Although cells migrate in a constrained 3D environment in vivo, in-vitro studies have mainly focused on the analysis of cells moving on 2D substrates. Under such conditions, the Golgi complex is ...always located towards the leading edge of the cell, suggesting that it is involved in the directional movement. However, several lines of evidence indicate that this location can vary depending on the cell type, the environment or the developmental processes. We have used micro contact printing (μCP) to study the migration of cells that have a geometrically constrained shape within a polarized phenotype. Cells migrating on micropatterned lines of fibronectin are polarized and migrate in the same direction. Under such conditions, the Golgi complex and the centrosome are located behind the nucleus. In addition, the Golgi complex is often displaced several micrometres away from the nucleus. Finally, we used the zebrafish lateral line primordium as an in-vivo model of cells migrating in a constrained environment and observe a similar localization of both the Golgi and the centrosome in the leading cells. We propose that the positioning of the Golgi complex and the centrosome depends on the geometrical constraints applied to the cell rather than on a precise migratory function in the leading region.
Pre-cordial thump (PT) relies on cardiac mechano-electric transduction to transform mechanically-delivered energy into an electrophysiologically relevant stimulus. Its use for emergency resuscitation ...has declined recent years, amidst concerns about effectiveness and side-effects. In addition, there is insufficient knowledge about bio-mechanical properties and mechanisms of PT. Using a PT-mechanics recorder, we measured PT off-patient among healthcare professionals (n = 58) in North-East Italy, and related this to retrospective information on self-reported PT outcomes. Impact-speed and peak-force were 4.7 ± 1.3 m s−1 (2.2–7.8 m s−1) and 394 ± 110 N (202–648 N), respectively. Average self-reported cardioversion rate by PT was 35%. No adverse events were stated. All but 3 of PT providers with self-reported cardioversion rates ≥50% had pre-impact fist-speeds of ≥3.7 m s−1. In comparison with previously-reported data from UK and US (n = 22 each), self-reported success-rates and pre-impact fist-speeds were more similar to US (PT-induced cardioversion rate 27.7%; fist-speed 4.17 ± 1.68 m s−1) than to UK participants (PT-induced cardioversion rate 13.3%; fist-speed 1.55 ± 0.68 m s−1). Small cohort-size, retrospective nature of data-gathering, and ‘self-selection bias’ (participants who have used PT on patients) limits the extent to which firm conclusions can be drawn. Observations are compatible, though, with the possibility that pre-impact fist-speed may affect success-rate of PT. Thus, where PT is used for acute resuscitation, it is delivered because it is immediately ‘at hand’. Negative side effects are rare or absent in witnessed cardiac arrest cases. Pre-impact fist-speed may be a determinant of outcome, and this could be trained using devices suitable for self-assessment.
Both mechanical induction and mechanical termination of arrhythmias have been reported in man. Examples include pre
-cordial impacts by sports implements (baseballs, pucks) that can trigger ...arrhythmias, including ventricular fibrillation, or via the so-called pre-cordial thump, used as an emergency resuscitation measure to convert arrhythmias to normal sinus node rhythm. These interventions have been partially reproduced in experimental studies on whole animals. Relating observations at the system's level to underlying mechanisms has been difficult, however, largely because of: (i) a deficit in efficient and affordable pharmacological agents to selectively target (sub-)cellular responses in whole animal studies, and (ii) the lack of suitable experimental models to study the above responses at intermediate levels of functional and structural integration, such as the isolated heart or cardiac tissue. This paper presents a soft tissue impact characterisation kit (STICK), suitable for quantitative investigations into the effects of acute mechanical stimulation on cardiac electro-mechanical function in rodent isolated heart or tissue preparations. The STICK offers independent control over a range of relevant biophysical parameters, such as impact location and energy, pre-impact projectile speed and contact area, as well as over the timing of a mechanical stimulus relative to the cardiac cycle (monitored via electrocardiogram, ECG, here recorded directly from the cardiac surface). Projectile deceleration upon interaction with the tissue is monitored, contact-free, with a resolution of 175
μm, providing information on tissue deformation dynamics, force, pressure and work of the mechanical intervention. In order to study functional effects of cardiac mechanical stimulation in the absence of tissue damage, impacts must be limited (for juvenile Guinea pig heart) to 2–2.5
mJ in the slack left ventricle (diastolic impact) and 5–10
mJ in contracture (systolic impact), as confirmed by enzyme assay and histological investigation. Impacts, timed to coincide with the early T-wave of the ECG, are capable of triggering short runs of ventricular fibrillation. Thus, the STICK is a suitable tool for the study of acute cardiac mechano-electric feedback effects, caused by short impulse-like mechanical stimulation, at the level of the isolated organ or tissue.
Evolutionary transitions from egg laying (oviparity) to live birth (viviparity) are common across various taxa. Many species also exhibit genetic variation in egg-laying mode or display an ...intermediate mode with laid eggs containing embryos at various stages of development. Understanding the mechanistic basis and fitness consequences of such variation remains experimentally challenging. Here, we report highly variable intra-uterine egg retention across 316
wild strains, some exhibiting strong retention, followed by internal hatching. We identify multiple evolutionary origins of such phenotypic extremes and pinpoint underlying candidate loci. Behavioral analysis and genetic manipulation indicates that this variation arises from genetic differences in the neuromodulatory architecture of the egg-laying circuitry. We provide experimental evidence that while strong egg retention can decrease maternal fitness due to in utero hatching, it may enhance offspring protection and confer a competitive advantage. Therefore, natural variation in
egg-laying behaviour can alter an apparent trade-off between different fitness components across generations. Our findings highlight underappreciated diversity in
egg-laying behavior and shed light on its fitness consequences. This behavioral variation offers a promising model to elucidate the molecular changes in a simple neural circuit underlying evolutionary shifts between alternative egg-laying modes in invertebrates.
Evolutionary transitions from egg laying (oviparity) to live birth (viviparity) are common across various taxa. Many species also exhibit genetic variation in egg-laying mode or display an ...intermediate mode with laid eggs containing embryos at various stages of development. Understanding the mechanistic basis and fitness consequences of such variation remains experimentally challenging. Here, we report highly variable intra-uterine egg retention across 316 Caenorhabditis elegans wild strains, some exhibiting strong retention, followed by internal hatching. We identify multiple evolutionary origins of such phenotypic extremes and pinpoint underlying candidate loci. Behavioral analysis and genetic manipulation indicates that this variation arises from genetic differences in the neuromodulatory architecture of the egg-laying circuitry. We provide experimental evidence that while strong egg retention can decrease maternal fitness due to in utero hatching, it may enhance offspring protection and confer a competitive advantage. Therefore, natural variation in C. elegans egg-laying behaviour can alter an apparent trade-off between different fitness components across generations. Our findings highlight underappreciated diversity in C. elegans egg-laying behavior and shed light on its fitness consequences. This behavioral variation offers a promising model to elucidate the molecular changes in a simple neural circuit underlying evolutionary shifts between alternative egg-laying modes in invertebrates.
The assembly of neurotransmitter receptors in the endoplasmic reticulum limits the number of receptors delivered to the plasma membrane, ultimately controlling neurotransmitter sensitivity and ...synaptic transfer function. In a forward genetic screen conducted in the nematode
, we identified
as a gene required for the synaptic expression of ionotropic acetylcholine receptors (AChR). We demonstrated that the CRLD-1A isoform is a membrane-associated ER-resident protein disulfide isomerase (PDI). It physically interacts with AChRs and promotes the assembly of AChR subunits in the ER. Mutations of
the human ortholog of
are responsible for developmental cardiac defects. We showed that
knockdown in mouse muscle cells decreased surface expression of AChRs and that expression of mouse
in
rescued
mutant phenotypes. Altogether these results identify a novel and evolutionarily-conserved maturational enhancer of AChR biogenesis, which controls the abundance of functional receptors at the cell surface.
Evolutionary transitions from oviparity to viviparity are frequent across diverse taxa. Some species also display intraspecific variation in parity mode, or they exhibit an intermediate mode by ...laying eggs containing embryos at variable, often advanced stages of development. How such natural quantitative variation in egg retention arises through differences in genetics, behaviour, and physiology – and how this variation ultimately connects to variation in specific fitness components – is not well-understood. Here, we study this problem by characterizing intraspecific variation in constitutive retention of fertilized eggs of the nematode Caenorhabditis elegans. Analysing a panel of ∼300 wild strains, we find highly variable intra-uterine retention of fertilized eggs, with a fraction of strains showing either strongly reduced or increased egg retention with partial viviparity. We provide evidence for multiple evolutionary origins of such phenotypic extremes and we identify candidate loci explaining this natural variation. Characterizing a subset of wild strains, we confirm that natural variation in egg-laying behaviour contributes to observed differences in egg retention. Using multiple neuromodulatory agents and controlled CRISPR-Cas9-mediated genetic manipulation of endogenous serotonin levels in 10 wild strains, we then show that this behavioural variation arises through an evolutionarily divergent neuromodulatory architecture of the egg-laying circuitry. Intraspecific variation in C. elegans neural circuit activity therefore connects with variation in reproductive strategy, including transitions from oviparity to partial viviparity. In a second objective, we asked why natural variation in C. elegans egg retention might be maintained. Examining potential fitness costs and benefits of this natural variation, we show that strong egg retention reduces maternal fertility and survival, mostly due to detrimental larval hatching in utero. On the other hand, such genotypes with strong egg retention can benefit from improved offspring protection against environmental insults and by gaining a competitive advantage as offspring exhibit a shortened extra-uterine developmental time to reproductive maturity. Observed natural variation in C. elegans egg-laying behaviour may therefore reflect modifications of a trade-off between alternative fitness components expressed across generations. Our study uncovers underappreciated natural diversity in the C. elegans egg-laying circuit and provides insights into the fitness consequences of this behavioural variation. We propose that intraspecific variation in nematode egg-laying behaviour can serve as an ideal system to pinpoint the molecular changes underlying evolutionary transitions between invertebrate ovi- and viviparity.
Auxiliary subunits regulate the trafficking, localization or gating kinetics of voltage- and ligand-gated ion channels by associating tightly and specifically with pore-forming subunits. However, no ...auxiliary subunits have been identified for members of the Cys-loop receptor superfamily. Here we identify MOLO-1, a positive regulator of levamisole-sensitive acetylcholine receptors (L-AChRs) at the Caenorhabditis elegans neuromuscular junction. MOLO-1 is a one-pass transmembrane protein that contains a single extracellular globular domain-the TPM domain, found in bacteria, plants and invertebrates, including nonvertebrate chordates. Loss of MOLO-1 impairs locomotion and renders worms resistant to the anthelmintic drug levamisole. In molo-1 mutants, L-AChR-dependent synaptic transmission is reduced by half, while the number and localization of receptors at synapses remain unchanged. In a heterologous expression system, MOLO-1 physically interacts with L-AChRs and directly enhances channel gating without affecting unitary conductance. The identification of MOLO-1 expands the mechanisms for generating functional and pharmacological diversity in the Cys-loop superfamily.
Genetic assimilation-the evolutionary process by which an environmentally induced phenotype is made constitutive-represents a fundamental concept in evolutionary biology. Thought to reflect adaptive ...phenotypic plasticity, matricidal hatching in nematodes is triggered by maternal nutrient deprivation to allow for protection or resource provisioning of offspring. Here, we report natural
populations harboring genetic variants expressing a derived state of near-constitutive matricidal hatching. These variants exhibit a single amino acid change (V530L) in KCNL-1, a small-conductance calcium-activated potassium channel subunit. This gain-of-function mutation causes matricidal hatching by strongly reducing the sensitivity to environmental stimuli triggering egg-laying. We show that reestablishing the canonical KCNL-1 protein in matricidal isolates is sufficient to restore canonical egg-laying. While highly deleterious in constant food environments, KCNL-1 V530L is maintained under fluctuating resource availability. A single point mutation can therefore underlie the genetic assimilation-by either genetic drift or selection-of an ancestrally plastic trait.
Bone formation and resorption are typically coupled, such that the efficacy of anabolic osteoporosis treatments may be limited by bone destruction. The multi-kinase inhibitor YKL-05-099 potently ...inhibits salt inducible kinases (SIKs) and may represent a promising new class of bone anabolic agents. Here, we report that YKL-05-099 increases bone formation in hypogonadal female mice without increasing bone resorption. Postnatal mice with inducible, global deletion of SIK2 and SIK3 show increased bone mass, increased bone formation, and, distinct from the effects of YKL-05-099, increased bone resorption. No cell-intrinsic role of SIKs in osteoclasts was noted. In addition to blocking SIKs, YKL-05-099 also binds and inhibits CSF1R, the receptor for the osteoclastogenic cytokine M-CSF. Modeling reveals that YKL-05-099 binds to SIK2 and CSF1R in a similar manner. Dual targeting of SIK2/3 and CSF1R induces bone formation without concomitantly increasing bone resorption and thereby may overcome limitations of most current anabolic osteoporosis therapies.
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