the IFB-2 molecule. The results are presented in new Figs. 5 and S5.
- No quantification of the morphological defects such as using fluorescent-labeled IF proteins as in previous studies is provided in the manuscript. The EM pictures are not sufficient to provide information on how often the IF network perturbations and morphology defects occur. Also, the rescue of the actual morphological gut defects was not quantified. The assessment of development time and arrest, body length, lifespan, oxidative stress resistance, and others should be related to intestinal tube defects. They are useful and important but are an indirect measure of intestine defects and rescue.
We provide the requested data on IF localization and intestinal morphology in new Figs. S2 and S3, respectively.
- It is not clear how exactly the mutant ifb-2 allele kc20 was identified. In the Materials and methods section, the authors provide information on the specific primers for the ifb-2 locus. But how did they know that the mutation lies within this region? Was there mutation mapping or whole-genome sequencing applied?
The requested information is included in the revised Result section (first paragraph).
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eLife Assessment:
Mutations in a variety of intermediate filament proteins and their regulators lead to abnormal development, reduced lifetime, and increased stress sensitivity. This manuscript rigorously demonstrates that such defects result from inappropriate assembly of intermediate filament networks, as mutations in a central intermediate filament protein prevent assembly of both the normal network and these inappropriate assemblages and largely rescue most of the defects. This has important implications for our understanding of the assembly of intermediate filament structures and for understanding and potentially treating diseases resulting from mutations in intermediate filament protein genes.
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Reviewer #1 (Public Review):
The article is a straightforward continuation of their previous 2016 study. The authors demonstrate an organism-level role of intermediate filaments (IFs) in C. elegans with a model highlighting intermediate filament functions in organism development, larval development, oxidative stress-resilience, size, and lifespan.
The study uses endotube morphogenesis in C. elegans as an elegant model to examine the effect of aberrant IF network morphogenesis on endotube morphology and how these effects are reflected in terms of progeny growth and development.
The study identifies the C. elegans IF protein IFB-2 as a core component of IF network morphogenesis where any mutation or dysfunction of IF interacting proteins such as SMA-5, IFO-1, and BBLN1 can be mostly rescued by silencing of IFB-2.
The observed mutations …
Reviewer #1 (Public Review):
The article is a straightforward continuation of their previous 2016 study. The authors demonstrate an organism-level role of intermediate filaments (IFs) in C. elegans with a model highlighting intermediate filament functions in organism development, larval development, oxidative stress-resilience, size, and lifespan.
The study uses endotube morphogenesis in C. elegans as an elegant model to examine the effect of aberrant IF network morphogenesis on endotube morphology and how these effects are reflected in terms of progeny growth and development.
The study identifies the C. elegans IF protein IFB-2 as a core component of IF network morphogenesis where any mutation or dysfunction of IF interacting proteins such as SMA-5, IFO-1, and BBLN1 can be mostly rescued by silencing of IFB-2.
The observed mutations cause a range of systemic and functional defects of which endotube-related defects that include luminal widening and cytoplasmic invaginations are regarded as the key parameters to observe the direct result of IF network perturbation in the study. Based on these parameters authors narrowed down on IFB-2 head domain as a critical interactor in IF network morphogenesis and function.
On the whole, very interesting findings and an elegant study with excellent data that would be of broad interest for cytoskeletal research. The study has clear ramifications also for the understanding of the evolutionary development and roles of IF, both IF aspects that are still very poorly understood.
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Reviewer #2 (Public Review):
The authors describe in the nematode C. elegans the effects of perturbed organization of Intermediate filaments (IFs), which form the cytoskeleton of animal cells together with actin filaments. They focus on a previously identified mutant of the kinase SMA-5, which when mutated leads to disorganized IF structure in intestinal cells of C. elegans. The authors found that the phenotypes caused by the mutated SMA-5 kinase concerning gut morphology and animal health can be reversed by removing IF network components such as the protein IFB-2. This finding is extended to other components of the IF network, which also display a certain degree of sma-5 phenotype alleviation when depleted.
Strength:
The finding that suppressing the intestinal phenotypes caused in sma-5 mutants can be suppressed by removing functional …Reviewer #2 (Public Review):
The authors describe in the nematode C. elegans the effects of perturbed organization of Intermediate filaments (IFs), which form the cytoskeleton of animal cells together with actin filaments. They focus on a previously identified mutant of the kinase SMA-5, which when mutated leads to disorganized IF structure in intestinal cells of C. elegans. The authors found that the phenotypes caused by the mutated SMA-5 kinase concerning gut morphology and animal health can be reversed by removing IF network components such as the protein IFB-2. This finding is extended to other components of the IF network, which also display a certain degree of sma-5 phenotype alleviation when depleted.
Strength:
The finding that suppressing the intestinal phenotypes caused in sma-5 mutants can be suppressed by removing functional IF components is an interesting observation. It confirms a previous study showing that bbln-1 mutation-caused IF phenotypes can be suppressed by depleting IFB-2.Weakness:
The finding of suppressing the intestinal phenotypes caused in sma-5 mutants can be considered a minor conceptual advancement. However, the study comes short of providing insight into the molecular processes of how deranged IF networks and its consequence can be rescued/suppressed by removing e.g. the IFB-2 filaments. Many statements concerning the relationship between SMA-5 and the IFs are based on assumptions. The study requires protein biochemical analysis to show whether SMA-5 phosphorylates the IF proteins - mainly the IFB-2 polypeptide. The relationship between SMA-5 / IFB-2 is a central aspect of this study but the main conclusions are based on the notion that IFB-2 and other IF proteins may be phosphorylated by SMA-5. Mutating putative phosphorylation sites of IFB-2 without having shown any proof that the modification occurs by SMA-5 is futile. This important open question needs to be addressed. And will allow statements whether the ifb-2(kc20) mutant allele-encoded shorter IFB-2 protein lacks phosphorylation or not.
No quantification of the morphological defects such as using fluorescent-labeled IF proteins as in previous studies is provided in the manuscript. The EM pictures are not sufficient to provide information on how often the IF network perturbations and morphology defects occur. Also, the rescue of the actual morphological gut defects was not quantified. The assessment of development time and arrest, body length, lifespan, oxidative stress resistance, and others should be related to intestinal tube defects. They are useful and important but are an indirect measure of intestine defects and rescue.
It is not clear how exactly the mutant ifb-2 allele kc20 was identified. In the Materials and methods section, the authors provide information on the specific primers for the ifb-2 locus. But how did they know that the mutation lies within this region? Was there mutation mapping or whole-genome sequencing applied?
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Reviewer #3 (Public Review):
This manuscript by Geisler and colleagues used suppressor genetics to identify suppressors of the sma-5(n678) allele, which results in a defective gut endotube (an IF layer just under the microvillar structure), small body size, slow development, and short life span. The authors identified an internal deletion allele in ifb-2, which stunningly rescues all of the phenotypes listed above (despite the apparent absence of an endotube). This suppression is also observed with a previously characterized knockout allele. Conversely, this allele also suppresses analogous defects that result from mutations in the ifo-1 gene and bbln-1.
This is an exceptionally rigorous set of experiments, beautifully described in a clear manuscript illustrated by nicely constructed figures. The overall finding, that some IF mutations …
Reviewer #3 (Public Review):
This manuscript by Geisler and colleagues used suppressor genetics to identify suppressors of the sma-5(n678) allele, which results in a defective gut endotube (an IF layer just under the microvillar structure), small body size, slow development, and short life span. The authors identified an internal deletion allele in ifb-2, which stunningly rescues all of the phenotypes listed above (despite the apparent absence of an endotube). This suppression is also observed with a previously characterized knockout allele. Conversely, this allele also suppresses analogous defects that result from mutations in the ifo-1 gene and bbln-1.
This is an exceptionally rigorous set of experiments, beautifully described in a clear manuscript illustrated by nicely constructed figures. The overall finding, that some IF mutations result in toxic aggregates that can be eliminated by the loss of a single IF protein is interesting both from a fundamental understanding of IF networks and its clinical implications. With one minor exception, the conclusions are well supported by the data presented.
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