Biology subjects

Huang, J.

Publications and source records attributed to Huang, J..

At least 361 records · Page 20Linked to original sources

bioRxiv2023

Characterization of mitotic phenotypes associated with a MYC synthetic lethal compound

Therapeutic targeting of MYC directly has proven difficult, but several means to target MYC indirectly using a synthetic lethal drug approach have been proposed. Synthetic lethal approaches for MYC have sought to take advantage of vulnerabilities MYC imposes related to either metabolic reprogramming, apoptotic signaling or the cycling of cancer cells. Here, we describe in detail the cell division phenotypes induced by a MYC synthetic lethal compound, dimethylfasudil (diMF). DiMF is a known ROCK inhibitor, but structurally related ROCK inhibitors are not synthetic lethal with MYC, so the activity of diMF is not related to blockade of this family of kinases. Instead, this compound induced multiple cell cycle-related liabilities. These included the early mitotic arrest of cycling cells followed by mitotic catastrophe-induced death and the induction of polyploidy in cells that do manage to pass through mitosis. As early as prometaphase, we noted diminished staining for BUB1 kinase, which binds to kinetochores and regulates the mitotic spindle checkpoint and chromosome congression. Kinetochore proteins, such as CENP-F, failed to localize at the metaphase plate, confirming a deficit in centromere assembly. This, presumably, contributed to the development of segregation anomalies in diMF-treated cells. In anaphase cells, the protein regulator of cytokinesis 1 (PRC1), failed to be recruited to the midzone, leading to a cascade of defects that included failed recruitment of the chromosomal passenger protein complex, the centralspindlin complex and polo-like-kinase 1 (PLK1). These observations correlate well with the cell death phenotypes induced by diMF, which may serve as a prototype MYC synthetic lethal compound to explore synthetic lethal therapy or as a scaffold upon which to build superior compounds. The phenotypes described here serve as examples of MYC synthetic lethal drug effects that can be used to explore and maximize drug discovery programs.

cancer biology↗

bioRxiv2023

Alphafold-multimer predicts cross-kingdom interactions at the plant-pathogen interface

Adapted plant pathogens from various microbial kingdoms produce hundreds of unrelated small secreted proteins (SSPs) with elusive roles. Some of these SSPs might be inhibitors targeting the most harmful hydrolases secreted by the host. Here, we used Alphafold-Multimer (AFM) to screen 1,879 SSPs of seven tomato pathogens for interacting with six defence-related hydrolases of tomato that accumulate to high levels in the apoplast during infection. This screen of 11,274 protein pairs identified 15 SSPs that are predicted to obstruct the active site of chitinases and proteases with an intrinsic fold. Four SSPs were experimentally verified to be inhibitors of pathogenesis-related subtilase P69B, including extracellular protein-36 (Ecp36) and secreted-into-xylem-15 (Six15) of the fungal tomato pathogens Cladosporium fulvum and Fusarium oxysporum, respectively. Together with a novel P69B inhibitor from the bacterial pathogen Xanthomonas perforans and the previously reported Kazal-like inhibitors of the oomycete pathogen Phytophthora infestans, P69B emerges as an important effector hub targeted by different microbial kingdoms, consistent with the presence of a hyper-variant residue in P69B orthologs and gene duplication and diversification of P69B paralogs that could avoid inhibitor binding. This study demonstrates the power of artificial intelligence to accurately predict novel cross-kingdom interactions at the plant-pathogen interface.

plant biology↗

bioRxiv2023

Molecular Mechanisms of Na+-driven Bile Acid Transport in Human NTCP

Human Na+ taurocholate co-transporting protein (hNTCP) is a key bile salt transporter to maintain enterohepatic circulation and is responsible for the recognition of hepatitis B and D viruses (HBV/HDV). Despite recent cryo-EM studies revealing open-pore and inward-facing states of NTCP stabilized by antibodies, the transport mechanism remains largely unknown. To address this knowledge gap, we used molecular dynamics (MD) and enhanced sampling Metadynamics simulations to elucidate the intrinsic mechanism of hNTCP-mediated taurocholate acid (TCA) transport driven by Na+-binding. We uncovered three TCA binding modes, including one that closely matched the limited cryo-EM density observed in the open-pore hNTCP. We also captured several key hNTCP conformations in the substrate transport cycle, particularly including an outward-facing, substrate-bound state. Furthermore, we provided thermodynamic evidence supporting that changes in the Na+-binding state drive the TCA transport by exploiting the amphiphilic nature of the substrate and modulating the protein environment, thereby enabling the TCA molecule to flip through. Understanding these mechanistic details of Na+-driven bile acid transport may aid in the development of hNTCP-targeted therapies for liver diseases.

biophysics↗

bioRxiv2023

The immune receptor SNC1 monitors helper NLRs targeted by a bacterial effector

Plants deploy intracellular receptors to counteract pathogen effectors that suppress cell-surface receptor-mediated immunity. To what extent pathogens manipulate also immunity mediated by intracellular receptors, and how plants tackle such manipulation, remains unknown. Arabidopsis thaliana encodes three very similar ADR1 class helper NLRs (ADR1, ADR1-L1 and ADR1-L2), which play key roles in plant immunity initiated by intracellular receptors. Here, we report that Pseudomonas syringae AvrPtoB, an effector with E3 ligase activity, can suppress ADR1-L1- and ADR1-L2-mediated cell death. ADR1, however, evades such suppression by diversification of two ubiquitination sites targeted by AvrPtoB. The intracellular sensor NLR SNC1 interacts with and guards the CCR domains of ADR1-L1 and ADR-L2. Removal of ADR1-L1 and ADR1-L2 or delivery of AvrPtoB activates SNC1, which then signals through ADR1 to trigger immunity. Our work not only uncovers the long sought-after physiological function of SNC1 in pathogen defense, but also that reveals how plants can use dual strategies, sequence diversification and a multiple layered guard-guardee system, to counteract pathogen attack on core immunity functions.

plant biology↗

bioRxiv2023

mRNA challenge predicts brain cancer immunogenicity and response to checkpoint inhibitors

To prospectively determine whether brain tumors will respond to immune checkpoint inhibitors (ICIs), we developed a novel mRNA vaccine as a viral mimic to elucidate cytokine release from brain cancer cells in vitro. Our results indicate that cytokine signatures following mRNA challenge differ substantially from ICI responsive versus non-responsive murine tumors. These findings allow for creation of a diagnostic assay to quickly assess brain tumor immunogenicity, allowing for informed treatment with ICI or lack thereof in poorly immunogenic settings.

cancer biology↗

bioRxiv2023

A general computational design strategy for stabilizing viral class I fusion proteins

Many pathogenic viruses, including influenza virus, Ebola virus, coronaviruses, and Pneumoviruses, rely on class I fusion proteins to fuse viral and cellular membranes. To drive the fusion process, class I fusion proteins undergo an irreversible conformational change from a metastable prefusion state to an energetically more favorable and stable postfusion state. An increasing amount of evidence exists highlighting that antibodies targeting the prefusion conformation are the most potent. However, many mutations have to be evaluated before identifying prefusion-stabilizing substitutions. We therefore established a computational design protocol that stabilizes the prefusion state while destabilizing the postfusion conformation. As a proof of concept, we applied this principle to the fusion protein of the RSV, hMPV, and SARS-CoV-2 viruses. For each protein, we tested less than a handful of designs to identify stable versions. Solved structures of designed proteins from the three different viruses evidenced the atomic accuracy of our approach. Furthermore, the immunological response of the RSV F design compared to a current clinical candidate in a mouse model. While the parallel design of two conformations allows identifying and selectively modifying energetically less optimized positions for one conformation, our protocol also reveals diverse molecular strategies for stabilization. We recaptured many approaches previously introduced manually for the stabilization of viral surface proteins, such as cavity-filling, optimization of polar interactions, as well as postfusion-disruptive strategies. Using our approach, it is possible to focus on the most impacting mutations and potentially preserve the immunogen as closely as possible to its native version. The latter is important as sequence re-design can cause perturbations to B and T cell epitopes. Given the clinical significance of viruses using class I fusion proteins, our algorithm can substantially contribute to vaccine development by reducing the time and resources needed to optimize these immunogens.

microbiology↗

bioRxiv2023

Epithelial galectin-3 induces mitochondrial complex inhibition and cell cycle arrest of CD8+ T Cells in severe/critical ill COVID-19

Several studies have identified the presence of functionally depleted CD8+ T cells in COVID-19 patients, and particularly abnormally reduced CD8+ T cells in severe/critical patients, which may be a major cause of disease progression and poor prognosis. In this study, a proliferating-depleted CD8+ T cell phenotype was observed in severe/critical COVID-19 patients through scRNA-seq and scTCR-seq analysis. These CD8+ T cells were subsequently found to be characterized by cell cycle arrest and downregulation of mitochondrial biogenesis and respiratory chain complex genes. Cellchat analysis revealed that the Galectin signaling pathways between infected lung epithelial cells and CD8+ T cells play the key role in inducing CD8+ T cell reduction and dysfunction in severe/critical COVID-19. We used SARS-COV-2 ORF3a to transfect A549 epithelial cells, and co-cultured with CD8+ T cells. The ex vivo experiments confirmed that galectin-3 inhibited the transcription of mitochondrial respiratory chain complex III/IV genes in CD8+ T cells by suppressing the nuclear translocation of nuclear respiratory factor 1 (NRF1). In addition, the regulatory effect of galectin-3 was correlated with the activation of ERK signaling and/or the inhibition of Akt signaling. Galectin-3 inhibitor, TD-139, promoted nuclear translocation of NRF1, and enhanced mitochondrial respiratory chain complex III/IV gene expression and mitochondrial biogenesis, then restore the expansion ability of CD8+ T cells. Our study improved the understanding the immunopathogenesis and provided new target for the prevention and treatment of severe/critical COVID-19.

immunology↗

bioRxiv2023

Transcriptional characterization of iPSC-derived microglia as a model for therapeutic development in neurodegeneration

BackgroundMicroglia are the resident immune cells in the brain that play a key role in driving neuroinflammation, a hallmark of neurodegenerative disorders. Inducible microglia-like cells have been developed as an in vitro platform for molecular and therapeutic hypothesis generation and testing. However, there has been no systematic assessment of similarity of these cells to primary human microglia along with their responsiveness to external cues expected of primary cells in the brain. MethodsIn this study, we performed transcriptional characterization of commercially available human inducible pluripotent stem cell (iPSC)-derived microglia-like (iMGL) cells by bulk and single cell RNA sequencing to assess their similarity with primary human microglia. To evaluate their stimulation responsiveness, iMGL cells were treated with Liver X Receptor (LXR) pathway agonists and their transcriptional responses characterized by bulk and single cell RNA sequencing. ResultsBulk transcriptome analyses demonstrate that iMGL cells have a similar overall expression profile to freshly isolated human primary microglia and express many key microglial transcription factors and functional and disease-associated genes. Notably, at the single-cell level, iMGL cells exhibit distinct transcriptional subpopulations, representing both homeostatic and activated states present in normal and diseased primary microglia. Treatment of iMGL cells with LXR pathway agonists induces robust transcriptional changes in lipid metabolism and cell cycle at the bulk level. At the single cell level, we observe heterogeneity in responses between cell subpopulations in homeostatic and activated states and deconvolute bulk expression changes into their corresponding single cell states. ConclusionsIn summary, our results demonstrate that iMGL cells exhibit a complex transcriptional profile and responsiveness, reminiscent of in vivo microglia, and thus represent a promising model system for therapeutic development in neurodegeneration.

genomics↗

bioRxiv2023

Inhibition mechanism and antiviral activity of an α-ketoamide based SARS-CoV-2 main protease inhibitor

SARS-CoV-2 has demonstrated extraordinary ability to evade antibody immunity by antigenic drift. Small molecule drugs may provide effective therapy while being part of a solution to circumvent SARS-CoV-2 immune escape. In this study we report an -ketoamide based peptidomimetic inhibitor of SARS-CoV-2 main protease (Mpro), RAY1216. Enzyme inhibition kinetic analysis established that RAY1216 is a slow-tight inhibitor with a Ki of 8.6 nM; RAY1216 has a drug-target residence time of 104 min compared to 9 min of PF-07321332 (nirmatrelvir), the antiviral component in Paxlovid, suggesting that RAY1216 is approximately 12 times slower to dissociate from the protease-inhibitor complex compared to PF-07321332. Crystal structure of SARS-CoV-2 Mpro:RAY1216 complex demonstrates that RAY1216 is covalently attached to the catalytic Cys145 through the -ketoamide warhead; more extensive interactions are identified between bound RAY1216 and Mpro active site compared to PF-07321332, consistent with a more stable acyl-enzyme inhibition complex for RAY1216. In cell culture and human ACE2 transgenic mouse models, RAY1216 demonstrates comparable antiviral activities towards different SARS-CoV-2 virus variants compared to PF-07321332. Improvement in pharmacokinetics has been observed for RAY1216 over PF-07321332 in various animal models, which may allow RAY1216 to be used without ritonavir. RAY1216 is currently undergoing phase III clinical trials (https://clinicaltrials.gov/ct2/show/NCT05620160) to test real-world therapeutic efficacy against COVID-19.

biochemistry↗

bioRxiv2023

Integrated single-cell analysis defines the epigenetic basis of castration-resistant prostate luminal cells

Understanding prostate response to castration and androgen receptor signaling inhibitors (ARSI) is critical to improving long-term prostate cancer (PCa) patient survival. Here we use a multi-omics approach on 229,794 single cells to create a mouse single-cell reference atlas better suited to interpreting mouse prostate biology and castration response. Our reference atlas refines single-cell annotations and provides chromatin context, which, when coupled with mouse lineage tracing demonstrates that the castration-resistant luminal cells are distinct from the pre-existent urethra- proximal stem/progenitor cells. Molecular pathway analysis and therapeutic studies further implicate JUN/FOS, WNT/{beta}-Catenin, FOXQ1, NF{kappa}B, and JAK/STAT pathways as the major drivers of castration- resistant luminal populations with high relevance to human PCa. Importantly, we demonstrate the utility of our datasets, which can be explored through an interactive portal (https://visportal.roswellpark.org/data/tang/), to aid in developing novel combination treatments with ARSI for advanced PCa patients. O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/530998v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@8bd384org.highwire.dtl.DTLVardef@1ebc004org.highwire.dtl.DTLVardef@8b4dbforg.highwire.dtl.DTLVardef@19d7052_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Angiotensin II receptor inhibition ameliorates liver fibrosis and enhances hepatocellular carcinoma infiltration by effector T cells

Although viral hepatocellular carcinoma (HCC) is declining, non-viral HCC, which often is the end-stage of non-alcoholic or alcoholic steatohepatitis (NASH, ASH), is on an upward trajectory. Immune checkpoint inhibitors (ICI) that block the T cell inhibitory receptor PD-1 were approved for treatment of all HCC types. However, only a small portion of HCC patients show a robust and sustained response to PD-1 blockade, calling for improved understanding of factors that negatively impact response rate and duration and the discovery of new adjuvant treatments that enhance ICI responsiveness. Using a mouse model of NASH-driven HCC, we identified peritumoral fibrosis as a potential obstacle to T cell mediated tumor regression and postulated that anti-fibrotic medications may increase ICI responsiveness. We now show that the angiotensin II receptor inhibitor losartan, a commonly prescribed and safe antihypertensive drug, reduced liver and peritumoral fibrosis and substantially enhanced anti-PD-1 induced tumor regression. Although losartan did not potentiate T cell reinvigoration, it substantially enhanced HCC infiltration by effector CD8+ T cells compared to PD-1 blockade alone. The beneficial effects of losartan correlated with inhibition of TGF-{beta} receptor signaling, collagen deposition and depletion of immunosuppressive fibroblasts. SignificanceImmune checkpoint inhibitors are used in HCC treatment but overall response rates for single agent PD-1/PD-L1 blockers have remained stubbornly low. Using a mouse model of NASH-driven HCC, we show that co-treatment with the safe and inexpensive angiotensin II receptor inhibitor losartan substantially enhanced anti-PD-1 triggered HCC regression. Although losartan did not influence the reinvigoration of exhausted CD8+ T cells it considerably enhanced their intratumoral invasion, which we postulated to be compromised by peritumoral fibrosis. Indeed, the beneficial effect of losartan correlated with inhibition of TGF-{beta} signaling and collagen deposition, and depletion of immunosuppressive fibroblasts. Losartan should be evaluated for its adjuvant activity in HCC patients undergoing PD-1/PD-L1 blocking therapy.

cancer biology↗

bioRxiv2023

A Simple Way to Incorporate Target Structural Information in Molecular Generative Models

Deep learning generative models are now being applied in various fields including drug discovery. In this work, we propose a novel approach to include target 3D structural information in molecular generative models for structure-based drug design. The method combines a message-passing neural network model that predicts docking scores with a generative neural network model as its reward function to navigate the chemical space searching for molecules that bind favorably with a specific target. A key feature of the method is the construction of target-specific molecular sets for training, designed to overcome potential transferability issues of surrogate docking models through a two-round training process. Consequently, this enables accurate guided exploration of the chemical space without reliance on the collection of prior knowledge about active and inactive compounds for the specific target. Tests on eight target proteins showed a 100-fold increase in hit generation compared to conventional docking calculations, and the ability to generate molecules similar to approved drugs or known active ligands for specific targets without prior knowledge. This method provides a general and highly efficient solution for structure-based molecular generation.

bioinformatics↗

bioRxiv2023

Three-Dimensional Bioconjugated Liquid-Like Solid (LLS) Enhance Characterization of Solid Tumor - Chimeric Antigen Receptor T cell Interactions

Cancer immunotherapy offers lifesaving treatments for cancers, but the lack of reliable preclinical models that could enable the mechanistic studies of tumor-immune interactions hampers the identification of new therapeutic strategies. We hypothesized 3D confined microchannels, formed by interstitial space between bio-conjugated liquid-like solids (LLS), enable CAR T dynamic locomotion within an immunosuppressive TME to carry out anti-tumor function. Murine CD70-specific CAR T cells cocultured with the CD70-expressing glioblastoma and osteosarcoma demonstrated efficient trafficking, infiltration, and killing of cancer cells. The anti-tumor activity was clearly captured via longterm in situ imaging and supported by upregulation of cytokines and chemokines including IFNg, CXCL9, CXCL10, CCL2, CCL3, and CCL4. Interestingly, target cancer cells, upon an immune attack, initiated an "immune escape" response by frantically invading the surrounding microenvironment. This phenomenon however was not observed for the wild-type tumor samples which remained intact and produced no relevant cytokine response. Single cells collection and transcriptomic profiling of CAR T cells at regions of interest revealed feasibility of identifying differential gene expression amongst the immune subpopulations. Complimentary 3D in vitro platforms are necessary to uncover cancer immune biology mechanisms, as emphasized by the significant roles of the TME and its heterogeneity.

bioengineering↗

bioRxiv2023

Ferroptosis is determined by chloride ions

Ferroptosis has emerged as a deliberate type of programmed cell death that manifests marked importance ubiquitously in health and diseases. However, after a decade of research, the mechanisms of ferroptosis execution remain unclear. Here we identify chloride ions (Cl-) as essential determinants of ferroptosis. Water homeostasis manipulated by extracellular solute concentration disrupts ferroptotic cell death. Hyperosmotic stress attenuates ferroptosis and endues cells with high lipid peroxidation. Analyses of a fluorescent chloride probe show that Cl- fluxes into the cytoplasm during ferroptosis, substantiating a role for Cl- to drive water flow. Depletion of extracellular chloride ions ([Cl-]o) from culture media congruously confers resistance to ferroptosis. The [Cl-]o-depleted ferroptotic cells fall into two populations: cells with low lipid peroxidation; cells with high lipid peroxidation but not cell swelling or cell rupture. Contrarily, solitary [Cl-]o overload is sufficient to elicit ferroptosis without canonical ferroptosis inducers. Further experiments show that ferroptotic cells depolarize and [Cl-]o is positively correlated with this process. Membrane depolarization upregulates the level of lipid peroxidation, suggesting that membrane potential may be a universal mechanism governing ferroptosis. Together, our findings reveal that ferroptosis is determined by chloride ions.

cell biology↗

bioRxiv2023

SUMOylation of PTEN promotes DNA end resection through directly dephosphorylating 53BP1 in homologous recombination repair

Homologous recombination (HR) repair for DNA double-strand breaks (DSBs) is critical for maintaining genome stability and cell survival. Nuclear PTEN plays a key role in HR repair, but the underlying mechanism remains largely elusive. We find that SUMOylated PTEN promotes HR repair but represses non-homologous end joining (NHEJ) repair by directly dephosphorylating 53BP1. During DNA damage responses (DDR), p14ARF was phosphorylated and then interacted efficiently with PTEN, thus promoting PTEN SUMOylation as an atypical SUMO E3 ligase. Interestingly, SUMOylated PTEN was subsequently recruited to the chromatin at DNA-break sites. This was because that SUMO1 conjugated to PTEN was recognized and bound by the SUMO-interacting motif (SIM) of BRCA1, which has been located to the core of 53BP1 foci on the chromatin during S/G2 stage. Further, these chromatin-loaded PTEN directly and specifically dephosphorylated pT543 of 53BP1, resulting in the dissociation of 53BP1-complex, which facilitated DNA end resection and ongoing HR repair. The SUMOylation-deficient PTENK254R mice also showed decreased DNA damage repair in vivo. Blocking PTEN SUMOylation pathway with either an SUMOylation inhibitor or a p14ARF(2-13) peptide sensitized tumor cells to chemotherapy. Our study therefore provides the new mechanistic understanding of PTEN in HR repair and clinical intervention of chemo-resistant tumors.

cell biology↗

bioRxiv2023

p53 and RB Cooperate to Suppress Transposable Elements

Accumulating data suggest that the long interspersed nuclear element 1 (LINE1), a major class of transposable elements, can promote tumorigenesis by causing deletions of tumor suppressor genes, amplifications of oncogenes, and chromosomal translocations(1). LINE1 and other transposable elements such as short interspersed nuclear elements (SINEs) can be activated during cancer development. Aberrant transposon expression has been correlated with loss of p53, however, how the loss of p53 or other tumor suppressors leads to activation of transposons during tumorigenesis remains to be fully understood. Here, we investigate the link between loss of the p53 and RB tumor suppressors and derepression of transposable elements. We observe that loss of p53 and RB in mouse embryonic fibroblasts leads to epigenetic changes, such as loss of DNA and histone methylation, which correlate with marked upregulation of LINE and SINE transposable elements. These results suggest that p53 and RB may utilize epigenetic mechanisms that work in tandem to suppress transposons, which may contribute to tumor suppression. SignificanceThe two most commonly mutated genes in human cancer are p53 and the retinoblastoma (RB) tumor suppressors. Together, these genes regulate complex interconnecting pathways responsible for the regulation of cell growth, cell death, and genomic integrity. Studies in genetically engineered mouse models demonstrate that the canonical functions of these two tumor suppressors fail to fully explain their tumor suppressive capabilities. Individually, p53 and RB have each been implicated in repressing transposons. Here we show that combined loss of p53 and RB, which rapidly transforms cells, also results in cooperative upregulation of transposable elements and coincides with decreased methylation of DNA and H3K9. Our results present a framework by which p53 and RB work together to restrain the expression of LINE1 and SINEs, which may contribute to tumor suppression.

cancer biology↗

bioRxiv2023

Real-Time Quantification of Cell Mechanics and Functions by Double Resonator Piezoelectric Cytometry -Theory and Study of Cellular Adhesion of HUVECs

Cell mechanics is closely associated with cellular structure and function. However, the inability to measure both cellular force and viscoelasticity of statistically significant number of cells noninvasively remains a challenge for quantitative characterizations of various cellular functions and practical applications. Here a double resonator piezoelectric cytometry (DRPC), using AT and BT cut quartz crystals of the same frequency and surface morphology is developed to simultaneously quantify the cells-generated forces ({Delta}S) and viscoelastic moduli (G', G'') of a population of isolated single cells or cells with different degrees of cell-cell interactions in a non-invasive and real time manner. DRPC captures the dynamic mechanical parameters {Delta}S and G', G'' during the adhesions of human umbilical vein endothelial cells (HUVECs) under different ligand densities of adhesion molecules fibronectin or Arg-Gly-Asp (RGD) modified on the gold surfaces of 9 MHz AT and BT cut quartz crystals, and different seeding densities of HUVECs. It is found that both the ligand density and cell seeding density affect the magnitudes of {Delta}S and G', G'' and their correlations are revealed for the first time by DRPC. The validity of DRPC is further verified by mechanical changes of the cells in response to treatments with cytoskeleton regulators.

biophysics↗

bioRxiv2023

EB1 decoration of microtubule lattice facilitates spindle-kinetochore lateral attachment in Plasmodium male gametogenesis

Faithful chromosome segregation of 8 duplicated haploid genomes into 8 daughter male gametes is essential for male gametogenesis and mosquito transmission of Plasmodium. Plasmodium evolves the endomitosis for this multinucleated cell division. However, the mechanism underlying the spindle-kinetochore attachment remains elusive. End-binding proteins (EBs) are the conserved microtubule (MT) plus-end binding proteins and play an important role in regulating MT plus-end dynamics. Here we report that Plasmodium EB1 is a unique orthologue distinct from the canonical eukaryotic EB1. Both in vitro and in vivo assays revealed that Plasmodium EB1 lost MT plus-end tracking but gained MT-lattice affinity. This MT-binding feature of EB1 is contributed by both the CH domain and the linker region. EB1-deficient parasites produce male gametocytes that develop to the anucleated male gametes, leading to defective mosquito transmission of parasite. EB1 is localized at the nucleoplasm of male gametocytes. Upon gametogenesis, EB1 decorates the full-length of spindle MTs and regulates spindle structure. The kinetochores attach to spindle MTs laterally throughout three rounds of endomitosis and this attachment is EB1-dependent. Consequently, impaired spindle-kinetochore attachment was observed in EB1-deficient parasites. These results indicate that a parasite-specific EB1 with MT-lattice affinity has evolved to fulfill the spindle-kinetochore lateral attachment in male gametogenesis.

microbiology↗