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Aster formation in eggs of Xenopus laevis. Induction by isolated basal bodies.
We have assayed various materials for their ability to induce aster formation by microinjection into unfertilized eggs of Xenopus laevis. We have found that purified basal bodies from Chlamydomonas reinhardtii and Tetrahymena pyriformis induce the formation of asters and irregular cleavage furrows within 1 h after injection. Other microtubule structures such as flagella, flagellar axonemes, cilia, and brain microtubules are completely ineffective at inducing asters or cleavage furrows in unfertilized eggs. When known amounts of sonicated Tetrahymena and Chlamydomonas preparations are injected into unfertilized eggs, 50% of the injected eggs show a furrowing response at approximately 3 cell equvalents for Chlamydomonas and 0.1 cell equivalent for Tetrahymena. These results are close to those expected if basal bodies were the effective astral-inducing agent in these cells. Other materials effective at inducing asters in unfertilized eggs, such as crude brain nuclei, sperm, and a particulate fraction from brain known to induce parthenogenesis in eggs of Rana pipiens, probably contain centrioles as the effective agent. Our experiments provide the first functional assay to indicate that centrioles play an active role in aster initiation. None of the injected materials effective in unfertilized eggs produced any observable response in fully grown oocytes. Oocytes and eggs were found to have equal tubulin pools as judged by colchicine-binding activity. Therefore, the inability of oocytes to form asters cannot be due to a lack of an organizing center or to a lack of tubulin. Experiments in which D2O was found to stimulate aster-like fibrous areas in eggs but not oocytes suggest that the inability of oocytes to form asters may be due to an inability of tubulin in oocytes to assemble.
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Distribution of membrane glycoproteins among the organelles of a single identified neuron of Aplysia. II. Isolation and characterization of a glycoprotein associated with vesicles.
Glycoprotein-I (mol.wt. 180,000) is associated with a vesicle fraction from the cytoplasm of R2, the giant cholinergic neuron in the abdominal ganglion. Electron microscopy has shown that R2 contains both lucent and compound vesicles. We have used anisomycin, an inhibitor of protein synthesis, to provide evidence that Component-I is a constituent of somatic compound vesicles. In the presence of the drug, [3H]N-acetylgalactosamine injected into R2 is incorporated almost exclusively into Component-I. Quantitative electron microscopic radioautography of treated cells shows a marked increase in the proportion of silver grains over compound vesicles and a decrease in labeling of other organelles compared with untreated cells. Analysis of Component-I, isolated using the chaotropic agent lithium diiodosalicylate, shows it to contain fucose, N-acetylgalactosamine, and N-acetylglucosamine. Proteolytic digestion with pronase yields a complex pattern of glycopeptides. The proportion of [3H]N-acetyl-galactosamine in these glycopeptides is altered in the presence of anisomycin. These results together with radioautographic analyses suggest that large carbohydrate chains are elaborated within the endoplasmic reticulum and that smaller chains are added in the Golgi region or on the membrane of the compound vesicle.
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Distribution of membrane glycoproteins among the organelles of a single identified neuron of Aplysia. I. Association of a [3H]glycoprotein with vesicles.
[3H]N-acetylgalactosamine injected into the cell body of R2, the giant cholinergic neuron in the abdominal ganglion, is rapidly incorporated into membrane glycoprotein and glycolipid. Incorporation, which is localized to the injected cells, occurs at a constant rate for approximately 15 h. By that time, 83% of the labeled macromolecules are associated with membranes. Quantitative electron microscopic radioautography of the cell body shows that labeling of membranous organelles is selective: the Golgi apparatus, endoplasmic reticulum, and lucent and compound vesicles are labeled, while the nucleus, end-stage lysosomes, and mitochondria are not. SDS-polyacrylamide gel electrophoresis of the total membranes from more than 40 R2s examined individually resolves reproducibly 5 major labeled glycoprotein components. In order to determine which of these area associated with vesicles, we isolated a labeled vesicle fraction from R2 using a combination of differential centrifugation and filtration on a column of glass beads with 200 nm pores. This fraction was consistently enriched in [3H]glycoproteins I (180,000 Daltons) and V (90,000 Daltons) relative to those fractions containing larger organelles. These experiments suggest that different organelles contain characteristic membrane components.
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A framework for neural organoids, assembloids and transplantation studies.
As the field of neural organoids and assembloids expands, there is an emergent need for guidance and advice on designing, conducting and reporting experiments to increase the reproducibility and utility of these models. In this Perspective, we present a framework for the experimental process that encompasses ensuring the quality and integrity of human pluripotent stem cells, characterizing and manipulating neural cells in vitro, transplantation techniques and considerations for modelling human development, evolution and disease. As with all scientific endeavours, we advocate for rigorous experimental designs tailored to explicit scientific questions as well as transparent methodologies and data sharing to provide useful knowledge for current research practices and for developing regulatory standards.
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Complex carbohydrate composition of large dense-cored vesicles from sympathetic nerve.
Highly purified noradrenergic, large, dense-cored vesicles were isolated from bovine sympathetic nerve endings by sucrose-D2O density gradient centrifugation. Their concentration of glycoprotein hexosamine and sialic acid was 6.6 and 3.9 mumol/100 mg lipid-free dry weight, respectively, values which are similar to those previously found in bovine chromaffin granules. However, whereas chromaffin granule glycoproteins are characterized by their high proportion of N-acetylgalactosamine-containing O-glycosidically-linked oligosaccharides (present in the chromogranins), such oligosaccharides accounted for only 17% of those in noradrenergic synaptic vesicle glycoproteins. Fractionation of N-3H-acetylated glycopeptides by sequential lectin affinity chromatography demonstrated that approximately two-thirds of the oligosaccharides were of the tri- and tetraantennary complex type, accompanied by 14% biantennary oligosaccharides and 3% high-mannose oligosaccharides. The vesicles had a relatively low concentration of chondroitin sulfate (less than 5% of that in chromaffin granules) but significant amounts of heparan sulfate (0.4 mumol N-acetylglucosamine/100 mg lipid-free dry weight). No hyaluronic acid was detected. The concentration of ganglioside sialic acid in the noradrenergic vesicles was approximately 1 mumol/100 mg lipid-free dry weight, which is significantly higher than that of a crude membrane mixture from which the vesicles were prepared; the ratio of N-acetyl- to N-glycolylneuraminic acid was 0.8. Several molecular species of gangliosides were detected by thin-layer chromatography, but most of these did not exactly comigrate with bovine brain gangliosides. Cholera toxin binding indicated that approximately half or less of the gangliosides belong to the gangliotetraose series.
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Fine structural changes in cat L7 ventral horn neurones after chronic sub LD50 DFP.
The fine structural changes in the ventral anterior horn of spinal segment L7, have been studied in adult cats after single and chronic sub LD50 (0.1 to 0.75 mg/kg SC with a cumulative range of 1.3 mg/kg to 10.5 mg/kg) low dose exposure to diisopropylfluorophosphate (DFP). Only the motoneurons of the chronically treated animals show an increase in the number of lysosomes, neurofilaments and vesicle-like structures. A large number of coated vesicles is observed within axons and axon terminals of both acute and chronically treated animals. Morphological evidence of axon and terminal degeneration is seen only in chronically treated animals. The present study shows that chronic sub LD50 low dose administration of DFP over periods from 5 to 21 days results in degenerative changes of presynaptic terminals and axons, with the severity of the changes being dependent on dose and duration of treatment. The data are interpreted by comparison with single high dose exposure reported in the literature with a discussion of acute and delayed neurotoxic effects of DFP, on the central nervous system.
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Ras-like Gem GTPase induced by Npas4 promotes activity-dependent neuronal tolerance for ischemic stroke.
Ischemic stroke, which results in loss of neurological function, initiates a complex cascade of pathological events in the brain, largely driven by excitotoxic Ca2+ influx in neurons. This leads to cortical spreading depolarization, which induces expression of genes involved in both neuronal death and survival; yet, the functions of these genes remain poorly understood. Here, we profiled gene expression changes that are common to ischemia (modeled by middle cerebral artery occlusion [MCAO]) and to experience-dependent activation (modeled by exposure to an enriched environment [EE]), which also induces Ca2+ transients that trigger transcriptional programs. We found that the activity-dependent transcription factor Npas4 was up-regulated under MCAO and EE conditions and that transient activation of cortical neurons in the healthy brain by the EE decreased cell death after stroke. Furthermore, both MCAO in vivo and oxygen-glucose deprivation in vitro revealed that Npas4 is necessary and sufficient for neuroprotection. We also found that this protection involves the inhibition of L-type voltage-gated Ca2+ channels (VGCCs). Next, our systematic search for Npas4-downstream genes identified Gem, which encodes a Ras-related small GTPase that mediates neuroprotective effects of Npas4. Gem suppresses the membrane localization of L-type VGCCs to inhibit excess Ca2+ influx, thereby protecting neurons from excitotoxic death after in vitro and in vivo ischemia. Collectively, our findings indicate that Gem expression via Npas4 is necessary and sufficient to promote neuroprotection in the injured brain. Importantly, Gem is also induced in human cerebral organoids cultured under an ischemic condition, revealing Gem as a new target for drug discovery.
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The mechanism of action of colchicine. Colchicine binding to sea urchin eggs and the mitotic apparatus.
Colchicine forms a complex in vivo with a protein present in fertilized or unfertilized sea urchin eggs; similar binding was obtained in vitro with the soluble fraction from egg homogenates. Kinetic parameters and binding equilibrium constant were essentially the same in vivo and in vitro. The binding site protein was shown to have a sedimentation constant of 6S by zone centrifugation. The protein was present in extracts of the isolated mitotic apparatus at a concentration which was several times higher than in whole-egg homogenates. It was extracted from the mitotic apparatus at low ionic strength under conditions which lead to the disappearance of microtubules. No binding could be detected to the 27S protein, previously described by Kane, which is a major protein component of the isolated mitotic apparatus. The properties of the colchicine-bindinG protein, (binding constant, sedimentation constant, Sephadex elution volume) are similar to those obtained with the protein from mammalian cells, sea-urchin sperm tails, and brain tissue, and thus support the conclusion that the protein is a subunit of microtubules.
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Structural Variants Create New Topological-Associated Domains and Ectopic Retinal Enhancer-Gene Contact in Dominant Retinitis Pigmentosa.
The cause of autosomal-dominant retinitis pigmentosa (adRP), which leads to loss of vision and blindness, was investigated in families lacking a molecular diagnosis. A refined locus for adRP on Chr17q22 (RP17) was delineated through genotyping and genome sequencing, leading to the identification of structural variants (SVs) that segregate with disease. Eight different complex SVs were characterized in 22 adRP-affected families with >300 affected individuals. All RP17 SVs had breakpoints within a genomic region spanning YPEL2 to LINC01476. To investigate the mechanism of disease, we reprogrammed fibroblasts from affected individuals and controls into induced pluripotent stem cells (iPSCs) and differentiated them into photoreceptor precursor cells (PPCs) or retinal organoids (ROs). Hi-C was performed on ROs, and differential expression of regional genes and a retinal enhancer RNA at this locus was assessed by qPCR. The epigenetic landscape of the region, and Hi-C RO data, showed that YPEL2 sits within its own topologically associating domain (TAD), rich in enhancers with binding sites for retinal transcription factors. The Hi-C map of RP17 ROs revealed creation of a neo-TAD with ectopic contacts between GDPD1 and retinal enhancers, and modeling of all RP17 SVs was consistent with neo-TADs leading to ectopic retinal-specific enhancer-GDPD1 accessibility. qPCR confirmed increased expression of GDPD1 and increased expression of the retinal enhancer that enters the neo-TAD. Altered TAD structure resulting in increased retinal expression of GDPD1 is the likely convergent mechanism of disease, consistent with a dominant gain of function. Our study highlights the importance of SVs as a genomic mechanism in unsolved Mendelian diseases.
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Microphysiological systems as a pillar of the Human Exposome Project.
The Human Exposome Project (HEP) aims to decode how lifelong environmental exposures shape health and disease, complementing genomic insights with a systems-level understanding of external influences. Achieving this vision requires experimental platforms that move beyond the limitations of animal models, which often lack human relevance and mechanistic resolution. Microphysiological systems (MPSs)-including organoids and organs-on-chips derived from human stem cells-offer such an opportunity. These engineered models recapitulate human tissue architecture and function under controlled conditions, enabling direct study of exposure-response relationships at the cellular and organ levels. In this review, we outline how MPS can serve as a foundation for exposome research by bridging epidemiological observations with mechanistic biology. We describe applications ranging from air pollutant toxicity to food contaminants, endocrine disruptors, and nanomaterials, highlighting how MPS integrated with omics technologies and artificial intelligence can reveal pathways of injury, identify biomarkers, and support the development of digital twins to simulate exposure-disease trajectories. We also discuss frameworks for validation, quality assurance, and transparent reporting, which are essential for reproducibility and regulatory acceptance. Finally, we consider ethical issues, such as donor rights, data sovereignty, and equitable access, underscoring the importance of anticipatory governance. Together, MPS represent more than alternatives to animal testing-they are strategic enablers of a human-relevant, artificial intelligence-empowered exposome science. By anchoring statistical associations in mechanistic data, MPS can accelerate translation into public health policies that are predictive, preventive, and personalized.