Biology subjects

Hibshman, J. D.

Publications and source records attributed to Hibshman, J. D..

10 recordsLinked to original sources

bioRxiv2026

A secreted LEA-like protein functions as a basement membrane stress protectant

Organisms have evolved to withstand a wide variety of environmental stresses. Survival requires individual cells to mount appropriate responses to combat molecular damage. In animals, multicellularity introduces an additional challenge: how is tissue integrity preserved? Our knowledge of extracellular tissue-level mechanisms of protection remains limited. Here, we report that a late embryogenesis abundant (LEA)-like protein, DUR-1, is secreted and then accumulates in basement membranes, where it acts to promote tissue integrity and stress survival. DUR-1 is strongly upregulated in the stress-tolerant dauer diapause stage of C. elegans and further upregulated upon desiccation. Loss of the protein sensitizes animals to desiccation and osmotic stress. Other LEA proteins have been regarded as strong desicco-protectants, but none have been recognized as components of basement membranes. We show that DUR-1 is important for maintaining the integrity of basement membrane-ensheathed tissues under mechanical stresses associated with drying or osmotic challenge. This work defines a mechanism for extracellular protection and emphasizes the importance of preserving tissue integrity during stress.

genetics↗

bioRxiv2023

Tardigrades dramatically upregulate DNA repair pathway genes in response to ionizing radiation

Tardigrades can survive remarkable doses of ionizing radiation, up to about 1000 times the lethal dose for humans. How they do so is incompletely understood. We found that the tardigrade Hypsibius exemplaris suffers DNA damage upon gamma irradiation, but damage is repaired. We show that tardigrades have a specific and robust response to ionizing radiation: irradiation induces a rapid, dramatic upregulation of many DNA repair genes. By expressing tardigrade genes in bacteria, we validate that increased expression of some repair genes can suffice to increase radiation tolerance. We show that at least one such gene is necessary for tardigrade radiation tolerance. Tardigrades ability to sense ionizing radiation and massively upregulate specific DNA repair pathway genes may represent an evolved solution for maintaining DNA integrity. One-Sentence SummaryTardigrades exposed to ionizing radiation survive DNA damage by massively upregulating DNA repair pathway genes.

molecular biology↗

bioRxiv2023

A bacterial expression cloning screen reveals tardigrade single-stranded DNA-binding proteins as potent desicco-protectants

Tardigrades are known for their ability to survive remarkable extremes, but relatively little is known about the molecular underpinnings that allow for their extraordinary stress tolerance. We devised an expression cloning approach to screen for novel protectants from desiccation-tolerant tardigrades. We expressed cDNA libraries from the tardigrades Hypsibius exemplaris and Ramazzottius varieornatus in E. coli and subjected the bacteria to desiccation. Sequencing the populations of surviving bacteria revealed enrichment of mitochondrial single-stranded DNA-binding proteins (mtSSBs) from both tardigrade species. Expression of mtSSBs in bacteria improved desiccation survival to similar levels as some of the best protectants known to date. The DNA-binding activity of the oligonucleotide/oligosaccharide binding (OB) fold domain of mtSSB was necessary and sufficient to improve desiccation tolerance. By comparing the efficacy of mtSSBs from multiple organisms, we provide evidence that desicco-protection is a conserved feature of mtSSBs. More broadly, any OB-fold containing protein may act similarly to promote desiccation tolerance, likely by coating ssDNA to limit damage during desiccation. These results identify single-stranded DNA-binding activity as a potent contributor to extreme desiccation tolerance.

molecular biology↗

bioRxiv2022

Tardigrade small heat shock proteins can limit desiccation-induced protein aggregation

Small heat shock proteins (sHSPs) are chaperones known for their response to heat stress. While sHSPs have well-characterized roles in heat tolerance, potential roles for sHSPs in desiccation tolerance have not been as thoroughly explored. We identified nine sHSPs from the genome of the tardigrade Hypsibius exemplaris, each containing a conserved alpha-crystallin domain flanked by disordered regions. Many of these sHSPs are highly expressed and in some cases are upregulated during desiccation. We found that tardigrade sHSPs were sufficient to improve desiccation tolerance when expressed in E. coli. Purification and subsequent analysis of two sHSPs, HSP21 and HSP24.6, revealed that these proteins can form large complexes in vitro, similar to oligomeric assemblies documented for other sHSPs. These proteins limited heat-induced aggregation of the model enzyme citrate synthase. Heterologous expression of HSP24.6 improved bacterial heat shock survival, and the protein significantly reduced heat-induced aggregation of soluble bacterial protein. Thus, HSP24.6 likely chaperones against protein aggregation to promote survival of heat stress. Furthermore, HSP21 and HSP24.6 also limited desiccation-induced aggregation and loss of function of citrate synthase. This suggests a mechanism by which tardigrade sHSPs promote desiccation tolerance: by limiting desiccation-induced protein aggregation, thereby maintaining proteostasis and supporting survival. These results suggest that sHSPs, classical chaperones, provide a mechanism of general stress resistance that can also be deployed to support survival during anhydrobiosis.

biochemistry↗

bioRxiv2021

In vivo analysis of LEA-1 in C. elegans desiccation tolerance

BackgroundCells and organisms typically cannot survive in the absence of water. However, there are some notable exceptions, including animals such as nematodes, tardigrades, rotifers, and some arthropods. One class of proteins known to play a role in desiccation resistance is the late embryogenesis abundant (LEA) proteins. These largely disordered proteins protect plants and animals from desiccation. A multitude of studies have characterized stress-protective capabilities of LEA proteins in vitro and in heterologous systems. However, the extent to which LEA proteins exhibit such functions in vivo, in their native contexts in animals, is unclear. Furthermore, little is known about the distribution of LEA proteins in multicellular organisms or tissue-specific requirements in conferring stress protection. ResultsTo study the endogenous function of an LEA protein in an animal, we created a true null mutant of C. elegans LEA-1, as well as endogenous fluorescent reporters of the protein. We confirmed that C. elegans lacking LEA-1 are sensitive to desiccation. LEA-1 mutant animals were also sensitive to heat and osmotic stress and were prone to protein aggregation. During desiccation, LEA-1 expression increased and became more widespread throughout the body. LEA-1 was required at high levels in body wall muscle for animals to survive desiccation and osmotic stress. We identified minimal motifs within C. elegans LEA-1 that are sufficient to increase desiccation survival of E. coli. To test whether such motifs are central to LEA-1s in vivo functions, we then replaced the sequence of lea-1 with these minimal motifs and found that C. elegans survived mild desiccation and osmotic stress at the same levels as worms with the full-length protein. ConclusionsOur results provide insights into the endogenous functions and expression dynamics of an LEA protein in a multicellular animal. The results show that LEA-1 buffers animals from a broad range of stresses. Our identification of LEA motifs that can function in both bacteria and in a multicellular organism suggests the possibility of engineering LEA-1-derived peptides for optimized desiccation protection.

genetics↗

bioRxiv2020

Early-life mitochondrial DNA damage results in lifelong deficits in energy production mediated by redox signaling in Caenorhabditis elegans

The consequences of damage to the mitochondrial genome (mtDNA) are poorly understood, although mtDNA is more susceptible to damage than nuclear DNA (nucDNA), and many environmental toxicants target the mitochondria. Reports from the toxicological literature suggest that exposure to early-life mitochondrial damage could lead to deleterious consequences later in life (the "Developmental Origins of Health and Disease" paradigm) but reports from other fields often report beneficial ("mitohormetic") responses to such damage. Here, we test the effects of low (causing no change in lifespan) levels of ultraviolet C (UVC)-induced, irreparable mtDNA damage during early development in Caenorhabditis elegans. This exposure led to life-long reductions in mtDNA copy number and steady-state ATP levels, accompanied by increased oxygen consumption and altered metabolite profiles, suggesting inefficient mitochondrial function. Exposed nematodes were also developmentally delayed, reached smaller adult size, and were rendered more susceptible to subsequent exposure to chemical mitotoxicants. Metabolomic and genetic analysis of key signaling and metabolic pathways supported redox signaling during early development as a mechanism for establishing these persistent alterations. Our results highlight the importance of early-life exposures to environmental pollutants, especially in the context of exposure to chemicals that target mitochondria.

cell biology↗

bioRxiv2020

Expanding the Caenorhabditis elegans auxin-inducible degron system toolkit with internal expression and degradation controls and improved modular constructs for CRISPR/Cas9-mediated genome editing

The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell-types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying an equimolar co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. We have also constructed a set of plasmids to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These templates can be produced through frequently used cloning systems (Gibson assembly or SapTrap) or through ribonucleoprotein complex-mediated insertion of PCR-derived, linear repair templates. We have generated a set of sgRNA plasmids carrying modifications shown to boost editing efficiency, targeting standardized transgene insertion sites on chromosomes I and II. Together these reagents should complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID* tagging of factors of interest. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for facile assembly of CRISPR/Cas9 repair templates for conditional protein depletion.

genetics↗

bioRxiv2019

Population selection and sequencing of C. elegans wild isolates identifies a region on chromosome III affecting starvation resistance

To understand the genetic basis of complex traits, it is important to be able to efficiently phenotype many genetically distinct individuals. In the nematode Caenorhabditis elegans, individuals have been isolated from diverse populations around the globe and whole-genome sequenced. As a result, hundreds of wild strains with known genome sequences can be used for genome-wide association studies (GWAS). However, phenotypic analysis of these strains can be laborious, particularly for quantitative traits requiring multiple measurements per strain. Starvation resistance is likely a fitness-proximal trait for nematodes, and it is related to metabolic disease risk in humans. However, natural variation in C. elegans starvation resistance has not been characterized, and precise measurement of the trait is time-intensive. Here, we developed a population selection and sequencing-based approach to phenotype starvation resistance in a pool of 96 wild strains. We used restriction site-associated DNA sequencing (RAD-seq) to infer the frequency of each strain among survivors in a mixed culture over time during starvation. We used manual starvation survival assays to validate the trait data, confirming that strains that increased in frequency over time are starvation-resistant relative to strains that decreased in frequency. These results document natural variation in starvation resistance. Further, we found that variation in starvation resistance is significantly associated with variation at a region on chromosome III. Using a near-isogenic line (NIL), we showed the importance of this genomic interval for starvation resistance. This study demonstrates the feasibility of using population selection and sequencing in an animal model for phenotypic analyses of quantitative traits, reveals natural variation of starvation resistance in C. elegans, and identifies a genomic region that contributes to such variation.

genetics↗

bioRxiv2018

Dauer diapause has transgenerational effects on starvation survival and gene expression plasticity

Phenotypic plasticity is facilitated by epigenetic regulation, and remnants of such regulation may persist after plasticity-inducing cues are gone. However, the relationship between plasticity and transgenerational epigenetic memory is not understood. Dauer diapause in Caenorhabditis elegans provides an opportunity to determine how a plastic response to the early-life environment affects traits later in life and in subsequent generations. We report that after extended diapause, post-dauer worms initially exhibit reduced reproductive success and greater inter-individual variation. In contrast, F3 progeny of post-dauers display increased starvation resistance and lifespan, revealing potentially adaptive transgenerational effects. Transgenerational effects are dependent on the duration of diapause, indicating an effect of extended starvation. In agreement, RNA-seq demonstrates a transgenerational effect on nutrient-responsive genes. Further, post-dauer F3 progeny exhibit reduced gene expression plasticity, suggesting a trade-off between plasticity and epigenetic memory. This work reveals complex effects of nutrient stress over different time scales in an animal that evolved to thrive in feast and famine.

evolutionary biology↗

bioRxiv2018

Intergenerational effects of dietary restriction on insulin/IGF signaling and reproductive development

The roundworm C. elegans transiently arrests larval development to survive extended starvation (1), but such early-life starvation reduces reproductive success (2, 3). Maternal dietary restriction (DR) buffers progeny from starvation, increasing reproductive success (4). It is unknown why early-life starvation decreases reproductive success and how maternal diet modifies this process. We show here that extended starvation in first-stage (L1) larvae followed by unrestricted feeding results in a variety of abnormalities in the reproductive system, including glp-1/Notch-sensitive germ-cell tumors and uterine masses that express neuronal and epidermal markers. We found that maternal DR reduces the penetrance of starvation-induced abnormalities, including tumors. Furthermore, we show that maternal DR reduces insulin/IGF signaling (IIS) in progeny, and that daf-16/FoxO and skn-1/Nrf, transcriptional effectors of IIS, are required in progeny for maternal DR to suppress abnormalities. daf-16/FoxO activity in somatic tissues is sufficient to suppress starvation-induced abnormalities, suggesting cell-nonautonomous regulation of reproductive system development. This work reveals complex inter- and intra-generational effects of nutrient availability mediated by IIS with consequences on developmental integrity and reproductive success.\n\nOne Sentence SummaryIntergenerational effects of diet on IIS

developmental biology↗