Biology subjects

Cram, E. J.

Publications and source records attributed to Cram, E. J..

16 recordsLinked to original sources

bioRxiv2026

The single-PDZ domain protein TXBP-3 regulates contractility in the C. elegans spermatheca

In the C. elegans reproductive system, oocyte transit through the spermatheca relies on parallel calcium and Rho-dependent signaling pathways to regulate actomyosin contractility. Here, we identify the conserved single-PDZ domain protein TXBP-3/TAX1BP3 as a dosage-dependent regulator of spermathecal contractility. TXBP-3 is expressed in contractile tissues, including the spermatheca and spermathecal-uterine (sp-ut) valve. Animals lacking txbp-3 do not display overt phenotypes or defects in ovulation. However, in nematodes expressing the genetically encoded calcium sensor GCaMP, deletion of txbp-3 results in more rapid transit of embryos through the spermatheca. Conversely, tissue-specific overexpression of TXBP-3 results in slower transit times due to hypercontraction of the spermathecal neck and sp-ut valve, causing oocyte severing and failures of embryos to exit the spermatheca. We find TXBP-3 promotes the active phosphorylated state of the calcium/calmodulin-dependent protein kinase II (CaMKII) UNC-43. When TXBP-3 is overexpressed or unc-43 is depleted, ovulations in which the embryo fails to exit show disrupted calcium signaling. Additionally, TXBP-3 functions independently of the RhoGEF RHGF-1 and in parallel with the RhoGAP SPV-1 to maintain the alignment of actin fiber bundles and protect embryo structural integrity. Together, our results establish TXBP-3 as a regulator of contractility, kinase activation, and cytoskeletal architecture in contractile tissues.

cell biology↗

bioRxiv2026

PES-8 is required for Cytoskeletal Organization and Contractility in the C. elegans Spermatheca

Proper regulation of contraction and relaxation in biological tubes is essential for organismal function. In C. elegans, the spermatheca, composed of smooth muscle-like cells, undergoes repeated stretching and contraction as oocytes pass through. Here we describe PES-8, a previously uncharacterized protein, as a regulator of spermatheca contractility. PES-8 contains a predicted extracellular zona pellucida-like domain and an unstructured cytoplasmic tail, suggesting dual roles in extracellular and cytoplasmic signaling. PES-8 localizes to the plasma membrane of the spermatheca, the spermathecal-uterine valve, and uterus. Functional analysis shows that PES-8 is essential for spermathecal function; its loss disrupts actomyosin fiber alignment, FLN-1/filamin localization, apical junction organization, and Ca{superscript 2} signaling, preventing oocyte transit. These findings identify PES-8 as a key regulator of cytoskeletal organization and calcium-mediated contractility in the C. elegans spermatheca.

cell biology↗

bioRxiv2025

A switch in collagen expression regulates cessation of distal tip cell migration in C. elegans

Development of the C. elegans gonad requires precise regulation of cell migration. The distal tip cell (DTC) guides the elongation of the gonad into its final U-shaped structure before halting in adulthood. How cessation of elongation is regulated remains unknown. Here, we analyze an RNA-seq data set isolated from stage-specific DTCs to uncover the temporal gene expression dynamics underlying this process. Collagens emerged as the most enriched gene family during the transition from migratory larval stages to adulthood. We identify distinct temporal waves of collagen expression, culminating in a core adult-specific module that coincides with migration cessation. Functional analysis by RNAi depletion revealed that many collagens upregulated in adulthood are required for timely migration arrest, while others affect gonad shape. Perturbation of collagen remodeling enzymes phenocopied these effects. Our findings uncover a stage-specific collagen program in the DTC and suggest that terminal migration arrest is actively reinforced by matrix remodeling.

developmental biology↗

bioRxiv2025

GOA-1 regulates spermathecal transits

G protein signaling regulates calcium dynamics and contractility in the C. elegans spermatheca. G protein-coupled receptors activate heterotrimeric G proteins, triggering downstream cascades, including the Gs-mediated activation of adenylyl cyclase and subsequent Protein Kinase A (PKA) activation. Our previous work identified GSA-1/Gs and PKA as key modulators of Ca2+ oscillations and tissue contractility within the C. elegans spermatheca. In this study, we show that the inhibitory Gi/o subunit GOA-1 regulates spermathecal transits. We employed TurboID proximity labeling and mass spectrometry to identify 16 candidate interactors of GOA-1. Depletion of these candidates by RNAi did not yield overt spermathecal transit defects.

cell biology↗

bioRxiv2025

Stage-specific transcriptomics of a leader cell reveals cell machineries driving collective invasion

Collective cell invasion underlies organ development, epithelial repair, and cancer metastasis. "Leader cells" remodel extracellular matrix, sense guidance cues, reorganize their cytoskeleton, and coordinate follower cells, but the molecular programs enabling these functions remain unclear. Here, we present a stage-specific transcriptomic dataset of the C. elegans gonadal leader cell, the distal tip cell (DTC), which invades basement membrane and guides germ cells to form U-shaped gonadal arms. Comparing invasive larval-stage DTCs with non-invasive adult-stage DTCs defines the molecular signature of an actively invading leader cell in vivo. Our dataset recapitulates known regulators of gonad morphogenesis and reveals numerous uncharacterized genes with potential roles in leader cell activity. As proof of concept, we identify vesicular trafficking proteins as enriched in invading DTCs, and demonstrate their importance for gonad development using endogenous tagging and DTC-specific RNAi. We also catalog diverse DTC-specific knockdown phenotypes. This resource establishes a molecular framework for leader cell activity and a platform to investigate conserved mechanisms of invasive migration.

developmental biology↗

bioRxiv2025

On-demand delivery of fibulin-1 enables repeated basement membrane stretching

Basement membrane (BM) extracellular matrices enwrap and structurally support tissues. Whether BMs are uniquely constructed to support tissues that undergo repetitive stretching and recoil events is unknown. During C. elegans ovulation, the spermathecal BM stretches [~]1.7-fold and then returns to its original shape every twenty minutes to passage hundreds of oocytes. Through live fluorescence microscopy, we discovered that ovulating oocytes secrete and deliver the fibulin-1 extracellular matrix protein to the spermathecal BM during stretching, where it forms a dynamic overlapping network with type IV collagen. Fibulin-1 depletion led to a breakdown in type IV collagen and BM organization, resulting in a more deformable BM and extended spermatheca. Moreover, perturbation to fibulin-1 network formation via mutagenesis was sufficient to disrupt organ shape. Together, our study reveals an on-demand fibulin-1 delivery system that protects the BM network when it is stretched, thereby allowing repeated rounds of organ expansion and recovery.

cell biology↗

bioRxiv2024

Tensions on the actin cytoskeleton and apical cell junctions in the C. elegans spermatheca are influenced by spermathecal anatomy, ovulation state and activation of myosin

Cells generate mechanical forces mainly through myosin motor activity on the actin cytoskeleton. In C. elegans, actomyosin stress fibers drive contractility of the smooth muscle-like cells of the spermatheca, a distensible, tube-shaped tissue in the hermaphrodite reproductive system and the site of oocyte fertilization. Stretching of the spermathecal cells by oocyte entry triggers activation of the small GTPase Rho. In this study, we asked how forces are distributed in vivo using the spermatheca, and explored how this tissue responds to alterations in myosin activity. Using laser ablation, we show that the basal actomyosin fibers are under tension in the occupied spermatheca. Reducing actomyosin contractility by depletion of the phospholipase C-{varepsilon}/PLC-1 or non-muscle myosin II/NMY-1, leads to distended spermathecae occupied by one or more embryos, but does not alter tension on the basal actomyosin fibers. This suggests that much of the tension on the basal actin fibers in the occupied spermatheca is due to the presence of the embryo. However, activating myosin through depletion of the Rho GAP SPV-1 increases tension on the actomyosin fibers, consistent with earlier studies showing Rho drives spermathecal contractility. On the inner surface of the spermathecal tube, tension on the apical junctions is decreased by depletion of PLC-1 and NMY-1. Surprisingly, when basal contractility is increased through SPV-1 depletion, the tension on apical junctions also decreases, with the most significant effect on the junctions aligned in perpendicular to the axis of the spermatheca. This suggests tension on the outer basal surface may compress the apical side, and suggests the three-dimensional shape of the spermatheca plays a role in force distribution and contractility during ovulation.

cell biology↗

bioRxiv2020

FLN-1/Filamin is required to anchor the actomyosin cytoskeleton and for global organization of sub-cellular organelles in a contractile tissue

Actomyosin networks are organized in space, direction, size, and connectivity to produce coordinated contractions across cells. We use the C. elegans spermatheca, a tube composed of contractile myoepithelial cells, to study how actomyosin structures are organized. FLN-1/filamin is required for the formation and stabilization of a regular array of parallel, contractile, actomyosin fibers in this tissue. Loss of fln-1 results in the detachment of actin fibers from the basal surface, which then accumulate along the cell junctions and are stabilized by spectrin. In addition, actin and myosin are captured at the nucleus by the linker of nucleoskeleton and cytoskeleton complex (LINC) complex, where they form large foci. Nuclear positioning and morphology, distribution of the endoplasmic reticulum and the mitochondrial network are also disrupted. These results demonstrate that filamin is required to prevent large actin bundle formation and detachment, to prevent excess nuclear localization of actin and myosin, and to ensure correct positioning of organelles.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsFRAPfluorescence recovery after photobleachingGFPgreen fluorescent proteinKASHKlarsicht/ANC-1/Syne homologyLINClinker of nucleoskeleton and cytoskeletonmoeABDmoesin actin binding domainRNAiRNA interferencesp-utspermatheca-uterineSUNSad1/UNC-84WTwild typeView Full Text

cell biology↗

bioRxiv2020

Addition of Multiple Introns to a Cas9 Gene Results in Dramatic Improvement in Efficiency for Generation of Gene Knockouts in Plants

The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by stably transforming a Cas9 expression construct into the plant genome. The efficiency with which mutations are obtained in genes of interest can vary considerably depending on specific features of the constructs, including the source and nature of the promoters and terminators used for expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency with which mutations could be obtained in target genes in Arabidopsis thaliana with the Cas9 nuclease, we have investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLS) and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on Cas9 activity and that two NLSs work better than one. However, the most important impact on the efficiency of the constructs was obtained by addition of 13 introns into the Cas9 coding sequence, which dramatically improved editing efficiencies of the constructs; none of the primary transformants obtained with a Cas9 lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of primary transformants generated with intronized Cas9 displayed mutant phenotypes. The intronized Cas9 was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.

molecular biology↗

bioRxiv2020

Gα/GSA-1 works upstream of PKA/KIN-1 to regulate calcium signaling and contractility in the Caenorhabditis elegans spermatheca

Correct regulation of cell contractility is critical for the function of many biological systems. The reproductive system of the hermaphroditic nematode C. elegans contains a contractile tube of myoepithelial cells known as the spermatheca, which stores sperm and is the site of oocyte fertilization. Regulated contraction of the spermatheca pushes the embryo into the uterus. Cell contractility in the spermatheca is dependent on actin and myosin and is regulated, in part, by Ca2+ signaling through the phospholipase PLC-1, which mediates Ca2+ release from the endoplasmic reticulum. Here, we describe a novel role for GSA-1/Gs, and protein kinase A, composed of the catalytic subunit KIN-1/PKA-C and the regulatory subunit KIN-2/PKA-R, in the regulation of Ca2+ release and contractility in the C. elegans spermatheca. Without GSA-1/Gs or KIN-1/PKA-C, Ca2+ is not released, and oocytes become trapped in the spermatheca. Conversely, when PKA is activated through either a gain of function allele in GSA-1 (GSA-1(GF)) or by depletion of KIN-2/PKA-R, Ca2+ is increased, and waves of Ca2+ travel across the spermatheca even in the absence of oocyte entry. In the spermathecal-uterine valve, loss of GSA-1/Gs or KIN-1/PKA-C results in sustained, high levels of Ca2+ and a loss of coordination between the spermathecal bag and sp-ut valve. Additionally, we show that depleting phosphodiesterase PDE-6 levels alters contractility and Ca2+ dynamics in the spermatheca, and that the GPB-1 and GPB-2 G{beta} subunits play a central role in regulating spermathecal contractility and Ca2+ signaling. This work identifies a signaling network in which Ca2+ and cAMP pathways work together to coordinate spermathecal contractility. Author SummaryOrganisms are full of biological tubes that transport substances such as food, liquids, and air through the body. Moving these substances in a coordinated manner, with the correct directionality, timing, and rate is critical for organism health. In this study we used Caenorhabditis elegans, a small transparent worm, to study how cells in biological tubes coordinate how and when they squeeze and relax. The C. elegans spermatheca is part of the reproductive system, which uses calcium signaling to drive the coordinated contractions that push fertilized eggs out into the uterus. Using genetic analysis and a calcium-sensitive fluorescent protein, we show that the G-protein GSA-1 functions with protein kinase A to regulate calcium release, and contraction of the spermatheca. These findings establish a link between G-protein and cAMP signaling that may apply to similar signaling pathways in other systems.

cell biology↗

bioRxiv2020

Atf-6 regulates lifespan through ER-mitochondrial calcium homeostasis

Functional crosstalk between organelles is critical for maintaining cellular homeostasis. Individually, dysfunction of both endoplasmic reticulum (ER) and mitochondria have been linked to cellular and organismal aging, but little is known about how mechanisms of inter-organelle communication might be targeted to extended longevity. The metazoan unfolded protein response (UPR) maintains ER health through a variety of mechanisms beyond its canonical role in proteostasis, including calcium storage and lipid metabolism. Here we provide evidence that in C. elegans, inhibition of the conserved UPR mediator, activating transcription factor (atf)-6 increases lifespan via modulation of calcium homeostasis and signaling to the mitochondria. Loss of atf-6 confers long life via downregulation of the ER calcium buffering protein, calreticulin. Function of the ER calcium release channel, the inositol triphosphate receptor (IP3R/itr-1), is required for atf-6 mutant longevity while a gain-of-function IP3R/itr-1 mutation is sufficient to extend lifespan. IP3R dysfunction leads to altered mitochondrial behavior and hyperfused morphology, which is sufficient to suppress long life in atf-6 mutants. Highlighting a novel and direct role for this inter-organelle coordination of calcium in longevity, the mitochondrial calcium import channel, mcu-1, is also required for atf-6 mutant longevity. Altogether this study reveals the importance of organellar coordination of calcium handling in determining the quality of aging, and highlights calcium homeostasis as a critical output for the UPR and atf-6 in particular.

cell biology↗

bioRxiv2019

Caenorhabditis elegans PIEZO Channel Coordinates Multiple Reproductive Tissues to Govern Ovulation

The PIEZO proteins are involved in a wide range of developmental and physiological processes. Human PIEZO1 and PIEZO2 are newly identified excitatory mechano-sensitive proteins; they are non-selective ion channels that exhibit a preference for calcium in response to mechanical stimuli. To further understand the function of these proteins, we investigated the roles of pezo-1, the sole PIEZO ortholog in C. elegans. pezo-1 is expressed throughout development in C. elegans, with strong expression in reproductive tissues. A number of deletion alleles as well as a putative gain-of-function mutant caused severe defects in reproduction. A reduced brood size was observed in the strains depleted of PEZO-1. In vivo observations show that oocytes undergo a variety of transit defects as they enter and exit the spermatheca during ovulation. Post ovulation oocytes were frequently damaged during spermathecal contraction. Calcium signaling in the spermatheca is normal during ovulation in pezo-1 mutants, however, pezo-1 interacts genetically with known regulators of calcium signaling. Lastly, loss of PEZO-1 caused defective sperm navigation after being pushed out of the spermatheca during ovulation. Mating with males rescued these reproductive deficiencies in our pezo-1 mutants. These findings suggest that PEZO-1 may act in different reproductive tissues to promote proper ovulation and fertilization in C. elegans.

developmental biology↗

bioRxiv2019

Redox signaling modulates Rho activity and tissue contractility in the C. elegans spermatheca

Actomyosin based contractility in smooth muscle and non-muscle cells is regulated by signaling through the small GTPase Rho and by calcium-activated pathways. We use the myoepithelial cells of the Caenorhabditis elegans spermatheca to study the mechanisms of coordinated myosin activation in vivo. Here, we demonstrate that redox signaling regulates RHO-1/Rho activity in this contractile tissue. Exogenous hydrogen peroxide treatment decreases spermathecal contractility by inhibiting RHO-1, which is mediated through a conserved cysteine in its active site (C20). Further, we identify a gradient of oxidation across the spermathecal tissue, which is regulated by the cytosolic superoxide dismutase, SOD-1. SOD-1 functions in the Rho pathway to inhibit RHO-1 through oxidation of C20. Our results suggest that SOD-1 functions to regulate the redox environment and to fine-tune Rho activity across the spermatheca.

cell biology↗

bioRxiv2019

Intercellular communication controls agonist-induced calcium oscillations independently of gap junctions in smooth muscle cells.

We report the existence of a unique mode of communication among human smooth muscle cells (SMCs) where they use force to frequency modulate long-range calcium waves. An important consequence of this mechanical signaling is that changes in stiffness of the underlying extracellular matrix can interfere with the frequency modulation of Ca2+ waves causing healthy SMCs to falsely perceive a much higher agonist dose than they actually received. This distorted sensing of contractile agonist dose on stiffer matrices is absent in isolated SMCs, even though the isolated cells can sense matrix rigidity. We show that intercellular communication that enables this collective Ca2+ response does not involve transport across gap junctions or extracellular diffusion of signaling molecules. The aberrant communication between cells that distorts the individual cell's perception of contractile stimulus can explain the sudden, exaggerated narrowing of the lumen when exposed to low dose of inhaled agonists in diseases like asthma.

biophysics↗

bioRxiv2018

The RhoGAP SPV-1 regulates calcium signaling to control the contractility of the C. elegans spermatheca during embryo transits.

Contractility of the non-muscle and smooth muscle cells that comprise biological tubing is regulated by the Rho-ROCK and calcium signaling pathways. Although many molecular details about these signaling pathways are known, less is known about how they are coordinated spatiotemporally in biological tubes. The spermatheca of the C. elegans reproductive system enables study of the signaling pathways regulating actomyosin contractility in live adult animals. The RhoGAP SPV-1 was previously identified as a negative regulator of RHO-1/Rho and spermathecal contractility. Here, we uncover a role for SPV-1 as a key regulator of calcium signaling. spv-1 mutants expressing the calcium indicator GCaMP in the spermatheca exhibit premature calcium release, elevated calcium levels, and disrupted spatial regulation of calcium signaling during spermathecal contraction. Although RHO-1 is required for spermathecal contractility, RHO-1 does not play a significant role in regulating calcium. In contrast, activation of CDC-42 recapitulates many aspects of spv-1 mutant calcium signaling. Depletion of cdc-42 by RNAi does not suppress the premature or elevated calcium signal seen in spv-1 mutants, suggesting other targets remain to be identified. Our results suggest SPV-1 works through both the Rho-ROCK and calcium signaling pathways to coordinate cellular contractility.\n\nHighlight SummaryThrough in vivo imaging of the calcium sensor GCaMP, we show that the RhoGAP SPV-1 is a key regulator of calcium signaling in the C. elegans spermatheca. Our data suggests SPV-1 acts at least partially through the small GTPase CDC-42 to modulate calcium signaling, while also acting on RHO-1 to modulate Rho-ROCK signaling. This places SPV-1 as a central regulator of cellular contractility.

cell biology↗

bioRxiv2018

Ancestral Roles of the Fam20C Family of Secreted Protein Kinases Revealed by Functional Analysis in C. elegans

Fam20C is a secreted protein kinase mutated in Raine syndrome, a human skeletal disorder. In vertebrates, bone and enamel proteins are major Fam20C substrates. However, Fam20 kinases are conserved in invertebrates lacking bone and enamel, suggesting other ancestral functions. We show that FAMK-1, the C. elegans Fam20C ortholog, contributes to fertility, embryogenesis, and development. These functions are not fulfilled when FAMK-1 is retained in the early secretory pathway. During embryogenesis, FAMK-1 maintains inter-cellular partitions and prevents multinucleation; notably, temperature elevation or lowering cortical stiffness reduces requirement for FAMK-1 in this context. FAMK-1 is expressed in multiple adult tissues that undergo repeated mechanical strain, and selective expression in the spermatheca restores fertility. Informatic, biochemical and functional analysis implicate lectins as FAMK-1 substrates. These findings suggest that FAMK-1 phosphorylation of substrates, including lectins, in the late secretory pathway is important in embryonic and tissue contexts where cells are subjected to mechanical strain.

cell biology↗