Common genetic variation impacts stress response in the brain.

To explain why individuals exposed to identical stressors experience divergent clinical outcomes, we determine how molecular encoding of stress modifies genetic risk for brain disorders. Analysis of post-mortem brain (n=304) revealed 8557 stress-interactive expression quantitative trait loci (eQTLs) that dysregulate expression of 915 eGenes in response to stress, and lie in stress-related transcription factor binding sites. Response to stress is robust across experimental paradigms: up to 50% of stress-interactive eGenes validate in glucocorticoid treated hiPSC-derived neurons (n=39 donors). Stress-interactive eGenes show brain region- and cell type-specificity, and, in post-mortem brain, implicate glial and endothelial mechanisms. Stress dysregulates long-term expression of disorder risk genes in a genotype-dependent manner; stress-interactive transcriptomic imputation uncovered 139 novel genes conferring brain disorder risk only in the context of traumatic stress. Molecular stress-encoding explains individualized responses to traumatic stress; incorporating trauma into genomic studies of brain disorders is likely to improve diagnosis, prognosis, and drug discovery.

Modeling gene × environment interactions in PTSD using human neurons reveals diagnosis-specific glucocorticoid-induced gene expression.

Post-traumatic stress disorder (PTSD) can develop following severe trauma, but the extent to which genetic and environmental risk factors contribute to individual clinical outcomes is unknown. Here, we compared transcriptional responses to hydrocortisone exposure in human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons and peripheral blood mononuclear cells (PBMCs) from combat veterans with PTSD (n = 19 hiPSC and n = 20 PBMC donors) and controls (n = 20 hiPSC and n = 20 PBMC donors). In neurons only, we observed diagnosis-specific glucocorticoid-induced changes in gene expression corresponding with PTSD-specific transcriptomic patterns found in human postmortem brains. We observed glucocorticoid hypersensitivity in PTSD neurons, and identified genes that contribute to this PTSD-dependent glucocorticoid response. We find evidence of a coregulated network of transcription factors that mediates glucocorticoid hyper-responsivity in PTSD. These findings suggest that induced neurons represent a platform for examining the molecular mechanisms underlying PTSD, identifying biomarkers of stress response, and conducting drug screening to identify new therapeutics.

Integrating deep learning and unbiased automated high-content screening to identify complex disease signatures in human fibroblasts.

Drug discovery for diseases such as Parkinson's disease are impeded by the lack of screenable cellular phenotypes. We present an unbiased phenotypic profiling platform that combines automated cell culture, high-content imaging, Cell Painting, and deep learning. We applied this platform to primary fibroblasts from 91 Parkinson's disease patients and matched healthy controls, creating the largest publicly available Cell Painting image dataset to date at 48 terabytes. We use fixed weights from a convolutional deep neural network trained on ImageNet to generate deep embeddings from each image and train machine learning models to detect morphological disease phenotypes. Our platform's robustness and sensitivity allow the detection of individual-specific variation with high fidelity across batches and plate layouts. Lastly, our models confidently separate LRRK2 and sporadic Parkinson's disease lines from healthy controls (receiver operating characteristic area under curve 0.79 (0.08 standard deviation)), supporting the capacity of this platform for complex disease modeling and drug screening applications.

The Kinase Function of MSK1 Regulates BDNF Signaling to CREB and Basal Synaptic Transmission, But Is Not Required for Hippocampal Long-Term Potentiation or Spatial Memory.

The later stages of long-term potentiation (LTP) in vitro and spatial memory in vivo are believed to depend upon gene transcription. Accordingly, considerable attempts have been made to identify both the mechanisms by which transcription is regulated and indeed the gene products themselves. Previous studies have shown that deletion of one regulator of transcription, the mitogen- and stress-activated kinase 1 (MSK1), causes an impairment of spatial memory. Given the ability of MSK1 to regulate gene expression via the phosphorylation of cAMP response element binding protein (CREB) at serine 133 (S133), MSK1 is a plausible candidate as a prime regulator of transcription underpinning synaptic plasticity and learning and memory. Indeed, prior work has revealed the necessity for MSK1 in homeostatic and experience-dependent synaptic plasticity. However, using a knock-in kinase-dead mouse mutant of MSK1, the current study demonstrates that, while the kinase function of MSK1 is important in regulating the phosphorylation of CREB at S133 and basal synaptic transmission in hippocampal area CA1, it is not required for metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD), two forms of LTP or several forms of spatial learning in the watermaze. These data indicate that other functions of MSK1, such as a structural role for the whole enzyme, may explain previous observations of a role for MSK1 in learning and memory.

In situ cell-matrix mechanics in tendon fascicles and seeded collagen gels: implications for the multiscale design of biomaterials.

Designing biomaterials to mimic and function within the complex mechanobiological conditions of connective tissues requires a detailed understanding of the micromechanical environment of the cell. The objective of our study was to measure the in situ cell-matrix strains from applied tension in both tendon fascicles and cell-seeded type I collagen scaffolds using laser scanning confocal microscopy techniques. Tendon fascicles and collagen gels were fluorescently labelled to simultaneously visualise the extracellular matrix and cell nuclei under applied tensile strains of 5%. There were significant differences observed in the micromechanics at the cell-matrix scale suggesting that the type I collagen scaffold did not replicate the pattern of native tendon strains. In particular, although the overall in situ tensile strains in the matrix were quite similar (∼2.5%) between the tendon fascicles and the collagen scaffolds, there were significant differences at the cell-matrix boundary with visible shear across cell nuclei of >1 µm measured in native tendon which was not observed at all in the collagen scaffolds. Similarly, there was significant non-uniformity of intercellular strains with relative sliding observed between cell rows in tendon which again was not observed in the collagen scaffolds where the strain environment was much more uniform. If the native micromechanical environment is not replicated in biomaterial scaffolds, then the cells may receive incorrect or mixed mechanical signals which could affect their biosynthetic response to mechanical load in tissue engineering applications. This study highlights the importance of considering the microscale mechanics in the design of biomaterial scaffolds and the need to incorporate such features in computational models of connective tissues.

Multi-unit sustained vibration loading platform for biological tissues: design, validation and experimentation.

The relationships between mechanical inputs and resulting biological tissue structure, composition, and metabolism are critical to detailing the nuances of tissue mechanobiology in both healthy and injured tissues. Developing a model system to test the mechanobiology of tissues ex-vivo is a complex task, as controlling chemical and mechanical boundary layers in-vitro are difficult to replicate. A novel multi-unit vibration loading platform for intervertebral discs was designed and validated with both independent electronic data and experimental loading of 6 bovine intervertebral discs (IVDs) and an equal number of unloaded controls. Sustained vibration was applied using closed-loop positional control of pushrods within four independent bioreactors with circulating phosphate buffered saline. The bioreactors were designed to be modular with removable components allowing for easy cleaning and replacement. The loading regime was chosen to maximize target mRNA expression as reported in previous research. Aggrecan, decorin, and versican mRNA all reported statistically significant increases above control levels. Biglycan, collagen type I and II showed no significant difference from the control group. Further study is required to determine the resulting effect of increased mRNA expressions on long-term disc health. However these results indicate that this research is past the proof of concept stage, supporting future studies of mechanobiology utilizing this new device. The next stage in developing this novel loading platform should consider modifying the tissue grips to explore the effects of different directional loading on different gene expression, and also loading different types of tissues.

MSK1 regulates homeostatic and experience-dependent synaptic plasticity.

The ability of neurons to modulate synaptic strength underpins synaptic plasticity, learning and memory, and adaptation to sensory experience. Despite the importance of synaptic adaptation in directing, reinforcing, and revising the behavioral response to environmental influences, the cellular and molecular mechanisms underlying synaptic adaptation are far from clear. Brain-derived neurotrophic factor (BDNF) is a prime initiator of structural and functional synaptic adaptation. However, the signaling cascade activated by BDNF to initiate these adaptive changes has not been elucidated. We have previously shown that BDNF activates mitogen- and stress-activated kinase 1 (MSK1), which regulates gene transcription via the phosphorylation of both CREB and histone H3. Using mice with a kinase-dead knock-in mutation of MSK1, we now show that MSK1 is necessary for the upregulation of synaptic strength in response to environmental enrichment in vivo. Furthermore, neurons from MSK1 kinase-dead mice failed to show scaling of synaptic transmission in response to activity deprivation in vitro, a deficit that could be rescued by reintroduction of wild-type MSK1. We also show that MSK1 forms part of a BDNF- and MAPK-dependent signaling cascade required for homeostatic synaptic scaling, which likely resides in the ability of MSK1 to regulate cell surface GluA1 expression via the induction of Arc/Arg3.1. These results demonstrate that MSK1 is an integral part of a signaling pathway that underlies the adaptive response to synaptic and environmental experience. MSK1 may thus act as a key homeostat in the activity- and experience-dependent regulation of synaptic strength.

Optimizing gelling parameters of gellan gum for fibrocartilage tissue engineering.

Gellan gum is an attractive biomaterial for fibrocartilage tissue engineering applications because it is cell compatible, can be injected into a defect, and gels at body temperature. However, the gelling parameters of gellan gum have not yet been fully optimized. The aim of this study was to investigate the mechanics, degradation, gelling temperature, and viscosity of low acyl and low/high acyl gellan gum blends. Dynamic mechanical analysis showed that increased concentrations of low acyl gellan gum resulted in increased stiffness and the addition of high acyl gellan gum resulted in greatly decreased stiffness. Degradation studies showed that low acyl gellan gum was more stable than low/high acyl gellan gum blends. Gelling temperature studies showed that increased concentrations of low acyl gellan gum and CaCl2 increased gelling temperature and low acyl gellan gum concentrations below 2% (w/v) would be most suitable for cell encapsulation. Gellan gum blends were generally found to have a higher gelling temperature than low acyl gellan gum. Viscosity studies showed that increased concentrations of low acyl gellan gum increased viscosity. Our results suggest that 2% (w/v) low acyl gellan gum would have the most appropriate mechanics, degradation, and gelling temperature for use in fibrocartilage tissue engineering applications.

Disc strain and resulting positive mRNA expression from application of a noninvasive treatment.

STUDY DESIGN: Bovine caudal intervertebral discs were exposed to a noninvasive vibrating intervention for 10 minutes at amplitudes of 0 or 0.5 to 5 g and frequencies of 0, 16, 50 to 80, and a combined 16+50 to 80 Hz treatment. Expression of mRNA for aggrecan, collagen type I, collagen type II, biglycan, decorin, and versican were assayed. OBJECTIVE: To determine if the intervention is effective in altering intervertebral disc gene expression. SUMMARY OF BACKGROUND DATA: Studies have variously suggested either an increased risk of disc degeneration with vibrations, no effect, analgesic effect, or even positive effects within certain loading parameters. The KKT intervention is in clinical use for spinal ailment pain reduction. METHODS: The intervention was applied in a clinic emulation set-up. Gene expression in the nucleus pulposus was assessed using real-time RT-PCR and SYBR Green chemistry. RESULTS: Expression of mRNAs for aggrecan, collagen type II, and versican were significantly effected by the intervention. Collagen type I, biglycan, and decorin were uneffected. CONCLUSION: Expression of the extracellular matrix genes were significantly up-regulated when vibrated with the intervention under specific loading patterns, indicating a potential therapeutic stimulus. Further studies on the protein-level and long-term effects are warranted. Previous studies have indicated a mixed effect of vibrations in the human spine. In this study, a clinical intervention using vibrations was applied to bovine intervertebral discs, and gene expression in the nucleus pulposus was measured. Several extracellular matrix genes were up-regulated, suggesting a potential therapeutic effect.

Free axial vibrations at 0 to 200 Hz positively affect extracellular matrix messenger ribonucleic acid expression in bovine nucleus pulposi.

STUDY DESIGN: Bovine caudal intervertebral discs (IVDs) were exposed to free axial vibration for 10 to 60 minutes at 0 to 0.5 g and 0 to 200 Hz. Expression of messenger ribonucleic acid for aggrecan, collagen type I, collagen type II, biglycan, decorin, and versican were assayed, as was apoptosis. OBJECTIVE: To determine the vibration conditions which are most effective in altering intervertebral disc IVD gene expression. SUMMARY OF BACKGROUND DATA: Various studies have suggested widely varying effects of vibration in the IVD, ranging from harmful (increased risk of degeneration) to beneficial (increased analgesia) to neutral (no effect). METHODS: Vibration was applied using a custom designed voice coil system, which generated controlled motion in the axial direction. Gene expression in the nucleus pulposus was assessed using RT-PCR and the SYBR green chemistry; apoptosis was assessed using TUNEL staining. RESULTS: Expression of messenger ribonucleic acids for biglycan, collagen type I, collagen type II, decorin, and versican were significantly affected by vibration duration, frequency, and amplitude. Aggrecan was unaffected. Of the 3 factors, amplitude had the largest and widest effect. CONCLUSION: Expression of extracellular matrix genes was significantly upregulated at high amplitudes (>0.4 g) in as little as 10 minutes. This may indicate a potential therapeutic stimulus; periodic application of controlled vibration could positively influence matrix maintenance. Further studies on the protein level and long-term effects are warranted.

Regulation of the miR-212/132 locus by MSK1 and CREB in response to neurotrophins.

Neurotrophins are growth factors that are important in neuronal development and survival as well as synapse formation and plasticity. Many of the effects of neurotrophins are mediated by changes in protein expression as a result of altered transcription or translation. To determine whether neurotrophins regulate the production of microRNAs (miRNAs), small RNA species that modulate protein translation or mRNA stability, we used deep sequencing to identify BDNF (brain-derived neurotrophic factor)-induced miRNAs in cultured primary cortical mouse neurons. This revealed that the miR-212/132 cluster contained the miRNAs most responsive to BDNF treatment. This cluster was found to produce four miRNAs: miR-132, miR-132*, miR-212 and miR-212*. Using specific inhibitors, mouse models and promoter analysis we have shown that the regulation of the transcription of the miR-212/132 miRNA cluster and the miRNAs derived from it are regulated by the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, via both MSK (mitogen and stress-activated kinase)-dependent and -independent mechanisms.

Is vibration truly an injurious stimulus in the human spine?

Epidemiological data at one time was taken to suggest that chronic vibrations--for example operating vehicles with low-quality seats--contributed to intervertebral disc degeneration and lower back pain. More recent discussions, based in part upon extended twin studies, have cast doubt upon this interpretation, and question how much of the vibration is actually transmitted to the spine during loading. This review summarizes our recent survey of the current state of knowledge. In particular, we note that current studies are lacking a detailed factorial exploration of frequency, amplitude, and duration; this may be the primary cause for inconclusive and/or contradictory studies. It is our conclusion that vibrations are still an important consideration in discogenic back pain, and further controlled studies are warranted to definitively examine the underlying hypothesis: that chronic vibration can influence IVD cell biology and tissue mechanics.

Chordoma and chondrosarcoma gene profile: implications for immunotherapy.

Chordoma and chondrosarcoma are malignant bone tumors characterized by the abundant production of extracellular matrix. The resistance of these tumors to conventional therapeutic modalities has prompted us to delineate the gene expression profile of these two tumor types, with the expectation to identify potential molecular therapeutic targets. Furthermore the transcriptional profile of chordomas and chrondrosarcomas was compared to a wide variety of sarcomas as well as to that of normal tissues of similar lineage, to determine whether they express unique gene signatures among other tumors of mesenchymal origin, and to identify changes associated with malignant transformation. A HG-U133A Affymetrix Chip platform was used to determine the gene expression signature in 6 chordoma and 14 chondrosarcoma lesions. Validation of selected genes was performed by qPCR and immunohistochemistry (IHC) on an extended subset of tumors. By unsupervised clustering, chordoma and chondrosarcoma tumors grouped together in a genomic cluster distinct from that of other sarcoma types. They shared overexpression of many extracellular matrix genes including aggrecan, type II & X collagen, fibronectin, matrillin 3, high molecular weight-melanoma associated antigen (HMW-MAA), matrix metalloproteinase MMP-9, and MMP-19. In contrast, T Brachyury and CD24 were selectively expressed in chordomas, as were Keratin 8,13,15,18 and 19. Chondrosarcomas are distinguished by high expression of type IX and XI collagen. Because of its potential usefulness as a target for immunotherapy, the expression of HMW-MAA was analyzed by IHC and was detected in 62% of chordomas and 48% of chondrosarcomas, respectively. Furthermore, western blotting analysis showed that HMW-MAA synthesized by chordoma cell lines has a structure similar to that of the antigen synthesized by melanoma cells. In conclusion, chordomas and chondrosarcomas share a similar gene expression profile of up-regulated extracellular matrix genes. HMW-MAA represents a potential useful target to apply immunotherapy to these tumors.

Development and validation of a system for the growth of cells and tissues under intermittent hydrostatic pressure.

Various cell populations have been shown to respond to hydrostatic pressure; however, many of the culture systems suffer from shortcomings in design or methodology. Of particular interest to us is the potential role of pressure and other environmental factors in modulating stem cell behavior in intervertebral disk repair. A system was developed for the growth of cells and tissues under intermittent hydrostatic pressure. The system was validated with NIH 3T3 fibroblasts for sterilizability and cytotoxicity. Further experiments were conducted with canine mesenchymal stem cells under various levels of pressure, oxygen, glucose, and conditioned medium. The culture system showed no cytotoxicity and was able to demonstrate that the proliferation and metabolism of mesenchymal stem cells are sensitive to medium glucose and oxygen concentration and hydrostatic pressure. The cells exposed to hydrostatic pressure differed in their morphology from nonexposed cells. The system is capable of supporting long-term cell culture and examining the role of mechanical and environmental stimulation in vivo.

Identifying mechanisms for therapeutic intervention in chordoma: c-Met oncoprotein.

STUDY DESIGN: A human sacral chordoma cell line, CCL3, was established and in vitro characterization of c-Met oncoprotein in chordoma cells was performed. OBJECTIVE: Determination of whether c-Met plays an important role in chordoma's malignancy. SUMMARY OF BACKGROUND DATA: Chordomas are malignant life-threatening tumors that arise from the remnants of the notochord. c-Met is an oncoprotein that is expressed by a variety of solid tumors, including chordomas, and HGF is its high affinity ligand. In the present study, we investigated c-Met and HGF expression, localization, and function in human chordoma cells. METHODS: SDS-PAGE, Western blotting, immunofluorescence techniques, and cell migration functional assays were used to asses c-Met and HGF expression, localization, and functional activity. RESULTS: Intracellular protein tyrosine phosphorylation was enhanced on HGF binding, and an increase in the amount of 50 kDa alpha-chain of c-Met was detected in HGF-stimulated cells. Immunostaining of c-Met and HGF revealed membrane/cytoplasmic localization in nonstimulated cells, and perinuclear colocalization in HGF-stimulated cells. Positive chemotactic and migration activity in response to HGF was also demonstrated. CONCLUSION: Our data supports our hypothesis that the c-Met oncoprotein plays a leading role in the metastatic process in chordomas, and that a c-Met-HGF pair is involved in chordoma malignancy. Taking into consideration the very limited treatment options and an extremely poor prognosis for the chordoma patients, our results are a valuable and promising addition to the current situation in managing chordomas.

The role of extracellular factors in human metastatic chordoma cell growth in vitro.

STUDY DESIGN: Human metastatic chordoma cells were isolated, and initial in vitro characterization was performed. Biochemical and physiologic changes were observed in response to pH, oxygen, and glucose. OBJECTIVE: The extracellular microenvironment directly affects metastatic chordoma cell phenotype in vitro. SUMMARY OF BACKGROUND DATA: Chordomas are primary bone tumors that usually occur in the spine or skull. Chordomas arise from embryonic notochordal remnants along the axial skeleton, most commonly the sacrum, followed by the base of the skull and the mobile spine. Due to a high degree of resistance to radiation and chemotherapy, chordomas eventually cause death by direct growth or by metastasizing to other organs. METHODS: Extracellular pH, oxygen, and glucose levels in the culture medium were controlled, and cell response was assessed using MTT staining, SDS-PAGE, Western blotting, tandem mass spectrometry, TUNEL, immunofluorescence, and flow cytometry. RESULTS: In this study, we present a new chordoma cell line established from metastatic tissue and novel data characterizing some aspects of chordoma cell phenotype in different conditions in vitro. Chordoma biologic markers were expressed in the new cell line. Alkaline pH dramatically increased intracellular protein tyrosine phosphorylation, metabolic activity, and albumin accumulation in the cells, while acidic pH caused apoptosis. CONCLUSION: The level of proliferation, apoptosis, and tyrosine phosphorylation, as well as the overall protein expression profile, strongly depended on extracellular media pH and oxygen/glucose levels. Chordoma's preferred extracellular microenvironment in vitro was rather alkaline, with an optimum at pH 8.5, and apoptotic changes were induced at acidic pH. We found that bovine serum albumin was accumulated by chordoma cells from the incubation media, and this accumulation depended on extracellular pH, with the highest accumulation at alkaline pH.

Bod1, a novel kinetochore protein required for chromosome biorientation.

We have combined the proteomic analysis of Xenopus laevis in vitro-assembled chromosomes with RNA interference and live cell imaging in HeLa cells to identify novel factors required for proper chromosome segregation. The first of these is Bod1, a protein conserved throughout metazoans that associates with a large macromolecular complex and localizes with kinetochores and spindle poles during mitosis. Small interfering RNA depletion of Bod1 in HeLa cells produces elongated mitotic spindles with severe biorientation defects. Bod1-depleted cells form syntelic attachments that can oscillate and generate enough force to separate sister kinetochores, suggesting that microtubule-kinetochore interactions were intact. Releasing Bod1-depleted cells from a monastrol block increases the frequency of syntelic attachments and the number of cells displaying biorientation defects. Bod1 depletion does not affect the activity or localization of Aurora B but does cause mislocalization of the microtubule depolymerase mitotic centromere- associated kinesin and prevents its efficient phosphorylation by Aurora B. Therefore, Bod1 is a novel kinetochore protein that is required for the detection or resolution of syntelic attachments in mitotic spindles.

The effect of novel nitrogen-rich plasma polymer coatings on the phenotypic profile of notochordal cells.

BACKGROUND: The loss of the notochordal cells from the nucleus pulposus is associated with ageing and disc degeneration. However, understanding the mechanisms responsible for the loss of these cells has been hampered in part due to the difficulty of culturing and maintaining their phenotype. Furthermore, little is known about the influence of the substratum on the molecular markers of notochordal cells. METHODS: Notochordal cells were isolated from lumbar spine of non-chondrodystrophoid dogs and cultured on N-rich plasma polymer layers, so-called "PPE:N" (N-doped plasma-polymerised ethylene, containing up to 36% [N]) surfaces, for 3, 7 or 14 days. Gene expression of vimentin (VIM), pleiotrophin (PTN), matrix Gla protein (MGP), cartilage oligomeric matrix protein (COMP), keratin 18 (KRT 18), aggrecan (AGG), collagen type 1 (COL1A2), collagen type 2 (COL2A1) was analyzed through semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Notochordal cells were maintained in culture on PPE:N for up to 14 days with no loss in cell viability. Except for VIM, gene expression varied depending on the culture periods and [N] concentration of the substratum. Generally, PPE:N surfaces altered gene expression significantly when cells were cultured for 3 or 7 days. CONCLUSION: The present study has shown that notochordal cells from dogs can attach to and grow on PPE:N surfaces. Analysis of the expression of different genes in these cells cultured on different N-functionalized surfaces indicates that cellular behaviour is gene-specific and time-dependent. Further studies are required to better understand the roles of specific surface functionalities on receptor sites, and their effects on cellular phenotypes.

Developing an alginate/chitosan hybrid fiber scaffold for annulus fibrosus cells.

A fibrous scaffold made of alginate or alginate/chitosan was fabricated for annulus fibrosus (AF) cell culture using a wet-spinning and lyophilization technique. The scaffolds were evaluated using several in vitro tests. Scanning electron microscopy showed the scaffold fibers generally aligned in one direction with individual fiber diameters varying between 40-100 microm. The alginate/chitosan hybrid scaffold exhibited a slower degradation rate, while both scaffold types did not display any cyto-toxicity to 3T3 fibroblasts and could maintain canine AF cell growth. The AF cells retained their spherical shape within the fibrous scaffold at the beginning of the culture period and formed into cell clusters at later times. Specific extracellular matrix molecules, including collagen I, collagen II, and aggrecan, could be detected in the AF cell clusters. These results demonstrate the feasibility of using this hybrid alginate/chitosan scaffold for AF cell culture, and the potential application for intervertebral disc tissue engineering.

Osmoregulatory function of large vacuoles found in notochordal cells of the intervertebral disc running title: an osmoregulatory vacuole.

The nucleus pulposi of many species contain residual cells from the embryonic notochord, which exhibit a very unusual appearance (large vacuoles occupying approximately 80% of the cell volume, surrounded by an actin cytoskeleton). While the vacuoles have been qualitatively described, their composition and function has remained elusive. Given that these cells are believed to generate and experience significant osmotic pressures in both the notochord and intervertebral disc, we hypothesized that the vacuoles may serve as osmoregulatory organelles. Using both experimental and theoretical means, we demonstrated that the vacuoles contain a low-osmolality solution, generated via ion pumps on the vacuolar membrane. During hypotonic stress the vacuoles release their contents into the cytoplasm, diluting the cytoplasm and restoring the osmotic balance across the cell membrane. Thus the vacuoles function to regulate the cell volume and tonicity during rapid osmotic stress, protecting the cells from potentially damaging swelling pressures.