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Single-molecule localization microscopy of expanded mammalian centrioles unravels the ultrastructural constitution of distal appendages

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Biophysical journal 2023年第3S1期122卷 17a页

作者： Chang, Ting-Jui B Hsu, Jimmy C-C Yang, Tony Physics National Taiwan University Taipei City Taiwan Nano Science and Technology Program Taiwan International Graduate Program Academia Sinica Taipei City Taiwan Electrical Engineering National Taiwan University Taipei City Taiwan Electrical Engineering National Taiwan University Taipei City Taiwan Graduate Institute of Biomedical Electronics and Bioinformatics National Taiwan University Taipei City Taiwan

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Development of label-free triboelectric nanosensors as screening platforms for anti-tumor drugs

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Nano Energy 2024年 125卷

作者： Cheng, Yu-Ying Ganguly, Anindita Cheng, Yi-Yun Ortiz, Christopher Llynard D. Pal, Arnab Shah, Pramod Kaswan, Kuldeep Yang, Lee-Wei Lin, Zong-Hong Department of Biomedical Engineering National Taiwan University Taipei10617 Taiwan Institute of Biomedical Engineering National Tsing Hua University Hsinchu30013 Taiwan Internet of Senses Research Center National Tsing Hua University Hsinchu30013 Taiwan Praexisio Taiwan Inc. New Taipei221425 Taiwan Institute of Bioinformatics and Structural Biology National Tsing Hua University Hsinchu300044 Taiwan Chemical Biology and Molecular Biophysics Program Taiwan International Graduate Program Institute of Biological Chemistry Academia Sinica Taipei115 Taiwan Department of Chemistry National Tsing Hua University Hsinchu300044 Taiwan International Intercollegiate PhD Program National Tsing Hua University Hsinchu300044 Taiwan Department of Power Mechanical Engineering National Tsing Hua University Hsinchu300044 Taiwan Biomedical Artificial Intelligence PhD Program National Tsing Hua University Hsinchu300044 Taiwan Physics Division National Center for Theoretical Sciences Taipei Taiwan

This study introduces a general label-free drug screening platform with potential to support high-throughput drug screening for any protein target. Its innovation lies in quantifying the affinity of molecule-molecule interactions via voltage output variation, which achieves unprecedented selectivity and sensitivity. It combines computational analysis complemented by experimental substantiation with a solid-liquid triboelectric nanosensor. Owing to its high binding affinity, FKBP-rapamycin is used as a model system to demonstrate the platform's sensitivity. Subsequently, the research is extended to study drug interactions with an oncogenic protein ATG4B. The findings unveil the binding of S130 and Tioconazole to ATG4B, a critical cysteine protease involved in autophagy, while revealing that Dexamethasone does not bind ATG4B. This binding exerts a specific inhibitory effect on autophagic flux and triggers cancer cell apoptosis. The results support the previously verified inhibitory effects of these drugs and the effectiveness of a newly developed self-powered drug screening platform, leveraging the principles of solid-liquid contact electrification. This approach adeptly confronts enduring challenges in traditional drug development methods where biochemical assays need to be designed for each individual protein target or only time-consuming and concentration-demanding molecular interaction measurements were made available. © 2024 Elsevier Ltd

关键词： Nanosensors

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Structural insights into the role of N-terminal integrity in PhoSL for core-fucosylated N-glycan recognition

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international Journal of Biological Macromolecules 2024年 255卷 128309页

作者： Lou, Yuan-Chao Tu, Cheng-Fen Chou, Chun-Chi Yeh, Hsin-Hong Chien, Chia-Yu Sadotra, Sushant Chen, Chinpan Yang, Ruey-Bing Hsu, Chun-Hua Institute of Biomedical Sciences Academia Sinica Taipei115 Taiwan Department of Agricultural Chemistry National Taiwan University Taipei106 Taiwan Chemical Biology and Molecular Biophysics Taiwan International Graduate Program Academia Sinica Taipei115 Taiwan Institute of Bioinformatics and Structural Biology National Tsing Hua University Hsinchu300 Taiwan Biomedical Translation Research Center Academia Sinica Taipei115 Taiwan Ph.D. Program in Drug Discovery and Development Industry College of Pharmacy Taipei Medical University Taipei110 Taiwan Genome and Systems Biology Degree Program National Taiwan University and Academia Sinica Taipei106 Taiwan Institute of Biochemical Sciences National Taiwan University Taipei106 Taiwan Center for Computational and Systems Biology National Taiwan University Taipei106 Taiwan

PhoSL (Pholiota squarrosa Lectin) has an exceptional binding affinity for biomolecules with core-fucosylated N-glycans. This modification involves the addition of fucose to the inner N-acetylglucosamine within the N-glycan structure and is known to influence many physiological processes. Nevertheless, the molecular interactions underlying high-affinity binding of native PhoSL to core-fucosylated N-glycans remain largely unknown. In this study, we devised a strategy to produce PhoSL with the essential structural characteristics of the native protein (n-PhoSL). To do so, a fusion protein was expressed in E. coli and purified. Then, enzymatic cleavage and incubation with glutathione were utilized to recapitulate the native primary structure and disulfide bonding pattern. Subsequently, we identified the residues crucial for n-PhoSL binding to core-fucosylated chitobiose (N2F) via NMR spectroscopy. Additionally, crystal structures were solved for both apo n-PhoSL and its N2F complex. These analyses suggested a pivotal role of the N-terminal amine in maintaining the integrity of the binding pocket and actively contributing to core-fucose recognition. In support of this idea, the inclusion of additional residues at the N-terminus considerably reduced binding affinity and PhoSL cytotoxicity toward breast cancer cells. Taken together, these findings can facilitate the utilization of PhoSL in basic research, diagnostics and therapeutic strategies. © 2023 Elsevier B.V.

关键词： Proteins

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Successive responses of three coral holobiont components (coral hosts, symbiotic algae, and bacteria) to daily temperature fluctuations

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Ecological Indicators 2024年 158卷

作者： Hsieh, Yunli Eric Lu, Chih-Ying Liu, Po-Yu Kao, Jia-Min Yang, Sung-Yin Wu, Chien-Yi Wong, Jing-Wen Michelle Shikina, Shinya Fan, Tung-Yung Yang, Shan-Hua Systems Biology and Mathematical Modelling group Max Planck Institute of Molecular Plant Physiology Potsdam Germany Institute of Biochemistry and Biology Bioinformatics Department University of Potsdam Potsdam Germany School of BioSciences The University of Melbourne ParkvilleVIC Australia Molecular and Biological Agricultural Sciences Program Taiwan International Graduate Program National Chung Hsing University and Academia Sinica Taipei Taiwan Graduate Institute of Biotechnology National Chung Hsing University Taichung Taiwan Biodiversity Research Center Academia Sinica Taipei Taiwan School of Medicine College of Medicine National Sun Yat-sen University Kaohsiung Taiwan Institute of Fisheries Science National Taiwan University Taipei Taiwan Department of Aquatic Bioscience National Chiayi University Chiayi Taiwan Institute of Marine Environment and Ecology National Taiwan Ocean University Keelung Taiwan National Museum of Marine Biology and Aquarium Pingtung Taiwan

Corals and their associated microbiota form a "holobiont," which includes symbiotic algae and other associated microbiota dominated by bacteria. The composition of holobiont is influenced by various environmental factors, such as increasing sea water temperatures. Previous studies of the effects of temperature changes on coral physiology and associated bacterial communities have been conducted based on stable water temperatures set by mean temperatures, or by slowly increasing/decreasing temperatures. However, the daily temperature fluctuations that corals experience in nature are not stable. The current understanding of the effects of large daily temperature fluctuations on coral and associated bacterial community dynamics is limited. Hence, we conducted a four-week tank experiment using different large daily temperature fluctuations (±5 °C and ±7 °C) accompanied by continuous warming conditions (26 °C to 29 °C) to investigate the effects on two common reef-building corals, Stylophora pistillata and Pocillopora acuta. During the experiment, the activity of coral host catalase and superoxide dismutase were measured, the photosynthetic ability of symbiotic algae was recorded, and the variation in bacterial communities was analyzed using the V6-V8 region of 16S rDNA. According to the results, different parts of the holobionts of two coral species exhibited varying response rates to the continuous warming conditions and diurnal temperature fluctuations. Additionally, it was found that diurnal temperature fluctuations may mitigate the heat stress on the host and reduce the changes in bacterial response to warming. Therefore, it is suggested that the holobionts of different coral species may adopt different adaptation and survival strategies in response to diurnal temperature fluctuations and warming. Finally, based on the response of these two coral species under the conditions of diurnal temperature fluctuations and continuous warming, Acinetobacter and Rhodobacteracea

关键词： Temperature distribution

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Improving quantitation accuracy in isobaric-labeling mass spectrometry experiments with spectral library searching and feature-based peptide-spectrum match filter

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Scientific Reports 2023年第1期13卷 1-10页

作者： Kuo, Tzu-Yun Wang, Jen-Hung Huang, Yung-Wen Sung, Ting-Yi Chen, Ching-Tai Department of Biochemical Science and Technology National Taiwan University Taipei 10617 Taiwan Bioinformatics Program Taiwan International Graduate Program Institute of Statistical Science Academia Sinica Taipei 11529 Taiwan Institute of Information Science Academia Sinica Taipei 11529 Taiwan Institute of Biomedical Informatics National Yang Ming Chiao Tung University Taipei 11221 Taiwan Department of Computer Science and Information Engineering National Taiwan University Taipei 10617 Taiwan Department of Bioinformatics and Biomedical Engineering Asia University Taichung 41354 Taiwan Center for Precision Health Research Asia University Taichung 41354 Taiwan

Isobaric labeling relative quantitation is one of the dominating proteomic quantitation technologies. Traditional quantitation pipelines for isobaric-labeled mass spectrometry data are based on sequence database searching. In this study, we present a novel quantitation pipeline that integrates sequence database searching, spectral library searching, and a feature-based peptide-spectrum-match (PSM) filter using various spectral features for filtering. The combined database and spectral library searching results in larger quantitation coverage, and the filter removes PSMs with larger quantitation errors, retaining those with higher quantitation accuracy. Quantitation results show that the proposed pipeline can improve the overall quantitation accuracy at the PSM and protein levels. To our knowledge, this is the first study that utilizes spectral library searching to improve isobaric labeling-based quantitation. For users to conveniently perform the proposed pipeline, we have implemented the feature-based filter being executable on both Windows and Linux platforms;its executable files, user manual, and sample data sets are freely available at https://***/comics/Software_*** . Furthermore, with the developed filter, the proposed pipeline is fully compatible with the Trans-Proteomic Pipeline. © 2023, Springer Nature Limited.

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Phosphoproteomic analysis reveals distinctive responses in Mangrovibacter phragmatis under high-salinity condition

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Biochemical and biophysical research communications 2024年 736卷 150514页

作者： Hong Soon Chin Narendrakumar Ravi Varadharajulu Zhi-Han Lin Chuan-Chih Hsu Steve S-F Yu Institute of Chemistry Academia Sinica Taipei Taiwan Chemical Biology and Molecular Biophysics Program Taiwan International Graduate Program Academia Sinica Taipei Taiwan Institute of Bioinformatics and Structural Biology National Tsing Hua University Hsinchu Taiwan. Molecular Science and Technology Program Taiwan International Graduate Program Academia Sinica Taipei Taiwan Department of Chemistry National Tsing Hua University Hsinchu Taiwan. Institute of Biochemical Sciences National Taiwan University Taipei Taiwan. Institute of Plant and Microbial Biology Academia Sinica Taipei Taiwan. Molecular Science and Technology Program Taiwan International Graduate Program Academia Sinica Taipei Taiwan. Electronic address: sfyu@gate.sinica.edu.tw.

We conducted a thorough genome-wide investigation of protein phosphorylation in the halotolerant bacterium Mangrovibacter phragmitis (MPH) ASIOC01, using the Fe-IMAC enrichment method combined with tandem mass spectrometry under low- and high-salinity conditions. The phosphoproteome comprises 86 unique phosphorylated proteins, crucially involving pathways such as glycolysis/gluconeogenesis, the citrate cycle, chaperones, ribosomal proteins, and cell division. This study represents the first and most extensive investigation to-date comparing the bacterial phosphoproteome under different osmotic conditions using a gel-free approach. We identified 45 unique phosphoproteins in MPH cultured in media containing 1 % NaCl, and 33 exclusive phosphoproteins in MPH cultured in media containing 5 % NaCl. Eight phosphoproteins were detected in both growth conditions. Analysis of high-confidence phosphosites reveals that phosphorylation predominantly occurs on serine residues (52.3 %), followed by threonine (35.1 %) and tyrosine (12.6 %) residues. Interestingly, 34 % of the phosphopeptides display multiple phosphosites. Currently, prokaryotic phosphorylation site prediction platforms like MPSite and NetPhosBac 1.0 demonstrate an average prediction accuracy of only 21 % when applied to our dataset. Fourteen phosphoproteins did not yield matches when compared against dbPSP 2.0 (database of Phosphorylation Sites in Prokaryotes), indicating that these proteins may be novel phosphoproteins. These unique proteins undergoing phosphorylation under high salinity growth conditions potentially enhance their adaptive capabilities to environmental challenges.

关键词： Halotolerance LC-MS/MS Mangrovibacter phragmitis Osmotic-stress Phosphoproteomes Ser/thr/tyr phosphorylation

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Environmental melamine exposure and adverse kidney outcomes in patients with type 2 diabetes mellitus

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Environmental Pollution 2023年第Part1期331卷 121883页

作者： Tsai, Yi-Chun Wu, Chia-Fang Hung, Wei-Wen Yu, Ping-Shaou Liu, Chia-Chu Hsieh, Tusty-Jiuan Chen, Chu-Chih Li, Sih-Syuan Chen, Jia-Jen Chiu, Yi-Wen Hwang, Shang-Jyh Wu, Ming-Tsang Division of Nephrology Department of Internal Medicine Kaohsiung Medical University Hospital Kaohsiung Medical University Kaohsiung Taiwan Division of General Medicine Department of Internal Medicine Kaohsiung Medical University Hospital Kaohsiung Medical University Kaohsiung Taiwan Center for Liquid Biopsy and Cohort Research Kaohsiung Medical University Kaohsiung Taiwan School of Medicine College of Medicine Kaohsiung Medical University Kaohsiung Taiwan International Master Program of Translational Medicine National United University Miaoli Taiwan Division of Endocrinology and Metabolism Kaohsiung Medical University Hospital Kaohsiung Medical University Kaohsiung Taiwan Kaohsiung Municipal CiJin Hospital Kaohsiung Taiwan Research Center for Precision Environmental Medicine Kaohsiung Medical University Kaohsiung Taiwan Department of Urology Kaohsiung Medical University Hospital Kaohsiung Medical University Kaohsiung Taiwan Graduate Institute of Medicine College of Medicine Kaohsiung Medical University Kaohsiung Taiwan Division of Biostatistics and Bioinformatics Institute of Population Health Sciences National Health Research Institutes Taiwan Department of Public Health College of Health Sciences Kaohsiung Medical University Kaohsiung Taiwan Department of Family Medicine Kaohsiung Medical University Hospital Kaohsiung Medical University Kaohsiung City Taiwan Program in Environmental and Occupational Medicine College of Medicine Kaohsiung Medical University Kaohsiung City Taiwan

The impact of melamine exposure on kidney outcomes in type 2 diabetes mellitus (T2D) patients remains unclear. In this prospective cohort study, 561 T2D patients during October 2016 and June 2020 were enrolled and followed until December 2021. Baseline one-spot urinary corrected melamine levels were measured by LC-MS/MS. Average daily intake (ADI) of melamine represented environmental melamine exposure in daily life, and was estimated using urinary corrected melamine level by creatinine excretion (CE)-based model. Primary kidney outcomes were defined as doubling of serum creatinine levels or end stage kidney disease (ESKD), and secondary kidney outcomes included rapid decline in kidney function as estimated glomerular filtration rate (eGFR) decline >5 ml/min/1.73 m2/year. Baseline median urinary corrected melamine levels and estimated DI of melamine were 0.8 μg/mmol and 0.3 μg/kg/day in 561 T2D patients. During 3.7 years of follow-up, urinary corrected melamine level was positively correlated with reaching composite outcomes of either doubling of serum creation levels or ESKD and rapid decline in kidney function. Those with the highest quartile of urinary corrected melamine had 2.96-fold risk of composite outcomes of either doubling of serum creation levels or ESKD and 2.47-fold risk of eGFR decline >5 ml/min/1.73 m2/year. Estimated ADI of melamine also had significant correlation with adverse kidney outcomes. Furthermore, the positive relationship between melamine exposure and rapid decline in kidney function was only found in T2D patients with male, baseline eGFR ≥60 ml/min/1.73 m2 or glycated hemoglobin ≤7%. In conclusion, melamine exposure is significantly associated with adverse kidney outcomes in T2D patients, especially in those with male, well sugar control or good baseline kidney function. © 2023 Elsevier Ltd

关键词： Melamine Kidney outcomes Type 2 diabetes mellitus

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Sex-specific interactive effect of melamine and DEHP on a marker of early kidney damage in taiwanese adults: A national population-based study from the taiwan Biobank

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Ecotoxicology and Environmental Safety 2023年第1期263卷 115208页

作者： Li, Sih-Syuan Chen, Jia-Jen Su, Ming-Wei Lin, Chien-Wei Chen, Chu-Chih Wang, Yin-Han Liu, Chia-Chu Tsai, Yi-Chun Hsieh, Tusty-Jiuan Wu, Ming-Tsang Wu, Chia-Fang Research Center for Precision Environmental Medicine Kaohsiung Medical University Kaohsiung Taiwan Taiwan Biobank Academia Sinica Taipei Taiwan Division of Biostatistics and Bioinformatics Institute of Population Health Sciences National Health Research Institutes Taiwan Department of Urology Kaohsiung Medical University Hospital Kaohsiung Medical University Kaohsiung Taiwan Division of Nephrology Department of Internal Medicine Kaohsiung Medical University Hospital Kaohsiung Medical University Kaohsiung Taiwan Graduate Institute of Medicine College of Medicine Kaohsiung Medical University Kaohsiung Taiwan Department of Family Medicine Kaohsiung Medical University Hospital Kaohsiung Medical University Kaohsiung Taiwan Ph.D. Program in Environmental and Occupational Medicine Kaohsiung Medical University Kaohsiung Taiwan Department of Public Health Kaohsiung Medical University Kaohsiung Taiwan International Master Program of Translational Medicine National United University Miaoli Taiwan

taiwan had the high incidence of chronic kidney disease (CKD) worldwide. Our objective was to examine associations between daily exposure of phthalates and melamine, two common nephrotoxins, and kidney damage risk in a well-established nationwide cohort. Study subjects were from taiwan Biobank (TWB) with existing data of questionnaire and biochemical examinations. Average daily intake (ADI) levels of melamine and seven parental phthalates, including DEHP (di-2-ethylhexylphthalate), DiBP (Dibutyl phthalate), DnBP (Di-n-butyl phthalate), BBzP (Butyl benzyl phthalate), DEP (Diethyl phthalate), and DMP (Dimethyl phthalate) were estimated using a creatinine excretion-based model from urine melamine and 10 phthalate metabolites. Urine microalbumin to creatinine ratio (ACR) was used to represent for the outcome of kidney damage. Two statistical strategies were used: First, a weighted quantile sum (WQS) regression model to select the most important exposure variables of ADI levels of phthalates and melamine associated with ACR;Second, to examine effects of those most important exposure variables on ACR in multivariable linear regression models. In total, 1153 eligible adults were left for analyses. Of them, 591 (51.3%) and 562 (48.7%) were men and women, respectively, with a median age of 49 years old. By WQS, a significant and positive association was found between ADI of melamine and phthalates and ACR (β = 0.14, p = 0.002). ADI levels of melamine had the highest weight (0.57), followed by DEHP (0.13). Next, examining the two most important exposures in association with ACR, we found that the higher the melamine and DEHP intakes, the higher the ACR levels were found. An interaction effect was also found between melamine and DEHP intakes on urine ACR (p = 0.015). This result was more prominent in men (p = 0.008) than in women (p = 0.651). Environmental co-exposure of melamine and DEHP can potentially affect ACR in the community-dwelling taiwanese adult population. © 2023 T

关键词： Chronic kidney disease (CKD) Interactive effect Melamine Phthalates Renal dysfunction Sex

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Astrocytic Cebpd Regulates Pentraxin 3 Expression to Promote Fibrotic Scar Formation After Spinal Cord Injury

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Molecular neurobiology 2023年第4期60卷 2200-2208页

作者： Shao-Ming Wang Jung-Yu C Hsu Chiung-Yuan Ko Hsiang-En Wu Yu-Wei Hsiao Ju-Ming Wang Graduate Institute of Biomedical Sciences China Medical University Taichung 404333 Taiwan. wangs@mail.cmu.edu.tw. Neuroscience and Brain Disease Center China Medical University Taichung Taiwan. wangs@mail.cmu.edu.tw. Department of Neurology China Medical University Hospital Taichung Taiwan. wangs@mail.cmu.edu.tw. Department of Cell Biology and Anatomy College of Medicine National Cheng Kung University Tainan Taiwan. Ph.D. Program in Medical Neuroscience College of Medical Science and Technology Taipei Medical University Taipei Taiwan. TMU Research Center of Neuroscience Taipei Medical University Taipei Taiwan. TMU Research Center of Cancer Translational Medicine Taipei Taiwan. Cellular Pathobiology Section Integrative Neuroscience Research Branch Intramural Research Program National Institute on Drug Abuse NIH/DHHS Suite 3512 333 Cassell Drive Baltimore MD 21224 USA. Department of Biotechnology and Bioindustry Sciences College of Bioscience and Biotechnology National Cheng Kung University Tainan Taiwan. Department of Biotechnology and Bioindustry Sciences College of Bioscience and Biotechnology National Cheng Kung University Tainan Taiwan. yumingw@mail.ncku.edu.tw. Graduate Institute of Medical Sciences College of Medicine Taipei Medical University Taipei Taiwan. yumingw@mail.ncku.edu.tw. International Research Center for Wound Repair and Regeneration National Cheng Kung University Tainan Taiwan. yumingw@mail.ncku.edu.tw. Graduate Institute of Medicine College of Medicine Kaohsiung Medical University Kaohsiung Taiwan. yumingw@mail.ncku.edu.tw. Institute of Bioinformatics and Biosignal Transduction College of Bioscience and Biotechnology National Cheng Kung University Tainan 701 Taiwan. yumingw@mail.ncku.edu.tw.

Astroglial-fibrotic scars resulted from spinal cord injury affect motor and sensory function, leading to paralysis. In particular, the fibrotic scar is a main barrier that disrupts neuronal regeneration after spinal cord injury. However, the association between astrocytes and fibrotic scar formation is not yet understood. We have previously demonstrated that the transcriptional factor Cebpd contributes to astrogliosis, which promotes glial scar formation after spinal cord injury. Herein, we show that fibrotic scar formation was decreased in the epicenter region in Cebpd mice after contusive spinal cord injury and astrocytic Cebpd promoted fibroblast migration through secretion of Ptx3. Furthermore, the expression of Mmp3 was increased under recombinant protein Ptx3 treatment in fibroblasts by observing microarray data, resulting in fibroblast migration. In addition, regulation of Mmp3 occurs through the NFκB signaling pathway by using an irreversible inhibitor of IκBα phosphorylation in pretreated fibroblasts. Of note, we used the synthetic peptide RI37, which blocks fibroblast migration and decreases fibroblast Mmp3 expression in IL-1β-treated astrocyte conditioned media. Collectively, our data suggest that fibroblast migration can be affected by astrocytic Cebpd through the Ptx3/NFκB/Mmp3 axis pathway and that the RI37 peptide may act as a therapeutic medicine to inhibit fibrotic scar formation after spinal cord injury.

关键词： Astrocyte Cebpd Fibrotic scar Ptx3 Spinal cord injury

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Single-molecule localization microscopy reveals the ultrastructural constitution of distal appendages in expanded mammalian centrioles

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Nature communications 2023年第1期14卷 1688页

作者： Ting-Jui Ben Chang Jimmy Ching-Cheng Hsu T Tony Yang Department of Physics National Taiwan University Taipei Taiwan. Department of Electrical Engineering National Taiwan University Taipei Taiwan. Nano Science and Technology Program Taiwan International Graduate Program Academia Sinica and National Taiwan University Taipei Taiwan. Department of Electrical Engineering National Taiwan University Taipei Taiwan. tonyyang@ntu.edu.tw. Graduate Institute of Biomedical Electronics and Bioinformatics National Taiwan University Taipei Taiwan. tonyyang@ntu.edu.tw.

Distal appendages (DAPs) are vital in cilia formation, mediating vesicular and ciliary docking to the plasma membrane during early ciliogenesis. Although numerous DAP proteins arranging a nine-fold symmetry have been studied using superresolution microscopy analyses, the extensive ultrastructural understanding of the DAP structure developing from the centriole wall remains elusive owing to insufficient resolution. Here, we proposed a pragmatic imaging strategy for two-color single-molecule localization microscopy of expanded mammalian DAP. Importantly, our imaging workflow enables us to push the resolution limit of a light microscope well close to a molecular level, thus achieving an unprecedented mapping resolution inside intact cells. Upon this workflow, we unravel the ultra-resolved higher-order protein complexes of the DAP and its associated proteins. Intriguingly, our images show that C2CD3, microtubule triplet, MNR, CEP90, OFD1, and ODF2 jointly constitute a unique molecular configuration at the DAP base. Moreover, our finding suggests that ODF2 plays an auxiliary role in coordinating and maintaining DAP nine-fold symmetry. Together, we develop an organelle-based drift correction protocol and a two-color solution with minimum crosstalk, allowing a robust localization microscopy imaging of expanded DAP structures deep into the gel-specimen composites.

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