ORCID Profile
0000-0003-2936-7853
Current Organisation
University of New South Wales
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Protein trafficking | Biochemistry and cell biology | Cellular interactions (incl. adhesion matrix cell wall) | Mechanobiology
Publisher: Springer Science and Business Media LLC
Date: 2021
Publisher: Cold Spring Harbor Laboratory
Date: 23-11-2022
DOI: 10.1101/2022.11.22.516823
Abstract: Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense, and compacted environment of the cytoplasm remains challenging. Here we have developed a novel cryogenic correlative light and electron microscopy (cryo-CLEM) workflow which combines thin cells grown on a mechanically defined substratum to rapidly analyse organelles and macromolecular complexes in the cell by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. We have developed a protocol where cells can be frozen, imaged by cryo-fluorescence microscopy and ready for batch cryo-ET within a day.
Publisher: Cold Spring Harbor Laboratory
Date: 20-12-2021
DOI: 10.1101/2021.12.19.473396
Abstract: Association with microtubules inhibits the fission of mitochondria in Schizosachharomyces pombe . Here we show that this attachment of mitochondria to microtubules is an important cell intrinsic factor in determining ision symmetry. By comparing mutant cells that exhibited enhanced attachment and no attachment of mitochondria to microtubules (Dnm1Δ and Mmb1Δ respectively), we show that microtubules in these mutants displayed aberrant dynamics compared to wild-type cells, which resulted in errors in nuclear positioning. This translated to cell ision asymmetry in a significant proportion of both Dnm1Δ and Mmb1Δ cells. Asymmetric ision in Dnm1Δ and Mmb1Δ cells resulted in unequal distribution of mitochondria, with the daughter cell that received more mitochondria growing faster than the other daughter. Taken together, we show the existence of homeostatic feedback controls between mitochondria and microtubules in fission yeast, which directly influence mitochondrial partitioning and thereby, cell growth.
Publisher: Cold Spring Harbor Laboratory
Date: 27-10-2023
Publisher: Springer Science and Business Media LLC
Date: 2021
Publisher: Bio-Protocol, LLC
Date: 2019
Publisher: The Company of Biologists
Date: 03-2023
DOI: 10.1242/JCS.260749
Abstract: Motor proteins are key players in exerting spatiotemporal control over the intracellular location of membrane-bound compartments, including endosomes containing cargo. In this Review, we focus on how motors and their cargo adaptors regulate positioning of cargoes from the earliest stages of endocytosis and through the two main intracellular itineraries: (1) degradation at the lysosome or (2) recycling back to the plasma membrane. In vitro and cellular (in vivo) studies on cargo transport thus far have typically focussed independently on either the motor proteins and adaptors, or membrane trafficking. Here, we will discuss recent studies to highlight what is known about the regulation of endosomal vesicle positioning and transport by motors and cargo adaptors. We also emphasise that in vitro and cellular studies are often performed at different scales, from single molecules to whole organelles, with the aim to provide a perspective on the unified principles of motor-driven cargo trafficking in living cells that can be learned from these differing scales.
Publisher: Springer Science and Business Media LLC
Date: 12-02-2021
Publisher: The Company of Biologists
Date: 2023
DOI: 10.1242/JCS.260705
Abstract: Association with microtubules inhibits the fission of mitochondria in Schizosaccharomyces pombe. Here, we show that this attachment of mitochondria to microtubules is an important cell-intrinsic factor in determining cell ision symmetry. By comparing mutant cells that exhibited enhanced attachment and no attachment of mitochondria to microtubules (Dnm1Δ and Mmb1Δ, respectively), we show that microtubules in these mutants displayed aberrant dynamics compared to wild-type cells, which resulted in errors in nuclear positioning. This translated to cell ision asymmetry in a significant proportion of both Dnm1Δ and Mmb1Δ cells. Asymmetric ision in Dnm1Δ and Mmb1Δ cells resulted in unequal distribution of mitochondria, with the daughter cell that received more mitochondria growing faster than the other daughter cell. Taken together, we show the existence of homeostatic feedback controls between mitochondria and microtubules in fission yeast, which directly influence mitochondrial partitioning and, thereby, cell growth. This article has an associated First Person interview with the first author of the paper.
Publisher: Elsevier
Date: 2015
DOI: 10.1016/BS.MCB.2014.10.001
Abstract: While early fluorescence microscopy experiments employing fluorescent probes afforded snapshots of the cell, the power of live-cell microscopy is required to understand complex dynamics in biological processes. The first successful cloning of green fluorescent protein in the 1990s paved the way for development of approaches that we now utilize for visualization in a living cell. In this chapter, we discuss a technique to observe fluorescently tagged single molecules in fission yeast. With a few simple modifications to the established total internal reflection fluorescence microscopy, cytoplasmic dynein molecules in the cytoplasm and on the microtubules can be visualized and their intracellular dynamics can be studied. We illustrate a technique to study motor behavior, which is not apparent in conventional ensemble studies of motors. In general, this technique can be employed to study single-molecule dynamics of fluorescently tagged proteins in the cell interior.
Publisher: Cold Spring Harbor Laboratory
Date: 21-08-2017
DOI: 10.1101/178913
Abstract: Mitochondria are organized as tubular networks in the cell and undergo fission and fusion. While several of the molecular players involved in mediating mitochondrial dynamics have been identified, the precise cellular cues that initiate fission or fusion remain largely unknown. In fission yeast, mitochondria are organized along microtubule bundles. Here, we employed deletions of kinesin-like proteins to perturb microtubule dynamics, and determined that cells with long microtubules exhibited long, but fewer mitochondria, whereas cells with short microtubules exhibited short, but several mitochondria due to reduced mitochondrial fission in the former and elevated fission in the latter. Correspondingly, upon onset of closed mitosis in fission yeast, wherein interphase microtubules assemble to form the spindle within the nucleus, we measured increased mitochondrial fission. We determined that the consequent rise in the mitochondrial copy number was necessary to reduce partitioning errors while stochastically partitioning mitochondria between daughter cells. We discovered that the association of mitochondria with microtubules physically impeded the assembly of the fission protein Dnm1 around mitochondria, resulting in inhibition of mitochondrial fission. Taken together, we demonstrate a novel mechanism for regulation of mitochondrial fission that is dictated by the interaction between mitochondria and the microtubule cytoskeleton.
Publisher: Rockefeller University Press
Date: 03-10-2019
Abstract: During sexual reproduction in eukaryotes, processes such as active degradation and dilution of paternal mitochondria ensure maternal mitochondrial inheritance. In the isogamous organism fission yeast, we employed high-resolution fluorescence microscopy to visualize mitochondrial inheritance during meiosis by differentially labeling mitochondria of the two parental cells. Remarkably, mitochondria, and thereby mitochondrial DNA from the parental cells, did not mix upon zygote formation but remained segregated at the poles by attaching to clusters of the anchor protein Mcp5 via its coiled-coil domain. We observed that this tethering of parental mitochondria to the poles results in uniparental inheritance of mitochondria, wherein two of the four spores formed subsequently contained mitochondria from one parent and the other spores contained mitochondria from the other parent. Further, the presence of dynein on an Mcp5 cluster precluded the attachment of mitochondria to the same cluster. Taken together, we reveal a distinct mechanism that achieves uniparental inheritance by segregation of parental mitochondria.
Publisher: The Company of Biologists
Date: 15-04-2020
DOI: 10.1242/JCS.245928
Abstract: The Molecular Motors, Transport and Trafficking (M2T2) meeting serves as a platform for both Indian and global scientists working on the cytoskeleton, cytoskeletal motors and membrane trafficking to gather and discuss the latest developments in the field. The 2019 edition of the meeting, held from 18–20 October at the National Brain Research Centre (NBRC), Manesar, India and organised by Mahak Sharma (Indian Institute of Science Education and Research, Mohali) and Anindya Ghosh Roy (NBRC), was witness to stimulating research on a range of topics related to the cytoskeleton, including cytoskeletal organization, motor protein function and regulation, mechanical forces and vesicular transport, and trafficking in health and disease.
Publisher: Cold Spring Harbor Laboratory
Date: 11-07-2023
DOI: 10.1101/2023.07.10.548473
Abstract: Protein aggregation is a common underlying feature of neurodegenerative disorders. Cells expressing neurodegeneration–associated mutant proteins show altered uptake of ligands, suggestive of impaired endocytosis, in a manner as yet unknown. Using live cell imaging, we show that clathrin-mediated endocytosis (CME) is affected due to altered actin cytoskeletal organization in the presence of Huntingtin aggregates. Additionally, we find that cells containing Huntingtin aggregates are stiffer and less viscous than their wild-type counterparts due to altered actin conformation, and not merely due to the physical presence of aggregate(s). We further demonstrate that CME and cellular viscosity can be rescued by overexpressing Hip1, Arp2/3 or transient LatrunculinA treatment. Examination of other pathogenic aggregates revealed that only a subset of these display defective CME, along with altered actin organization and increased stiffness. Together, our results point to an intimate connection between functional CME, actin organization and cellular stiffness in the context of neurodegeneration.
Publisher: American Chemical Society (ACS)
Date: 24-02-2017
Publisher: Royal Society of Chemistry (RSC)
Date: 2022
DOI: 10.1039/D2SM00155A
Abstract: Mitochondrial populations in cells are maintained by cycles of fission and fusion events, which are linked to microtubule dynamics.
Publisher: Springer Science and Business Media LLC
Date: 25-04-2023
Publisher: Proceedings of the National Academy of Sciences
Date: 14-03-2017
Abstract: Molecular motors perform a variety of functions such as muscle movement and nuclear positioning during cell ision. One such motor protein, cytoplasmic dynein, is responsible for oscillations of the nucleus during meiosis of the unicellular eukaryote, fission yeast. In this study, we used genetics, live-cell fluorescence microscopy, and image processing to identify the key players that keep dynein at the cortex while it performs the function of back-and-forth nuclear movement by producing force on the microtubule. We discovered that the membrane phospholipid PI(4,5)P 2 and, surprisingly, the myosin I motor Myo1 are both indispensable for nuclear oscillations in fission yeast. This study thus demonstrates one of the few instances of interplay between different classes of motor proteins in the cell.
Publisher: Wiley
Date: 03-05-2016
Abstract: Cytoplasmic dynein is the major minus-end-directed motor protein in eukaryotes, and has functions ranging from organelle and vesicle transport to spindle positioning and orientation. The mode of regulation of dynein in the cell remains elusive, but a tantalising possibility is that dynein is maintained in an inhibited, non-motile state until bound to cargo. In vivo, stable attachment of dynein to the cell membrane via anchor proteins enables dynein to produce force by pulling on microtubules and serves to organise the nuclear material. Anchor proteins of dynein assume erse structures and functions and differ in their interaction with the membrane. In yeast, the anchor protein has come to the fore as one of the key mediators of dynein activity. In other systems, much is yet to be discovered about the anchors, but future work in this area will prove invaluable in understanding dynein regulation in the cell.
Publisher: Optica Publishing Group
Date: 02-01-2014
DOI: 10.1364/OE.22.000210
Publisher: Elsevier BV
Date: 03-2019
Publisher: Cold Spring Harbor Laboratory
Date: 05-04-2021
DOI: 10.1101/2021.04.05.438428
Abstract: Cytoplasmic dynein 1 (dynein) is the primary minus end-directed motor protein in most eukaryotic cells. Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin and a cargo adaptor. How this process of dynein activation occurs is unclear, since it entails the formation of a three-protein complex inside the crowded environs of a cell. Here, we employed live-cell, single-molecule imaging to visualise and track fluorescently tagged dynein. First, we observed that only ~30% of dynein molecules that bound to the microtubule (MT) engaged in minus end-directed movement, and that too for a short duration of ~0.6 s. Next, using high-resolution imaging in live and fixed cells, and using correlative light and electron microscopy, we discovered that dynactin and endosomal cargo remained in proximity to each other and to MTs. We then employed two-colour imaging to visualise cargo movement effected by single motor binding. Finally, we performed long-term imaging to show short movements are sufficient to drive cargo to the perinuclear region of the cell. We then used these discoveries as the basis for a stochastic model incorporating dynamic motors binding to cargo located along MTs, and also developed a coarse-grained 3-state run- and-tumble particle (RTP) model for the cargo that quantitatively recapitulates the emergent statistics of cargo movement. Taken together, we discovered a search mechanism that is facilitated by dynein’s frequent MT binding-unbinding kinetics: (1) in a futile event when dynein does not encounter cargo anchored in proximity to the MT, dynein dissociates and diffuses into the cytoplasm, (2) when dynein encounters cargo and dynactin upon MT-binding, it moves cargo in a short run. Several of these short runs are undertaken in succession for long-range directed movement. In conclusion, we demonstrate that dynein activation and cargo capture are coupled in a step that relies on the reduction of dimensionality to enable minus end-directed transport in cellulo , and that complex cargo behaviour emerges from stochastic motor-cargo interactions.
Publisher: Springer Science and Business Media LLC
Date: 2010
Publisher: American Chemical Society (ACS)
Date: 08-10-2019
DOI: 10.1021/ACS.BIOCHEM.9B00772
Abstract: Cytoplasmic dynein, the major minus end-directed motor protein in several cell types, transports a variety of intracellular cargo upon forming a processive tripartite complex with its activator dynactin and cargo adaptors such as Hook3 and BicD2. Our current understanding of dynein regulation stems from a combination of
Publisher: Cold Spring Harbor Laboratory
Date: 19-01-2019
DOI: 10.1101/525196
Abstract: During sexual reproduction in eukaryotes, processes such as active degradation and dilution of paternal mitochondria ensure maternal mitochondrial inheritance. In the isogamous organism fission yeast, we employed high-resolution fluorescence microscopy to visualize mitochondrial inheritance during meiosis by differentially labeling mitochondria of the two parental cells. Remarkably, mitochondria, and thereby, mitochondrial DNA from the parental cells did not mix upon zygote formation, but remained segregated at the poles by attaching to clusters of the dynein anchor Mcp5 via its coiled-coil domain. We observed that this tethering of parental mitochondria to the poles results in uniparental inheritance of mitochondria, wherein two of the four spores formed subsequently contained mitochondria from one parent and the other spores, mitochondria from the other parent. Further, the presence of dynein on an Mcp5 cluster precluded the attachment of mitochondria to the same cluster. Taken together, we reveal a distinct mechanism that achieves uniparental inheritance by segregation of parental mitochondria.
Publisher: Springer Science and Business Media LLC
Date: 03-06-2020
Publisher: Elsevier BV
Date: 02-2023
Location: Germany
Location: India
Start Date: 2023
End Date: 12-2023
Amount: $1,009,078.00
Funder: Australian Research Council
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