ORCID Profile
0000-0002-9929-2909
Current Organisation
Monash University
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Developmental Genetics (incl. Sex Determination) | Biochemistry and Cell Biology | Biochemistry and Cell Biology not elsewhere classified | Regenerative Medicine (incl. Stem Cells and Tissue Engineering) |
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Medical and Health Sciences
Publisher: Springer Science and Business Media LLC
Date: 30-05-2023
DOI: 10.1038/S41467-023-38436-2
Abstract: The spatial sorting of RNA transcripts is fundamental for the refinement of gene expression to distinct subcellular regions. Although, in non-mammalian early embryogenesis, differential RNA localisation presages cell fate determination, in mammals it remains unclear. Here, we uncover apical-to-basal RNA asymmetries in outer blastomeres of 16-cell stage mouse preimplantation embryos. Basally directed RNA transport is facilitated in a microtubule- and lysosome-mediated manner. Yet, despite an increased accumulation of RNA transcripts in basal regions, higher translation activity occurs at the more dispersed apical RNA foci, demonstrated by spatial heterogeneities in RNA subtypes, RNA-organelle interactions and translation events. During the transition to the 32-cell stage, the biased inheritance of RNA transcripts, coupled with differential translation capacity, regulates cell fate allocation of trophectoderm and cells destined to form the pluripotent inner cell mass. Our study identifies a paradigm for the spatiotemporal regulation of post-transcriptional gene expression governing mammalian preimplantation embryogenesis and cell fate.
Publisher: MyJove Corporation
Date: 30-11-2021
DOI: 10.3791/63290
Abstract: The microtubule cytoskeleton forms the framework of a cell and is fundamental for intracellular transport, cell ision, and signal transduction. Traditional pharmacological disruption of the ubiquitous microtubule network using, for instance, nocodazole can have devastating consequences for any cell. Reversibly photoswitchable microtubule inhibitors have the potential to overcome the limitations by enabling drug effects to be implemented in a spatiotemporally-controlled manner. One such family of drugs is the azobenzene-based photostatins (PSTs). These compounds are inactive in dark conditions, and upon illumination with UV light, they bind to the colchicine-binding site of β-tubulin and block microtubule polymerization and dynamic turnover. Here, the application of PSTs in the 3-dimensional (3D) live preimplantation mouse embryo is set out to disrupt the microtubule network on a subcellular level. This protocol provides instructions for the experimental setup, as well as light activation and deactivation parameters for PSTs using live-cell confocal microscopy. This ensures reproducibility and enables others to apply this procedure to their research questions. Innovative photoswitches like PSTs may evolve as powerful tools to advance the understanding of the dynamic intracellular microtubule network and to non-invasively manipulate the cytoskeleton in real-time. Furthermore, PSTs may prove useful in other 3D structures such as organoids, blastoids, or embryos of other species.
Publisher: Wiley
Date: 21-05-2014
DOI: 10.1002/GLIA.22696
Abstract: Peripheral myelin protein 2 (Pmp2, P2 or Fabp8), a member of the fatty acid binding protein family, was originally described together with myelin basic protein (Mbp or P1) and myelin protein zero (Mpz or P0) as one of the most abundant myelin proteins in the peripheral nervous system (PNS). Although Pmp2 is predominantly expressed in myelinated Schwann cells, its role in glia is currently unknown. To study its function in PNS biology, we have generated a complete Pmp2 knockout mouse (Pmp2(-/-) ). Comprehensive characterization of Pmp2(-/-) mice revealed a temporary reduction in their motor nerve conduction velocity (MNCV). While this change was not accompanied by any defects in general myelin structure, we detected transitory alterations in the myelin lipid profile of Pmp2(-/-) mice. It was previously proposed that Pmp2 and Mbp have comparable functions in the PNS suggesting that the presence of Mbp can partially mask the Pmp2(-/-) phenotype. Indeed, we found that Mbp lacking Shi(-/-) mice, similar to Pmp2(-/-) animals, have preserved myelin structure and reduced MNCV, but this phenotype was not aggravated in Pmp2(-/-) /Shi(-/-) mutants indicating that Pmp2 and Mbp do not substitute each other's functions in the PNS. These data, together with our observation that Pmp2 binds and transports fatty acids to membranes, uncover a role for Pmp2 in lipid homeostasis of myelinating Schwann cells.
Publisher: Elsevier BV
Date: 08-2022
DOI: 10.1016/J.CEB.2022.102114
Abstract: Mammalian preimplantation embryogenesis depends on the spatio-temporal dynamics of the microtubule cytoskeleton to enable exceptionally fast changes in cell number, function, architecture, and fate. Microtubule organising centres (MTOCs), which coordinate the remodelling of microtubules, are therefore of fundamental significance during the first days of a new life. Despite its indispensable role during early mammalian embryogenesis, the origin of microtubule growth remains poorly understood. In this review, we summarise the most recent discoveries on microtubule organisation and function during early human embryogenesis and compare these to innovative studies conducted in alternative mammalian models. We emphasise the differences and analogies of centriole inheritance and their role during the first cleavage. Furthermore, we highlight the significance of non-centrosomal MTOCs for embryo viability and discuss the potential of novel in vitro models and light-inducible approaches towards unravelling microtubule formation in research and assisted reproductive technologies.
Publisher: The Company of Biologists
Date: 15-10-2021
DOI: 10.1242/DEV.199909
Abstract: With the advancement of cutting-edge live imaging technologies, microtubule remodelling has evolved as an integral regulator for the establishment of distinct differentiated cells. However, despite their fundamental role in cell structure and function, microtubules have received less attention when unravelling the regulatory circuitry of pluripotency. Here, we summarise the role of microtubule organisation and microtubule-dependent events required for the formation of pluripotent cells in vivo by deciphering the process of early embryogenesis: from fertilisation to blastocyst. Furthermore, we highlight current advances in elucidating the significance of specific microtubule arrays in in vitro culture systems of pluripotent stem cells and how the microtubule cytoskeleton serves as a highway for the precise intracellular movement of organelles. This Review provides an informed understanding of the intrinsic role of subcellular architecture of pluripotent cells and accentuates their regenerative potential in combination with innovative light-inducible microtubule techniques.
Publisher: Springer Science and Business Media LLC
Date: 29-06-2017
Abstract: Probing transcription factor (TF)-DNA interactions remains challenging in complex in vivo systems such as mammalian embryos, especially when TF copy numbers and fluorescence background are high. To address this difficulty, fluorescence correlation spectroscopy (FCS) can be combined with the use of photoactivatable fluorescent proteins to achieve selective photoactivation of a subset of tagged TF molecules. This approach, termed paFCS, enables FCS measurements within single cell nuclei inside live embryos, and obtains autocorrelation data of a quality previously only attainable in simpler in vitro cell culture systems. Here, we present a protocol demonstrating the applicability of paFCS in developing mouse embryos by outlining its implementation on a commercial laser-scanning microscope. We also provide procedures for optimizing the photoactivation and acquisition parameters and determining key parameters describing TF-DNA binding. The entire procedure can be performed within ∼2 d (excluding embryo culture time), although the acquisition of each paFCS data set takes only ∼10 min. This protocol can be used to noninvasively reveal cell-to-cell variation in TF dynamics, as well as critical, fate-predicting changes over the course of early embryonic development.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 09-2017
Abstract: Cell functions ranging from cell ision to morphogenesis rely on microtubules, with microtubule-organizing centers serving as anchoring sites for their outgrowth. Although the centrosome organizes the microtubule cytoskeleton in most animal cells, this organelle is absent in early development. Using live-cell imaging, Zenker et al. found that the cells of the early mouse embryo are connected by stable microtubule bridges to direct the growth of microtubules within them. Microtubules emanating from the bridges help to guide transport of key proteins, including E-cadherin, to the cell membrane to control cell polarization during early development. Science , this issue p. 925
Publisher: Public Library of Science (PLoS)
Date: 10-04-2015
Publisher: Elsevier BV
Date: 09-2021
DOI: 10.1016/J.TCB.2021.03.010
Abstract: Delta (δ-) and epsilon (ε-) tubulin are lesser-known cousins of alpha (α-) and beta (β-) tubulin. They are likely to regulate centriole function in a broad range of species however, their in vivo role and mechanism of action in mammals remain mysterious. In unicellular species and mammalian cell lines, mutations in δ- and ε-tubulin cause centriole destabilization and atypical mitosis and, in the most severe cases, cell death. Beyond the centriole, δ- and ε-tubulin localize to the manchette during murine spermatogenesis and interact with katanin-like 2 (KATNAL2), a protein with microtubule (MT)-severing properties, indicative of novel non-centriolar functions. Herein we summarize the current knowledge surrounding δ- and ε-tubulin, identify pathways for future research, and highlight how and why spermatogenesis and embryogenesis are ideal systems to define δ- and ε-tubulin function in vivo.
Publisher: Oxford University Press (OUP)
Date: 15-05-2014
DOI: 10.1093/HMG/DDU238
Abstract: The microtubule cytoskeleton is critical for the generation and maturation of neurons in the developing mammalian nervous system. We have previously shown that mutations in the β-tubulin gene TUBB5 cause microcephaly with structural brain abnormalities in humans. While it is known that TUBB5 is necessary for the proper generation and migration of neurons, little is understood of the role it plays in neuronal differentiation and connectivity. Here, we report that perturbations to TUBB5 disrupt the morphology of cortical neurons, their neuronal complexity, axonal outgrowth, as well as the density and shape of dendritic spines in the postnatal murine cortex. The features we describe are consistent with defects in synaptic signaling. Cellular-based assays have revealed that TUBB5 substitutions have the capacity to alter the dynamic properties and polymerization rates of the microtubule cytoskeleton. Together, our studies show that TUBB5 is essential for neuronal differentiation and dendritic spine formation in vivo, providing insight into the underlying cellular pathology associated with TUBB5 disease states.
Publisher: Springer Science and Business Media LLC
Date: 17-03-2021
Publisher: Springer Science and Business Media LLC
Date: 26-04-2017
DOI: 10.1007/S10571-017-0494-0
Abstract: The protein component of the myelin layer is essential for all aspects of peripheral nerves, and its deficiency can lead to structural and functional impairment. The presence of peripheral myelin protein 2 (P2, PMP2, FABP8, M-FABP) in Schwann cells has been known for decades and shown recently to be involved in the lipid homeostasis in the peripheral neural system. However, its precise role during de- and remyelination has yet to be elucidated. To this end, we assessed remyelination after sciatic nerve crush injury in vivo, and in an experimental de/remyelination ex vivo myelinating culture model in P2-deficient (P2
Publisher: Cold Spring Harbor Laboratory
Date: 22-10-2022
DOI: 10.1101/2022.10.22.513321
Abstract: Circular RNAs (circRNAs) are covalently closed single stranded RNAs that are produced by RNA back-splicing. A small number of circRNAs have been implicated as functional, however, we still lack systematic understanding of cellular processes and signalling pathways that are regulated by circRNAs. A major gap in understanding circRNA function is the ability to define pathways that are regulated by circRNAs. Here, we generated a pooled shRNA library targeting the back-splice junction of 3,354 human circRNAs that are expressed at low to high levels in humans. We used this library for loss of function proliferation screens in a panel of 18 cancer cell lines from four tissue types that harbour mutations leading to constitutive activity of defined pathways. Using this dataset, we identify context specific and non-specific circRNAs. We validated these observations with a secondary screen and uncovered a role for circRERE , a cell essential circRNA that regulates ferroptosis. Furthermore, we characterised the functional roles of pathway-specific circRNA, circSMAD2 , a novel regulator of the WNT pathway and circMTO1 , a regulator of MAPK signalling in a PTEN dependent manner. Our work sheds light on molecular pathways regulated by circRNAs and provides a catalogue of circRNAs with a measurable function in human cells.
Start Date: 2015
End Date: 2018
Funder: Human Frontier Science Program
View Funded ActivityStart Date: 2019
End Date: 2021
Funder: Azrieli Foundation
View Funded ActivityStart Date: 2014
End Date: 2014
Funder: Deutsche Forschungsgemeinschaft
View Funded ActivityStart Date: 2013
End Date: 2013
Funder: Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
View Funded ActivityStart Date: 2021
End Date: 2024
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2021
End Date: 2022
Funder: Canadian Institute for Advanced Research
View Funded ActivityStart Date: 05-2022
End Date: 12-2022
Amount: $800,000.00
Funder: Australian Research Council
View Funded Activity