Biology subjects

Mei, L.

Publications and source records attributed to Mei, L..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

A transcriptome atlas of leg muscles from healthy human volunteers reveals molecular and cellular signatures associated with muscle location

Skeletal muscles support the stability and mobility of the skeleton but differ in biomechanical properties and physiological functions. The intrinsic factors that regulate muscle-specific characteristics are poorly understood. To study these, we constructed a large atlas of RNA-seq profiles from six leg muscles and two locations from one muscle, using biopsies from 20 healthy young males. We identified differential expression patterns and cellular composition across the seven tissues using three bioinformatics approaches confirmed by large-scale newly developed quantitative immune-histology procedures. With all three procedures, the muscle samples clustered into three groups congruent with their anatomical location. Concomitant with genes marking oxidative metabolism, genes marking fast- or slow-twitch myofibers differed between the three groups. The groups of muscles with higher expression of slow-twitch genes were enriched in endothelial cells and showed higher capillary content. In addition, expression profiles of Homeobox (HOX) transcription factors differed between the three groups and were confirmed by spatial RNA hybridization. We created an open-source graphical interface to explore and visualize the leg muscle atlas (https://tabbassidaloii.shinyapps.io/muscleAtlasShinyApp/). Our study reveals molecular specialization of human leg muscles and provides a novel resource to study muscle-specific molecular features, which could be linked with (patho)physiological processes.

neuroscience↗

bioRxiv2022

ATP6AP2-to-MMP14, a key pathway for osteoblast to osteocyte transition

Osteocytes, derived from osteoblast (OB)-lineage, occupy lacunae within bone matrix and exhibit unique morphology with dendrite-like projections to form an inter-connected network. Such a network is essential for osteocytes to monitor and orchestrate bone homeostasis. Thus, it is of considerable interest to investigate how osteocytes are formed and how they built the network. Here we provide evidence for ATP6AP2, an accessory subunit of V-ATPase, in OB-lineage cells to be critical for OB-to-osteocyte transition, and osteocyte distribution, maturation, and morphogenesis. Mice (ATP6AP2Ocn-Cre) that selectively deplete ATP6AP2 in OB-lineage cells results in altered osteocyte distribution and morphology, impaired osteocyte maturation and dendrite-like processes, increased osteocyte cell death and cortical woven bone formation. Further mechanistic studies identify MMP14 (matrix metalloproteinase-14) as a critical downstream of ATP6AP2 for osteocyte differentiation. ATP6AP2 interacts with MMP14 and promotes MMP14 surface distribution largely in immature- or osteoid-osteocytes, where this pathway regulates bone matrix remodeling and osteocyte differentiation. Expression of MMP14 into ATP6AP2 knock out OB-lineage cells in the mouse cortical bone could diminish the deficits in osteocyte maturation, dendrite-like process formation, and survival. These results thus demonstrate an un-recognized function of ATP6AP2 in promoting OB-to-osteocyte transition and uncover a pathway from ATP6AP2-to-MMP14 in osteocyte differentiation.

developmental biology↗

bioRxiv2022

Evaluating Client Protein Recovery by the Hsp40s DNAJB8 and DNAJB1 with AP-MS

Hsp40s, also termed J-domain proteins, play a central role in cellular protein homeostasis by promiscuously surveying the proteome for misfolded proteins. We have exploited this property to develop Hsp40 affinity profiling as a method for identifying proteins that misfold in response to cellular stresses. In this assay, we use the Hsp40 FlagDNAJB8H31Q as our recognition element for misfolded proteins. This protein is exogenously introduced into cells, promoting interactions without regard for native protein clients. Herein, we evaluate potential approaches to improve the performance of this assay. We find that although intracellular crosslinking increases recovery of protein interactors, this is not enough to overcome the relative drop in DNAJB8 recovery. While the J-domain promotes Hsp70 association, it does not affect the yield of protein association with DNJAB8 under basal conditions. By contrast, crosslinking and J-domain ablation both substantially increase relative protein interactor recovery with the structurally distinct Class B Hps40 DNAJB1 but are completely compensated by poorer yield of DNAJB1 itself. Cellular thermal stress promotes increased affinity between DNAJB8H31Q and interacting proteins, as expected for interactions driven by recognition of misfolded proteins. DNAJB8WT does not demonstrate such a property, suggesting that under stress misfolded proteins are handed off to Hsp70. Hence, we find that DNAJB8H31Q is still our most effective recognition element for the recovery of destabilized client proteins following cellular stress. Raw data is accessible through the PRIDE Archive at PXD030633.

biochemistry↗

bioRxiv2022

A toolkit of highly selective and sensitive genetically encoded neuropeptide sensors

Neuropeptides are key signaling molecules in the endocrine and nervous systems that regulate many critical physiological processes, including energy balance, sleep and circadian rhythms, stress, and social behaviors. Understanding the functions of neuropeptides in vivo requires the ability to monitor their dynamics with high specificity, sensitivity, and spatiotemporal resolution; however, this has been hindered by the lack of direct, sensitive and non-invasive tools. Here, we developed a series of GRAB (G protein-coupled receptor activation-based) sensors for detecting somatostatin (SST), cholecystokinin (CCK), corticotropin-releasing factor (CRF), neuropeptide Y (NPY), neurotensin (NTS), and vasoactive intestinal peptide (VIP). These fluorescent sensors utilize the corresponding GPCRs as the neuropeptide-sensing module with the insertion of a circular-permutated GFP as the optical reporter. This design detects the binding of specific neuropeptides at nanomolar concentration with a robust increase in fluorescence. We used these GRAB neuropeptide sensors to measure the spatiotemporal dynamics of endogenous SST release in isolated pancreatic islets and to detect the release of both CCK and CRF in acute brain slices. Moreover, we detect endogenous CRF release induced by stressful experiences in vivo using fiber photometry and 2-photon imaging in mice. Together, these new sensors establish a robust toolkit for studying the release, function, and regulation of neuropeptides under both physiological and pathophysiological conditions.

neuroscience↗

bioRxiv2022

Compartmental Neuropeptide Release Measured Using a New Oxytocin Sensor

As a peptide hormone and neuromodulator, oxytocin (OT) plays a critical role in a variety of physiological and pathophysiological processes in both the central nervous system and the periphery. However, the processes that regulate spatial OT release in the brain remain enigmatic. Here, we developed a genetically encoded GPCR activation-based (GRAB) OT sensor called GRABOT1.0. Using this sensor, we directly visualized stimulation-induced OT release from specific compartments of OT neurons in acute brain slices, and discovered that N-type calcium channels predominantly mediate axonal OT release, while L-type calcium channels mediate somatodendritic OT release. In addition, we found that components in the fusion machinery of OT release differ between axon terminals versus somata and dendrites. Finally, we demonstrated the sensor responses to the activation of OT neurons in various brain regions in vivo and revealed region specific OT release during male courtship behavior. Taken together, these results provide key insights regarding the role of compartmental OT release in the control of physiological and behavioral functions.

neuroscience↗

bioRxiv2021

Structural mechanism for bi-directional actin crosslinking by T-plastin

To fulfill the cytoskeletons diverse functions in cell mechanics and motility, actin networks with specialized architectures are built by crosslinking proteins, which bridge filaments to control micron-scale network geometry through nanoscale binding interactions via poorly defined structural mechanisms. Here, we introduce a machine-learning enabled cryo-EM pipeline for visualizing active crosslinkers, which we use to analyze human T-plastin, a member of the evolutionarily ancient plastin/fimbrin family of tandem calponin-homology domain (CHD) proteins. We define a sequential bundling mechanism which enables T-plastin to bridge filaments in both parallel and anti-parallel orientations. Our structural, biochemical, and cell biological data highlight inter-CHD linkers as key structural elements underlying flexible but stable crosslinking which are likely to be disrupted by mutations causing hereditary bone diseases. Beyond revealing how plastins are evolutionary optimized to crosslink dense actin networks with mixed polarity, our cryo-EM workflow will broadly enable analysis of the structural mechanisms underlying cytoskeletal network construction. One sentence summaryCryo-EM, biochemical, and cellular studies reveal how the crosslinking protein T-plastin bridges actin filaments in two opposing orientations.

biophysics↗

bioRxiv2021

Monitoring Protein Import into the Endoplasmic Reticulum in Living Cells with Proximity Labeling

The proper trafficking of eukaryotic proteins is essential to cellular function. Genetic, environmental, and other stresses can induce protein mistargeting, and in turn threaten cellular protein homeostasis. Current methods for measuring protein mistargeting are difficult to translate to living cells, and thus the role of cellular signaling networks in stress-dependent protein mistargeting processes, such as ER pre-emptive quality control (ER pQC), are difficult to parse. Herein, we use genetically encoded peroxidases to characterize protein import into the endoplasmic reticulum (ER). We show that the ERHRP/cytAPEX pair provides good selectivity and sensitivity for identifying protein mistargeting, using the known ER pQC substrate transthyretin (TTR). Although ERHRP labeling induces formation of detergent-resistant TTR aggregates, this is minimized by using low ERHRP expression, without loss of labeling efficiency. cytAPEX labeling recovers TTR that is mistargeted as a consequence of Sec61 inhibition or ER stress-induced ER pQC. Furthermore, we demonstrate that stress-free activation of the ER stress-associated transcription factor ATF6 recapitulates the TTR import deficiency of ER pQC. Hence, proximity labeling is an effective strategy for characterizing factors that influence ER protein import in living cells.

molecular biology↗

bioRxiv2021

Hsp40 Affinity to Identify Proteins Destabilized by Cellular Toxicant Exposure

Environmental toxins and toxicants can damage proteins and threaten cellular proteostasis. Most current methodologies to identify misfolded proteins in cells survey the entire proteome for sites of changed reactivity. We describe and apply a quantitative proteomics methodology to identify destabilized proteins based on their binding to the human Hsp40 chaperone DNAJB8. These protein targets are validated by an orthogonal limited proteolysis assay using parallel reaction monitoring. We find that brief exposure of HEK293T cells to meta-arsenite increases the affinity of two dozen proteins to DNAJB8, including known arsenite-sensitive proteins. In particular, arsenite treatment destabilizes both the pyruvate dehydrogenase complex E1 subunit and several RNA-binding proteins. This platform can be used to explore how environmental toxins impact cellular proteostasis, and to identify the susceptible proteome.

biochemistry↗

bioRxiv2021

Detecting Phytophthora cinnamomi associated with dieback disease on Carya cathayensis using loop-mediated isothermal amplification

Chinese hickory (Carya cathayensis Sarg.) is an economically and ecologically important nut plant in China. Dieback and basal stem necrosis have been observed in the plants since 2016, and its recent spread has significantly affected plant growth and nut production. Therefore, a survey was conducted to evaluate the disease incidence at five sites in Linan County, China. The highest incidence was recorded at the Tuankou site at up to 11.39% in 2019. The oomycete, Phytophthora cinnamomi, was isolated from symptomatic plant tissue and plantation soil using baiting and selective media-based detection methods and identified. Artificial infection with the representative P. cinnamomi ST402 isolate produced vertically elongated discolorations in the outer xylem and necrotic symptoms in C. cathayensis seedlings in a greenhouse trial. Molecular detections based on loop-mediated isothermal amplification (LAMP) specific to P. cinnamomi ST402 were conducted. Result indicated that LAMP detection showed a high coherence level with the baiting assays for P. cinnamomi detection in the field. This study provides the evidence of existence of high-pathogenic P. cinnamomi in the C. cathayensis plantation soil in China and the insights into a convenient tool developed for conducting field monitoring of this aggressive pathogen.

plant biology↗

bioRxiv2021

The Consequences of Differential Origin Licensing Dynamics

MCM complexes are loaded onto chromosomes to license DNA replication origins in G1 phase of the cell cycle, but it is not yet known how mammalian MCM complexes are adequately distributed to both euchromatin and heterochromatin. To address this question, we combined time-lapse live-cell imaging with fixed cell immunofluorescence imaging of single human cells to quantify the relative rates of MCM loading in heterochromatin and euchromatin at different times within G1. We report here that MCM loading in euchromatin is faster than in heterochromatin in very early G1, but surprisingly, heterochromatin loading accelerates relative to euchromatin loading in middle and late G1. These different loading dynamics require ORCA-dependent differences in ORC distribution during G1. A consequence of heterochromatin origin licensing dynamics is that cells experiencing a truncated G1 phase from premature cyclin E expression enter S phase with under-licensed heterochromatin, and DNA damage accumulates preferentially in heterochromatin in the subsequent S/G2 phase. Thus G1 length is critical for sufficient MCM loading, particularly in heterochromatin, to ensure complete genome duplication and to maintain genome stability.

molecular biology↗

bioRxiv2021

Early functional and cognitive declines measured by auditory evoked cortical potentials in Alzheimer's disease mice

Alzheimers disease (AD) is characterized with a progressive loss of memory and cognitive decline. However, assessment of AD-associated functional and cognitive changes is still a big challenge. Auditory evoked cortical potential (AECP) is an event-related potential reflecting not only neural activation in the auditory cortex but also cognitive activity in the brain. In this study, we used the subdermal needle electrodes with the same electrode setting as the auditory brainstem response (ABR) recording and recorded AECP in normal aging CBA/CaJ mice and APP/PS1 AD mice. AECP in mice usually appeared three positive peaks, i.e., P1, P2, and P3, and three corresponding negative peaks N1, N2, and N3. In normal aging CBA mice, the early sensory peaks P1, N1, and P2 were reduced as age increased, whereas the later cognitive peaks N2, P3, and N3 were increased or had no changes with aging. Moreover, the latency of P1 peak was increased as age increased, although the latencies of later peaks had significant reduction with aging. In AD mice, peak P1 was significantly reduced in comparison with wild-type (WT) littermates at young ages, proceeding AD phenotype presentation. In particular, the later cognitive peak P3 was diminished after 3 months old, different from normal aging effect. However, the latencies of AECP peaks in AD mice generally had no significant delay or changes with aging. Finally, consistent with AECP changes, the accumulation of amyloid precursor protein (APP) at the auditory cortex was visible in AD mice as early as 2 months old. These data suggest that AECP could serve as an early, noninvasive, and objective biomarker for detecting AD and AD-related dementia.

neuroscience↗

bioRxiv2020

Efferent control of hearing sensitivity and protection via inner ear supporting cells

It is well-known that medial olivocochlear (MOC) efferent nerves innervate outer hair cells (OHCs) and inhibit OHC electromotility to control hearing sensitivity. Here, we report that the MOC nerve fibers associated with MOC neurotransmitter acetylcholine (ACh) receptors and presynaptic vesicular acetylcholine transporters (VAChT) also innervated the cochlear supporting cells (SCs). Application of ACh could evoke inward currents in SCs and reduced gap junctions (GJs) between them with long-lasting effect. We further found that uncoupling of GJs between SCs shifted OHC electromotility to the left hyperpolarization direction and enhanced the direct effect of ACh on OHC electromotility. Diminution of this control by deficiency of GJs between SCs compromised the regulation of active cochlear amplification and increased susceptibility to noise. These data suggest that the MOC efferent system has functional innervations with SCs. Such efferent innervation may have an important role in the protection of hearing from noise and slow efferent effect.

neuroscience↗

bioRxiv2020

Novel gene-specific translation mechanism of dysregulated, chronic inflammation reveals promising, multifaceted COVID-19 therapeutics

Hyperinflammation and lymphopenia provoked by SARS-CoV-2-activated macrophages contribute to the high mortality of Coronavirus Disease 2019 (COVID-19) patients. Thus, defining host pathways aberrantly activated in patient macrophages is critical for developing effective therapeutics. We discovered that G9a, a histone methyltransferase that is overexpressed in COVID-19 patients with high viral load, activates translation of specific genes that induce hyperinflammation and impairment of T cell function or lymphopenia. This noncanonical, pro-translation activity of G9a contrasts with its canonical epigenetic function. In endotoxin-tolerant (ET) macrophages that mimic conditions which render patients with pre-existing chronic inflammatory diseases vulnerable to severe symptoms, our chemoproteomic approach with a biotinylated inhibitor of G9a identified multiple G9a-associated translation regulatory pathways that were upregulated by SARS-CoV-2 infection. Further, quantitative translatome analysis of ET macrophages treated progressively with the G9a inhibitor profiled G9a-translated proteins that unite the networks associated with viral replication and the SARS-CoV-2-induced host response in severe patients. Accordingly, inhibition of G9a-associated pathways produced multifaceted, systematic effects, namely, restoration of T cell function, mitigation of hyperinflammation, and suppression of viral replication. Importantly, as a host-directed mechanism, this G9a-targeted, combined therapeutics is refractory to emerging antiviral-resistant mutants of SARS-CoV-2, or any virus, that hijacks host responses.

systems biology↗

bioRxiv2020

Mechanical Tension in Syndecan-1 is Regulated by Extracellular Mechanical Cues and Fluidic Shear Stress

The endothelium plays a central role in regulating vascular homeostasis and is key in determining the response to materials implanted in the vascular system. Endothelial cells are uniquely sensitive to biophysical cues from applied forces and their local cellular microenvironment. The glycocalyx is a layer of proteoglycans, glycoproteins and glycosaminoglycans that lines the luminal surface of the vascular endothelium, interacting directly with the components of the blood and the forces of blood flow. In this work, we examined the changes in mechanical tension of syndecan-1, a cell surface proteoglycan that is an integral part of the glycocalyx, in response to substrate stiffness and fluidic shear stress. Our studies demonstrate that syndecan-1 is mechanically responsive to extracellular mechanical cues and alters its association with cytoskeletal and adhesion-related proteins in response to substrate stiffness and physiological flow.

bioengineering↗

bioRxiv2020

Molecular mechanism for direct actin force-sensing by α-catenin

The actin cytoskeleton mediates mechanical coupling between cells and their tissue microenvironments. The architecture and composition of actin networks are modulated by force, but it is unclear how interactions between actin filaments (F-actin) and associated proteins are mechanically regulated. Here, we employ both optical trapping and biochemical reconstitution with myosin motor proteins to show force greater than one piconewton applied solely to F-actin enhances binding by the essential cell-cell adhesion protein E-catenin, but not its homolog vinculin. Near atomic-resolution cryo-electron microscopy structures of both proteins bound to F-actin reveal unique rearrangements that facilitate their flexible C-termini refolding to engage distinct interfaces. Truncating -catenins C-terminus eliminates force-activated F-actin binding, and addition of this motif to vinculin confers force-activated binding, demonstrating that -catenins C-terminus is a modular detector of F-actin tension. Our studies establish that piconewton force on F-actin can enhance partner binding, which we propose mechanically regulates cellular adhesion through -catenin.

biophysics↗

bioRxiv2020

Myosin X interaction with KIF13B, a crucial pathway for Netrin-1-induced axonal development

Myosin X (Myo X) transports cargos to the tip of filopodia for cell adhesion, migration, and neuronal axon guidance. Deleted in Colorectal Cancer (DCC) is one of Myo X cargos essential for Netrin-1-regulated axon pathfinding. Myo Xs function in axon development in vivo and the underlying mechanisms remain poorly understood. Here, we provide evidence for Myo Xs function in Netrin-1-DCC regulated axon development in mouse neocortex. Knocking-out (KO) or knocking-down (KD) Myo X in embryonic cortical neurons impairs axon initiation and contralateral branching/targeting. Similar axon deficits are detected in Netrin-1-KO or DCC-KD cortical neurons. Myo X interacts with KIF13B (a kinesin family motor protein), which is induced by Netrin-1. Netrin-1 promotes anterograde transportation of Myo X into axons in KIF13B dependent manner. KIF13B-KD cortical neurons exhibit similar axon deficits. These results suggest Myo X-KIF13B as a critical pathway for Netrin-1 promoted axon initiation and branching/targeting.

developmental biology↗

bioRxiv2019

Critical role for Slam/SAP signaling in the thymic developmental programming of IL-17- and IFN-γ-producing γδ T cells

During thymic development, {gamma}{delta} T cells commit to either an IFN-{gamma}- or an IL-17-producing phenotype through mechanisms that remain unclear. Here, we investigated whether the SLAM/SAP signaling pathway played a role in the functional programming of thymic {gamma}{delta} T cells. Characterization of SLAM family receptor expression revealed that thymic {gamma}{delta} T cell subsets were each marked by distinct co-expression profiles of SLAMF1, SLAMF4, and SLAMF6. In the thymus, immature CD24hi V{gamma}1 and V{gamma}4 {gamma}{delta} T cells were largely contained within a SLAMF1+SLAMF6+ double positive (DP) population, while mature CD24low subsets were either SLAMF1+ or SLAMF6+ single positive (SP) cells. In the periphery, SLAMF1 and SLAMF6 expression on V{gamma}1, V{gamma}4, and V{gamma}6 T cells distinguished IL-17- and IFN-{gamma}-producing subsets, respectively. Disruption of SLAM family receptor signaling through deletion of SAP resulted in impaired thymic {gamma}{delta} T cell maturation at the CD24hiSLAMF1+SLAMF6+ DP stage that was associated with a decreased frequency of CD44+ROR{gamma}t+ {gamma}{delta} T cells. These defects were in turn associated with impaired {gamma}{delta} T cell IL-17 and IFN-{gamma} production in both the thymus as well as in peripheral tissues. The role for SAP was subset-specific, as V{gamma}1, V{gamma}4, V{gamma}5, but not V{gamma}6 subsets were SAP-dependent. Together, these data suggest that the SLAM/SAP signaling pathway regulates a critical checkpoint in the functional programming of IL-17 and IFN-{gamma}-producing {gamma}{delta} T cell subsets during thymic development.

immunology↗

bioRxiv2018

FAM83D directs protein kinase CK1α to the mitotic spindle for proper spindle positioning

The concerted action of many protein kinases helps orchestrate the error-free progression through mitosis of mammalian cells. The roles and regulation of some prominent mitotic kinases, such as cyclin-dependent kinases, are well-established. However, these and other known mitotic kinases alone cannot account for the extent of protein phosphorylation that has been reported during mammalian mitosis. Here we demonstrate that CK1, of the casein kinase 1 family of protein kinases, localises to the spindle and is required for proper spindle-positioning and timely cell division. CK1 is recruited to the spindle by FAM83D, and cells devoid of FAM83D, or those harbouring CK1-binding-deficient FAM83DF283A/F283A knockin mutation, display pronounced spindle-positioning defects, and a prolonged mitosis. Restoring FAM83D at the endogenous locus in FAM83D-/- cells, or artificially delivering CK1 to the spindle in FAM83DF283A/F283A cells, rescues these defects. These findings implicate CK1 as new mitotic kinase that orchestrates the kinetics and orientation of cell division.

cell biology↗