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BioRxiv : the Preprint Server For... Jun 2024Matching arousal level to the motor activity of an animal is important for efficiently allocating cognitive resources and metabolic supply in response to behavioral...
Matching arousal level to the motor activity of an animal is important for efficiently allocating cognitive resources and metabolic supply in response to behavioral demands, but how the brain coordinates changes in arousal and wakefulness in response to motor activity remains an unclear phenomenon. We hypothesized that the locus coeruleus (LC), as the primary source of cortical norepinephrine (NE) and promoter of cortical and sympathetic arousal, is well-positioned to mediate movement-arousal coupling. Here, using a combination of physiological recordings, fiber photometry, optogenetics, and behavioral tracking, we show that the LC activation is tightly coupled to the return of organized movements during waking from an anesthetized state. Moreover, in an awake animal, movement initiations are coupled to LC activation, while movement arrests, to LC deactivation. We also report that LC activity covaries with the depth of anesthesia and that LC photoactivation leads to sympathetic activation, consistent with its role in mediating increased arousal. Together, these studies reveal a more nuanced, modulatory role that LC plays in coordinating movement and arousal.
PubMed: 38948871
DOI: 10.1101/2024.06.18.599619 -
BioRxiv : the Preprint Server For... Jun 2024Nucleoli are large nuclear sub-compartments where vital processes, such as ribosome assembly, take place. Technical obstacles still limit our understanding of the...
Nucleoli are large nuclear sub-compartments where vital processes, such as ribosome assembly, take place. Technical obstacles still limit our understanding of the biological functions of nucleolar proteins in cell homeostasis and cancer pathogenesis. Since most nucleolar proteins are essential, their abrogation cannot be achieved through conventional approaches. Additionally, the biological activities of many nucleolar proteins are connected to their physiological concentration. Thus, artificial overexpression might not fully recapitulate their endogenous functions. Proteolysis-based approaches, such as the Auxin Inducible Degron (AID) system paired with CRISPR/Cas9 knock-in gene-editing, have the potential to overcome these limitations, providing unprecedented characterization of the biological activities of endogenous nucleolar proteins. We applied this system to endogenous nucleolin (NCL), one of the most abundant nucleolar proteins, and characterized the impact of its acute depletion on Triple-Negative Breast Cancer (TNBC) cell behavior. Abrogation of endogenous NCL reduced proliferation and caused defective cytokinesis, resulting in bi-nucleated tetraploid cells. Bioinformatic analysis of patient data, and quantitative proteomics using our experimental NCL-depleted model, indicated that NCL levels are correlated with the abundance of proteins involved in chromosomal segregation. In conjunction with its effects on sister chromatid dynamics, NCL abrogation enhanced the anti-proliferative effects of chemical inhibitors of mitotic modulators such as the Anaphase Promoting Complex. In summary, using the AID system in combination with CRISPR/Cas9 for endogenous gene editing, our findings indicate a novel role for NCL in supporting the completion of the cell division in TNBC models, and that its abrogation could enhance the therapeutic activity of mitotic-progression inhibitors.
PubMed: 38948867
DOI: 10.1101/2024.06.17.599429 -
BioRxiv : the Preprint Server For... Jun 2024Cellular mechanical properties influence cellular functions across pathological and physiological systems. The observation of these mechanical properties is limited in...
Cellular mechanical properties influence cellular functions across pathological and physiological systems. The observation of these mechanical properties is limited in part by methods with a low throughput of acquisition or with low accessibility. To overcome these limitations, we have designed, developed, validated, and optimized a microfluidic cellular deformation system (MCDS) capable of mechanotyping suspended cells on a population level at a high throughput rate of ∼300 cells pers second. The MCDS provides researchers with a viable method for efficiently quantifying cellular mechanical properties towards defining prognostic implications of mechanical changes in pathology or screening drugs to modulate cytoskeletal integrity.
PubMed: 38948841
DOI: 10.1101/2024.06.17.599307 -
BioRxiv : the Preprint Server For... Jun 2024The inner ear houses two sensory modalities: the hearing organ, located in the cochlea, and the balance organs, located throughout the vestibular regions of the ear....
UNLABELLED
The inner ear houses two sensory modalities: the hearing organ, located in the cochlea, and the balance organs, located throughout the vestibular regions of the ear. Both hearing and vestibular sensory regions are composed of similar cell types, including hair cells and associated supporting cells. Recently, we showed that is required for maintaining supporting cell survival postnatally during cochlear maturation. However, it is not known whether plays a similar role in the balance organs of the inner ear. To characterize the role of Notch during vestibular maturation, we conditionally deleted from -expressing cells of the vestibular organs in the mouse at P0/P1. Histological analyses showed a dramatic loss of supporting cells accompanied by an increase in type II hair cells without cell death, indicating the supporting cells are converting to hair cells in the maturing vestibular regions. Analysis of 6-week old animals indicate that the converted hair cells survive, despite the reduction of supporting cells. Interestingly, measurements of vestibular sensory evoked potentials (VsEPs), known to be generated in the striolar regions of the vestibular afferents in the maculae, failed to show a response, indicating that NOTCH1 expression is critical for striolar function postnatally. Consistent with this, we find that the specialized type I hair cells in the striola fail to develop the complex calyces typical of these cells. These defects are likely due to the reduction in supporting cells, which have previously been shown to express factors critical for the striolar region. Similar to other mutants that lack proper striolar development, mutants do not exhibit typical vestibular behaviors such as circling and head shaking, but do show difficulties in some vestibular tests, including the balance beam and forced swim test. These results indicate that, unlike the hearing organ in which the supporting cells undergo cell death, supporting cells in the balance regions retain the ability to convert to hair cells during maturation, which survive into adulthood despite the reduction in supporting cells.
SIGNIFICANCE STATEMENT
Notch signaling regulates the cell fate choices between hair cells and supporting cells during inner ear development. However, little is known about how Notch functions in the mammalian vestibular sensory organs once cell fate has been determined. Here, we examine the role of in the maturing balancing organs. We show that deletion of results in vestibular physiological and behavioral dysfunction by 3 months of age. Histological analyses reveal supporting cells are converting to type II hair cells in the utricle, and despite a loss of supporting cells, the hair cells survive to adulthood. Additionally, the striolar type I hair cells important for generating a VsEP response are decreased in number and not innervated properly. These results show that Notch continues to function in maintaining supporting cell identity in the vestibular organs postnatally, which may be important in strategies for hair cell regeneration.
PubMed: 38948821
DOI: 10.1101/2024.06.21.600098 -
BioRxiv : the Preprint Server For... Jun 2024G protein-coupled receptors (GPCRs) modulate various physiological functions by re-wiring cellular gene expression in response to extracellular signals. Control of gene...
G protein-coupled receptors (GPCRs) modulate various physiological functions by re-wiring cellular gene expression in response to extracellular signals. Control of gene expression by GPCRs has been studied almost exclusively at the transcriptional level, neglecting an extensive amount of regulation that takes place translationally. Hence, little is known about the nature and mechanisms of gene-specific post-transcriptional regulation downstream of receptor activation. Here, we apply an unbiased multiomics approach to delineate an extensive translational regulatory program initiated by the prototypical beta2-adrenergic receptor (β2-AR) and provide mechanistic insights into how these processes are orchestrated. Using ribosome profiling (Ribo-seq), we identify nearly 120 novel gene targets of adrenergic receptor activity which expression is exclusively regulated at the level of translation. We next show that all translational changes are induced selectively by endosomal β2-ARs. We further report that this proceeds through activation of the mammalian target of rapamycin (mTOR) pathway. Specifically, within the set of translational GPCR targets we discover significant enrichment of genes with 5' terminal oligopyrimidine (TOP) motifs, a gene class classically known to be translationally regulated by mTOR. We then demonstrate that endosomal β2-ARs are required for mTOR activation and subsequent mTOR-dependent TOP mRNA translation. Together, this comprehensive analysis of drug-induced translational regulation establishes a critical role for location-biased GPCR signaling in fine-tuning the cellular protein landscape.
PubMed: 38948806
DOI: 10.1101/2024.06.17.599400 -
BioRxiv : the Preprint Server For... Jun 2024During fertilization, mammalian sperm undergo a winnowing selection process that reduces the candidate pool of potential fertilizers from ∼10 -10 cells to 10 -10...
During fertilization, mammalian sperm undergo a winnowing selection process that reduces the candidate pool of potential fertilizers from ∼10 -10 cells to 10 -10 cells (depending on the species). Classical sperm competition theory addresses the positive or 'stabilizing' selection that acts on sperm phenotypes within populations of organisms but does not strictly address the developmental consequences of sperm traits among individual organisms that are under purifying selection during fertilization. It is the latter that is of utmost concern for improving assisted reproductive technologies (ART) because 'low fitness' sperm may be inadvertently used for fertilization during interventions that rely heavily on artificial sperm selection, such as intracytoplasmic sperm injection (ICSI). Importantly, some form of sperm selection is used in nearly all forms of ART (e.g., differential centrifugation, swim-up, or hyaluronan binding assays, etc.). To date, there is no unifying quantitative framework (i.e., theory of sperm selection) that synthesizes causal mechanisms of selection with observed natural variation in individual sperm traits. In this report, we reframe the physiological function of sperm as a collective diffusive search process and develop multi-scale computational models to explore the causal dynamics that constrain sperm 'fitness' during fertilization. Several experimentally useful concepts are developed, including a probabilistic measure of sperm 'fitness' as well as an information theoretic measure of the magnitude of sperm selection, each of which are assessed under systematic increases in microenvironmental selective pressure acting on sperm motility patterns.
PubMed: 38948799
DOI: 10.1101/2024.06.17.599386 -
BioRxiv : the Preprint Server For... Jun 2024The physiological response of a cell to stimulation depends on its proteome configuration. Therefore, the abundance variation of regulatory proteins across unstimulated...
The physiological response of a cell to stimulation depends on its proteome configuration. Therefore, the abundance variation of regulatory proteins across unstimulated single cells can be associatively linked with their response to stimulation. Here we developed an approach that leverages this association across individual cells and nuclei to systematically identify potential regulators of biological processes, followed by targeted validation. Specifically, we applied this approach to identify regulators of nucleocytoplasmic protein transport in macrophages stimulated with lipopolysaccharide (LPS). To this end, we quantified the proteomes of 3,412 individual nuclei, sampling the dynamic response to LPS treatment, and linking functional variability to proteomic variability. Minutes after the stimulation, the protein transport in individual nuclei correlated strongly with the abundance of known protein transport regulators, thus revealing the impact of natural protein variability on functional cellular response. We found that simple biophysical constraints, such as the quantity of nuclear pores, partially explain the variability in LPS-induced nucleocytoplasmic transport. Among the many proteins newly identified to be associated with the response, we selected 16 for targeted validation by knockdown. The knockdown phenotypes confirmed the inferences derived from natural protein and functional variation of single nuclei, thus demonstrating the potential of (sub-)single-cell proteomics to infer functional regulation. We expect this approach to generalize to broad applications and enhance the functional interpretability of single-cell omics data.
PubMed: 38948785
DOI: 10.1101/2024.06.17.599449 -
BioRxiv : the Preprint Server For... Jun 2024Sickle cell disease is caused by a mutation in the beta subunit of hemoglobin (HbSS) that drives Hb fiber formation when the protein is in the deoxygenated (tense, T)...
UNLABELLED
Sickle cell disease is caused by a mutation in the beta subunit of hemoglobin (HbSS) that drives Hb fiber formation when the protein is in the deoxygenated (tense, T) state. The drug voxelotor was recently approved to treat sickle cell disease by preventing HbSS fiber formation. Voxelotor acts as an allosteric inhibitor of polymerization by maintaining the HbSS protein in the relaxed (R) conformation, limiting polymerization of T-state fibers. Normal blood cells contain small amounts of natural Hb fibers and a few percent of the Fe ferric form, metHb, incapable of binding oxygen. Although the drug Voxelotor is now in use, the effect of the drug on the oxidized metHb state has not been reported. Here we assessed the influence of voxelotor on normal human metHb. We compared the aggregation of metHb at two pH values (5.5 and 7.1). MetHb is known to form organized fiber structures at or below pH 5.5. We find that voxelotor significantly enhances fiber formation of metHb R-state at pH 5.5, consistent with the mode of action for this drug in maintaining the Hb R conformation. The opposite effect is observed at physiological pH values. Voxelotor significantly decreases the rate of metHb aggregate formation at pH 7.1 but did not affect protein stability. Notably, drug binding drives metHb into novel spherical particles with a morphology never seen before for Hb. The formation of these particles should be considered in patients being treated for sickle cell disease with voxelotor.
WHY IT MATTERS
Voxelotor is an FDA-approved drug for sickle cell anemia, known to prevent hemoglobin fiber formation. Here, we investigate its effect on methemoglobin, the form of hemoglobin in which iron takes on the ferric Fe state. Our study examines voxelotor's impact on methemoglobin aggregation and stability. At pH 7.1, we found voxelotor to have an effect on methemoglobin solubility as evidenced by the formation of novel methemoglobin spherical structures. We observe that voxelotor significantly increases methemoglobin fiber formation at pH 5.5 but, notably, reduces methemoglobin aggregation at physiological pH levels. Minimal impact on methemoglobin thermodynamic stability is noted. These findings suggest voxelotor's potential therapeutic efficacy for various hemoglobinopathies, including conditions characterized by Heinz body formation.
PubMed: 38948767
DOI: 10.1101/2024.06.16.599216 -
BioRxiv : the Preprint Server For... Jun 2024Pain is a prominent and debilitating symptom in myotonic disorders, yet its physiological mechanisms remain poorly understood. This study assessed preclinical pain-like...
Pain is a prominent and debilitating symptom in myotonic disorders, yet its physiological mechanisms remain poorly understood. This study assessed preclinical pain-like behavior in murine models of pharmacologically induced myotonia and myotonic dystrophy type 1 (DM1). In both myotonia congenita and DM1, impairment of the gene, which encodes skeletal muscle voltage-gated CLC-1 chloride channels, reduces chloride ion conductance in skeletal muscle cells, leading to prolonged muscle excitability and delayed relaxation after contraction. We used the CLC-1 antagonist anthracene-9- carboxylic acid (9-AC) at intraperitoneal doses of 30 or 60 mg/kg and HSA LR20b DM1 mice to model CLC-1-induced myotonia. Our experimental approach included pain behavioral testing, calcium imaging, and whole-cell current-clamp electrophysiology in mouse dorsal root ganglion (DRG) neurons. A single injection of 9-AC induced myotonia in mice, which persisted for several hours and resulted in long-lasting allodynic pain-like behavior. Similarly, HSA LR20b mice exhibited both allodynia and hyperalgesia. Despite these pain-like behaviors, DRG neurons did not show signs of hyperexcitability in either myotonic model. These findings suggest that myotonia induces nociplastic pain-like behavior in preclinical rodents, likely through central sensitization mechanisms rather than peripheral sensitization. This study provides insights into the pathophysiology of pain in myotonic disorders and highlights the potential of using myotonic mouse models to explore pain mechanisms and assess novel analgesics. Future research should focus on the central mechanisms involved in myotonia-induced pain and develop targeted therapies to alleviate this significant clinical burden.
PubMed: 38948724
DOI: 10.1101/2024.06.19.599732 -
BioRxiv : the Preprint Server For... Oct 2023Low nephron endowment at birth is a risk factor for chronic kidney disease. The prevalence of this condition is increasing due to higher survival rates of preterm...
Low nephron endowment at birth is a risk factor for chronic kidney disease. The prevalence of this condition is increasing due to higher survival rates of preterm infants and children with multi- organ birth defect syndromes that affect the kidney and urinary tract. We created a mouse model of congenital low nephron number due to deletion of in nephron progenitor cells. is a core component of the Nucleosome Remodeling and Deacetylase (NuRD) chromatin remodeling complex. These mice developed albuminuria at 4 weeks of age followed by focal segmental glomerulosclerosis (FSGS) at 8 weeks, with progressive kidney injury and fibrosis. Our studies reveal that altered mitochondrial metabolism in the post-natal period leads to accumulation of neutral lipids in glomeruli at 4 weeks of age followed by reduced mitochondrial oxygen consumption. We found that NuRD cooperated with Zbtb7a/7b to regulate a large number of metabolic genes required for fatty acid oxidation and oxidative phosphorylation. Analysis of human kidney tissue also supported a role for reduced mitochondrial lipid metabolism and ZBTB7A/7B in FSGS and CKD. We propose that an inability to meet the physiological and metabolic demands of post-natal somatic growth of the kidney promotes the transition to CKD in the setting of glomerular hypertrophy due to low nephron endowment.
PubMed: 38948707
DOI: 10.1101/2023.10.18.562984