Biology subjects

Chen, Z.

Publications and source records attributed to Chen, Z..

At least 361 records · Page 20Linked to original sources

bioRxiv2024

METTL3 inhibition-activated cGAS/STING axis enhances immunotherapy and PARP inhibitor sensitivity of lung carcinoma

The cGAS/STING-mediated type I interferon response can augment antitumor activity, while the regulatory factors within this innate immune response remain elusive. Herein we found that the RNA m6A methyltransferase METTL3 was upregulated in lung carcinoma tissues. Elevated METTL3 level was correlated with diminished CD8+ T cell infiltration and cancer progression in lung carcinoma patients. METTL3 deficiency exacerbated nuclear DNA leakage into the cytoplasm, activating the cGAS pathway and thereby enhancing anti-tumor immunity. Mechanistically, METTL3 deficiency reduced the homologous recombination repair efficacy via downregulation of MSH5, a mutS family protein involved in DNA mismatch repair, leading to increased cytosolic DNA levels. m6A methylation of A2521 of MSH5 stabilized its mRNA via binding with IGF2BP2. On the other hand, m6A methylation of A1545 at the CDS of cGAS decreased mRNA stability and regulates its protein expression. Functionally, knockdown of METTL3 sensitized lung carcinoma cells to the PARP inhibitors. In vivo and clinical data confirmed the positive roles of METTL3 inhibition-activated cGAS/STING axis in tumor growth and lung adenocarcinoma progression. Collectively, METTL3 inhibition activates the cGAS/STING-mediated anti-tumor immunity via induction of cytosolic DNA and cGAS expression, which in turn regulate PARP inhibitor response and cancer progression in lung carcinoma.

cancer biology↗

bioRxiv2024

Complete Chloroplast Genome Sequence and Phylogenetic Analysis of Camellia sinensis sp. Baihaozao

Baihaozao (Camellia sinensis sp. Baihaozao) is esteemed in the domain of tea plant cultivation for its early harvest period, profusion of bud trichomes, and exceptional suitability for tea processing. Nevertheless, the intricate phylogenetic relationships among species within this genus remain ambiguous, primarily due to the limited availability of genomic data. This study endeavors to comprehensively investigate the genomic resources of Baihaozao by sequencing, assembling, and annotating its entire chloroplast (cp) genome. The sequencing results indicated that the total length of the Baihaozao cp genome is 157,052 base pairs (bp), with an overall guanine-cytosine (GC) content of 37.30%. The genome exhibits a typical quadripartite structure, consisting of a large single-copy region (LSC) of 86,586 bp, a small single-copy region (SSC) of 18,227 bp, and a pair of inverted repeats (IRs) totaling 52,162 bp. A total of 133 genes were identified within this genome, including 8 ribosomal RNA (rRNA) genes, 37 transfer RNA (tRNA) genes, 87 protein-coding genes, and 1 pseudogene. Furthermore, the study identified 157 simple sequence repeats (SSRs) and 90 long repeat sequences. Analysis of codon usage bias indicated that codons encoding leucine (Leu) were the most frequently utilized, whereas those encoding cysteine (Cys) were the least utilized. Examination of nucleotide diversity within the Baihaozao cp genome revealed five highly variable regions with elevated Pi values (rps19, rpl32, ndhF, rpl22, matK), suggesting their potential utility as molecular markers. Phylogenetic analysis of 20 cp genomes of the Theaceae family indicated a close evolutionary relationship between Baihaozao and Camellia sinensis OL450428.1. This study not only provides valuable data support for elucidating the maternal origin of cultivated Camellia species but also holds significant scientific implications for further exploring the phylogenetic relationships and germplasm resource utilization of Camellia plants.

genomics↗

bioRxiv2024

Microbial Weathering Analysis of Anshun Tunbao Artifacts

AbstractsCultural heritages are the crystallization of human technology, culture and embody the efforts of many craftsmen in ancient times. Wooden cultural heritages are affected by their own materials, and are very susceptible to microbial invasion under suitable temperature and humidity conditions. This project mainly studies the microbial weathering of the core wood carving masks and wooden ancient stage in Anshun Tunpu cultural heritage, and uses scanning electron microscopy, high-throughput sequencing and traditional culture methods to analyze the characteristics of their microbial communities, and finds that the bacteria represented by Pseudomonas, Paenibacillus and Stenotrophomonas, and the fungi represented by Cladosporium, Alternaria and Aspergillus, are the core microorganisms shared by indoor and outdoor cultural heritage. The dominant genera such as Pseudomonas, Stenotrophomonas, and Cladosporium showed lignocellulose deradation ability. By cultivating insect eggs extracted from sampled specimens and analyzing the correlation with surface microbiota, we verified the role of insect eggs as vectors in dispersing key microbial communities. We speculate that these insects are likely to be Anobiidae family. Furthermore, by comparing the microbial compositions under different climatic conditions, we validated the influence of temperature, humidity, vegetation diversity, and microbial intrinsic structures on microbial growth. Therefore, monitoring the surrounding environment is crucial in microbial weathering studies and cultural heritage preservation efforts. This study pioneered the investigation of microbial weathering aspects on unique heritage of the Tuenbao culture, emphasizing the importance of preserving distinctive ethnic cultures. Additionally, it presented a case study on the microbial weathering of wooden artifacts in karst landscape environments.

microbiology↗

bioRxiv2024

Evolution of the Spatial transcriptomic landscape during the progression of high-grade pancreatic intraductal papillary mucinous neoplasms to invasive cancer

Intraductal papillary mucinous neoplasms (IPMN) is one of the known precancerous lesions. Patients prognosis is aggravated as IPMN transforms into invasive Pancreatic Ductal Adenocarcinoma (PDAC). The molecular mechanisms underlying this progression lack effective experimental models and urgently need to be elaborated. We performed spatial transcriptomics (ST) on fresh tissue samples from the same patient including normal pancreas, high-grade IPMN, and invasive PDAC, and described the step-by-step development of transcriptional landscape including clone evolution and adjacent TME feature variation. Our findings identified the master transcript factors and critical signaling pathways promoting IPMN progression to invasive PDAC. Additionally, both IPMN and PDAC harbored the ELF3, MYC, and KLF4 amplification. The Spatial CNV profile demonstrated significant heterogeneity among PDAC in their spatial distribution compared to IPMN, with seven distinct subclones showing diverse functions, such as hypoxia, oxidative phosphorylation, and epithelial-mesenchymal Transition. We observed a marked shift in the immune landscape, with the depletion of CD4+ T-cells and dendritic cells and an increase in immune-suppressive M2 macrophages in invasive PDAC, indicating a transition to an immune-evasive microenvironment. Additionally, cancer-associated fibroblasts (CAFs), particularly myofibroblastic CAFs, were enriched adjacent to invasive PDAC, suggesting their active role in tumor progression. By leveraging spatial transcriptomic analysis, our study provides comprehensive insights into the intricate molecular landscape that underlies the progression of IPMNs to invasive PDAC. These findings not only enhance our understanding of this complex process but also offer valuable knowledge for early diagnosis and intervention. HighlightsO_LISpatial CNV analysis reveals clonal evolution and distinct subclones in PDAC. C_LIO_LIKey drivers like ELF3, MYC, and KLF4 are amplified in both IPMN and invasive PDAC. C_LIO_LIImmune landscape shifts from pro-inflammatory in IPMN to immune-evasive in PDAC. C_LIO_LIEnrichment of myofibroblastic CAFs suggests their role in tumor progression C_LI

cancer biology↗

bioRxiv2024

Steering cell-state and phenotype transitions by causal disentanglement learning

Understanding and manipulating cell-state and phenotype transitions is essential for advancing biological research and therapeutic treatment. We introduce CauFinder, an advanced framework designed to accurately identify causal regulators of these transitions and further precisely steer such transitions by integrating causal disentanglement modelling with network control based solely on observed data. By leveraging do-calculus and optimizing information flow metrics, CauFinder can distinguish causal factors from spurious ones, ensuring precise control over desired state transitions. One significant advantage of CauFinder is its ability to identify those variables causally affecting the cell-state/phenotype transitions among all observed variables, both theoretically and computationally, leading to the identification of their master regulators when combined with network control. Consequently, by employing a counterfactual algorithm, CauFinder is able effectively to facilitate desirable state transitions or steer these transitional trajectories/paths by modulating these causal drivers. Beyond its theoretical advantages, CauFinder outperforms existing approaches computationally in both simulated and real-world settings. CauFinder is able to not only reveal natural biological transition processes such as (a) cell differentiation, (b) lung adenocarcinoma (LUAD) to lung squamous cell carcinoma (LUSC) transdifferentiation and (c) drug-sensitive to drug-resistant transitions but also identify the causal regulators of their reverse transition processes, such as (A) cell dedifferentiation, (B) LUSC to LUAD transdifferentiation and (C) drug-resistant to drug-sensitive transitions. These findings highlight its superior ability to causally uncover essential regulatory mechanisms and accurately steer cell-state/phenotype transitions, thus providing novel therapeutic strategies.

bioinformatics↗

bioRxiv2024

Single-Molecule Barcoding Technology for Single-Cell Genomics

Recent advances in barcoding technologies have significantly enhanced the scalability of single-cell genomic experiments. However, large-scale experiments are still rare due to high costs, complex logistics, and laborintensive procedures. To facilitate the routine application of the largest scalability, it is critical to simplify the production and use of barcoding reagents. Here, we introduce AmpliDrop, a technology that initiates the barcoding process using a pool of inexpensive single-copy barcodes and integrates barcode multiplicity generation with tagging of cellular content into a single reaction driven by DNA polymerase during library preparation. The barcoding reactions are compartmentalized using an electronic pipette or a robotic or standalone liquid handling system. These innovations eliminate the need for barcoded beads and complex combinatorial indexing workflows and provide flexibility for a wide range of scales and tube formats, as well as compatibility with automation. We show that AmpliDrop is capable of capturing transcriptomes and chromatin accessibility, and it can also be adapted for user-customized applications, including antibody-based protein detection, bacterial or viral DNA detection, and CRISPR perturbations without dual guide RNA-expression vectors. We validated AmpliDrop by investigating the influence of short-term static culturing on cell composition in human forebrain organoids, revealing metabolic reprogramming in lineage progenitors.

molecular biology↗

bioRxiv2024

Therapeutic targeting of Syndecan-1 axis overcomes acquired resistance to KRAS-targeted therapy in gastrointestinal cancers

The therapeutic benefit of recently developed mutant KRAS (mKRAS) inhibitors has been limited by the rapid onset of resistance. Here, we aimed to delineate the mechanisms underlying acquired resistance to mKRAS inhibition and identify actionable targets for overcoming this clinical challenge. Previously, we identified Syndecan-1 (SDC1) as a key effector for pancreatic cancer progression whose surface expression is driven by mKRAS. By leveraging both pancreatic and colorectal cancer models, we found that surface SDC1 expression was initially diminished upon mKRAS inhibition, but recovered in tumor cells that bypass mKRAS dependency. Functional studies showed that these tumors depended on SDC1 for survival, further establishing SDC1 as a driver for the acquired resistance to mKRAS inhibition. Mechanistically, we revealed that the YAP1-SDC1 axis was the major driving force for bypassing mKRAS dependency to sustain nutrient salvage machinery and tumor maintenance. Specifically, YAP1 activation mediated the recovery of SDC1 localization on cell surface that sustained macropinocytosis and enhanced the activation of multiple RTKs, promoting resistance to KRAS-targeted therapy. Overall, our study has provided the rationale for targeting the YAP-SDC1 axis to overcome resistance to mKRAS inhibition, thereby revealing new therapeutic opportunities for improving the clinical outcome of patients with KRAS-mutated cancers.

cancer biology↗

bioRxiv2024

Salidroside Mitigates Malignant Arrhythmias by Restoring Sodium Channel Function During Ultra-Acute Myocardial Infarction

BackgroundThe ultra-acute phase (Phase 1a) of acute myocardial infarction (AMI) is marked by a high incidence of malignant arrhythmias, often occurring during the prehospital period. Currently, there are no effective treatment options available for managing these arrhythmias at this early stage. Methods and ResultsUsing dual-channel optical mapping, we simultaneously recorded membrane potentials and calcium transients during acute myocardial infarction. Calcium transient duration maps accurately localized the infarcted region, and action potential activation time maps revealed conduction heterogeneity in the infarcted zone. Patch-clamp recordings showed that Salidroside (Sal) (1 {micro}g/mL) significantly increased sodium current density from -59.27 {+/-} 2.15 pA/pF to -83.46 {+/-} 3.19 pA/pF (P<0.01) and shifted the Nav1.5 activation curve leftward (V1/2 from -37.27 {+/-} 0.5 mV to -44.55 {+/-} 0.7 mV, P<0.01). In rat and rabbit AMI models, Sal pre-treatment reduced conduction heterogeneity and arrhythmia incidence compared to controls. Optical mapping showed improved conduction velocity and uniformity in the Sal group. ConclusionsSal restores electrophysiological function in damaged myocardium by modulating sodium currents, reducing conduction heterogeneity, and decreasing malignant arrhythmia incidence during the ultra-acute phase of AMI. These findings suggest a novel therapeutic strategy for AMI, addressing a critical unmet need in antiarrhythmic therapy. What is New?O_LIThis study identifies Salidroside (Sal) as a novel agent that enhances sodium channel currents (Nav1.5), distinguishing it from traditional antiarrhythmic drugs which primarily target potassium channels or {beta}-adrenergic receptors. Sal improves conduction uniformity in the infarcted myocardium by restoring the electrophysiological function of damaged cardiac cells, eliminating slow conduction pathways, and reducing conduction heterogeneity. C_LIO_LIThis research introduces Sal as a promising candidate for preventing and treating arrhythmias during the critical early stages of MI, potentially improving patient outcomes. Sal administration during the ultra-acute phase (phase 1a) of myocardial infarction (MI) significantly reduces the incidence of malignant arrhythmias, a critical period characterized by high extracellular potassium and increased arrhythmia risk. C_LIO_LIUtilizing calcium transient imaging and optical mapping, this study provides precise localization of ischemic regions and detailed electrophysiological characterization, offering a robust methodology for assessing therapeutic efficacy. C_LI

pharmacology and toxicology↗

bioRxiv2024

Functional gene embeddings improve rare variant polygenic risk scores

Rare variant association testing is a powerful strategy for identifying effector genes underlying common traits. However, its effectiveness is limited by the scarcity of high-impact rare allele carriers, posing challenges for sensitivity and robustness. Here, we introduce FuncRVP, a rare variant association framework addressing this issue by leveraging functional information across genes. FuncRVP models the effects of rare variants as a weighted sum of gene impairment scores, with weights regularized through a prior based on functional gene embeddings. Modeling 41 quantitative traits from unrelated UK Biobank participants showed that FuncRVP consistently outperformed linear regressions on significantly associated genes and did so more effectively for traits with higher burden heritability. The framework demonstrated versatility, yielding consistent improvements across diverse gene embeddings. Moreover, FuncRVP generated more robust gene effect estimates and yielded more gene discoveries, especially among genetically constrained genes. These findings demonstrate the value of integrating functional information in rare variant association studies and showcase FuncRVP as a promising tool for enhancing phenotype prediction and gene discovery.

genetics↗

bioRxiv2024

Benchmarking algorithms for spatially variable gene identification in spatial transcriptomics

The rapid development of spatial transcriptomics has underscored the importance of identifying spatially variable genes. As a fundamental task in spatial transcriptomic data analysis, spatially variable gene identification has been extensively studied. However, the lack of comprehensive benchmark makes it difficult to validate the effectiveness of various algorithms scattered across a large number of studies with real-world datasets. In response, this article proposes a benchmark framework to evaluate algorithms for identifying spatially variable genes through the analysis of synthesized and real-world datasets, aiming to identify the best algorithms and their corresponding application scenarios. This framework can assist medical and life scientists in selecting suitable algorithms for their research, while also aid bioinformatics scientists in developing more powerful and efficient computational methods in spatial transcriptomic research.

bioinformatics↗

bioRxiv2024

Adult-neurogenesis allows for representational stability and flexibility in early olfactory system

In the olfactory system, adult-neurogenesis results in the continuous reorganization of synaptic connections and network architecture throughout the animals life. This poses a critical challenge: How does the olfactory system maintain stable representations of odors amidst this ongoing circuit instability? Utilizing a detailed spiking network model of early olfactory circuits, we uncovered dual roles for adult-neurogenesis: one that both supports representational stability to faithfully encode odor information and also one that facilitates plasticity to allow for learning and adaptation. In the main olfactory bulb, adult-neurogenesis affects neural codes in individual mitral and tufted cells but preserves odor representations at the neuronal population level. By contrast, in the olfactory piriform cortex, both individual cell responses and overall population dynamics undergo progressive changes due to adult-neurogenesis. This leads to representational drift, a gradual alteration in stimulus-evoked activity patterns. Both processes are dynamic and depend on experience such that repeated exposure to specific odors reduces the drift due to adult-neurogenesis; thus, when the odor environment is stable over the course of adult-neurogenesis, it is spike-timing-dependent plasticity that leads representations to remain stable in piriform cortex; when those olfactory environments change, adult-neurogenesis allows cortical representations to track environmental change. Whereas perceptual stability and plasticity due to learning are often thought of as two distinct, often contradictory processes in neuronal coding, we find that adult-neurogenesis serves as a shared mechanism for both. In this regard, the quixotic presence of adult-neurogenesis in the mammalian olfactory bulb that has been the focus of considerable investigation in chemosensory neuroscience may be the mechanistic underpinning behind an array of complex computations.

neuroscience↗

bioRxiv2024

Reversible ubiquitination of integrated domain controls paired NLR immune receptor complex homeostasis

AbstractPlant intracellular NLR immune receptors can function individually or in pairs to detect pathogen effectors and activate immune responses. NLR homeostasis has to be tightly regulated to ensure proper defense without triggering autoimmunity. However, in contrast to singleton NLRs, the mechanisms controlling the paired NLRs complex homeostasis are less understood. The paired Arabidopsis RRS1/RPS4 immune receptor complex confers disease resistance through effector recognition mediated by the integrated WRKY domain of RRS1. Here, through proximity labelling, we reveal a ubiquitination-deubiquitination cycle that controls the homeostasis of the RRS1/RPS4 complex. E3 ligase RARE directly binds and ubiquitinates RRS1s WRKY domain to promote its proteasomal degradation, thereby destabilizing RPS4 indirectly and compromising the stability and function of the RRS1/RPS4 complex. Conversely, the deubiquitinating enzymes UBP12/UBP13 deubiquitinate RRS1s WRKY domain, counteracting RAREs effects. Interestingly, the abundance of WRKY transcription factors WRKY70 and WRKY41 is also regulated by RARE and UBP12/UBP13. Phylogenetic analysis suggests this regulation likely transferred from WRKY70/WRKY41 to RRS1 upon WRKY domain integration. Our findings improve our understanding of homeostatic regulation of paired NLR complex and uncover a new paradigm whereby domain integration can co-opt preexisting post-translational modification to regulate novel protein functions.

plant biology↗

bioRxiv2024

A Drug-Elicitable Alternative-Splicing Module (DreAM) for Tunable AAV Expression and Controlled Myocardial Regeneration

Adeno-associated virus (AAV) is a major vector for gene therapy. A technique to fine-tune the time and level of AAV expression is lacking, which greatly restricts indication choice, therapeutic efficacy and safety. To solve this problem, here we developed a drug-elicitable alternative-splicing module (DreAM) responsive to risdiplam, an FDA-approved alternative splicing modulator. Risdiplam activates DreAM-regulated AAV expression in a dose-dependent manner with an over 2000-fold inducible change depending on the inducer dosage and the organ of interests. With a temporally resolution of a couple of days, DreAM could repeatedly activate AAV expression, determined by the frequency and duration of risdiplam administration. As an example of DreAM-realized gene therapy, this technique was used to control AAV-based cardiac delivery of YAP5SA, a potent cardiomyocyte regeneration factor. DreAM transiently activated AAV-YAP5SA, establishing the dedifferentiation-proliferation-redifferentiation cycle in cardiomyocytes. Consequently, DreAM fulfilled the regenerative capacity of AAV-YAP5SA in myocardial infarction while circumventing toxicity associated with prolonged AAV-YAP5SA expression. Together, these data demonstrated a tremendous potential of DreAM to enhance the efficacy, safety and applicable scope of gene therapy.

bioengineering↗

bioRxiv2024

Single-cell analysis of human diversity in circulating immune cells

Lack of diversity and proportionate representation in genomics datasets and databases contributes to inequity in healthcare outcomes globally1,2. The relationships of human diversity with biological and biomedical phenotypes are pervasive3, yet remain understudied, particularly in a single-cell genomics context. Here we present the Asian Immune Diversity Atlas (AIDA), a multi-national single-cell RNA-sequencing (scRNA-seq) healthy reference atlas of human immune cells. AIDA comprises 1,265,624 circulating immune cells from 619 healthy donors and 6 controls, spanning 7 population groups across 5 countries. AIDA is one of the largest healthy blood datasets in terms of number of cells, and also the most diverse in terms of number of population groups. Though population groups are frequently compared at the continental level, we identified a pervasive impact of sub-continental diversity on cellular and molecular properties of immune cells. These included cell populations and genes implicated in disease risk and pathogenesis as well as those relevant for diagnostics. We detected single-cell signatures of human diversity not apparent at the level of cell types, as well as modulation of the effects of age and sex by self-reported ethnicity. We discovered functional genetic variants influencing cell type-specific gene expression, including context-dependent effects, which were under-represented in analyses of non-Asian population groups, and which helped contextualise disease-associated variants. We validated our findings using multiple independent datasets and cohorts. AIDA provides fundamental insights into the relationships of human diversity with immune cell phenotypes, enables analyses of multi-ancestry disease datasets, and facilitates the development of precision medicine efforts in Asia and beyond.

genomics↗

bioRxiv2024

High efficient chromatin conformation capture without pre-enrichment (HiChew) in single cells

This study presents HiChew, a cutting-edge technique for high-efficiency chromatin conformation capture in single cells, without the need for pre-enrichment. This unique approach minimizes the risk of cell or DNA loss. When compared to Dip-C, HiChew captures valid pairs with 4-8 times more efficiency, reducing wastage and saving significant sequencing budget. Furthermore, HiChew delivers a lower false positive ratio, ensuring data accuracy. It also achieves more contacts per cell, enhancing resolution in single cell. HiChews superior performance not only enhances single-cell Hi-C but also streamlines conventional Hi-C, making it more robust than conventional HiC methods. This study also unveils a fascinating mechanism of gene activation in the B compartment of chromatin, providing insight into the elusive aspect of gene expression within this region.

genomics↗

bioRxiv2024

Exploring DNA movement through the application of droplet based high efficient chromatin conformation capture (DropHiChew) and loop velocity

This study presents a novel approach to understanding DNA movement dynamics through the development of a droplet-based, high-efficient chromatin conformation capture method, known as DropHiChew, and a new algorithm, loop velocity. DropHiChew, a user-friendly and cost-effective technique, employs the 10X single-cell systems allowing for easy experimental implementation. The loop velocity algorithm, on the other hand, enables the estimation of the speed and direction of cell development, providing a dynamic perspective on chromatin movement. Even with shallow sequencing, our loop velocity algorithm accurately gauges the trajectory of DNA motion and cellular states. The combined use of DropHiChew and loop velocity offers potential for a wide array of applications in future chromatin capture studies, including disease modeling, cellular differentiation studies, and developmental biology.

genomics↗

bioRxiv2024

BEACON: Benchmark for Comprehensive RNA Tasks and Language Models

RNA plays a pivotal role in translating genetic instructions into functional outcomes, underscoring its importance in biological processes and disease mechanisms. Despite the emergence of numerous deep learning approaches for RNA, particularly universal RNA language models, there remains a significant lack of standardized benchmarks to assess the effectiveness of these methods. In this study, we introduce the first comprehensive RNA benchmark BEACON (BEnchmArk for COmprehensive RNA Task and Language Models). First, BEACON comprises 13 distinct tasks derived from extensive previous work covering structural analysis, functional studies, and engineering applications, enabling a comprehensive assessment of the performance of methods on various RNA understanding tasks. Second, we examine a range of models, including traditional approaches like CNNs, as well as advanced RNA foundation models based on language models, offering valuable insights into the task-specific performances of these models. Third, we investigate the vital RNA language model components from the tokenizer and positional encoding aspects. Notably, our findings emphasize the superiority of single nucleotide tokenization and the effectiveness of Attention with Linear Biases (ALiBi) over traditional positional encoding methods. Based on these insights, a simple yet strong baseline called BEACON-B is proposed, which can achieve outstanding performance with limited data and computational resources. The datasets and source code of our benchmark are available at https://github.com/terry-r123/RNABenchmark.

bioinformatics↗

bioRxiv2024

CCDC32 stabilizes clathrin-coated pits and drives their invagination

Clathrin-mediated endocytosis (CME) is essential for maintaining cellular homeostasis. Previous studies have reported more than 50 CME accessory proteins; however, the mechanism driving the invagination of clathrin-coated pits (CCPs) remains elusive. We show by quantitative live cell imaging that siRNA-mediated knockdown of CCDC32, a poorly characterized endocytic accessory protein, leads to the accumulation of unstable flat clathrin assemblies. CCDC32 interacts with the -appendage domain (AD) of AP2 in vitro and with full length AP2 complexes in cells. Deletion of aa78-98 in CCDC32, corresponding to a predicted -helix, abrogates AP2 binding and CCDC32s early function in CME. Furthermore, clinically observed nonsense mutations in CCDC32, which result in C-terminal truncations that lack aa78-98, are linked to the development of cardio-facio-neuro-developmental syndrome (CFNDS). Overall, our data demonstrate the function of a novel endocytic accessory protein, CCDC32, in regulating CCP stabilization and invagination, critical early stages of CME. SummaryWe show that CCDC32, a poorly studied and functionally ambiguous protein, binds to AP2 and regulates CCP stabilization and invagination. Clinically observed mutations in CCDC32 lose their ability to interact with AP2 likely contributing to the development of cardio-facio-neuro-developmental syndrome.

cell biology↗