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Sun, S. Y.

Publications and source records attributed to Sun, S. Y..

9 recordsLinked to original sources
bioRxiv2025

In situ cryo-ET reveals restricted docking of intraflagellar transport at the base of the trypanosome flagellum

Cilia and flagella are conserved organelles essential for sensory and motility functions and are assembled and maintained by bidirectional intraflagellar transport (IFT) along the axoneme. In Trypanosoma brucei, IFT is concentrated at the flagellar base near the transition zone and is restricted to four microtubule doublets (MTDs) as it traverses from the transition zone to the axoneme before reaching the extra-axonemal structure, the paraflagellar rod (PFR), associated region. To gain an insight on its initial working model before IFT restriction occurs, we revealed the in situ architecture of the IFT complex during the initial and complete landing to the transition zone using cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET). IFT proteins assemble into polymeric trains upon landing. However, in the proximal portion of the flagellum, subtomogram averaging revealed that each single train is associated with individual MTDs at variable A- or B-tubule positions. By using miniaturized and enucleated T. brucei zoids that preserve the full circular arrangement of MTDs without milling, we revealed an alternating but spatially restricted IFT train pattern during the landing process, resembling the anterograde train scaffold known in Chlamydomonas. Magnify expansion microscopy further confirmed this restricted distribution of IFT proteins at the basal pool. Depletion of the IFT component IFT46 protein reduced the number of simultaneously docked trains without altering the restricted landing spatial pattern. These findings reveal a previously unrecognized spatial regulation governing IFT train initiation at the flagellar base, which could operate independently of IFT-B structural integrity, bridging the gap between the IFT architecture and doublet-spatial docking prior to anterograde transport.

cell biology↗
bioRxiv2025

An Essential Adaptor for Apicoplast Fission and Inheritance in Malaria Parasites

Blood-stage Plasmodium falciparum parasites rely on a non-photosynthetic plastid, the apicoplast, for survival, making it an attractive target for antimalarial intervention. Like the mitochondrion, the apicoplast cannot be generated de novo and must be inherited by daughter parasites during cell division. This inheritance relies on coordinated apicoplast positioning and fission, but the molecular mechanisms controlling these processes remain poorly understood. Here, we identify a previously uncharacterized P. falciparum protein (Pf3D7_0613600), which we name PfAnchor, as a key regulator of apicoplast fission. Using Ultrastructure Expansion Microscopy (U-ExM), we show that PfAnchor localizes to the apicoplast throughout the asexual blood-stage. Conditional depletion disrupts apicoplast fission, leading to incomplete cytokinesis and parasite death. Notably, loss of the apicoplasts elongated branched structure via azithromycin treatment rescues these defects, underscoring Anchors specific role in apicoplast fission. Immunoprecipitation identified an interaction with the dynamin-like GTPase PfDyn2, a key mediator of both apicoplast and mitochondrial fission, establishing PfAnchor as the first apicoplast-specific dynamin adaptor protein. Our findings define PfAnchor as an essential factor for apicoplast fission and inheritance in P. falciparum blood-stage parasites, highlighting parasite-specific organelle division as a potential vulnerability for therapeutic intervention.

cell biology↗
bioRxiv2024

Detection of genome structural variation in normal cells and tissues by single molecule sequencing

Rare genome structural-variants (SVs) in somatic cells and tissues are difficult to measure in bulk DNA because library preparation can create chimeric junctions that mimic authentic events. Based on our previous Single-Molecule Mutation sequencing (SMM-seq) assay, we developed a new method, SMM-SV-seq, which combines a chimera-suppressing library preparation with duplex confirmation of breakpoint-supporting molecules. Using libraries from mixtures of human and Drosophila DNA, we show a remaining artifact rate of 1.52 +/- 0.53 detectable cross-species chimeras per billion bases, providing an empirical background for correcting breakpoint frequencies. In a benchmark of evaluable homozygous germline deletions in the GM24385 cell line, SMM-SV-seq achieved 81.4% precision and 85.7% recall, exceeding the precision of established short-read callers while maintaining comparable recall. The assay detected dose-associated increases in SV-breakpoint frequencies in cells treated with bleomycin, a known clastogen. Biological relevance was demonstrated by a significantly greater irradiation-associated increase in translocation-like breakpoint frequencies in the BRCA1 185delAG-heterozygous mammary epithelial clone than in its isogenic wild-type control. These results establish SMM-SV-seq as a practical assay for calibrated, duplex-confirmed detection of rare somatic SV breakpoints directly in bulk DNA from non-clonal cell populations.

genomics↗
bioRxiv2023

shRNA drop-out screen identifies BRD4 targeting transcription from RNA polymerase II system to activate β-catenin to promote soft-tissue tumor proliferations

BRD4 (Bromodomain containing protein 4) is a chromatin reader binds to acetylated lysine residues on histones interacting with RNA Pol II, p-TFeb. PDGF-BB was presented here, in soft-tissue tumor, as an oncogenic factor driving cell proliferation, and aberrant BRD4 knockdown significantly reduced tumor aggressiveness and unfavorable prognosis in soft-tissue tumors. To identify suppressive key drivers impeding demoid tumor growth, shRNA drop-out screen analysis identified signature of "transcription from RNA polymerase II promoter" including DDX, Stat3, SMARCA, ATM, SIRT1, cMyc that were recruited with BRD4 interation in activating {beta}-catenin, which is a major key driver mutated in soft-tissue tumor, and its depletion ceased soft-tissue tumor cell growth. Sepcifically, BRD4 mediated PDGF-BB signaling in GSK stimulation through transcriptional regulation from RNA polymerase II activity with PI3K as target, and thus not only canonical {beta} -catenin/TCF4 signaling, but also non-canonical {beta} -catenin conjunction complex response was activated by BRD4 in nucleus involved in promoting cell proliferation. Our study delineated a signaling axis that may allow soft-tissue tumor cells to escape apoptosis during colonization by activating PDGFBB-BRD4-GSK-{beta} -catenin and non-canonical-{beta}-catenin pathway through BRD4 in cancer cells. An efficient treatment for soft-tissue tumors could be accomplished by targeting PDGF and BRD4 survival pathways on soft-tissue tumor cells.

cancer biology↗
bioRxiv2023

Arl3 regulates ODA16-mediated intraflagellar transport in motile cilia biogenesis

Arl13b and Arl3 are ciliary GTPases implicated in human Joubert Syndrome, affecting ciliary membrane and axoneme organization. Although the mechanism of Arl13b as a guanine nucleotide exchange factor (GEF) of Arl3 and the function of Arl13b and Arl3 in ciliary membrane protein transport are well established, their role in axoneme biogenesis is unclear. In Trypanosoma brucei, TbArl13 acts as a GEF for two distinct TbArl3 proteins, TbArl3A and TbArl3C. Here, we identified the T. brucei homolog of ODA16, a cargo adapter facilitating intraflagellar transport (IFT) of motile ciliary components, as an effector of both TbArl3A and TbArl3C. Depletion of TbArl3 GTPases stabilized TbODA16 interaction with IFT, while active TbArl3 variants displaced TbODA16 from IFT, demonstrating a mechanism of TbArl3 in motile ciliary cargo transport. One-sentence summaryArl3 acts as a displacement factor and releases ODA16 from IFT

cell biology↗
bioRxiv2022

Cryogenic electron tomography reveals novel structures in the apical complex of Plasmodium falciparum

Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. In this paper, we perform cryogenic electron tomography (cryo-ET) on isolated merozoites to visualize the apical machinery. Through tomography reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry-secretory-apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly patterning. These results provide new insight into the essential features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells.

cell biology↗
bioRxiv2022

Ionophore-stimulation promotes re-organization of the invasion machinery of Toxoplasma gondii

Host cell invasion by intracellular, eukaryotic parasites within the phylum Apicomplexa, is a remarkable and active process involving the coordinated action of apical organelles and other structures. To date, capturing how these structures interact during invasion has been difficult to observe in detail. Here, we used cryogenic electron tomography to image the apical complex of Toxoplasma gondii tachyzoites under conditions that mimic resting parasites and those primed to invade through stimulation with calcium ionophore. Through the application of Mixed Scale Dense networks for image-processing, we developed a highly efficient pipeline for annotation of tomograms, enabling us to identify and extract densities of relevant subcellular organelles and accurately analyze features in 3D. The results reveal a dramatic change in the shape of the anteriorly located apical vesicle upon its apparent fusion with a rhoptry, that occurs only in the stimulated parasites. We also present information indicating that this vesicle originates from the vesicles that parallel the intraconoidal microtubules and that the latter two structures are linked by a novel tether. We show that a rosette structure previously proposed to be involved in rhoptry secretion is associated with apical vesicles beyond just the most anterior one. This result, suggesting multiple vesicles are primed to enable rhoptry secretion, may shed light on the mechanisms Toxoplasma employs to enable repeated invasion attempts. Using the same approach, we examine Plasmodium falciparum merozoites and show that they too possess an apical vesicle just beneath a rosette, demonstrating evolutionary conservation of this overall subcellular organization. Significance StatementParasites in the phylum Apicomplexa are responsible for some of the most important parasitic diseases of humans, such as malaria and toxoplasmosis. Invasion by these obligatory, intracellular parasites depends on protein injection into the host cell. Using cryogenic electron tomography, we reveal evolutionarily conserved features shared by the invasive forms of Plasmodium falciparum and Toxoplasma gondii. By comparing resting Toxoplasma tachyzoites with those primed to invade we also gain new insight into the very first steps in invasion. For this work, we take an interdisciplinary approach, adopting a mixed-scale dense neural network that enables efficient and objective processing of the data. Combined, the results provide new information on how these important parasites accomplish the essential step of invasion.

cell biology↗
bioRxiv2021

Cryo-ET reveals two major tubulin-based cytoskeleton structures in Toxoplasma gondii

In the obligate intracellular parasite, Toxoplasma gondii, the subpellicular microtubules (SPMTs) help maintain shape, while the apical conoid (also tubulin-based) is implicated in invasion. Here, we use cryo-electron tomography to determine the molecular structures of the SPMTs and the conoid-fibrils (CFs) in vitrified and detergent-lysed parasites. Subvolume densities from detergent-extracted parasites yielded averaged density maps at subnanometer resolutions, and these were related back to their architecture in situ. An intraluminal spiral (IS) lines the interior of the 13-protofilament SPMTs, revealing a preferred orientation of these microtubules relative to the parasites long axis. Each CF is composed of 9 tubulin protofilaments, that produce a comma-shaped cross-section, plus additional associated components. Conoid protrusion, a crucial step in invasion, is associated with an altered pitch of each CF. The use of basic building blocks of protofilaments and different accessory proteins in one organism, illustrates the versatility of these critical structures.

molecular biology↗