Exophers to feed them all.

Cells release extracellular vesicles to remove damaged components and communicate with other cells via packets of proteins, lipids, and RNAs. Neuronal cells in the nematode C. elegans release particularly large extracellular vesicles, known as exophers, to rid themselves of damaged organelles and protein aggregates. Turek et al now demonstrate a new role for these vesicles: Embryos in the uterus stimulate body wall muscle cells to release exophers laden with yolk, which are taken up by oocytes to nourish the next set of embryos (Turek et al, 2021).

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/Gαs, 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/Gαs 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, the transit times and total numbers, although not frequencies, of Ca2+ pulses are increased, and Ca2+ propagates across the spermatheca even in the absence of oocyte entry. In the spermathecal-uterine valve, loss of GSA-1/Gαs 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 Gß 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 contractions for successful ovulations.

Stretch-induced actomyosin contraction in epithelial tubes: Mechanotransduction pathways for tubular homeostasis.

Many tissues in our body have a tubular shape and are constantly exposed to various stresses. Luminal pressure imposes tension on the epithelial and myoepithelial or smooth muscle cells surrounding the lumen of the tubes. Contractile forces generated by actomyosin assemblies within these cells oppose the luminal pressure and must be calibrated to maintain tube diameter homeostasis and tissue integrity. In this review, we discuss mechanotransduction pathways that can lead from sensation of cell stretch to activation of actomyosin contractility, providing rapid mechanochemical feedback for proper tubular tissue function.

The DSL ligand APX-1 is required for normal ovulation in C. elegans.

DSL ligands activate the Notch receptor in many cellular contexts across metazoa to specify cell fate. In addition, Notch receptor activity is implicated in post-mitotic morphogenesis and neuronal function. In C. elegans, the DSL family ligand APX-1 is expressed in a subset of cells of the proximal gonad lineage, where it can act as a latent proliferation-promoting signal to maintain proximal germline tumors. Here we examine apx-1 in the proximal gonad and uncover a role in the maintenance of normal ovulation. Depletion of apx-1 causes an endomitotic oocyte (Emo) phenotype and ovulation defects. We find that lag-2 can substitute for apx-1 in this role, that the ovulation defect is partially suppressed by loss of ipp-5, and that lin-12 depletion causes a similar phenotype. In addition, we find that the ovulation defects are often accompanied by a delay of spermathecal distal neck closure after oocyte entry. Although calcium oscillations occur in the spermatheca, calcium signals are abnormal when the distal neck does not close completely. Moreover, oocytes sometimes cannot properly transit through the spermatheca, leading to fragmentation of oocytes once the neck closes. Finally, abnormal oocytes and neck closure defects are seen occasionally when apx-1 or lin-12 activity is reduced in adult animals, suggesting a possible post-developmental role for APX-1 and LIN-12 signaling in ovulation.

CACN-1 is required in the Caenorhabditis elegans somatic gonad for proper oocyte development.

CACN-1/Cactin is a conserved protein identified in a genome-wide screen for genes that regulate distal tip cell migration in the nematode Caenorhabditis elegans. In addition to possessing distal tip cells that migrate past their correct stopping point, animals depleted of cacn-1 are sterile. In this study, we show that CACN-1 is needed in the soma for proper germ line development and maturation. When CACN-1 is depleted, sheath cells are absent and/or abnormal. When sheath cells are absent, hermaphrodites produce sperm, but do not switch appropriately to oocyte production. When sheath cells are abnormal, some oocytes develop but are not successfully ovulated and undergo endomitotic reduplication (Emo). Our previous proteomic studies show that CACN-1 interacts with a network of splicing factors. Here, these interactors were screened using RNAi. Depletion of many of these factors led to missing or abnormal sheath cells and germ line defects, particularly absent and/or Emo oocytes. These results suggest CACN-1 is part of a protein network that influences somatic gonad development and function through alternative splicing or post-transcriptional gene regulation.

Filamin and phospholipase C-ε are required for calcium signaling in the Caenorhabditis elegans spermatheca.

The Caenorhabditis elegans spermatheca is a myoepithelial tube that stores sperm and undergoes cycles of stretching and constriction as oocytes enter, are fertilized, and exit into the uterus. FLN-1/filamin, a stretch-sensitive structural and signaling scaffold, and PLC-1/phospholipase C-ε, an enzyme that generates the second messenger IP3, are required for embryos to exit normally after fertilization. Using GCaMP, a genetically encoded calcium indicator, we show that entry of an oocyte into the spermatheca initiates a distinctive series of IP3-dependent calcium oscillations that propagate across the tissue via gap junctions and lead to constriction of the spermatheca. PLC-1 is required for the calcium release mechanism triggered by oocyte entry, and FLN-1 is required for timely initiation of the calcium oscillations. INX-12, a gap junction subunit, coordinates propagation of the calcium transients across the spermatheca. Gain-of-function mutations in ITR-1/IP3R, an IP3-dependent calcium channel, and loss-of-function mutations in LFE-2, a negative regulator of IP3 signaling, increase calcium release and suppress the exit defect in filamin-deficient animals. We further demonstrate that a regulatory cassette consisting of MEL-11/myosin phosphatase and NMY-1/non-muscle myosin is required for coordinated contraction of the spermatheca. In summary, this study answers long-standing questions concerning calcium signaling dynamics in the C. elegans spermatheca and suggests FLN-1 is needed in response to oocyte entry to trigger calcium release and coordinated contraction of the spermathecal tissue.

Optimizing the transient Fast Agro-mediated Seedling Transformation (FAST) method in Catharanthus roseus seedlings.

KEY MESSAGE: An Agro-mediated transformation method has been adapted in Catharanthus roseus seedlings for transient overexpression. Our results suggest that Agro-mediated methods may induce defense-related genes, which should be considered in its application. The Fast Agro-mediated Seedling Transformation (FAST) method, which involves the co-cultivation and transient transformation of young seedlings with Agrobacterium, was adapted and optimized in Catharanthus roseus. We investigated the optimal conditions for Gus expression by varying the Agrobacterium density (OD600 = 0.29 and 0.50), A. rhizogenes strain (15834 and R1000), and co-cultivation time in liquid (2, 12, or 24 h) followed by incubation time on solid media (1 or 2 days). Transformation efficiency was assessed quantitatively in terms of average GUS intensity per cotyledon surface area and percentage of cotyledons transformed. GUS staining was observed in 100% of cotyledons co-cultivated with A. rhizogenes (OD600 = 0.50) co-transformed with the Mas promoter-driven Gus and pSoup helper plasmids, in the presence of 0.01% v/v Silwet L-77 for 24 h in liquid followed by 2-days on solid media. In addition, we observed that co-cultivation with Agrobacterium strongly induced Zct1 and Orca3, two transcription factors known to regulate defense-related alkaloid biosynthesis in C. roseus. Homologous transcription factors regulate defense responses in many plant species. Therefore, possible induction of defense-related genes by Agro-mediated transformation should be a consideration in experimental design.

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-ε/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.

An integrin binding motif in TLN-1/talin plays a minor role in motility and ovulation.

TLN-1/talin is a conserved focal adhesion protein that forms part of the linkage between the cytoplasmic tail of integrin and the actin cytoskeleton. In C. elegans , TLN-1 is expressed strongly in striated muscle and the gonadal sheath cells. Here, we report that a CRISPR-generated TLN-1 allele TLN-1(W387A), predicted to affect binding of talin to integrins, results in mild phenotypes, including motility defects and ovulation defects. The arrangement of the actin cytoskeleton in the body wall muscles, spermatheca, and sheath appears identical in wild type and TLN-1(W387A) animals. This analysis suggests that W387 in TLN-1 does not have a major effect on the binding of talin to integrin in vivo .

Tension-dependent RHGF-1 recruitment to stress fibers drives robust spermathecal tissue contraction.

Contractile epithelial tubes are found in various organs, such as lung airways and blood capillaries. Their ability to sense luminal pressure and respond with adequate contractility is essential for their physiology, and its mis-regulation results in diseases such as asthma and hypertension. Here, we describe a mechanoresponsive regulatory pathway downstream of tissue stretching that controls contraction of the C. elegans spermatheca, a tubular structure where fertilization occurs. Using live-imaging, we show that ovulation-induced stretching of spermathecal cells leads to recruitment of the RhoGEF RHGF-1 to stress fibers, which activates RHO-1 and myosin II in a positive feedback loop. Through deletion analysis, we identified the PDZ domain of RHGF-1 as responsible for F-actin binding, and genetic epistasis analysis with the RhoGAP spv-1 demonstrated that tension-dependent recruitment of RHGF-1 to F-actin is required for robust spermathecal contractility. Our study illustrates how mechanosensitive regulators of Rho GTPases provide epithelial tubes the ability to tune their contractility in response to internal pressure.

Worm development: Push not pull in gonad morphogenesis.

How does tissue elongation occur? A recent paper identifies a new mechanism: elongation of the Caenorhabditis elegans hermaphrodite gonad is driven by pressure from proliferating germ cells confined within a tube. The distal tip cell, which caps the tube, remodels the extracellular matrix and adjusts cell-matrix adhesion to guide the way.

Functional Insights into Protein Kinase A (PKA) Signaling from C. elegans.

Protein kinase A (PKA), which regulates a diverse set of biological functions downstream of cyclic AMP (cAMP), is a tetramer consisting of two catalytic subunits (PKA-C) and two regulatory subunits (PKA-R). When cAMP binds the PKA-R subunits, the PKA-C subunits are released and interact with downstream effectors. In Caenorhabditis elegans (C. elegans), PKA-C and PKA-R are encoded by kin-1 and kin-2, respectively. This review focuses on the contributions of work in C. elegans to our understanding of the many roles of PKA, including contractility and oocyte maturation in the reproductive system, lipid metabolism, physiology, mitochondrial function and lifespan, and a wide variety of behaviors. C. elegans provides a powerful genetic platform for understanding how this kinase can regulate an astounding variety of physiological responses.

EASI Transformation Protocol: An Agrobacterium-Mediated Transient Transformation Protocol for Catharanthus roseus Seedlings.

Catharanthus roseus produces medicinal terpenoid indole alkaloids, including the critical anti-cancer compounds vinblastine and vincristine in its leaves. Recently, we developed a highly efficient transient expression method relying on Agrobacterium-mediated transformation of seedlings to facilitate rapid and high-throughput studies on the regulation of terpenoid indole alkaloid biosynthesis in C. roseus . We detail our optimized protocol known as efficient Agrobacterium-mediated seedling infiltration method (EASI), including the development of constructs used in EASI and an example experimental design that includes appropriate controls. We applied our EASI method to rapidly screen and evaluate transcriptional activators and repressors and promoter activity. Our EASI method can be used for promoter transactivation studies or transgene overexpression paired with downstream analyses like quantitative PCR or metabolite analysis. Our protocol takes about 16 days from sowing seeds to obtaining the results of the experiment.

FLN-1/filamin is required for maintenance of actin and exit of fertilized oocytes from the spermatheca in C. elegans.

Filamin, known primarily for its actin cross-linking function, is a stretch-sensitive structural and signaling scaffold that binds transmembrane receptors and a wide variety of intracellular signaling proteins. The Caenorhabditis elegans filamin ortholog, FLN-1, has a well conserved overall structure, including an N-terminal actin-binding domain, and a series of 20 immunoglobulin (Ig)-like repeats. FLN-1 partially colocalizes with actin filaments in spermathecal and uterine cells. Analysis of phenotypes resulting from a deletion allele and RNAi depletion indicates FLN-1 is required to maintain the actin cytoskeleton in the spermatheca and uterus, and to allow the exit of embryos from the spermatheca. FLN-1 deficient animals accumulate embryos in the spermatheca, lay damaged and unfertilized eggs, and consequently exhibit dramatically reduced brood sizes. The phospholipase PLC-1 is also required for the exit of embryos from the spermatheca, and analysis of doubly mutant animals suggests that PLC-1 and FLN-1 act in the same pathway to promote proper transit of embryos from the spermatheca to the uterus. Given the modular protein structure, subcellular localization, genetic interaction with PLC-1, and known mechanosensory functions of filamin, we postulate that FLN-1 may be required to convert mechanical information about the presence of the oocyte into a biochemical signal, thereby allowing timely exit of the embryo from the spermatheca.

CACN-1/Cactin interacts genetically with MIG-2 GTPase signaling to control distal tip cell migration in C. elegans.

The two specialized C. elegans distal tip cells (DTCs) provide an in vivo model system for the study of developmentally regulated cell migration. We identified cacn-1/cactin, a well-conserved, novel regulator of cell migration in a genome-wide RNAi screen for regulators of DTC migration. RNAi depletion experiments and analysis of the hypomorphic allele cacn-1(tm3126) indicate that CACN-1 is required during DTC migration for proper pathfinding and for cessation of DTC migration at the end of larval morphogenesis. Strong expression of CACN-1 in the DTCs, and data from cell-specific RNAi depletion experiments, suggest that CACN-1 is required cell-autonomously to control DTC migration. Importantly, genetic interaction data with Rac GTPase activators and effectors suggest that CACN-1 acts specifically to inhibit the mig-2/Rac pathway, and in parallel to ced-10/Rac, to control DTC pathfinding.

Analysis of conserved residues in the betapat-3 cytoplasmic tail reveals important functions of integrin in multiple tissues.

Integrin cytoplasmic tails contain motifs that link extracellular information to cell behavior such as cell migration and contraction. To investigate the cell functions mediated by the conserved motifs, we created mutations in the Caenorhabditis elegans betapat-3 cytoplasmic tail. The beta1D (799FK800), NPXY, tryptophan (784W), and threonine (797TT798) motifs were disrupted to identify their functions in vivo. Animals expressing integrins with disrupted NPXY motifs were viable, but displayed distal tip cell migration and ovulation defects. The conserved threonines were required for gonad migration and contraction as well as tail morphogenesis, whereas disruption of the beta1D and tryptophan motifs produced only mild defects. To abolish multiple conserved motifs, a beta1C-like variant, which results in a frameshift, was constructed. The betapat-3(beta1C) transgenic animals showed cold-sensitive larval arrests and defective muscle structure and gonad migration and contraction. Our study suggests that the conserved NPXY and TT motifs play important roles in the tissue-specific function of integrin.

ZYX-1/Zyxin plays a minor role in oocyte transit through the spermatheca in C. elegans.

In C. elegans, oocytes are ovulated into the spermatheca, where they are fertilized before being pushed into the uterus. Contraction in the C. elegans spermatheca is driven by circumferential acto-myosin fibers. The C. elegans zyxin homolog, zyx-1, is expressed in the body wall muscle, pharynx and spermatheca. To our surprise, a CRISPR-generated zyx-1 deletion allele results in no overt developmental phenotypes, and the spermathecal actin cytoskeleton appears wild type, however, oocyte transit through the spermatheca is slower than in wild type animals. This suggests ZYX-1/Zyxin may regulate spermathecal contraction magnitude or timing of spermathecal bag contraction and/or spermathecal-uterine valve dilation.

High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns.

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 the stable transformation of a Cas9 expression construct into the plant genome. The efficiency of introducing mutations in genes of interest can vary considerably depending on the specific features of the constructs, including the source and nature of the promoters and terminators used for the expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency of the Cas9 nuclease in generating mutations in target genes in Arabidopsis thaliana, we investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLSs), and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than one. However, the highest efficiency of the constructs was achieved by the addition of 13 introns into the Cas9 coding sequence, which dramatically improved the editing efficiency of the constructs. None of the primary transformants obtained with a Cas9 gene lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of the primary transformants generated with the intronized Cas9 gene displayed mutant phenotypes. The intronized Cas9 gene was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.

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

Actomyosin-based contractility in smooth muscle and nonmuscle 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 show that redox signaling modulates RHO-1/Rho activity in this contractile tissue. Exogenously added as well as endogenously generated hydrogen peroxide decreases spermathecal contractility by inhibition of RHO-1, which depends on a conserved cysteine in its nucleotide binding site (C20). Further, we identify an endogenous gradient of H2O2 across the spermathecal tissue, which depends on the activity of cytosolic superoxide dismutase, SOD-1. Collectively, we show that SOD-1-mediated H2O2 production regulates the redox environment and fine tunes Rho activity across the spermatheca through oxidation of RHO-1 C20.

Atf-6 Regulates Lifespan through ER-Mitochondrial Calcium Homeostasis.

Individually, dysfunction of both the endoplasmic reticulum (ER) and mitochondria has been linked to aging, but how communication between these organelles might be targeted to promote longevity is unclear. Here, we provide evidence that, in Caenorhabditis elegans, inhibition of the conserved unfolded protein response (UPRER) mediator, activating transcription factor (atf)-6, increases lifespan by modulating calcium homeostasis and signaling to mitochondria. Atf-6 loss confers longevity via downregulation of the ER calcium buffer, calreticulin. ER calcium release via the inositol triphosphate receptor (IP3R/itr-1) is required for longevity, while IP3R/itr-1 gain of function is sufficient to extend lifespan. Highlighting coordination between organelles, the mitochondrial calcium import channel mcu-1 is also required for atf-6 longevity. IP3R inhibition leads to impaired mitochondrial bioenergetics and hyperfusion, which is sufficient to suppress long life in atf-6 mutants. This study reveals the importance of organellar calcium handling as a critical output for the UPRER in determining the quality of aging.