Biology subjects

Rothman, J. H.

Publications and source records attributed to Rothman, J. H..

9 recordsLinked to original sources

bioRxiv2026

Evidence that local viscosity and NOX-dependent ROS increases render the tardigrade H. exemplaris resilient to extreme physical force

Biological phase changes provoked by stress, such as vitrification or gel-sol transitions, enable many organisms, including extremotolerant tardigrades, to enter quiescent states and survive extreme environmental conditions. Protein-driven phase transitions are hypothesized to produce large-scale changes in intracellular viscosity, allowing tardigrades to survive extreme stresses such as desiccation. We report that the tardigrade Hypsibius exemplaris undergoes both large-scale and local increases in intracellular viscosity following exposure to anoxic and hyperosmotic stress. Such dramatic shifts in cellular viscosity would be expected to enhance cellular resilience to physical force. Indeed, we found that tardigrades can survive, behave normally, and reproduce after exposure to the highest simulated hypergravity (HG) achievable in an ultracentrifuge (one million times Earths gravity). In contrast, Caenorhabditis elegans, a similarly sized animal, does not survive these extreme forces owing to loss of cellular integrity. Remarkably, tardigrades frozen during exposure to extreme hypergravitational force show minimal disruption of fine cellular ultrastructure and little evidence of stratification of cellular components whose density varies by nearly a factor of two. Further, exposure to anoxia, hyperosmotic stress, and HG all result in a large increase in reactive oxygen species (ROS), which is required for survival under these extreme environments. Inhibition of NADPH oxidase (NOX) suppresses survival both to HG and hyperosmotic stress. Our findings suggest that intracellular viscosity changes in response to multiple extreme stresses may underlie the resilience of these animals to extraordinary physical stress, and that survival in or recovery from these states relies on ROS signaling via NADPH oxidase. Significance StatementTardigrades are renowned for surviving conditions that are lethal to nearly all other life forms. We reveal two mechanisms that support this resilience: intracellular viscosity changes and NADPH oxidase-mediated ROS signaling. Through direct assessment of the effects of altered cellular material properties, found that tardigrades are resilient to forces up to one million times Earths gravity, establishing them as the most hypergravity-resistant animal currently known.

physiology↗

bioRxiv2025

Selective targeting of oncogenic KRAS G12D using peptide nucleic acid oligomers attached to cell-penetrating peptides

KRAS is a proto-oncogene that contains activating mutations in up to 30% of tumors. Many conventional therapies inhibit both cancerous and normal cells, which may cause toxicity. Thus, programmable mutant-selective targeted inhibitors are needed. Peptide nucleic acids (PNAs) incorporate base sequences analogous to DNA, with modified peptide backbones instead of ribose-phosphate backbones, allowing PNAs to hybridize with DNA with high avidity to suppress transcription. Here, we developed KRAS G12D-selective PNA oligomers with novel cell-penetrating flanking regions. Fluorescein-labeled PNA oligomers displayed high uptake rates in cells and nuclei. Exposure to PNA-delivery peptide conjugates resulted in repression of KRAS G12D mRNA and protein expression within 2 hours and lasting up to 48 hours. Varying cell-penetrating peptide (CPP) compositions and lengths of complementary KRAS sequences were tested using dose-response cell viability assays. These experiments identified configurations that were effective at selectively preventing growth of on-target KRAS G12D cells, while relatively sparing off-target KRAS G12C cells. Electrophoretic mobility shift assays demonstrated in vitro binding and selectivity for KRAS G12D DNA sequences. CPP-PNA-G12D-1 was effective against a panel of pancreatic ductal adenocarcinoma cell lines and patient-derived xenografts in vivo. These results show promise for an enhanced PNA-delivery peptide conjugate strategy as both a tool for studying tumors driven by oncogenic point mutations and as a potential therapeutic strategy to selectively target mutant cancer cells.

cancer biology↗

bioRxiv2024

Single-animal, single-tube RNA extraction for quantitative analysis of transcripts in the tardigrade Hypsibius exemplaris

The tardigrade Hypsibius exemplaris is an emerging model organism renowned for its ability to survive environmental extremes. To explore the molecular mechanisms and genetic basis of such extremotolerance, many studies rely on RNA-sequencing (RNA-seq), which can be performed on populations ranging from large cohorts to individual animals. Reverse Transcription Polymerase Chain Reaction (RT-PCR) and RNA interference (RNAi) are subsequently used to confirm RNA-seq findings and assess the genetic requirements for candidate genes, respectively. Such studies require an efficient, accurate, and affordable method for RNA extraction and measurement of relative transcript levels by quantitative RT-PCR (qRT-PCR). This work presents an efficient single-tardigrade, single-tube RNA extraction method (STST) that not only reliably isolates RNA from individual tardigrades but also reduces the required time and cost for each extraction. This RNA extraction method yields quantities of cDNA that can be used to amplify and detect multiple transcripts by quantitative PCR (qRT-PCR). The method is validated by analyzing dynamic changes in the expression of genes encoding two heat-shock-regulated proteins, Heat-Shock Protein 70 {beta}2 (HSP70 {beta}2) and Heat-Shock Protein 90 (HSP90), making it possible to assess their relative expression levels in heat-exposed individuals using qRT-PCR. STST effectively complements existing bulk and single tardigrade RNA extraction methods, permitting rapid and affordable examination of individual tardigrade transcriptional levels by qRT-PCR. SUMMARYThis work presents a rapid RNA extraction and transcript level comparison method for analyzing gene expression in the tardigrade Hypsibius exemplaris. Using physical lysis, this high-throughput method requires a single tardigrade as the starting material and results in robust production of cDNA for quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR).

molecular biology↗

bioRxiv2022

Parallel Mechanosensory Systems are Required for Negative Gravitaxis in C. elegans

The ability to sense Earths gravitational pull is essential for orientation, navigation, and proprioception in many organisms. We report here that C. elegans exhibits pronounced negative gravitaxis, or movement away from the Earths center, independent of orientation of the geomagnetic field. This behavior is antagonized by light and electromagnetic fields, suggesting that it is integrated with other sensory inputs. We found that MEC-5/7/12, and TRPA-1, but not MEC-4/10 DEG/ENaC channels or other proteins involved in gentle touch transduction, are essential for negative gravitaxis, suggesting that the sensory system for detecting and responding to gravity is separable from touch sensation. We also found that the PVD neurons but not touch receptor neurons (TRNs) are required for this behavior. These findings implicate an interconnected mechanism for gravity sensation involving an ion channel that is also present in the mammalian vestibular system, suggesting possible homology in gravity sensing across animal phylogeny.

neuroscience↗

bioRxiv2021

Regulation of defective mitochondrial DNA accumulation and transmission in C. elegans by the programmed cell death and aging pathways

The heteroplasmic state of eukaryotic cells allows for cryptic accumulation of defective mitochondrial genomes (mtDNA). "Purifying selection" mechanisms operate to remove such dysfunctional mtDNAs. We found that activators of programmed cell death (PCD), including the CED-3 and CSP-1 caspases, the BH3-only protein CED-13, and PCD corpse engulfment factors, are required in C. elegans to attenuate germline abundance of a 3.1 kb mtDNA deletion mutation, uaDf5, which is normally stably maintained in heteroplasmy with wildtype mtDNA. In contrast, removal of CED-4/Apaf1 or a mutation in the CED-4-interacting prodomain of CED-3, do not increase accumulation of the defective mtDNA, suggesting induction of a non-canonical germline PCD mechanism or non-apoptotic action of the CED-13/caspase axis. We also found that the abundance of germline mtDNAuaDf5 reproducibly increases with age of the mothers. This effect is transmitted to the offspring of mothers, with only partial intergenerational removal of the defective mtDNA. In mutants with elevated mtDNAuaDf5 levels, this removal is enhanced in older mothers, suggesting an age-dependent mechanism of mtDNA quality control. Indeed, we found that both steady-state and age-dependent accumulation rates of uaDf5 are markedly decreased in long-lived, and increased in short-lived, mutants. These findings reveal that regulators of both PCD and the aging program are required for germline mtDNA quality control and its intergenerational transmission.

genetics↗

bioRxiv2021

Recursive feedforward regulatory logic underlies robustness of the specification-to-differentiation transition and fidelity of terminal cell fate during C. elegans endoderm development

Development is driven by gene regulatory networks (GRNs) that progressively dictate specification and differentiation of cell fates. The architecture of GRNs directly determines the specificity and accuracy of developmental outcomes. We report here that the core regulatory circuitry for endoderm development in C. elegans is comprised of a recursive series of interlocked feedforward modules linking a cascade of six sequentially expressed GATA-type transcription factors. This structure results in a reiterated sequential redundancy, in which removal of a single factor or alternate factors in the cascade results in no, or a mild, effect on endoderm development and gut differentiation, while elimination of any two factors that are sequentially deployed in the cascade invariably results in a strong phenotype. The strength of the observed phenotypes is successfully predicted by a computational model based on the timing and levels of transcriptional states. The feedforward regulatory logic in the GRN appears to ensure timely onset of terminal differentiation genes and allows rapid and robust lockdown of cell fate during early embryogenesis. We further found that specification-to-differentiation transition is linked through a common regulator, the END-1 GATA factor that straddles the two processes. Finally, we revealed roles for key GATA factors in establishing spatial regulatory state domains by acting as transcriptional repressors that appear to define the boundaries of the digestive tract. Our findings support a comprehensive model of the core gene network that describes how robust endoderm development is achieved during C. elegans embryogenesis. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/457588v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@8515d6org.highwire.dtl.DTLVardef@15ca234org.highwire.dtl.DTLVardef@9d0fe7org.highwire.dtl.DTLVardef@17f28db_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2020

Early C. elegans embryos modulate cell division timing to compensate for, and survive, the discordant conditions of a severe temperature gradient

Despite a constant barrage of intrinsic and environmental noise, embryogenesis is remarkably reliable, suggesting the existence of systems that ensure faithful execution of this complex process. We report that early C. elegans embryos, which normally show a highly reproducible lineage and cellular geometry, can compensate for deviations imposed by the discordant conditions of a steep temperature gradient generated in a microfluidic device starting at the two-cell stage. Embryos can survive a gradient of up to 7.5{degrees}C across the 50-micron axis through at least three rounds of division. This response is orientation-dependent: survival is higher when the normally faster-dividing anterior daughter of the zygote, AB, but not its sister, the posterior P1, is warmer. We find that temperature-dependent cellular division rates in the early embryo can be effectively modeled by a modification of the Arrhenius equation. Further, both cells respond to the gradient by dramatically reducing division rates compared to the predicted rates for the temperature experienced by the cell even though the temperature extremes are well within the range for normal development. This finding suggests that embryos may sense discordance and slow development in response. We found that in the cohort of surviving embryos, the cell on the warmer side at the two-cell stage shows a greater average decrease in expected division rate than that on the cooler side, thereby preserving the normal cellular geometry of the embryo under the discordant conditions. A diminished average slow-down response correlated with lethality, presumably owing to disruption of normal division order and developmental fidelity. Remarkably, some inviable embryos in which the canonical division order was reversed nonetheless proceeded through relatively normal morphogenesis, suggesting a subsequent compensation mechanism independent of cell division control. These findings provide evidence for a previously unrecognized process in C. elegans embryos that may serve to compensate for deviations imposed by aberrant environmental conditions, thereby resulting in a high-fidelity output.

developmental biology↗

bioRxiv2019

Natural cryptic epigenetic variation in an embryonic gene regulatory network

Gene regulatory networks (GRNs) that direct animal embryogenesis must respond to varying environmental and physiological conditions to ensure robust construction of organ systems. While GRNs are evolutionarily modified by natural genomic variation, the roles of epigenetic processes in shaping plasticity of GRN architecture are not well-understood. The endoderm GRN in C. elegans is initiated by the maternally supplied SKN-1/Nrf2 bZIP transcription factor; however, the requirement for SKN-1 in endoderm specification varies widely among distinct C. elegans wild isotypes owing to rapid developmental system drift driven by accumulation of cryptic genetic variants. We report here that heritable epigenetic factors that are stimulated by transient developmental diapause also underlie cryptic variation in the requirement for SKN-1 in endoderm development. This epigenetic memory is inherited from the maternal germline, apparently through a nuclear, rather than cytoplasmic, signal, resulting in a parent-of-origin effect (POE), in which the phenotype of the progeny resembles that of the maternal founder. The occurrence and persistence of POE varies between different parental pairs, perduring for at least ten generations in one pair. This long-perduring POE requires piwi-piRNA function and the germline nuclear RNAi pathway, as well as MET-2 and SET-32, which direct histone H3K9 trimethylation and drive heritable epigenetic modification. Such non-genetic cryptic variation may provide a resource of additional phenotypic diversity through which adaptation may facilitate evolutionary changes and shape developmental regulatory systems.

genetics↗

bioRxiv2019

Intertwined Functions of Separase and Caspase in Cell Division and Programmed Cell Death

Timely sister chromatid separation, promoted by separase, is essential for faithful chromosome segregation. Separase is a member of the CD clan of cysteine proteases, which also includes the pro-apoptotic enzymes known as caspases. We report that the C. elegans separase SEP-1, primarily known for its role in cell division, is required for apoptosis when the predominant pro-apoptotic caspase CED-3 is compromised. Loss of SEP-1 results in extra surviving cells in a weak ced-3(-) mutant, and suppresses the embryonic lethality of a mutant defective for the apoptotic suppressor ced-9/Bcl-2. We also report apparent non-apoptotic roles for CED-3 in promoting germ cell proliferation and germline meiotic chromosome disjunction and the normal rate of embryonic development. Moreover, loss of the soma-specific (CSP-3) and germline-specific (CSP-2) caspase inhibitors results in CED-3-dependent suppression of embryonic lethality and meiotic chromosome non-disjunction respectively, when separase function is compromised. Thus, while caspases and separases have evolved different substrate specificities associated with their specialized functions in apoptosis and cell division respectively, they appear to have retained the residual ability to participate in both processes, supporting the view that co-option of components in cell division may have led to the innovation of programmed cell suicide early in metazoan evolution.

genetics↗