Biology subjects

Reiner, D. J.

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

14 recordsLinked to original sources

bioRxiv2026

Aging, dauer, and stature phenotypes are conferred by structure-directed missense mutations in the endogenous AGE-1/phosphatidylinositol 3-kinase catalytic subunit.

Phosphatidylinositol 3-kinase (PI3K) integrates insulin/IGF signaling (IIS) and Ras inputs to control lifespan, metabolism and growth, yet the organismal consequences of selective structural perturbations remain poorly understood. Using structure-guided CRISPR/Cas9-dependent genome editing, we dissected functions of AGE-1, the sole Class IA PI3K catalytic subunit in Caenorhabditis elegans. An endogenously tagged AGE-1, containing a long flexible linker, epitope and fluorescent tag, retained full activity, enabling visualization of native protein dynamics in vivo. A constitutively active E630K substitution, modeled on oncogenic p110 alleles, markedly shortened lifespan and enhanced Ras-dependent induction of primary vulval precursor cell (VPC) fate, confirming evolutionary conservation of PI3K activation mechanisms that directly modulate longevity and development. Structural modeling further guided mutation of AGE-1 residues predicted to mediate Ras binding. Unexpectedly, a putative AGE-1 variant defective in Ras association, together with a complementary Ras effector-binding mutation, produced enlarged animals with reduced dauer formation. These phenotypes reveal a previously unrecognized Ras>PI3K signaling axis that restrains somatic growth and promotes entry into diapause, counter to canonical IIS models. Together, these structure-informed alleles show that discrete PI3K structural perturbations can differentially uncouple lifespan, growth, and developmental outcomes in vivo. By combining structural modeling with genome editing in a tractable aging model, this work establishes a framework for dissecting conserved signaling enzymes at single-residue resolution and uncovers unexpected organismal roles for PI3K structure in coordinating growth and longevity.

developmental biology↗

bioRxiv2025

The Ral small GTPase is an essential regulator of Exocyst complex function in secretion.

Ral GTPases have long been proposed as regulators of the metazoan Exocyst, a conserved secretory vesicle-tethering complex, but direct evidence for this role has been scarce. In contrast, the well-studied yeast Exocyst relies on multiple Rab GTPases to regulate function, but yeast do not encode Ral. Using Caenorhabditis elegans we demonstrate that endogenous RAL-1 directly engages the Exocyst through conserved binding sites in its subunits. Loss of RAL-1 disrupts dendritic arborization of PVD sensory neurons, impairs vesicle trafficking, and causes broad developmental defects, acting both cell-autonomously in neurons and non-autonomously through supporting epithelial cells. Structure-guided genome editing of RAL-1-Exocyst interfaces produced synthetic phenotypes, underscoring the physiological importance of these contacts. Taken together, our findings establish RAL-1 as a bona fide regulator of the metazoan Exocyst in vivo and suggest that Ral-Exocyst interactions operate in parallel with other secretory pathways. More broadly, this work positions C. elegans as a powerful system to dissect Ral- Exocyst mechanisms across molecular, cellular, and developmental scales.

cell biology↗

bioRxiv2025

Caenorhabditis elegans Uses Canonical and Non-canonical Hippo signaling

Hippo signaling is a conserved regulator of tissue homeostasis across metazoans. The Ste20 family kinase Hippo/MST activates the NDR family kinase Warts/LATS to inhibit the transcriptional coactivator Yorkie/YAP/TAZ and its transcription factor partner Scalloped/TEAD. In Caenorhabditis elegans, cell lineages and organ sizes are largely invariant, and classical Hippo phenotypes such as tissue overgrowth are absent. Nevertheless, WTS-1, YAP-1, and the TEAD-like transcription factor EGL-44 form a conserved core module required for larval development past the L2 stage. Crucially, a direct role for Hippo signaling remains unestablished. To address this question, we generated a fluorescently tagged endogenous YAP-1 as a live biomarker of pathway activity. Upon WTS-1 loss, endogenous YAP-1 translocated from cytosol to nucleus in epithelium and intestine. Tissue-specific depletion revealed that intestinal, but not epithelial, WTS-1 is essential for progression past L2. The duplicated Hippo-related kinases CST-1 and CST-2 repressed YAP-1 nuclear localization in the epithelium but not intestine, indicating that intestinal WTS-1 functions without CST-1/2. The Ste20 kinase MIG-15, orthologous to Drosophila Misshapen and mammalian MAP4K4/6/7/8, was redundant with CST-1/2 for larval progression. Yet deficient MIG-15 uniquely increased YAP-1 abundance without driving nuclear localization. By contrast, the Ste20 kinase GCK-2, orthologous to Drosophila Happyhour and mammalian MAP4K1/2/3/5, had no detectable role. Our findings establish C. elegans as a model for Hippo signaling, with a canonical cascade active in the epithelium and noncanonical inputs controlling WTS-1 in the intestine. In this context, YAP-1/EGL-44 outputs are repurposed from growth control to non-proliferative developmental functions.

developmental biology↗

bioRxiv2025

FGF-dependent, polarized SOS activity orchestrates directed migration of C. elegans muscle progenitors independently of canonical effectors in vivo

Directed cell migration is essential for animal development, tissue maintenance, regeneration, and disease states. Cells often migrate towards, or away from, sources of secreted signaling proteins that impart spatial information. How migrating cells interpret extracellular signals to orient and navigate within living animals is a fundamental question in biology. Receptor Tyrosine Kinase (RTK) signaling plays critical roles in cell migration, and aberrant RTK pathway activity is a key driver of multiple types of cancers. Yet, how RTKs control cell migration in living animals remains unclear, in part due to essential, pleiotropic roles of key proteins in development. To elucidate how RTK signaling controls cell migration in vivo, we dissected spatial and temporal requirements for key signal transduction and cytoskeletal regulatory proteins using C. elegans muscle progenitor migration as a tractable model. Cell type-specific depletion of endogenously tagged proteins revealed that homologs of FGFR, GRB2, SOS, and Ras control cell migration independently of their canonical ERK, PI3K, Akt, mTOR, and PLC{psi} effectors. Instead, we found that FGF-dependent, polarized SOS-1 orients migrating cells towards an FGF source, and mislocalizing SOS activity within migrating cells severely disrupts migration independent of ERK. Cell type-specific, gain-of-function experiments demonstrated that activated Ras is largely permissive for anterior migration in this context, and an intragenic revertant identified in a screen for suppressors of activated Ras/let-60 revealed that signal transduction in migrating muscle progenitors can be genetically uncoupled from Ras-ERK-dependent developmental processes. We found that conserved regulators of branched actin assembly control SM protrusive dynamics but are not essential for accurate, FGF-directed migration. Our findings provide a novel mechanism for RTK-directed cell migration in vivo and highlight the importance of cell type-specific approaches to elucidate signal transduction mechanisms in physiologically relevant contexts. Our work also outlines a comprehensive framework for investigating RTK-dependent processes in a multicellular organism and introduces a versatile genetic toolkit for dissecting spatial and temporal signaling dynamics fundamental to development, homeostasis, and disease.

cell biology↗

bioRxiv2022

Effect of selective lesions of nucleus accumbens μ-opioid receptor-expressing cells on heroin self-administration in male and female rats: a study with novel Oprm1-Cre knock-in rats

The brain {micro}-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell-type specific genetic access to MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to study the role of nucleus accumbens (NAc) MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and fluorescence in situ hybridization chain reaction (HCR-FISH), we found no differences in Oprm1 expression in NAc, dorsal striatum (DS), and dorsal hippocampus, or MOR receptor density (except DS) or function between Oprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed that iCre is highly co-expressed with Oprm1 (95-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells and report sex-specific effects: the lesions decreased acquisition of heroin self-administration in male Oprm1-Cre rats and had a stronger inhibitory effect on the effort to self-administer heroin in female Oprm1-Cre rats. The validation of an Oprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study with these rats suggests a sex-specific role of NAc MOR-expressing cells in heroin self-administration. Significance statementThe brain {micro}-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell-type specific genetic access to brain MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to show a potential sex-specific role of nucleus accumbens MOR-expressing cells in heroin self-administration. The new Oprm1-Cre rats can be used to study both the general and sex-specific role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.

neuroscience↗

bioRxiv2022

Identifying the C. elegans vulval transcriptome

Development of the C. elegans vulva is a classic model of organogenesis. This system, which starts with six equipotent cells, encompasses diverse types of developmental event, including developmental competence, multiple signaling events to control precise and faithful patterning of three cell fates, execution and proliferation of specific cell lineages, and a series of sophisticated morphogenetic events. Early events have been subjected to extensive mutational and genetic investigations and later events to cell biological analyses. We infer the existence of dramatically changing profiles of gene expression that accompanies the observed changes in development. Yet except from serendipitous discovery of several transcription factors expressed in dynamic patterns in vulval lineages, our knowledge of the transcriptomic landscape during vulval development is minimal. This study describes the composition of a vulva-specific transcriptome. We used tissue specific harvesting of mRNAs via immunoprecipitation of epitope-tagged poly(A) binding protein, PAB-1, heterologously expressed by a promoter known to express GFP in vulval cells throughout their development. The identified transcriptome was small but tightly interconnected. From this data set we identified several genes with identified functions in development of the vulva and validated more with promoter-GFP reporters of expression. For one target, lag-1, promoter-GFP expression was limited but fluorescent tag of the endogenous protein revealed extensive expression. Thus, we have identified a transcriptome of the C. elegans as a launching pad for exploration of functions of these genes in organogenesis.

genetics↗

bioRxiv2021

Lack of effect of different pain-related manipulations on opioid self-administration, reinstatement of opioid seeking, and opioid choice in rats

Rationale and Objective: Pain-related factors increase risk for opioid addiction, and opioid-induced pain relief may function as a negative reinforcer to increase opioid taking and seeking. However, experimental pain-related manipulations generally do not increase opioid self-administration in rodents. This discrepancy may reflect insufficient learning of pain-relief contingencies or confounding effects of pain-related behavioral impairments. Here we determined if pairing noxious stimuli with opioid self-administration would promote pain-related reinstatement of opioid seeking or increase opioid choice over food. Methods: In Experiment 1, rats self-administered fentanyl in the presence or absence of repeated intraplantar capsaicin injections in distinct contexts to model context-specific exposure to cutaneous nociception. After capsaicin-free extinction in both contexts, we tested if capsaicin would reinstate fentanyl seeking. In Experiment 2, rats self-administered heroin after intraperitoneal (i.p.) lactic acid injections to model acute visceral inflammatory pain. After lactic acid-free extinction, we tested if lactic acid would reinstate heroin seeking. In Experiment 3, we tested if repeated i.p. lactic acid or intraplantar Complete Freund Adjuvant (CFA; to model sustained inflammatory pain) would increase fentanyl choice over food. Results: In Experiments 1-2, neither capsaicin nor lactic acid reinstated opioid seeking after extinction, and lactic acid did not increase heroin-induced reinstatement. In Experiment 3, lactic acid and CFA decreased reinforcement rate without affecting fentanyl choice. Conclusions: Results extend the range of conditions across which pain-related manipulations fail to increase opioid seeking in rats and suggest that enhanced opioid-addiction risk in humans with chronic pain involves factors other than enhanced opioid reinforcement and relapse.

neuroscience↗

bioRxiv2021

Engineering Threshold-Based Selection Systems

Using model organisms to identify novel therapeutic targets is frequently constrained by pre-existing genetic toolkits. To expedite positive selection for identification of novel downstream effectors, we engineered conditional expression of activated CED-10/Rac to disrupt C. elegans embryonic morphogenesis, titrated to 100% lethality. The strategy of engineering thresholds for positive selection using experimental animals was validated with pharmacological and genetic suppression and is generalizable to diverse molecular processes and experimental systems.

genetics↗

bioRxiv2021

Parallel Rap1>RalGEF>Ral and Ras signals sculpt the C. elegans nervous system

Ras is the most commonly mutated oncogene in humans and uses three oncogenic effectors: Raf, PI3K, and RalGEF activation of Ral. Understanding the importance of RalGEF>Ral signaling in cancer is hampered by the paucity of knowledge about their function in animal development, particularly in cell movements. We found that mutations that disrupt function of RalGEF or Ral enhance migration phenotypes of mutations in genes with established roles in cell migration. We used as a model the migration of the canal associated neurons (CANs), and validated our results in HSN cell migration, neurite guidance, and general animal locomotion. These functions of RalGEF and Ral are specific to their control of Ral signaling output rather than other published functions of these proteins. In this capacity Ral functions cell autonomously as a permissive developmental signal. In contrast, we observed Ras, the canonical activator of RalGEF>Ral signaling in cancer, to function as an instructive signal. Furthermore, we unexpectedly identified a function for the close Ras relative, Rap1, consistent with activation of RalGEF>Ral. These studies define functions of RalGEF>Ral, Rap1 and Ras signaling in morphogenetic processes that fashion the nervous system. We have also defined a model for studying how small GTPases partner with downstream effectors. Taken together, this analysis defines novel molecules and relationships in signaling networks that control cell movements during development of the nervous system.

developmental biology↗

bioRxiv2021

Nuclear translocation of tagged endogenous ERK/MPK-1 MAP Kinase denotes a subset of activation events in C. elegans development

The extracellular signal-regulated kinase (ERK) MAP kinase is utilized downstream of Ras>Raf>MEK signaling to control activation of a wide array of targets. Activation of ERK is elevated in Ras-driven tumors and RASopathies, and is thus a target for pharmacological inhibition. Regulatory mechanisms of ERK activation has been studied extensively in vitro and in cultured cells but little in living animals. We used CRISPR to tag the 3 end of the C. elegans ERK-encoding gene, mpk-1. Endogenous MPK-1 protein is ubiquitously expressed with elevated expression in certain tissues. We detected cytosol-to-nuclear translocation of MPK-1 in maturing oocytes and hence validated nuclear translocation as a reporter of some activation events. During developmental patterning of the six vulval precursor cells, MPK-1 is necessary and sufficient for the central cell, P6.p, to assume 1{degrees} fate. We observed MPK-1 to be recruited to the nuclei of all six VPCs in a temporal and concentration gradient centered on P6.p. This observation contrasts with previous results using the ERK-nKTR reporter of substrate activation, raising questions about mechanisms and indicators of MPK-1 activation. This system and reagent promise to provide critical insights into regulation of MPK-1 activation within a complex intercellular signaling network.

developmental 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↗

bioRxiv2018

Developmental fidelity imposed by the RGL-1 balanced switch mediating opposing signals

The six C. elegans vulval precursor cells (VPCs) are induced to form the 30-30-20-{Gamma}-20-3{degrees} pattern of cell fates with high fidelity. In response to EGF signal, the LET-60/Ras-LIN-45/Raf-MEK-2/MEK-MPK-1/ERK canonical MAP kinase cascade is necessary to induce 1{degrees} fate and synthesis of DSL ligands. In turn, LIN-12/Notch signal is necessary to induce neighboring cells to become 2{degrees}. We previously showed that, in response to lower dose of EGF signal, the modulatory LET-60/Ras-RGL-1/RalGEF-RAL-1/Ral signal promotes 2{degrees} fate in support of LIN-12. In this study we identify two key differences between RGL-1 and RAL-1 functions. First, deletion of RGL-1 confers no overt developmental defects, while previous studies showed RAL-1 to be essential for viability and fertility. From this observation we hypothesize that the developmentally essential functions of RAL-1 are independent of upstream activation. Second, RGL-1 plays opposing and genetically separable roles in VPC fate patterning. RGL-1 promotes 2{degrees} fate via canonical GEF-dependent activation of RAL-1 and 1{degrees} fate via a non-canonical GEF-independent activity. Our genetic epistasis experiments are consistent with RGL-1 functioning in the modulatory 1{degrees}-promoting AGE-1/PI3-Kinase-PDK-1-AKT-1 cascade. Additionally, animals without RGL-1 experience 15-fold higher rates of VPC patterning errors compared to the wild type. Yet VPC patterning in RGL-1 deletion mutants is not more sensitive to environmental perturbations. We propose that RGL-1 functions as a \"Balanced Switch\" that orchestrates opposing 1{degrees}- and 2{degrees}-promoting modulatory cascades to decrease inappropriate fate decisions. We speculate that such switches are broadly conserved but mostly masked by paralog redundancy or essential genes.

genetics↗

bioRxiv2018

Ral signals through a MAP4 Kinase-p38 MAP kinase cascade in C. elegans cell fate patterning.

C. elegans vulval precursor cell (VPC) fates are patterned by an EGF gradient. High dose EGF induces 1{degrees} VPC fate, while lower dose EGF contributes to 2{degrees} fate in support of LIN-12/Notch. We previously showed that the EGF 2{degrees}-promoting signal is mediated by LET-60/Ras switching effectors, from the canonical Raf-MEK-ERK MAP kinase cascade that promotes 1{degrees} fate to the non-canonical RalGEF-Ral that promotes 2{degrees} fate. Of oncogenic Ras effectors, RalGEF-Ral is by far the least well-understood. We use genetic analysis to identify an effector cascade downstream of C. elegans RAL-1/Ral, starting with an established Ral binding partner, Exo84 of the exocyst complex. Additionally, RAL-1 signals through GCK-2, a CNH domain-containing MAP4 kinase, and PMK-1/p38 MAP kinase cascade to promote 2{degrees} fate. Our study delineates a Ral-dependent developmental signaling cascade in vivo, thus providing the mechanism by which lower EGF dose is transduced.

developmental biology↗

bioRxiv2018

C. elegans RAP-1 reinforces LET-60/Ras induction of cell fate

The notoriety of the small GTPase Ras as the most mutated oncoprotein has led to a well-characterized signaling network largely conserved across metazoans. Yet the role of its close relative Rap1 (Ras Proximal), which shares 100% identity between their core effector binding sequences, remains unclear. A long-standing controversy in the field is whether Rap1 also functions to activate the canonical Ras effector, the S/T kinase Raf. We used the developmentally simpler Caenorhabditis elegans, which lacks the extensive paralog redundancy of vertebrates, to examine the role of RAP-1 in two distinct LET-60/Ras-dependent cell fate patterning events: induction of 1{ring} vulval precursor cell (VPC) fate and of the excretory duct cell. Fluorescently tagged endogenous RAP-1 is localized to plasma membranes and is expressed ubiquitously, with even expression levels across the VPCs. RAP-1 and its activating GEF PXF-1 function cell autonomously and are necessary for maximal induction of 1{ring} VPCs. Critically, mutationally activated endogenous RAP-1 is sufficient both to induce ectopic 1{ring}s and duplicate excretory duct cells. Like endogenous RAP-1, before induction GFP expression from the pxf-1 promoter is uniform across VPCs. However, unlike endogenous RAP-1, after induction GFP expression is increased in presumptive 1{ring}s and decreased in presumptive 2{ring}s. We conclude that RAP-1 is a positive regulator that promotes Ras-dependent inductive fate decisions. We hypothesize that PXF-1 activation of RAP-1 serves as a minor parallel input into the major LET-60/Ras signal through LIN-45/Raf.

genetics↗