ORCID Profile
0000-0001-7364-3945
Current Organisation
University of Technology Sydney
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Biomechanical Engineering | Biomedical Engineering | Fluidisation and Fluid Mechanics
Scientific Instruments | Expanding Knowledge in the Biological Sciences | Expanding Knowledge in Technology |
Publisher: Springer Science and Business Media LLC
Date: 12-07-2021
DOI: 10.1186/S13059-021-02424-W
Abstract: Metazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Although various genetic and epigenetic signatures have been linked to the replication efficiency of origins, there is no consensus on how the selection of origins is determined. We apply dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within in idual topologically associating domains (TADs). We find that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase. Intriguingly, while both high-efficiency and low-efficiency origins are distributed homogeneously in the TAD during the G1 phase, high-efficiency origins relocate to the TAD periphery before the S phase. Origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observe that the replication machinery protein PCNA forms immobile clusters around TADs at the G1/S transition, explaining why origins at the TAD periphery are preferentially fired. Our work reveals a new origin selection mechanism that the replication efficiency of origins is determined by their physical distribution in the chromatin domain, which undergoes a transcription-dependent structural re-organization process. Our model explains the complex links between replication origin efficiency and many genetic and epigenetic signatures that mark active transcription. The coordination between DNA replication, transcription, and chromatin organization inside in idual TADs also provides new insights into the biological functions of sub-domain chromatin structural dynamics.
Publisher: Cold Spring Harbor Laboratory
Date: 29-03-2022
DOI: 10.1101/2022.03.29.486198
Abstract: Antibiotic resistance has been a global threat to public health. Majority of antibiotics kill bacteria by inducing the DNA damage. However, bacteria can repair DNA damage via a series of intrinsic pathways including the SOS response. The master regulator of the SOS response is RecA, which was shown to be involved in an enhanced evolution of resistance to fluoroquinolone. Until very recently, the relationship between the evolution of β-lactam resistance and the SOS response remains undefined. Here, we find a superfast evolution of β-lactam resistance (20-fold MIC) following the deletion of RecA in Escherichia coli and treatment with a single dose of β-lactams in 8 hours. Importantly, once this type of resistance being established, it was stable and heritable. Controversially to previous findings, our results indicate this process is completely orthogonal to the SOS response but dependent on the hindrance of DNA repair. In addition, we observe explosive appearance of drug-specific mutations of the bacterial genome in 8 hours of exposure to icillin, such as the acrB mutations which is responsible for multi-drug resistance. Together, these findings demonstrate that hindrance of DNA repair not only generally antagonizes cells fitness, but also provides bacteria with genetic plasticity to adapt to erse stressful environments and can dramatically accelerates the evolution of antibiotic resistance in DNA repair deficiency cells. The evolution of antibiotic resistance can be induced by long-term exposure to antibiotics. However, we for the first time report a superfast evolution of multi-drug resistance induced by a single treatment with β-lactam in DNA repair deficiency Escherichia coli . More importantly, this type of evolutionary trajectory can cause a more rapid spread of drug-resistant bacteria in the community, because once the resistance being established, it was stable and heritable. In addition, from a clinical perspective, our finding significantly highlights the possibility that the synergistic drug combination between β-lactam and inhibitors targeting DNA repair system especially in the patients with cancer treatment can lead to a superfast evolution of multi-drug resistance.
Publisher: Cold Spring Harbor Laboratory
Date: 29-03-2022
DOI: 10.1101/2022.03.29.486199
Abstract: A recent work reports that the single treatment of β-lactams can cause a SOS-independent superfast evolution of multi-drug resistance in the DNA repair deficiency Escherichia coli ( E. coli ), but the mechanism is not yet clear. Here, we find that the induction of PinR, a lambdoid prophage Rac, is involved in this process and facilitates the evolution of antibiotic resistance in DNA repair deficiency bacteria through the repression on the transcription of antioxidative genes and the thereafter ROS burst in cells. It is highlighted that the bacteriophage PinR can orchestrate the mutagenesis induced by the overaccumulation of ROS in cells. More importantly, we for the first time demonstrate that the deletion of pinR can avoid the rapid evolution of antibiotic resistance induced by either the single or long-term exposure to antibiotics, while strategies to target RecA, e . g ., the inactivation on RecA, can be safely implemented to disarm the bacterial resistance to other antibiotics. Therefore, from a drug development perspective, our work suggests future studies on the “evolutionary potentiators” towards a safe and more effective strategy to be developed for infectious disease treatment.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 31-10-2023
Publisher: Cold Spring Harbor Laboratory
Date: 21-01-2019
DOI: 10.1101/525915
Abstract: Mammalian DNA replication is initiated at numerous replication origins, which are clustered into thousands of replication domains (RDs) across the genome. However, it remains unclear whether the replication origins within each RD are activated stochastically. To understand how replication is regulated at the sub-RD level, we directly visualized the spatio-temporal organization, morphology, and in situ epigenetic signatures of in idual replication foci (RFi) across S-phase using super-resolution stochastic optical reconstruction microscopy (STORM). Importantly, we revealed a hierarchical radial pattern of RFi propagation that reverses its directionality from early to late S-phase, and is diminished upon caffeine treatment or CTCF knockdown. Together with simulation and bioinformatic analyses, our findings point to a ‘CTCF-organized REplication Propagation’ (CoREP) model. The CoREP model suggests a non-random selection mechanism for replication activation mediated by CTCF at the sub-RD level, as well as the critical involvement of local chromatin environment in regulating replication in space and time.
Publisher: Springer Science and Business Media LLC
Date: 04-05-2019
Publisher: Springer US
Date: 2021
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C8SC01023D
Abstract: Ligand competition directs heterogeneous bio-chemistry surface and self-assembly for upconversion nanoparticles.
Publisher: Wiley
Date: 15-05-2015
DOI: 10.1111/OMI.12100
Abstract: Streptococcus mutans is a primary pathogen responsible for dental caries. It has an outstanding ability to form biofilm, which is vital for virulence. Previous studies have shown that knockout of Wall-associated protein A (WapA) affects cell chain and biofilm formation of S. mutans. As a surface protein, the distribution of WapA remains unknown, but it is important to understand the mechanism underlying the function of WapA. This study applied the fluorescence protein mCherry as a reporter gene to characterize the dynamic distribution of WapA in S. mutans via time-lapse and super-resolution fluorescence imaging. The results revealed interesting subcellular distribution patterns of WapA in single, iding and long chains of S. mutans cells. It appears at the middle of the cell and moves to the poles as the cell grows and ides. In a cell chain, after each round of cell ision, such dynamic relocation results in WapA distribution at the previous cell ision sites, resulting in a pattern where WapA is located at the boundary of two adjacent cell pairs. This WapA distribution pattern corresponds to the breaking segmentation of wapA deletion cell chains. The dynamic relocation of WapA through the cell cycle increases our understanding of the mechanism of WapA in maintaining cell chain integrity and biofilm formation.
Publisher: Future Medicine Ltd
Date: 08-2021
Publisher: Wiley
Date: 03-10-2022
DOI: 10.1002/CYTO.A.24504
Abstract: Sensitive and quantitative detection of molecular biomarkers is crucial for the early diagnosis of diseases like metabolic syndrome and cancer. Here we present a single‐molecule sandwich immunoassay by imaging the number of single nanoparticles to diagnose aggressive prostate cancer. Our assay employed the photo‐stable upconversion nanoparticles (UCNPs) as labels to detect the four types of circulating antigens in blood circulation, including glypican‐1 (GPC‐1), leptin, osteopontin (OPN), and vascular endothelial growth factor (VEGF), as their serum concentrations indicate aggressive prostate cancer. Under a wide‐field microscope, a single UCNP doped with thousands of lanthanide ions can emit sufficiently bright anti‐Stokes' luminescence to become quantitatively detectable. By counting every single streptavidin‐functionalized UCNP which specifically labeled on each sandwich immune complex across multiple fields of views, we achieved the Limit of Detection (LOD) of 0.0123 ng/ml, 0.2711 ng/ml, 0.1238 ng/ml, and 0.0158 ng/ml for GPC‐1, leptin, OPN and VEGF, respectively. The serum circulating level of GPC‐1, leptin, OPN, and VEGF in a mixture of 10 healthy normal human serum was 25.17 ng/ml, 18.04 ng/ml, 11.34 ng/ml, and 1.55 ng/ml, which was within the assay dynamic detection range for each analyte. Moreover, a 20% increase of GPC‐1 and OPN was observed by spiking the normal human serum with recombinant antigens to confirm the accuracy of the assay. We observed no cross‐reactivity among the four biomarker analytes, which eliminates the false positives and enhances the detection accuracy. The developed single upconversion nanoparticle‐assisted single‐molecule assay suggests its potential in clinical usage for prostate cancer detection by monitoring tiny concentration differences in a panel of serum biomarkers.
Publisher: American Chemical Society (ACS)
Date: 25-03-2020
Publisher: Springer Science and Business Media LLC
Date: 25-03-2019
Publisher: Springer Science and Business Media LLC
Date: 08-09-2020
DOI: 10.1038/S41467-020-18202-4
Abstract: A human cell contains hundreds to thousands of mitochondrial DNA (mtDNA) packaged into nucleoids. Currently, the segregation and allocation of nucleoids are thought to be passively determined by mitochondrial fusion and ision. Here we provide evidence, using live-cell super-resolution imaging, that nucleoids can be actively transported via KIF5B-driven mitochondrial dynamic tubulation (MDT) activities that predominantly occur at the ER-mitochondria contact sites (EMCS). We further demonstrate that a mitochondrial inner membrane protein complex MICOS links nucleoids to Miro1, a KIF5B receptor on mitochondria, at the EMCS. We show that such active transportation is a mechanism essential for the proper distribution of nucleoids in the peripheral zone of the cell. Together, our work identifies an active transportation mechanism of nucleoids, with EMCS serving as a key platform for the interplay of nucleoids, MICOS, Miro1, and KIF5B to coordinate nucleoids segregation and transportation.
Publisher: Springer Science and Business Media LLC
Date: 24-07-2015
DOI: 10.1038/CR.2015.89
Publisher: Cold Spring Harbor Laboratory
Date: 19-02-2022
DOI: 10.1101/2022.02.16.480800
Abstract: The intracellular metabolism of organelles, like lysosomes and mitochondria, are highly coordinated spatiotemporally and functionally. The activities of lysosomal enzymes significantly rely on the cytoplasmic temperature, and heat is constantly released by mitochondria as the byproduct of ATP generation during active metabolism. Here, we develop temperature-sensitive LysoDots and MitoDots to monitor the in situ thermodynamics of lysosomes and mitochondria. The design is based on upconversion nanoparticles (UCNPs) with high-density surface modifications to achieve the exceptionally high sensitivity of 2.7% K -1 and accuracy of 0.8 K for nanothermometry to be used in living cells. We show the measurement is independent of the intracellular ion concentrations- and pH values. With Ca 2+ ion shock, the temperatures of both lysosomes and mitochondria increased by 2∼4 °C. Intriguingly, with Chloroquine treatment, the lysosomal temperature was observed to decrease by up to ∼3 °C, while mitochondria remained relatively stable. Lastly, with oxidative phosphorylation inhibitor treatment, we observed a 3∼7 °C thermal increase and transition from mitochondria to lysosomes. These observations indicate different metabolic pathways and thermal transitions between lysosomes and mitochondria inside HeLa cells. The nanothermometry probes provide a powerful tool for multi-modality functional imaging of subcellular organelles and interactions with high spatial, temporal and thermal dynamics resolutions. Cascade organelle-targeted nano-thermometers based on upconversion LysoDots and MitoDots.
Publisher: American Chemical Society (ACS)
Date: 06-02-2021
Publisher: Proceedings of the National Academy of Sciences
Date: 02-11-2022
Abstract: The intracellular metabolism of organelles, like lysosomes and mitochondria, is highly coordinated spatiotemporally and functionally. The activities of lysosomal enzymes significantly rely on the cytoplasmic temperature, and heat is constantly released by mitochondria as the byproduct of adenosine triphosphate (ATP) generation during active metabolism. Here, we developed temperature-sensitive LysoDots and MitoDots to monitor the in situ thermal dynamics of lysosomes and mitochondria. The design is based on upconversion nanoparticles (UCNPs) with high-density surface modifications to achieve the exceptionally high sensitivity of 2.7% K −1 and low uncertainty of 0.8 K for nanothermometry to be used in living cells. We show the measurement is independent of the ion concentrations and pH values. With Ca 2+ ion shock, the temperatures of both lysosomes and mitochondria increased by ∼2 to 4 °C. Intriguingly, with chloroquine (CQ) treatment, the lysosomal temperature was observed to decrease by up to ∼3 °C, while mitochondria remained relatively stable. Lastly, with oxidative phosphorylation inhibitor treatment, we observed an ∼3 to 7 °C temperature increase and a thermal transition from mitochondria to lysosomes. These observations indicate different metabolic pathways and thermal transitions between lysosomes and mitochondria inside HeLa cells. The nanothermometry probes provide a powerful tool for multimodality functional imaging of subcellular organelles and interactions with high spatial, temporal, and thermal dynamics resolutions.
Publisher: Frontiers Media SA
Date: 23-06-2020
Publisher: American Chemical Society (ACS)
Date: 02-05-0018
DOI: 10.1021/ACS.NANOLETT.2C00724
Abstract: Cancer-derived small extracellular vesicles (sEVs) are potential circulating biomarkers in liquid biopsies. However, their small sizes, low abundance, and heterogeneity in molecular makeups pose major technical challenges for detecting and characterizing them quantitatively. Here, we demonstrate a single-sEV enumeration platform using lanthanide-doped upconversion nanoparticles (UCNPs). Taking advantage of the unique optical properties of UCNPs and the background-eliminating property of total internal reflection fluorescence (TIRF) imaging technique, a single-sEV assay recorded a limit of detection 1.8 × 10
Publisher: Springer New York
Date: 2019
DOI: 10.1007/978-1-4939-8873-0_9
Abstract: Autophagic lysosome reformation (ALR) is the terminal step of autophagy. ALR functions to recycle lysosomal membranes and maintain lysosome homeostasis. Maintaining a functional lysosome pool is critical for generating autolysosomes, in which cellular components are degraded and turned over during autophagy. This unit describes methods to visualize ALR in cells. In addition, this unit provides detailed protocols to establish in vitro systems which can be used to reconstitute ALR as well as to reconstitute mitochondrial tubulation/network formation, another process that is driven by motor proteins.
Publisher: Springer Science and Business Media LLC
Date: 17-07-2014
DOI: 10.1038/NCOMMS5443
Abstract: Imaging the location and dynamics of in idual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space. Here we develop a new method combining bimolecular fluorescence complementation and photoactivated localization microscopy for super-resolution imaging and single-molecule tracking of specific protein–protein interactions. The method is used to study the interaction of two abundant proteins, MreB and EF-Tu, in Escherichia coli cells. The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB–EF-Tu pairs as a subpopulation of total EF-Tu. The single-molecule tracking of MreB, EF-Tu and MreB–EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB–EF-Tu interactions.
Publisher: Elsevier BV
Date: 04-2021
Publisher: EMBO
Date: 27-01-2020
Publisher: Proceedings of the National Academy of Sciences
Date: 19-09-2016
Abstract: Myosin-5a is a molecular motor that functions as a cargo transporter in cells. The motor function of myosin-5a is regulated by calcium via the calmodulin bound to the first isoleucine-glutamine (IQ) motif (IQ1) of myosin-5a. Here, we solve the crystal structure of a truncated myosin-5a containing the motor domain and the IQ1 complexed with calcium-bound calmodulin. Comparison of the structures of the IQ1 complexed with calmodulin with or without bound calcium reveals the calcium-induced conformational changes of calmodulin. We demonstrated that calmodulin continuously associates with the IQ1 during that calcium transition and that the IQ1 binding substantially changes the thermodynamic and kinetics of calcium transition in calmodulin. These findings provide insight into the mechanism by which calcium regulates myosin-5a.
Publisher: Springer Science and Business Media LLC
Date: 10-04-2017
DOI: 10.1038/NCOMMS14951
Abstract: Kinesins hydrolyse ATP to transport intracellular cargoes along microtubules. Kinesin neck linker (NL) functions as the central mechano-chemical coupling element by changing its conformation through the ATPase cycle. Here we report the crystal structure of kinesin-6 Zen4 in a nucleotide-free, apo state, with the NL initial segment (NIS) adopting a backward-docked conformation and the preceding α6 helix partially melted. Single-molecule fluorescence resonance energy transfer (smFRET) analyses indicate the NIS of kinesin-1 undergoes similar conformational changes under tension in the two-head bound (2HB) state, whereas it is largely disordered without tension. The backward-docked structure of NIS is essential for motility of the motor. Our findings reveal a key missing conformation of kinesins, which provides the structural basis of the stable 2HB state and offers a tension-based rationale for an optimal NL length to ensure processivity of the motor.
Publisher: Wiley
Date: 03-2018
DOI: 10.1002/CPCB.44
Abstract: Autophagy is a lysosome-based degradation pathway. Autophagic lysosome reformation (ALR) is a lysosomal membrane recycling process that marks the terminal step of autophagy. During ALR, LAMP1-positive tubules, named reformation tubules, are extruded from autolysosomes, and nascent lysosomes are generated from these tubules. By combining proteomic analysis of purified autolysosomes and RNA interference screening of identified candidates, we systematically elucidated the ALR pathway at the molecular level. Based on the key components clathrin, PtdIns(4,5)P
Publisher: Cold Spring Harbor Laboratory
Date: 19-11-0011
DOI: 10.1101/2020.11.29.402818
Abstract: Temperature dynamics reflect the physiological conditions of cells and organisms. Mitochondria regulates temperature dynamics in living cells, as they oxidize the respiratory substrates and synthesize ATP, with heat being released as a by-product of active metabolism. Here, we report an upconversion nanoparticles based thermometer that allows in situ thermal dynamics monitoring of mitochondria in living cells. We demonstrate that the upconversion nanothermometers can efficiently target mitochondria and the temperature responsive feature is independent of probe concentration and medium conditions. The relative sensing sensitivity of 3.2% K −1 in HeLa cells allows us to measure the mitochondrial temperature difference through the stimulations of high glucose, lipid, Ca 2+ shock and the inhibitor of oxidative phosphorylation. Moreover, cells display distinct response time and thermal dynamic profiles under different stimulations, which highlights the potential applications of this thermometer to study in situ vital processes related to mitochondrial metabolism pathways and interactions between organelles.
Publisher: Elsevier BV
Date: 05-2016
DOI: 10.1016/J.DEVCEL.2016.04.014
Abstract: Autophagic lysosome reformation (ALR) plays an important role in maintaining lysosome homeostasis. During ALR, lysosomes are reformed by recycling lysosomal components from autolysosomes. The most noticeable step of ALR is autolysosome tubulation, but it is currently unknown how the process is regulated. Here, using an approach combining in vivo studies and in vitro reconstitution, we found that the kinesin motor protein KIF5B is required for autolysosome tubulation and that KIF5B drives autolysosome tubulation by pulling on the autolysosomal membrane. Furthermore, we show that KIF5B directly interacts with PtdIns(4,5)P2. Kinesin motors are recruited and clustered on autolysosomes via interaction with PtdIns(4,5)P2 in a clathrin-dependent manner. Finally, we demonstrate that clathrin promotes formation of PtdIns(4,5)P2-enriched microdomains, which are required for clustering of KIF5B. Our study reveals a mechanism by which autolysosome tubulation was generated.
Publisher: Elsevier BV
Date: 09-2021
Publisher: Springer Science and Business Media LLC
Date: 07-04-2016
DOI: 10.1038/SREP24002
Abstract: Intracellular membrane nanotube formation and its dynamics play important roles for cargo transportation and organelle biogenesis. Regarding the regulation mechanisms, while much attention has been paid on the lipid composition and its associated protein molecules, effects of the vesicle size has not been studied in the cell. Giant unilamellar vesicles (GUVs) are often used for in vitro membrane deformation studies, but they are much larger than most intracellular vesicles and the in vitro studies also lack physiological relevance. Here, we use lysosomes and autolysosomes, whose sizes range between 100 nm and 1 μm, as model systems to study the size effects on nanotube formation both in vivo and in vitro . Single molecule observations indicate that driven by kinesin motors, small vesicles (100–200 nm) are mainly transported along the tracks while a remarkable portion of large vesicles (500–1000 nm) form nanotubes. This size effect is further confirmed by in vitro reconstitution assays on liposomes and purified lysosomes and autolysosomes. We also apply Atomic Force Microscopy (AFM) to measure the initiation force for nanotube formation. These results suggest that the size-dependence may be one of the mechanisms for cells to regulate cellular processes involving membrane-deformation, such as the timing of tubulation-mediated vesicle recycling.
Publisher: Springer Science and Business Media LLC
Date: 18-08-2018
Publisher: Springer Science and Business Media LLC
Date: 17-08-2018
DOI: 10.1038/S41467-018-05842-W
Abstract: Multiphoton fluorescence microscopy (MPM), using near infrared excitation light, provides increased penetration depth, decreased detection background, and reduced phototoxicity. Using stimulated emission depletion (STED) approach, MPM can bypass the diffraction limitation, but it requires both spatial alignment and temporal synchronization of high power (femtosecond) lasers, which is limited by the inefficiency of the probes. Here, we report that upconversion nanoparticles (UCNPs) can unlock a new mode of near-infrared emission saturation (NIRES) nanoscopy for deep tissue super-resolution imaging with excitation intensity several orders of magnitude lower than that required by conventional MPM dyes. Using a doughnut beam excitation from a 980 nm diode laser and detecting at 800 nm, we achieve a resolution of sub 50 nm, 1/20th of the excitation wavelength, in imaging of single UCNP through 93 μm thick liver tissue. This method offers a simple solution for deep tissue super resolution imaging and single molecule tracking.
Publisher: Springer Science and Business Media LLC
Date: 03-08-2015
DOI: 10.1038/NCB3215
Abstract: A fundamental question regarding autophagosome formation is how the shape of the double-membrane autophagosomal vesicle is generated. Here we show that in mammalian cells assembly of an actin scaffold inside the isolation membrane (the autophagosomal precursor) is essential for autophagosomal membrane shaping. Actin filaments are depolymerized shortly after starvation and actin is assembled into a network within the isolation membrane. When formation of actin puncta is disrupted by an actin polymerization inhibitor or by knocking down the actin-capping protein CapZβ, isolation membranes and omegasomes collapse into mixed-membrane bundles. Formation of actin puncta is PtdIns(3)P dependent, and inhibition of PtdIns(3)P formation by treating cells with the PI(3)K inhibitor 3-MA, or by knocking down Beclin-1, abolishes the formation of actin puncta. Binding of CapZ to PtdIns(3)P, which is enriched in omegasomes, stimulates actin polymerization. Our findings illuminate the mechanism underlying autophagosomal membrane shaping and provide key insights into how autophagosomes are formed.
Publisher: Springer Science and Business Media LLC
Date: 25-09-2015
DOI: 10.1038/CR.2015.115
Start Date: 12-2020
End Date: 12-2023
Amount: $469,000.00
Funder: Australian Research Council
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