ORCID Profile
0000-0003-4855-8284
Current Organisation
UNSW Sydney
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Publisher: Elsevier BV
Date: 02-2011
DOI: 10.1016/J.SEMCDB.2010.12.003
Abstract: The actin cytoskeleton in eukaryotic cells provides cell structure and organisation, and allows cells to generate forces against membranes. As such it is a central component of a variety of cellular structures involved in cell motility, cytokinesis and vesicle trafficking. In multicellular organisms these processes contribute towards embryonic development and effective functioning of cells of all types, most obviously rapidly moving cells like lymphocytes. Actin also defines and maintains the architecture of complex structures such as neuronal synapses and stereocillia, and is required for basic housekeeping tasks within the cell. It is therefore not surprising that misregulation of the actin cytoskeleton can cause a variety of disease pathologies, including compromised immunity, neurodegeneration, and cancer spread. Dictyostelium discoideum has long been used as a tool for dissecting the mechanisms by which eukaryotic cells migrate and chemotax, and recently it has gained precedence as a model organism for studying the roles of conserved pathways in disease processes. Dictyostelium's unusual lifestyle, positioned between unicellular and multicellular organisms, combined with ease of handling and strong conservation of actin regulatory machinery with higher animals, make it ideally suited for studying actin-related diseases. Here we address how research in Dictyostelium has contributed to our understanding of immune deficiencies and neurological defects in humans, and briefly discuss its future prospects for furthering our understanding of neurodegenerative disorders.
Publisher: Elsevier BV
Date: 2022
Publisher: Cold Spring Harbor Laboratory
Date: 31-08-2020
DOI: 10.1101/2020.08.31.243592
Abstract: To advance our understanding of cardiomyocyte identity and function, we need appropriate tools to isolate pure primary cardiomyocytes. We have developed a label-free method to purify viable cardiomyocytes from mouse neonatal hearts using a simple inertial microfluidics biochip. Cardiomyocytes were sorted from neonatal hearts and isolated to % purity and their physico-mechanical properties were evaluated using real time deformability cytometry. Purified cardiomyocytes were viable and retained their identity and function as depicted by expression of cardiac specific markers and contractility. Furthermore, we showed that cardiomyocytes have a distinct physico-mechanical phenotype that could be used as an intrinsic biophysical marker to distinguish these cells from other cell types within the heart. Taken together, this cardiomyocyte isolation and phenotyping method could serve as a valuable tool to progress our understanding of cardiomyocyte identity and function, which will ultimately benefit many diagnostic development and cardiac treatment studies.
Publisher: Ivyspring International Publisher
Date: 2021
DOI: 10.7150/THNO.63763
Publisher: Rockefeller University Press
Date: 23-05-2011
Abstract: WASP and SCAR homologue (WASH) is a recently identified and evolutionarily conserved regulator of actin polymerization. In this paper, we show that WASH coats mature Dictyostelium discoideum lysosomes and is essential for exocytosis of indigestible material. A related process, the expulsion of the lethal endosomal pathogen Cryptococcus neoformans from mammalian macrophages, also uses WASH-coated vesicles, and cells expressing dominant negative WASH mutants inefficiently expel C. neoformans. D. discoideum WASH causes filamentous actin (F-actin) patches to form on lysosomes, leading to the removal of vacuolar adenosine triphosphatase (V-ATPase) and the neutralization of lysosomes to form postlysosomes. Without WASH, no patches or coats are formed, neutral postlysosomes are not seen, and indigestible material such as dextran is not exocytosed. Similar results occur when actin polymerization is blocked with latrunculin. V-ATPases are known to bind avidly to F-actin. Our data imply a new mechanism, actin-mediated sorting, in which WASH and the Arp2/3 complex polymerize actin on vesicles to drive the separation and recycling of proteins such as the V-ATPase.
Publisher: Cold Spring Harbor Laboratory
Date: 12-12-2021
DOI: 10.1101/2021.12.12.472297
Abstract: Characterisation of I37R – a novel mutation in the lasso motif of ABC-transporter CFTR, a chloride channel – was conducted by theratyping using CFTR potentiators which increase channel gating activity and correctors which repair protein trafficking defects. I37R-CFTR function was characterised using intestinal current measurements (ICM) in rectal biopsies, forskolin-induced swelling (FIS) in intestinal organoids and short circuit current measurements (I sc ) in organoid-derived monolayers from an in idual with I37R/F508del CFTR genotype. We demonstrated that the I37R-CFTR mutation results in a residual function defect amenable to treatment with potentiators and type III, but not to type I, correctors. Molecular dynamics of I37R-CFTR using an extended model of the phosphorylated, ATP-bound human CFTR identified an altered lasso motif conformation which results in an unfavourable strengthening of the interactions between the lasso motif, the regulatory (R) domain and the transmembrane domain two (TMD2). In conclusion, structural and functional characterisation of the I37R- CFTR mutation increases understanding of CFTR channel regulation and provides a potential pathway to access CFTR modulator treatments for in iduals with CF caused by ultra-rare CFTR mutations.
Publisher: Springer New York
Date: 22-09-2015
DOI: 10.1007/978-1-4939-1752-5_18
Abstract: A significant number of biological processes occur at, or involve cellular membranes, including cell adhesion, migration, endocytosis, signal transduction, and many biochemical reactions involving membrane anchored scaffolds. Each process involves a complex arrangement of interacting molecules whose location in space and time influence the outcome of the event. In this protocol we discuss the application of fluorescence recovery after photobleaching (FRAP) to study the dynamics of membrane associated molecules. We discuss the principles, acquisition and the analysis of FRAP data and address issues surrounding its interpretation.
Publisher: Springer Science and Business Media LLC
Date: 23-04-2018
DOI: 10.1038/S41467-018-04088-W
Abstract: Endocytosis of surface receptors and their polarized recycling back to the plasma membrane are central to many cellular processes, such as cell migration, cytokinesis, basolateral polarity of epithelial cells and T cell activation. Little is known about the mechanisms that control the organization of recycling endosomes and how they connect to receptor endocytosis. Here, we follow the endocytic journey of the T cell receptor (TCR), from internalization at the plasma membrane to recycling back to the immunological synapse. We show that TCR triggering leads to its rapid uptake through a clathrin-independent pathway. Immediately after internalization, TCR is incorporated into a mobile and long-lived endocytic network demarked by the membrane-organizing proteins flotillins. Although flotillins are not required for TCR internalization, they are necessary for its recycling to the immunological synapse. We further show that flotillins are essential for T cell activation, supporting TCR nanoscale organization and signaling.
Publisher: Elsevier BV
Date: 07-2021
Publisher: Publiverse Online S.R.L
Date: 2015
Publisher: Springer Science and Business Media LLC
Date: 26-09-2019
DOI: 10.1038/S41467-019-12352-W
Abstract: The targeted endocytic recycling of the T cell receptor (TCR) to the immunological synapse is essential for T cell activation. Despite this, the mechanisms that underlie the sorting of internalised receptors into recycling endosomes remain poorly understood. To build a comprehensive picture of TCR recycling during T cell activation, we developed a suite of new imaging and quantification tools centred on photoactivation of fluorescent proteins. We show that the membrane-organising proteins, flotillin-1 and -2, are required for TCR to reach Rab5-positive endosomes immediately after endocytosis and for transfer from Rab5- to Rab11a-positive compartments. We further observe that after sorting into in Rab11a-positive vesicles, TCR recycles to the plasma membrane independent of flotillin expression. Our data suggest a mechanism whereby flotillins delineate a fast Rab5-Rab11a endocytic recycling axis and functionally contribute to regulate the spatial organisation of these endosomes.
Publisher: Wiley
Date: 28-12-2020
Publisher: American Association for Cancer Research (AACR)
Date: 07-2020
DOI: 10.1158/1541-7786.MCR-19-1122
Abstract: Simultaneously inhibiting Tpm3.1-containing actin filaments and microtubules is a promising strategy to potentiate the anticancer activity of low-dose vincristine.
Publisher: Cold Spring Harbor Laboratory
Date: 11-08-2021
DOI: 10.1101/2021.08.11.456003
Abstract: A significant challenge to making targeted CFTR modulator therapies accessible to all in iduals with cystic fibrosis (CF) are many mutations in the CFTR gene that can cause CF, most of which remain uncharacterized. Here, we characterized the structural and functional defects of the rare CFTR mutation R352Q – with potential role contributing to intrapore chloride ion permeation – in patient-derived cell models of the airway and gut. CFTR function in differentiated nasal epithelial cultures and matched intestinal organoids was assessed using ion transport assay and forskolin-induced swelling (FIS) assay respectively. Two CFTR potentiators (VX-770, GLPG1837) and a corrector (VX-809) were tested. Data from R352Q-CFTR were compared to that of participants with mutations with known impact on CFTR function. R352Q-CFTR has residual CFTR function which was restored to functional CFTR activity by CFTR potentiators but not the corrector. Molecular dynamics (MD) simulations of R352Q-CFTR were carried out which indicated the presence of a chloride conductance defect, with little evidence supporting a gating defect. The combination approach of in vitro patient-derived cell models and in silico MD simulations to characterize rare CFTR mutations can improve the specificity and sensitivity of modulator response predictions and aid in their translational use for CF precision medicine.
Publisher: The Company of Biologists
Date: 15-11-2011
DOI: 10.1242/JCS.080986
Abstract: The actin cytoskeleton provides scaffolding and physical force to effect fundamental processes such as motility, cytokinesis and vesicle trafficking. The Arp2/3 complex nucleates actin structures and contributes to endocytic vesicle invagination and trafficking away from the plasma membrane. Internalisation and directed recycling of integrins are major driving forces for invasive cell motility and potentially for cancer metastasis. Here, we describe a direct requirement for WASH and Arp2/3-mediated actin polymerisation on the endosomal membrane system for α5β1 integrin recycling. WASH regulates the trafficking of endosomal α5β1 integrin to the plasma membrane and is fundamental for integrin-driven cell morphology changes and integrin-mediated cancer cell invasion. Thus, we implicate WASH and Arp2/3-driven actin nucleation in receptor recycling leading to invasive motility.
Publisher: American Thoracic Society
Date: 07-2022
Publisher: Elsevier BV
Date: 2013
DOI: 10.1016/J.DEVCEL.2012.12.014
Abstract: WASH causes actin to polymerize on vesicles involved in retrograde traffic and exocytosis. It is found within a regulatory complex, but the physiological roles of the other four members are unknown. Here we present genetic analysis of the subunits' in idual functions in Dictyostelium. Mutants in each subunit are completely blocked in exocytosis. All subunits except FAM21 are required to drive actin assembly on lysosomes. Without actin, lysosomes never recycle vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) or neutralize to form postlysosomes. However, in FAM21 knockout lysosomes, WASH generates excessive, dynamic streams of actin. These successfully remove V-ATPase, neutralize, and form huge postlysosomes. The distinction between WASH and FAM21 phenotypes is conserved in human cells. Thus, FAM21 and WASH act at different steps of a cyclical pathway in which FAM21 mediates recycling of the complex back to acidic lysosomes. Recycling is driven by FAM21's interaction with capping protein, which couples the WASH complex to dynamic actin on vesicles.
Publisher: Frontiers Media SA
Date: 26-02-2016
No related grants have been discovered for Michael Carnell.