Biology subjects

Lin, J.

Publications and source records attributed to Lin, J..

At least 217 records · Page 12Linked to original sources

bioRxiv2023

In vivo proximity proteomics uncovers palmdelphin (PALMD) as a Z-line-associated mitigator of isoproterenol-induced cardiac injury

Z-lines are core ultrastructural organizers of cardiomyocytes that modulate many facets of cardiac pathogenesis. Yet a comprehensive proteomic atlas of Z-line-associated components remain incomplete. Here, we established an adeno-associated virus (AAV)-delivered, cardiomyocyte-specific, proximity-labeling approach to characterize the Z-line proteome in vivo. We found palmdelphin (PALMD) as a novel Z-line-associated protein in both adult murine cardiomyocytes and human pluripotent stem cell-derived cardiomyocytes. Germline and cardiomyocyte-specific palmd knockout mice were grossly normal at baseline but exhibited compromised cardiac hypertrophy and aggravated cardiac injury upon long-term isoproterenol treatment. By contrast, cardiomyocyte-specific PALMD overexpression was sufficient to mitigate isoproterenol-induced cardiac injury. PALMD ablation perturbed transverse tubules (T-tubules) and their association with sarcoplasmic reticulum, which formed the Z-line-associated junctional membrane complex (JMC) essential for calcium handling and cardiac function. These phenotypes were associated with disrupted localization of T-tubule markers caveolin-3 (CAV3) and junctophilin-2 (JPH2) and the reduction of nexilin (NEXN) protein, a crucial Z-line-associated protein that is essential for both Z-line and JMC structures and functions. PALMD was found to interact with NEXN and enhance its protein stability while the Nexn mRNA level was not affected. Together, this study discovered PALMD as a potential target for myocardial protection and highlighted in vivo proximity proteomics as a powerful approach to nominate novel players regulating cardiac pathogenesis. HighlightsO_LIIn vivo proximity proteomics uncover novel Z-line components that are undetected in in vitro proximity proteomics in cardiomyocytes. C_LIO_LIPALMD is a novel Z-line-associated protein that is dispensable for baseline cardiomyocyte function in vivo. C_LIO_LIPALMD mitigates cardiac dysfunction and myocardial injury after repeated isoproterenol insults. C_LIO_LIPALMD stabilizes NEXN, an essential Z-line-associated regulator of the junctional membrane complex and cardiac systolic function. C_LI

molecular biology↗

bioRxiv2023

Spatially resolved molecular and cellular atlas of the mouse brain

A comprehensive atlas of genes, cell types, and their spatial distribution across a whole mammalian brain is fundamental for understanding function of the brain. Here, using snRNA-seq and Stereo-seq techniques, we generated a mouse brain atlas with spatial information for 308 cell clusters with single-cell resolution involving over 6 million cells as well as for 29,655 genes. We have identified new astrocyte clusters, and demonstrated that distinct cell clusters exhibit preference for cortical subregions. In addition, we identified 155 genes exhibiting regional specificity in the brainstem, and 513 long non-coding RNA exhibited regional specificity in the adult brain. Parcellation of brain regions based on spatial transcriptomic information showed large overlap with that by traditional method. Furthermore, we have uncovered 411 transcription factor regulons with spatiotemporal specificity during development. Thus, our study has discovered genes and regulon with spatiotemporal specificity, and provided a high-resolution spatial transcriptomic atlas of the mouse brain.

neuroscience↗

bioRxiv2023

Chaperone-mediated autophagy is an overlooked pathway for mutant α1 antitrypsin Z degradation

Chaperone-mediated autophagy (CMA) is a specific form of autophagy that selectively targets proteins containing a KFERQ-like motif and relies on the chaperone protein HSC70 for substrate recognition. In 1-antitrypsin deficiency (AATD), a disease characterized by the hepatic build-up of 1-Antitrypsin Z mutant (ATZ), CMAs role had been unclear. This work demonstrates the critical role that CMA plays in preventing ATZ accumulation; suppressing CMA worsens ATZ accumulation, whilst activating it through chemical stimulation or LAMP2A overexpression promotes ATZ breakdown. Specifically, ATZs 121QELLR125 motif is critical for HSC70 recognition and LAMP2As charged C-terminal cytoplasmic tail is vital for substrate binding, facilitating CMA-mediated degradation of ATZ. This selective activation of CMA operates independently from other autophagy pathways and alleviate ATZ aggregates caused cellular stress. These findings highlight CMAs critical function in cellular protein quality control of ATZ and place it as a novel target for AATD treatment approaches.

cell biology↗

bioRxiv2023

Phenology across scales: an intercontinental analysis of leaf-out dates in temperate deciduous tree communities

AimTo quantify the intra-community variability of leaf-out (ICVLo) among dominant trees in temperate deciduous forests, assess its links with specific and phylogenetic diversity, identify its environmental drivers, and deduce its ecological consequences with regard to radiation received and exposure to late frost. LocationEastern North America (ENA) and Europe (EUR). Time period2009-2022 Major taxa studiedTemperate deciduous forest trees. MethodsWe developed an approach to quantify ICVLo through the analysis of RGB images taken from phenological cameras. We related ICVLo to species richness, phylogenetic diversity and environmental conditions. We quantified the intra-community variability of the amount of radiation received and of exposure to late frost. ResultsLeaf-out occurred over a longer time interval in ENA than in EUR. The sensitivity of leaf-out to temperature was identical in both regions (-3.4 days per {degrees}C). The distributions of ICVLo were similar in EUR and ENA forests, despite the latter being more species-rich and phylogenetically diverse. In both regions, cooler conditions and an earlier occurrence of leaf-out resulted in higher ICVLo. ICVLo resulted in a ca. 8% difference of radiation absorption over spring among individual trees. Forest communities in ENA had shorter safety margins as regards the exposure to late frosts, and were actually more frequently exposed to late frosts. Main conclusionsWe conducted the first intercontinental analysis of the variability of leaf-out at the scale of tree communities. North American and European forests showed similar ICVLo, in spite of their differences in terms of species richness and phylogenetic diversity, highlighting the relevance of environmental controls on ICVLo. We quantified two ecological implications of ICVLo (difference in terms of radiation absorption and exposure to late frost), which should be explored in the context of ongoing climate change, which affects trees differently according to their phenological niche.

ecology↗

bioRxiv2023

The hidden function of nonspecifically DNA-bound RNA polymerases: a buffer of resource competition in bacteria

RNA polymerase (RNAP) is the workhorse of bacterial gene expression, transcribing rRNA and mRNA. Experiments found that a significant fraction of RNAPs in bacteria are nonspecifically bound to DNA, which is puzzling as these idle RNAPs could have produced more RNAs. Whether nonspecifically DNA-bound RNAPs have any function or are merely a consequence of passive interaction between RNAP and DNA is unclear. In this work, we propose that nonspecifically DNA-bound RNAPs buffer the free RNAP concentration and mitigate the crosstalk between rRNA and mRNA transcription. We verify our theory using mean-field models and an agent-based model of transcription, showing that the buffering effects are robust against the interaction between RNAPs and sigma factors and the spatial fluctuation and temporal noise of RNAP concentration. We analyze the relevant parameters of Escherichia coli and find that the buffering effects are significant across different growth rates at a low cost, suggesting that nonspecifically DNA-bound RNAPs are evolutionarily advantageous.

microbiology↗

bioRxiv2023

Genetic deficiency of ribosomal rescue factor HBS1L causes retinal dystrophy associated with Pelota and EDF1 depletion

Inherited retinal diseases (IRDs) encompass a genetically diverse group of conditions in which mutations in genes critical to retinal function lead to progressive loss of photoreceptor cells and subsequent visual impairment. A handful of ribosome-associated genes have been implicated in retinal disorders alongside neurological phenotypes. This study focuses on the HBS1L gene, encoding HBS1 Like Translational GTPase which has been recognized as a critical ribosomal rescue factor. Previously, we have reported a female child carrying biallelic HBS1L mutations, manifesting growth restriction, developmental delay, and hypotonia. In this study, we describe her ophthalmologic findings, compare them with the Hbs1ltm1a/tm1a hypomorph mouse model, and evaluate the underlying microscopic and molecular perturbations. The patient was noted to have impaired visual function observed by electroretinogram (ERG), with dampened amplitudes of a- and b-waves in both rod- and cone-mediated responses. Hbs1ltm1a/tm1a mice exhibited profound retinal thinning of the entire retina, specifically of the outer retinal photoreceptor layer, detected using in vivo imaging of optical coherence tomography (OCT) and retinal cross sections. TUNEL assay revealed retinal degeneration due to extensive photoreceptor cell apoptosis. Loss of HBS1L resulted in comprehensive proteomic alterations in mass spectrometry analysis, with169 proteins increased and 480 proteins decreased including many critical IRD-related proteins. GO biological process and GSEA analyses reveal that these downregulated proteins are primarily involved in photoreceptor cell development, cilium assembly, phototransduction, and aerobic respiration. Furthermore, apart from the diminished level of PELO, a known partner protein, HBS1L depletion was accompanied by reduction in translation machinery associated 7 homolog (Tma7), and Endothelial differentiation-related factor 1(Edf1) proteins, the latter of which coordinates cellular responses to ribosome collisions. This novel connection between HBS1L and ribosome collision sensor (EDF1) further highlights the intricate mechanisms underpinning ribosomal rescue and quality control that are essential to maintain homeostasis of key proteins of retinal health, such as rhodopsin.

genetics↗

bioRxiv2023

Dual Ethanolamine Head Groups in Ionizable Lipids Facilitate Phospholipid-free Stable Nanoparticle Formulation for Augmented and Safer mRNA Delivery

The accelerated advent of mRNA-based therapeutics and vaccines, highlighted by the battle against SARS-CoV-2, underscores the urgency to refine lipid nanoparticles (LNPs) for efficient mRNA delivery. In this work, we introduce a novel series of ionizable lipids characterized by double ethanolamine head groups, significantly amplifying mRNA binding affinity. A succinct three-component formulation is subsequently delineated, obviating the conventional dependency on phospholipids inherent in traditional four-component LNPs. Intriguingly, this formulation enables particle formation under neutral pH conditions, a notable departure from the acidic milieu traditionally required, attributable to the enhanced nonionic interactions predominating in mRNA encapsulation. The resultant particles exhibit exceptional stability, superior mRNA encapsulation efficiency, and maintain robust delivery efficacy. When deployed as a vaccine platform, the formulation elicited pronounced humoral and T-cell immune responses, concurrently exhibiting a favorable toxicity profile with a reduced induction of pro-inflammatory cytokines such as IL-6. Our exploration suggests that by fine-tuning the non-electrostatic interactions between the ionizable lipid and mRNA, the dynamics of particle formation can be considerably divergent from the prevailing paradigms of mRNA-LNP formation, hinting at a broader horizon for lipid optimization within the realm of mRNA delivery systems.

bioengineering↗

bioRxiv2023

Missing view completion for multi-timepoints omics data via representation disentanglement and temporal knowledge transfer

Longitudinal multi-view omics data offer unique insights into the temporal dynamics of individual-level physiology, which provides opportunities to advance personalized healthcare. However, the common occurrence of incomplete views makes extrapolation tasks difficult, and there is a lack of tailored methods for this critical issue. Here, we introduce LEOPARD, an innovative approach specifically designed to complete missing views in multi-timepoint omics data. By disentangling longitudinal omics data into content and temporal representations, LEOPARD transfers the temporal knowledge to the omics-specific content, thereby completing missing views. The effectiveness of LEOPARD is validated on three benchmark datasets constructed with data from the MGH COVID study and the KORA cohort, spanning periods from 3 days to 14 years. Compared to conventional imputation methods, such as missForest, PMM, GLMM, and cGAN, LEOPARD yields the most robust results across the benchmark datasets. LEOPARD-imputed data also achieve the highest agreement with observed data in our analyses for age-associated metabolites detection, estimated glomerular filtration rate-associated proteins identification, and chronic kidney disease prediction. Our work takes the first step toward a generalized treatment of missing views in longitudinal omics data, enabling comprehensive exploration of temporal dynamics and providing valuable insights into personalized healthcare.

bioinformatics↗

bioRxiv2023

Theta mediated dynamics of human hippocampal-neocortical learning systems in memory formation and retrieval

Episodic memory arises as a function of dynamic interactions between the hippocampus and the neocortex, yet the mechanisms have remained elusive. Here, using human intracranial recordings during a mnemonic discrimination task, we report that 4-5 Hz (theta) power is differentially recruited during discrimination vs. overgeneralization, and its phase supports hippocampal-neocortical when memories are being formed and correctly retrieved. Interactions were largely bidirectional, with small but significant net directional biases; a hippocampus-to-neocortex bias during acquisition of new information that was subsequently correctly discriminated, and a neocortex-to-hippocampus bias during accurate discrimination of new stimuli from similar previously learned stimuli. The 4-5 Hz rhythm may facilitate the initial stages of information acquisition by neocortex during learning and the recall of stored information from cortex during retrieval. Future work should further probe these dynamics across different types of tasks and stimuli and computational models may need to be expanded accordingly to accommodate these findings.

neuroscience↗

bioRxiv2023

MAGIK: A rapid and efficient method to create lineage-specific reporters in human pluripotent stem cells

Precise insertion of a fluorescent protein into a lineage-specific gene in human pluripotent stem cells (hPSCs) presents challenges due to the low knockin efficiency and difficulties in selecting the correctly targeted cells. Here we introduce the ModRNA-based Activation for Gene Insertion and Knockin (MAGIK) approach to enhance knockin efficacy in hPSCs. MAGIK operates in two steps: first, it employs a Cas9-2A-p53DD modRNA with a mini-donor plasmid (without a drug-selection cassette) to significantly enhance efficiency; second, a dCas9 activator modRNA and a dgRNA are used to temporarily activate the successfully targeted gene, allowing for live cell sorting without single cell cloning. Consequently, MAGIK eliminates the need for drug selection cassettes or labor-intensive single cell colony screening, expediting precise genetic integration. We have demonstrated that MAGIK can be utilized to insert fluorescent proteins into various genes, including SOX17, NKX6.1, NKX2.5 and PDX1, across multiple hPSC lines, showcasing its robustness. This innovative MAGIK approach streamlines the process and provides a promising solution for targeted genetic modifications in hPSCs.

bioengineering↗

bioRxiv2023

Unraveling the Structure of Meclizine Dihydrochloride with MicroED

Meclizine (Antivert, Bonine) is a first-generation H1 antihistamine used in the treatment of motion sickness and vertigo. Despite its wide medical use for over 70 years, its crystal structure and the details of protein-drug interactions remained unknown. In this study, we used microcrystal electron diffraction (MicroED) to determine the three-dimensional (3D) crystal structure of meclizine dihydrochloride directly from a seemingly amorphous powder. Two racemic enantiomers (R/S) were found in the unit cell, which packed as repetitive double layers in the crystal lattice. The packing was made of multiple strong N-H{middle dot}{middle dot}{middle dot}Cl- hydrogen bonding interactions and weak interactions like C-H{middle dot}{middle dot}{middle dot}Cl- and pi-stacking. Molecular docking revealed the binding mechanism of meclizine to the histamine H1 receptor. A comparison of the docking complexes between histamine H1 receptor and meclizine or levocetirizine (a second-generation antihistamine) showed the conserved binding sites. This research illustrates the combined use of MicroED and molecular docking in unraveling protein-drug interactions for precision drug design and optimization.

biochemistry↗

bioRxiv2023

Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA-binding proteins with prion-like domains

RNA-binding proteins with prion-like domains, such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs (24-48 nucleotides) that prevent FUS fibrillization by promoting liquid phases, and distinct short RNAs that prevent and, remarkably, reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. Importantly, we define a short RNA that dissolves aberrant cytoplasmic FUS condensates, restores nuclear FUS, and mitigates FUS proteotoxicity in optogenetic models and human motor neurons. Another short RNA dissolves aberrant cytoplasmic TDP-43 condensates, restores nuclear TDP-43, and mitigates TDP-43 proteotoxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have therapeutic utility for ALS/FTD and related disorders.

biochemistry↗

bioRxiv2023

A defined diet for pre-adult Drosophila melanogaster

Drosophila melanogaster is unique among animal models because it has a fully defined synthetic diet available to study nutrient-gene interactions. However, use of this diet is limited to adult studies due to impaired larval development and survival. Here, we provide an adjusted formula that improves larval growth, development rate, and rescues survival. We demonstrate its use for exploring pre-adult diet compositions of therapeutic potential in a model of an inherited metabolic disorder.

genetics↗

bioRxiv2023

PQS and pyochelin in Pseudomonas aeruginosa share inner membrane transporters to mediate iron uptake

Bacteria uptake different forms of iron through various channels to meet their needs. Our previous studies have shown that TseF, a type VI secretion system effector for Fe uptake, facilitates the delivery of outer membrane vesicle (OMV)-associated PQS-Fe3+ to bacterial cells by involving the Fe(III) pyochelin receptor FptA and the porin OprF. However, the form in which the PQS-Fe3+ complex enters the periplasm and how it is taken up into the cytoplasm remain unclear. Here, we first demonstrate that the PQS-Fe3+ complex enters the cell directly through FptA or OprF. Next, we find that inner membrane transporters such as FptX, PchHI, and FepBCDG are not only necessary for P. aeruginosa to uptake PQS-Fe3+ and PCH-Fe3+, but also necessary for the virulence of P. aeruginosa toward Galleria mellonella larvae. Furthermore, we suggest that the function of PQS-Fe3+ (but not PQS)-mediated quorum-sensing regulation is dependent on FptX, PchHI, and FepBCDG. Additionally, the findings indicate that, unlike FptX, both FepBCDG and PchHI play no role in the autoregulatory loop involving PchR, but further deletion of fepBCDG and pchHI can reverse the inactive PchR phenotype caused by fptX deletion and reactivate the expression of the PCH pathway genes under iron-limited conditions. Finally, this work detected the interaction between FptX, PchHI, and FepBCDG, indicating that a larger complex could be formed to mediate uptake of PQS-Fe3+ and PCH-Fe3+. These results pave the way for a better understanding of the PQS and PCH iron uptake pathway, and provide future directions to tackle P. aeruginosa infections. IMPORTANCEIron is a key factor for P. aeruginosa to break through the hosts defense system and successfully infect. To acquire the necessary iron from the host, P. aeruginosa has evolved a number of strategies, the most common being the synthesis, secretion, and uptake of siderophores such as pyoverdine, pyochelin, and the quorum-sensing signaling molecule PQS. However, despite intensive studies of the siderophore uptake pathways of P. aeruginosa, our understanding of how siderophores transport iron across the inner membrane into the cytoplasm is still far from complete. Here, we reveal that PQS and pyochelin in P. aeruginosa share inner membrane transporters such as FptX, PchHI and FepBCDG to mediate iron uptake. Meanwhile, PQS and pyochelin-mediated signaling operates to a large extent via these inner membrane transporters. Our study revealed an interesting phenomenon of shared uptake pathways between PQS and pyochelin, which will lead us to reexamine the role of these two molecules in the iron uptake and virulence of P. aeruginosa.

microbiology↗

bioRxiv2023

Searching the mechanisms of mRNA buffering: the releasing-shuttling model

Many experiments showed that eukaryotic cells maintain a constant mRNA concentration upon various perturbations by actively regulating mRNA production and degradation rates, known as mRNA buffering. However, the underlying mechanism is still unknown. Here, we propose a mechanistic model of mRNA buffering: the releasing-shuttling (RS) model. The model incorporates two crucial factors, X and Y, which play key roles in the transcription, exportation, and degradation processes. The model explains the constant mRNA concentration under genome-wide genetic perturbations and cell volume changes. Moreover, it quantitatively explains the slowed-down mRNA degradation after Pol II depletion and the temporal transcription dynamics after Xrn1 depletion. The RS model suggests that X and Y are likely composed of multiple molecules possessing redundant functions. We also present a list of X and Y candidates, and an experimental method to identify X. Our work uncovers a possible coupling mechanism between transcription, exportation, and degradation.

systems biology↗

bioRxiv2023

Single-molecule analysis unveils the phosphorylation of FLS2 regulates its spatiotemporal dynamics and immunity

The Arabidopsis thaliana FLAGELLIN-SENSITIVE2 (FLS2), a typical receptor kinase, recognizes the conserved 22 amino acid sequence in the N-terminal region of flagellin (flg22) to initiate plant defense pathways, which was intensively studied in the past decades. However, the dynamic regulation of FLS2 phosphorylation at the plasma membrane after flg22 recognition needs further elucidation. Through single-particle tracking, we demonstrated that upon flg22 treatment, the phosphorylation of Ser-938 in FLS2 impacts its spatiotemporal dynamics and lifetime. Following forster resonance energy transfer-fluorescence lifetime (FRET-FLIM) imaging microscopy and protein proximity indexes (PPI) assays revealed that flg22 treatment increased the co-localization of GFP-tagged FLS2/FLS2S938D but not FLS2S938A with AtRem1.3-mCherry, a sterol-rich lipid marker, indicating that the phosphorylation of FLS2S938 affects FLS2 sorting efficiency to AtRem1.3-associated nanodomains. Importantly, we found that the phosphorylation of Ser-938 enhanced flg22-induced FLS2 internalization and immune responses, demonstrating that the phosphorylation may activate flg22-triggered immunity through partitioning FLS2 into functional AtRem1.3-associated nanodomains, which fills the gap between the FLS2S938 phosphorylation and FLS2-mediated immunity.

cell biology↗

bioRxiv2023

KDM6A facilitates Xist upregulation at the onset of X inactivation

X chromosome inactivation (XCI) is a female-specific process in which one X chromosome is silenced to balance X-linked gene expression between the sexes. XCI is initiated in early development by upregulation of the lncRNA Xist on the future inactive X (Xi). A subset of X-linked genes escape silencing and thus have higher expression in females, suggesting female-specific functions. One of these genes is the highly conserved gene Kdm6a, which encodes a histone demethylase that removes methyl groups at H3K27 to facilitate gene expression. Here, we investigate the role of KDM6A in the regulation of Xist. We observed impaired upregulation of Xist during early stages of differentiation in hybrid mouse ES cells following CRISPR/Cas9 knockout of Kdm6a. This is associated with reduced Xist RNA coating of the Xi, suggesting diminished XCI potency. Indeed, Kdm6a knockout results in aberrant overexpression of genes from the Xi after differentiation. KDM6A binds to the Xist promoter and knockout cells show an increase in H3K27me3 at Xist. These results indicate that KDM6A plays a role in the initiation of XCI through histone demethylase-dependent activation of Xist during early differentiation.

molecular biology↗

bioRxiv2023

UBAP2L drives scaffold assembly of nuclear pore complexes at the intact nuclear envelope

Assembly of macromolecular complexes at correct cellular sites is crucial for cell function. Nuclear pore complexes (NPCs) are large cylindrical assemblies with eightfold rotational symmetry, built through hierarchical binding of nucleoporins (Nups) forming distinct subcomplexes. Here, we uncover a direct role of ubiquitin-associated protein 2-like (UBAP2L) in the biogenesis of properly organized and functional NPCs at the intact nuclear envelope (NE) in human cells. UBAP2L localizes to the nuclear pores and drives the formation of the Y-complex, an essential scaffold component of the NPC, and its localization to the NE. UBAP2L facilitates the interaction of the Y-complex with POM121 and Nup153, the critical upstream factors in a well-defined sequential order of Nups assembly onto NE during interphase. Timely localization of the cytoplasmic Nup transport factor fragile X-related protein 1 (FXR1) to the NE and its interaction with the Y-complex are likewise dependent on UBAP2L. Thus, this NPC biogenesis mechanism integrates the cytoplasmic and the nuclear NPC assembly signals and ensures efficient nuclear transport, adaptation to nutrient stress and cellular proliferative capacity, highlighting the importance of NPC homeostasis at the intact nuclear envelope. TeaserLiao et al. show how UBAP2L drives the assembly of the scaffold elements into symmetrical and functional NPCs at the nuclear envelope in human cells.

cell biology↗