ORCID Profile
0000-0002-3880-4106
Current Organisation
University of New South Wales
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Biochemistry and Cell Biology | Signal Transduction | Cell Development, Proliferation and Death | Protein Trafficking | Stochastic Analysis and Modelling | Biomechanical Engineering | Systems Biology |
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in Technology
Publisher: Elsevier BV
Date: 08-2023
Publisher: Cold Spring Harbor Laboratory
Date: 21-03-2022
DOI: 10.1101/2022.03.21.22269988
Abstract: Cancer cells invoke phenotypic plasticity programs to drive disease progression and evade chemotherapeutic insults, yet until now there have been no validated clinical therapies targeting this process. Here, we identify a phenotypic plasticity signature associated with poor survival in basal/triple-negative breast cancer, in which androgen signalling is prominent. We establish that anti-androgen therapies block cancer stem cell function and prevent chemotherapy-induced emergence of new cancer stem cells. In particular, the anti-androgen agent seviteronel synergizes with chemotherapy to improve chemotherapeutic inhibition of primary and metastatic tumour growth and prevent the emergence of chemotherapy-resistant disease. We validate cytoplasmic AR expression as a clinical phenotypic plasticity biomarker that predicts poor survival and poor response to chemotherapy, and positive response to seviteronel plus chemotherapy. This new targeted combination therapy validates modulating phenotypic plasticity as an effective strategy to prevent and treat chemotherapy-resistant cancers with transformative clinical potential. There are currently no curative therapies for patients with chemotherapy-resistant cancer. We demonstrate that modulating phenotypic plasticity prevents the emergence of chemotherapy-resistant disease in triple-negative breast cancer. This represents the first known validated clinical therapy leveraging phenotypic plasticity. Moreover, we identify a highly effective anti-androgen drug and a biomarker to select and treat patients best-suited to this new therapy. A clinical trial is underway ( NCT04947189 ). Blocking phenotypic plasticity is an effective targeted therapeutic strategy to treat cance
Publisher: Wiley
Date: 24-09-2009
DOI: 10.1002/JCP.21932
Abstract: Estrogen effects on mammary gland development and differentiation are mediated by two receptors (ERalpha and ERbeta). Estrogen-bound ERalpha induces proliferation of mammary epithelial and cancer cells, while ERbeta is important for maintenance of the differentiated epithelium and inhibits proliferation in different cell systems. In addition, the normal breast contains higher ERbeta levels compared to the early stage breast cancers, suggesting that loss of ERbeta could be important in cancer development. Analysis of ERbeta-/- mice has consistently revealed reduced expression of cell adhesion proteins. As such, ERbeta is a candidate modulator of epithelial homeostasis and metastasis. Consequently, the aim of this study was to analyze estrogenic effects on adhesion of breast cancer cells expressing ERalpha and ERbeta. As ERbeta is widely found in breast cancer but not in cell lines, we used ERalpha positive T47-D and MCF-7 human breast cancer cells to generate cells with inducible ERbeta expression. Furthermore, the colon cancer cell lines SW480 and HT-29 were also used. Integrin alpha1 mRNA and protein levels increased following ERbeta expression. Integrin beta1-the unique partner for integrin alpha1-increased only at the protein level. ERbeta expression enhanced the formation of vinculin containing focal complexes and actin filaments, indicating a more adhesive potential. This was confirmed by adhesion assays where ERbeta increased adhesion to different extracellular matrix proteins, mostly laminin. In addition, ERbeta expression was associated to less cell migration. These results indicate that ERbeta affects integrin expression and clustering and consequently modulates adhesion and migration of breast cancer cells.
Publisher: Springer Science and Business Media LLC
Date: 10-08-2023
DOI: 10.1007/S11864-023-01121-Z
Abstract: Prostate cancer (PCa) is the second most diagnosed malignant neoplasm and is one of the leading causes of cancer-related death in men worldwide. Despite significant advances in screening and treatment of PCa, given the heterogeneity of this disease, optimal personalized therapeutic strategies remain limited. However, emerging predictive and prognostic biomarkers based on in idual patient profiles in combination with computer-assisted diagnostics have the potential to guide precision medicine, where patients may benefit from therapeutic approaches optimally suited to their disease. Also, the integration of genotypic and phenotypic diagnostic methods is supporting better informed treatment decisions. Focusing on advanced PCa, this review discusses polygenic risk scores for screening of PCa and common genomic aberrations in androgen receptor (AR), PTEN-PI3K-AKT, and DNA damage response (DDR) pathways, considering clinical implications for diagnosis, prognosis, and treatment prediction. Furthermore, we evaluate liquid biopsy, protein biomarkers such as serum testosterone levels, SLFN11 expression, total alkaline phosphatase (tALP), neutrophil-to-lymphocyte ratio (NLR), tissue biopsy, and advanced imaging tools, summarizing current phenotypic biomarkers and envisaging more effective utilization of diagnostic and prognostic biomarkers in advanced PCa. We conclude that prognostic and treatment predictive biomarker discovery can improve the management of patients, especially in metastatic stages of advanced PCa. This will result in decreased mortality and enhanced quality of life and help design a personalized treatment regimen.
Publisher: Springer Science and Business Media LLC
Date: 28-09-2022
DOI: 10.1038/S41598-022-20079-W
Abstract: Androgen receptor variant 7 (AR-V7) is an important biomarker to guide treatment options for castration-resistant prostate cancer (CRPC) patients. Its detectability in circulating tumour cells (CTCs) opens non-invasive diagnostic avenues. While detectable at the transcript level, AR-V7 protein detection in CTCs may add additional information and clinical relevance. The aim of this study was to compare commercially available anti-AR-V7 antibodies and establish reliable AR-V7 immunocytostaining applicable to CTCs from prostate cancer (PCa) patients. We compared seven AR-V7 antibodies by western blotting and immmunocytostaining using a set of PCa cell lines with known AR/AR-V7 status. The emerging best antibody was validated for detection of CRPC patient CTCs enriched by negative depletion of leucocytes. The anti-AR-V7 antibody, clone E308L emerged as the best antibody in regard to signal to noise ratio with a specific nuclear signal. Moreover, this antibody detects CRPC CTCs more efficiently compared to an antibody previously shown to detect AR-V7 CTCs. We have determined the best antibody for AR-V7 detection of CTCs, which will open future studies to correlate AR-V7 subcellular localization and potential co-localization with other proteins and cellular structures to patient outcomes.
Publisher: Elsevier BV
Date: 06-2018
Publisher: Wiley
Date: 13-04-2004
Publisher: Elsevier BV
Date: 05-2010
DOI: 10.1016/J.YEXCR.2010.04.001
Abstract: Dynamic cellular processes occurring in time and space are fundamental to all physiology and disease. To understand complex and dynamic cellular processes therefore demands the capacity to record and integrate quantitative multiparametric data from the four spatiotemporal dimensions within which living cells self-organize, and to subsequently use these data for the mathematical modeling of cellular systems. To this end, a raft of complementary developments in automated fluorescence microscopy, cell microarray platforms, quantitative image analysis and data mining, combined with multivariate statistics and computational modeling, now coalesce to produce a new research strategy, "systems microscopy", which facilitates systems biology analyses of living cells. Systems microscopy provides the crucial capacities to simultaneously extract and interrogate multiparametric quantitative data at resolution levels ranging from the molecular to the cellular, thereby elucidating a more comprehensive and richly integrated understanding of complex and dynamic cellular systems. The unique capacities of systems microscopy suggest that it will become a vital cornerstone of systems biology, and here we describe the current status and future prospects of this emerging field, as well as outlining some of the key challenges that remain to be overcome.
Publisher: Elsevier BV
Date: 11-2019
DOI: 10.1016/J.CELS.2019.09.002
Abstract: Although F-actin has a large number of binding partners and regulators, the number of phenotypic states available to the actin cytoskeleton is unknown. Here, we quantified 74 features defining filamentous actin (F-actin) and cellular morphology in >25 million cells after treatment with a library of 114,400 structurally erse compounds. After reducing the dimensionality of these data, only ∼25 recurrent F-actin phenotypes emerged, each defined by distinct quantitative features that could be machine learned. We identified 2,003 unknown compounds as inducers of actin-related phenotypes, including two that directly bind the focal adhesion protein, talin. Moreover, we observed that compounds with distinct molecular mechanisms could induce equivalent phenotypes and that initially ergent cellular responses could converge over time. These findings suggest a conceptual parallel between the actin cytoskeleton and gene regulatory networks, where the theoretical plasticity of interactions is nearly infinite, yet phenotypes in vivo are constrained into a limited subset of practicable configurations.
Publisher: Proceedings of the National Academy of Sciences
Date: 04-03-2008
Abstract: The transmembrane precursor of tumor necrosis factor-α (TNF) exits the trans -Golgi network (TGN) in tubular carriers for subsequent trafficking and delivery to the cell surface however, the molecular machinery responsible for Golgi export is unknown. We previously reported that members of the TGN golgin family are associated with subdomains and tubules of the TGN. Here, we show that the TGN golgin, p230/golgin-245 (p230), is essential for intracellular trafficking and cell surface delivery of TNF in transfected HeLa cells and activated macrophages. Live-cell imaging revealed that TNF transport from the TGN is mediated selectively by tubules and carriers marked by p230. Significantly, LPS activation of macrophages resulted in a dramatic increase of p230-labeled tubules and carriers emerging from the TGN, indicating that macrophages up-regulate the transport pathway for TNF export. Depletion of p230 in LPS-stimulated macrophages reduced cell surface delivery of TNF by -fold compared with control cells. To determine whether p230 depletion blocked TNF secretion in vivo , we generated retrogenic mice expressing a microRNA-vector to silence p230. Bone-marrow stem cells were transduced with recombinant retrovirus containing microRNA constructs and transplanted into irradiated recipients. LPS-activated peritoneal macrophages from p230 miRNA retrogenic mice were depleted of p230 and had dramatically reduced levels of cell surface TNF. Overall, these studies have identified p230 as a key regulator of TNF secretion and have shown that LPS activation of macrophages results in increased Golgi carriers for export. Also, we have demonstrated a previously undescribed approach to control cytokine secretion by the specific silencing of trafficking machinery.
Publisher: Wiley
Date: 07-12-2012
Publisher: Rockefeller University Press
Date: 09-04-2018
Abstract: Integrins are the core constituents of cell–matrix adhesion complexes such as focal adhesions (FAs) and play key roles in physiology and disease. Integrins fluctuate between active and inactive conformations, yet whether the activity state influences the spatial organization of integrins within FAs has remained unclear. In this study, we address this question and also ask whether integrin activity may be regulated either independently for each integrin molecule or through locally coordinated mechanisms. We used two distinct superresolution microscopy techniques, stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion microscopy (STED), to visualize active versus inactive β1 integrins. We first reveal a spatial hierarchy of integrin organization with integrin molecules arranged in nanoclusters, which align to form linear substructures that in turn build FAs. Remarkably, within FAs, active and inactive β1 integrins segregate into distinct nanoclusters, with active integrin nanoclusters being more organized. This unexpected segregation indicates synchronization of integrin activities within nanoclusters, implying the existence of a coordinate mechanism of integrin activity regulation.
Publisher: The Company of Biologists
Date: 15-10-2010
DOI: 10.1242/JCS.069609
Abstract: Phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] is a key regulator of cell signaling that acts by recruiting proteins to the cell membrane, such as at the leading edge during cell migration. Here, we show that PtdIns (3,4,5)P3 plays a central role in filopodia formation via the binding of myosin-X (Myo10), a potent promoter of filopodia. We found that the second pleckstrin homology domain (Myo10-PH2) of Myo10 specifically binds to PtdIns(3,4,5)P3, and that disruption of this binding led to impairment of filopodia and partial re-localization of Myo10 to microtubule-associated Rab7-positive endosomal vesicles. Given that the localization of Myo10 was dynamically restored to filopodia upon reinstatement of PtdIns(3,4,5)P3-binding, our results indicate that PtdIns(3,4,5)P3 binding to the Myo10-PH2 domain is involved in Myo10 trafficking and regulation of filopodia dynamics.
Publisher: American Society for Cell Biology (ASCB)
Date: 10-2010
Abstract: Cell-to-extracellular matrix adhesion is regulated by a multitude of pathways initiated distally to the core cell–matrix adhesion machinery, such as via growth factor signaling. In contrast to these extrinsically sourced pathways, we now identify a regulatory pathway that is intrinsic to the core adhesion machinery, providing an internal regulatory feedback loop to fine tune adhesion levels. This autoinhibitory negative feedback loop is initiated by cell adhesion to vitronectin, leading to PAK4 activation, which in turn limits total cell–vitronectin adhesion strength. Specifically, we show that PAK4 is activated by cell attachment to vitronectin as mediated by PAK4 binding partner integrin αvβ5, and that active PAK4 induces accelerated integrin αvβ5 turnover within adhesion complexes. Accelerated integrin turnover is associated with additional PAK4-mediated effects, including inhibited integrin αvβ5 clustering, reduced integrin to F-actin connectivity and perturbed adhesion complex maturation. These specific outcomes are ultimately associated with reduced cell adhesion strength and increased cell motility. We thus demonstrate a novel mechanism deployed by cells to tune cell adhesion levels through the autoinhibitory regulation of integrin adhesion.
Publisher: Rockefeller University Press
Date: 17-06-2019
Abstract: An understanding of the mechanisms whereby cell adhesion complexes (ACs) relay signals bidirectionally across the plasma membrane is necessary to interpret the role of adhesion in regulating migration, differentiation, and growth. A range of AC types has been defined, but to date all have similar compositions and are dependent on a connection to the actin cytoskeleton. Recently, a new class of AC has been reported that normally lacks association with both the cytoskeleton and integrin-associated adhesome components, but is rich in components of the clathrin-mediated endocytosis machinery. The characterization of this new type of adhesion structure, which is emphasized by mitotic cells and cells in long-term culture, identifies a hitherto underappreciated link between the adhesion machinery and clathrin structures at the plasma membrane. While this discovery has implications for how ACs are assembled and disassembled, it raises many other issues. Consequently, to increase awareness within the field, and stimulate research, we explore a number of the most significant questions below.
Publisher: Wiley
Date: 26-08-2010
DOI: 10.1002/IJC.25223
Abstract: Kindlin-2 is a novel integrin-interacting focal adhesion protein that belongs to the Kindlin family. Focal adhesion proteins control cytoskeleton dynamics and promote cancer cell growth, survival, migration and metastasis. Little is known, however, about expression of Kindlin-2 in association with human cancer. We now reveal high Kindlin-2 expression in malignant mesothelioma (MM) cell lines using an affinity-purified anti-Kindlin-2 antibody. Furthermore, we show by immunohistochemistry that Kindlin-2 is highly expressed in 92 of 102 (90%) MMs with epitheliod sarcomatoid, biphasic and poorly differentiated morphologies. In addition, Kindlin-2 expression correlates to cell proliferation, suggesting a role for Kindlin-2 in tumor growth. We also detect increased expression of Kindlin-2 at the invasion front of tumors concurrent with increased expression of integrin-linked kinase, a Kindlin-binding protein. Besides the high expression of Kindlin-2 in pleural MMs, pleural metastases of lung adenocarcinoma also express large amounts of Kindlin-2, but not Kindlin-1. Notably, in vitro, when endogenous Kindlin-2 was knocked down with RNAi in MM cells, this impaired cell spreading, adhesion and migration. Overall, our study suggests that heightened expression of Kindlin-2 might contribute to tumor progression in MM.
Publisher: The Company of Biologists
Date: 2012
DOI: 10.1242/JCS.105270
Abstract: Cell migration and invasion require regulated turnover of integrin-dependent adhesion complexes. RIAM is an adaptor protein mediating talin recruitment to the cell membrane, whose depletion leads to defective melanoma cell migration and invasion. Here we investigated the potential involvement of RIAM in focal adhesion (FA) dynamics. RIAM-depleted melanoma and breast carcinoma cells displayed an increased number, size and stability of FAs, which accumulated centrally located at the ventral cell surface, a phenotype caused by defective FA disassembly. Impairment in FA disassembly due to RIAM knocking down correlated with deficient integrin-dependent MEK-Erk1/2 activation, and importantly, overexpression of constitutively active MEK resulted in rescue of FA disassembly and recovery of cell invasion. Furthermore, RIAM-promoted RhoA activation following integrin engagement was needed for subsequent Erk1/2 activation, and RhoA overexpression partially rescued the FA phenotype in RIAM-depleted cells, suggesting a functional role also for RhoA downstream of RIAM, but upstream of Erk1/2. In addition, RIAM knock down led to enhanced phosphorylation of paxillin Tyr118 and Tyr31. However, expression of phosphomimetic and non-phosphorylatable mutants at these paxillin residues indicated that paxillin hyper-phosphorylation is a subsequent consequence of the blockade of FA disassembly, but does not cause the FA phenotype. RIAM depletion also weakened association between FA proteins, suggesting that it may play important adaptor roles for the correct assembly of adhesion complexes. Our data indicate that integrin-triggered, RIAM-dependent MEK activation may represent a key feed-back event required for efficient FA disassembly, which may contribute to explain the role of RIAM in cell migration and invasion.
Publisher: Springer Science and Business Media LLC
Date: 29-06-2007
Abstract: Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca 2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP). Here we report significant progress on the development of the latter type of Ca 2+ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca 2+ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca 2+ -free and Ca 2+ -saturated forms. We demonstrated the high potential of these sensors on various ex les, including monitoring of Ca 2+ response to a prolonged glutamate treatment in cortical neurons. We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.
Publisher: EMBO
Date: 08-2018
Publisher: Elsevier BV
Date: 02-2008
DOI: 10.1016/J.SEMCANCER.2007.10.001
Abstract: Cell-matrix adhesion complexes (CMACs) are foci of cellular attachment to the extracellular matrix (ECM). This attachment, mediated by integrins and adaptor proteins, provides both physical and regulatory links between the ECM and the cellular microfilament system. Through continual regulation and rearrangement of both ECM adhesion and actin structures, CMACs constitute core machineries of cell migration. To fulfill this role, CMACs are exceptionally flexible and dynamic complexes, and their components undergo rapid and regulated turn-over to maintain delicately balanced streams of mechanical and chemical information. Besides the critical role of CMACs in cell migration, signaling through these complexes provides influence over virtually every major cellular function, including for ex le cell survival, cell differentiation and cell proliferation. This review depicts the roles of CMACs in cell migration and discusses how CMACs integrate with other sub-cellular systems involved in cell motility. Importantly, we also present a rationalized view of CMACs as information handling machines, and suggest strategies that may facilitate better understanding of the complex cell migration phenomenon as a whole, through quantitative and integrative (systems biology) approaches.
Publisher: American Association for Cancer Research (AACR)
Date: 23-06-2022
DOI: 10.1158/2159-8290.CD-21-0282
Abstract: Phenotypic plasticity describes the ability of cancer cells to undergo dynamic, nongenetic cell state changes that lify cancer heterogeneity to promote metastasis and therapy evasion. Thus, cancer cells occupy a continuous spectrum of phenotypic states connected by trajectories defining dynamic transitions upon a cancer cell state landscape. With technologies proliferating to systematically record molecular mechanisms at single-cell resolution, we illuminate manifold learning techniques as emerging computational tools to effectively model cell state dynamics in a way that mimics our understanding of the cell state landscape. We anticipate that “state-gating” therapies targeting phenotypic plasticity will limit cancer heterogeneity, metastasis, and therapy resistance. Nongenetic mechanisms underlying phenotypic plasticity have emerged as significant drivers of tumor heterogeneity, metastasis, and therapy resistance. Herein, we discuss new experimental and computational techniques to define phenotypic plasticity as a scaffold to guide accelerated progress in uncovering new vulnerabilities for therapeutic exploitation.
Publisher: Hindawi Limited
Date: 30-03-2020
DOI: 10.1155/2020/7938280
Abstract: Prostate cancer (PCa) is initially driven by excessive androgen receptor (AR) signaling with androgen deprivation therapy (ADT) being a major therapeutic approach to its treatment. However, the development of drug resistance is a significant limitation on the effectiveness of both first-line and more recently developed second-line ADTs. There is a need then to study AR signaling within the context of other oncogenic signaling pathways that likely mediate this resistance. This review focuses on interactions between AR signaling, the well-known phosphatidylinositol-3-kinase/AKT pathway, and an emerging mediator of these pathways, the Hippo/YAP1 axis in metastatic castrate-resistant PCa, and their involvement in the regulation of epithelial-mesenchymal transition (EMT), a feature of disease progression and ADT resistance. Analysis of these pathways in circulating tumor cells (CTCs) may provide an opportunity to evaluate their utility as biomarkers and address their importance in the development of resistance to current ADT with potential to guide future therapies.
Publisher: Public Library of Science (PLoS)
Date: 06-08-2015
Publisher: Oxford University Press (OUP)
Date: 04-06-2014
Abstract: This manuscript describes technical advances allowing manipulation and quantitative analyses of human DC migratory behavior in lung epithelial tissue. DCs are hematopoietic cells essential for the maintenance of tissue homeostasis and the induction of tissue-specific immune responses. Important functions include cytokine production and migration in response to infection for the induction of proper immune responses. To design appropriate strategies to exploit human DC functional properties in lung tissue for the purpose of clinical evaluation, e.g., candidate vaccination and immunotherapy strategies, we have developed a live-imaging assay based on our previously described organotypic model of the human lung. This assay allows provocations and subsequent quantitative investigations of DC functional properties under conditions mimicking morphological and functional features of the in vivo parental tissue. We present protocols to set up and prepare tissue models for 4D (x, y, z, time) fluorescence-imaging analysis that allow spatial and temporal studies of human DCs in live epithelial tissue, followed by flow cytometry analysis of DCs retrieved from digested tissue models. This model system can be useful for elucidating incompletely defined pathways controlling DC functional responses to infection and inflammation in lung epithelial tissue, as well as the efficacy of locally administered candidate interventions.
Publisher: Springer New York
Date: 2018
DOI: 10.1007/978-1-4939-7701-7_10
Abstract: Cell migration is a dynamic process that emerges from fine-tuned networks coordinated in three-dimensional space, spanning molecular, subcellular, and cellular scales, and over multiple temporal scales, from milliseconds to days. Understanding how cell migration arises from this complexity requires data collection and analyses that quantitatively integrate these spatial and temporal scales. To meet this need, we have combined quantitative live and fixed cell fluorescence microscopy, customized image analysis tools, multivariate statistical methods, and mathematical modeling. Collectively, this constitutes the systems microscopy strategy that we have applied to dissect how cells organize themselves to migrate. In this overview, we highlight key principles, concepts, and components of our systems microscopy methodology, and exemplify what we have learnt so far and where this approach may lead.
Publisher: Public Library of Science (PLoS)
Date: 28-02-2014
Publisher: Springer Science and Business Media LLC
Date: 22-10-2018
DOI: 10.1038/S41556-018-0220-2
Abstract: Adhesion to the extracellular matrix persists during mitosis in most cell types. However, while classical adhesion complexes, such as focal adhesions, do and must disassemble to enable mitotic rounding, the mechanisms of residual mitotic cell-extracellular matrix adhesion remain undefined. Here, we identify 'reticular adhesions', a class of adhesion complex that is mediated by integrin αvβ5, formed during interphase, and preserved at cell-extracellular matrix attachment sites throughout cell ision. Consistent with this role, integrin β5 depletion perturbs mitosis and disrupts spatial memory transmission between cell generations. Reticular adhesions are morphologically and dynamically distinct from classical focal adhesions. Mass spectrometry defines their unique composition, enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P
Publisher: Wiley
Date: 28-09-2005
DOI: 10.1111/J.1600-0854.2005.00349.X
Abstract: E-cadherin is a cell-cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane-bound carriers for the post-Golgi exocytosis of E-cadherin have not been characterized. Green fluorescent protein (GFP)-tagged E-cadherin (Ecad-GFP) is transported from the trans-Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. Here, we examine the association of golgins with tubular carriers containing E-cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full-length proteins and were coexpressed with Ecad-GFP. The GRIP domains of p230/golgin-245 and golgin-97 had overlapping but nonidentical distributions on the TGN both domains were on TGN-derived tubules but only the golgin-97 GRIP domain coincided with Ecad-GFP tubules in live cells. When the Arl1-binding endogenous golgins, p230/golgin-245 and golgin-97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad-GFP was inhibited. siRNA knockdown of golgin-97 also inhibited trafficking of Ecad-GFP. Thus, the GRIP domains of p230/golgin-245 and golgin-97 bind discriminately to distinct membrane subdomains of the TGN. Golgin-97 is identified as a selective and essential component of the tubulovesicular carriers transporting E-cadherin out of the TGN.
Publisher: Cold Spring Harbor Laboratory
Date: 14-12-2017
DOI: 10.1101/234237
Abstract: Adhesion to the extracellular matrix (ECM) persists during mitosis in most cell types. Yet, classical adhesion complexes (ACs), such as focal adhesions and focal complexes, do and must disassemble to enable cytoskeletal rearrangements associated with mitotic rounding. Given this paradox, mechanisms of mitotic cell-ECM adhesion remain undefined. Here, we identify ‘reticular adhesions’, a new class of AC that is mediated by integrin αvβ5, formed during interphase and preserved at cell-ECM attachment sites throughout cell ision. Consistent with this role, integrin β5 depletion perturbs mitosis and disrupts spatial memory transmission between cell generations. Quantitative imaging reveals reticular adhesions to be both morphologically and dynamically distinct from classic focal adhesions, while mass spectrometry defines their unique composition lacking virtually all consensus adhesome components. Indeed, remarkably, reticular adhesions are functionally independent of both talin and F-actin, yet are promoted by phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). Overall, the distinct characteristics of reticular adhesions provide a unique solution to the problem of maintaining cell-ECM attachment during mitotic rounding and ision.
Publisher: Wiley
Date: 05-2001
DOI: 10.1034/J.1600-0854.2001.002005336.X
Abstract: Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi membranes. By immuno-electron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230GRIP fusion protein, we show binding specifically to a subset of membranes of the trans-Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230GRIP was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230GRIP-transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230GRIP binds to a specific population of vesicles distinct from those labelled for beta-COP or gamma-adaptin. The GFP-p230GRIP fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans-Golgi network tubulo-vesicular carriers.
Publisher: Springer International Publishing
Date: 2021
Publisher: University of Queensland Library
Date: 2022
DOI: 10.14264/40C2222
Publisher: American Society for Cell Biology (ASCB)
Date: 04-2005
Abstract: E-cadherin plays an essential role in cell polarity and cell-cell adhesion however, the pathway for delivery of E-cadherin to the basolateral membrane of epithelial cells has not been fully characterized. We first traced the post-Golgi, exocytic transport of GFP-tagged E-cadherin (Ecad-GFP) in unpolarized cells. In live cells, Ecad-GFP was found to exit the Golgi complex in pleiomorphic tubulovesicular carriers, which, instead of moving directly to the cell surface, most frequently fused with an intermediate compartment, subsequently identified as a Rab11-positive recycling endosome. In MDCK cells, basolateral targeting of E-cadherin relies on a dileucine motif. Both E-cadherin and a targeting mutant, ΔS1-E-cadherin, colocalized with Rab11 and fused with the recycling endosome before erging to basolateral or apical membranes, respectively. In polarized and unpolarized cells, coexpression of Rab11 mutants disrupted the cell surface delivery of E-cadherin and caused its mistargeting to the apical membrane, whereas apical ΔS1-E-cadherin was unaffected. We thus demonstrate a novel pathway for Rab11 dependent, dileucine-mediated, μ1B-independent sorting and basolateral trafficking, exemplified by E-cadherin. The recycling endosome is identified as an intermediate compartment for the post-Golgi trafficking and exocytosis of E-cadherin, with a potentially important role in establishing and maintaining cadherin-based adhesion.
Publisher: Springer Science and Business Media LLC
Date: 21-07-2023
DOI: 10.1186/S13578-023-01083-7
Abstract: Metastatic cancer cells exploit Epithelial-mesenchymal-transition (EMT) to enhance their migration, invasion, and resistance to treatments. Recent studies highlight that elevated levels of copper are implicated in cancer progression and metastasis. Clinical trials using copper chelators are associated with improved patient survival however, the molecular mechanisms by which copper depletion inhibits tumor progression and metastasis are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Here, we propose that copper chelation inhibits metastasis by reducing TGF-β levels and EMT signaling. Given that many drugs targeting TGF-β have failed in clinical trials, partly because of severe side effects arising in patients, we hypothesized that copper chelation therapy might be a less toxic alternative to target the TGF-β/EMT axis. Our cytokine array and RNA-seq data suggested a link between copper homeostasis, TGF-β and EMT process. To validate this hypothesis, we performed single-cell imaging, protein assays, and in vivo studies. Here, we used the copper chelating agent TEPA to block copper trafficking. Our in vivo study showed a reduction of TGF-β levels and metastasis to the lung in the TNBC mouse model. Mechanistically, TEPA significantly downregulated canonical (TGF-β/SMAD2& ) and non-canonical (TGF-β/PI3K/AKT, TGF-β/RAS/RAF/MEK/ERK, and TGF-β/WNT/β-catenin) TGF-β signaling pathways. Additionally, EMT markers of MMP-9, MMP-14, Vimentin, β-catenin, ZEB1, and p-SMAD2 were downregulated, and EMT transcription factors of SNAI1, ZEB1, and p-SMAD2 accumulated in the cytoplasm after treatment. Our study suggests that copper chelation therapy represents a potentially effective therapeutic approach for targeting TGF-β and inhibiting EMT in a erse range of cancers.
Publisher: Elsevier BV
Date: 08-2019
DOI: 10.1016/J.CBPA.2019.02.013
Abstract: The actin cytoskeleton is dysregulated in cancer, yet this critical cellular machinery has not translated as a druggable clinical target due to cardio-toxic side-effects. Many actin regulators are also considered undruggable, being structural proteins lacking clear functional sites suitable for targeted drug design. In this review, we discuss opportunities and challenges associated with drugging the actin cytoskeleton through its structural regulators, taking tropomyosins as a target ex le. In particular, we highlight emerging data acquisition and analysis trends driving phenotypic, imaging-based compound screening. Finally, we consider how the confluence of these trends is now bringing functionally integral machineries such as the actin cytoskeleton, and associated structural regulatory proteins, into an expanded repertoire of druggable targets with previously unexploited clinical potential.
Publisher: Elsevier BV
Date: 2018
DOI: 10.2139/SSRN.3299445
Publisher: Wiley
Date: 03-2003
DOI: 10.1034/J.1600-0854.2003.00106.X
Abstract: Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans-Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.
Publisher: Cold Spring Harbor Laboratory
Date: 06-10-2022
DOI: 10.1101/2022.10.03.510707
Abstract: Copper is a trace element essential to cellular function with elevated levels implicated in cancer progression. Clinical trials using copper chelators are associated with improved patient survival, however, the molecular mechanisms by which copper depletion inhibits tumor progression are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Epithelial-mesenchymal transition (EMT) is often exploited by malignant cells to promote growth and metastasis. Transforming growth factor (TGF)- β is a master regulator of EMT and facilitates cancer progression through changes in the tumor and its microenvironment. Herein, we report that a reduction of copper with the chelating agent tetraethylenepentamine (TEPA) inhibited EMT in vitro in three erse cancer cell types human triple-negative breast cancer (TNBC), neuroblastoma (NB), and diffuse intrinsic pontine glioma (DIPG) cell lines. Single-molecule imaging demonstrated EMT markers including Vimentin, β -catenin, ZEB1, and p-SMAD2 had increased expression with copper treatment and this pro-mesenchymal shift was rescued by the addition of TEPA. Moreover, SNAI1, ZEB1, and p-SMAD2 demonstrated increased accumulation in the cytoplasm after treating with TEPA. Transcriptomic analyses revealed a significant downregulation of the EMT pathway, including canonical (TGF- β /SMAD2& ) and non-canonical (TGF- β /PI3K/AKT and TGF- β /RAS/RAF/MEK/ERK) TGF signaling pathways. Matrix metalloproteinases MMP-9 and MMP-14 proteins which activate latent TGF- β complexes were also downregulated by TEPA treatment. These molecular changes are consistent with reduced plasma levels of TGF- β we observed in cancer models treated with TEPA. Importantly, copper chelation reduced metastasis to the lung in a TNBC orthotopic syngeneic mouse model. Our studies suggest copper chelation therapy can be used to inhibit EMT-induced metastasis by targeting TGF- β signalling. Because on-target anti-TGF- β therapies are failing in the clinic, copper chelation presents itself as a potential therapy for targeting TGF- β in cancer.
Publisher: Cold Spring Harbor Laboratory
Date: 29-03-2023
DOI: 10.1101/2023.03.28.534644
Abstract: While single-cell technologies have allowed scientists to characterize cell states that emerge during cancer progression through temporal s ling, connecting these s les over time and inferring gene-gene relationships that promote cancer plasticity remains a challenge. To address these challenges, we developed TrajectoryNet, a neural ordinary differential equation network that learns continuous dynamics via interpolation of population flows between s led timepoints. By running causality analysis on the output of TrajectoryNet, we compute rich and complex gene-gene networks that drive pathogenic trajectories forward. Applying this pipeline to scRNAseq data generated from in vitro models of breast cancer, we identify and validate a refined CD44 hi EPCAM + CAV1 + marker profile that improves the identification and isolation of cancer stem cells (CSCs) from bulk cell populations. Studying the cell plasticity trajectories emerging from this population, we identify comprehensive temporal regulatory networks that drive cell fate decisions between an epithelial-to-mesenchymal (EMT) trajectory, and a mesenchymal-to-epithelial (MET) trajectory. Through these studies, we identify and validate estrogen related receptor alpha as a critical mediator of CSC plasticity. We further apply TrajectoryNet to an in vivo xenograft model and demonstrate it’s ability to elucidate trajectories governing primary tumor metastasis to the lung, identifying a dominant EMT trajectory that includes elements of our newly-defined temporal EMT regulatory network. Demonstrated here in cancer, the TrajectoryNet pipeline is a transformative approach to uncovering temporal molecular programs operating in dynamic cell systems from static single-cell data.
Start Date: 2021
End Date: 2021
Funder: Australian Research Council
View Funded ActivityStart Date: 2022
End Date: 2024
Funder: Australian Research Council
View Funded ActivityStart Date: 2017
End Date: 2019
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2017
End Date: 2019
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2017
End Date: 2019
Funder: Australian Research Council
View Funded ActivityStart Date: 2018
End Date: 2018
Funder: Australian Research Council
View Funded ActivityStart Date: 2022
End Date: 12-2024
Amount: $611,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2017
End Date: 12-2019
Amount: $398,500.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2018
End Date: 12-2018
Amount: $600,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2021
End Date: 12-2021
Amount: $900,000.00
Funder: Australian Research Council
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