Biology subjects

Freeman, M.

Publications and source records attributed to Freeman, M..

35 records · Page 2Linked to original sources

bioRxiv2021

iRhom2 regulates ERBB signalling to promote KRAS-driven oncogenesis

Dysregulation of the ERBB/EGFR signalling pathway causes multiple types of cancer (1, 2). Accordingly, ADAM17, the primary shedding enzyme that releases and activates ERBB ligands, is tightly regulated. It has recently become clear that iRhoms, inactive members of the rhomboid-like superfamily, are regulatory cofactors for ADAM17 (3, 4). Here we show that oncogenic KRAS mutants target the cytoplasmic domain of iRhom2 to induce ADAM17-dependent shedding and the release of ERBB ligands. Activation of ERK1/2 by oncogenic KRAS induces the phosphorylation of iRhom2, recruitment of the phospho-binding 14-3-3 proteins, and consequent ADAM17-dependent shedding of ERBB ligands. In addition, cancer-associated mutations in iRhom2 act as sensitisers in this pathway by further increasing KRAS-induced shedding of ERBB ligands. This mechanism is conserved in lung cancer cells, where iRhom activity is required for tumour xenograft growth. In this context, the activity of oncogenic KRAS is modulated by the iRhom2-dependent release of ERBB ligands, thus placing iRhom2 as a central component of a positive feedback loop in lung cancer cells. Overall, the cytoplasmic domain of iRhom2 is a critical component of KRAS-induced oncogenesis of lung cancer cells. Both ADAM17 and iRhom2 have also been implicated in a wide range of other cancers (5-10), so the mechanism we have revealed may also have wider oncogenic significance.

cancer biology↗

bioRxiv2021

RHBDL4 protects against endoplasmic reticulum stress by regulating the morphology and distribution of ER sheets

In metazoans, the architecture of the endoplasmic reticulum (ER) differs between cell types, and undergoes major changes through the cell cycle and according to physiological needs. Although much is known about how the different ER morphologies are generated and maintained, especially the ER tubules, how context dependent changes in ER shape and distribution are regulated and the factors involved are less characterized. Here, we show that RHBDL4, an ER-resident rhomboid protease, modulates the shape and distribution of the ER, especially under conditions that require rapid changes in the ER sheet distribution, including ER stress. RHBDL4 interacts with CLIMP-63, a protein involved in ER sheet stabilisation, and with the cytoskeleton. Mice lacking RHBDL4 are sensitive to ER stress and develop liver steatosis, a phenotype associated with unresolved ER stress. Our data introduce a new physiological role of RHBDL4 and also imply that this function does not require its enzymatic activity.

cell biology↗

bioRxiv2021

Cap-dependent translation initiation monitored in living cells

Despite extensive biochemical, genetic, and structural studies, a complete understanding of mRNA translation initiation is still lacking. Imaging methodologies able to resolve the binding dynamics of translation factors at single-cell and single-molecule resolution are necessary to fully elucidate regulation of this paramount process. We fused tags suitable for live imaging to eIF4E, eIF4G1 and 4E-BP1 without affecting their function. We combined Fluorescence Correlation Spectroscopy (FCS) and Single-Particle Tracking (SPT) to interrogate the binding dynamics of initiation factors to the 5cap. Both FCS and SPT were able to detect eIF4E:eIF4G1 binding to the mRNA in the cytoplasm of proliferating cells and neuronal processes. Upon inhibition of phosphorylation by mTOR, 4E-BP1:eIF4E complexes rapidly dissociated from the 5cap followed by eIF4G1 dissociation. Imaging of the binding dynamics of individual translation factors in living cells revealed the temporal regulation of translation at unprecedented resolution.

cell biology↗

bioRxiv2021

An ELISA-based method for rapid genetic screens in Drosophila

Drosophila is a powerful model in which to perform genetic screens, but screening assays that are both rapid and can be used to examine a wide variety of cellular and molecular pathways are limited. Drosophila offer an extensive toolbox of GFP-based transcriptional reporters, GFP-tagged proteins, and driver lines which can be used to express GFP in numerous subpopulations of cells. Thus, a tool that can rapidly and quantitatively evaluate GFP levels in Drosophila tissue would provide a broadly applicable screening platform. To quantify GFP levels from Drosophila lysates, we developed a GFP-based ELISA assay. We demonstrate that this assay can detect membrane localized GFP in a variety of neuronal and glial cell populations and validate that it can identify genes that change the morphology of these cells. This assay was also able to detect STAT transcriptional activity after injury. We found that this assay can detect endogenously GFP-tagged proteins, including Draper and Cryptochrome, and it is able to report developmental and circadian changes in the expression of these proteins. Finally, we validated that the assay can be used to detect changes in synapse elimination upon genetic manipulation of astrocytes. We then used the assay to perform a small-scale screen, which identified Syntaxins as novel regulators of astrocyte-mediated synapse elimination. Together, these studies establish an ELISA as a rapid, easy and quantitative in vivo screening method to assay a wide breadth of fundamental questions in neurobiology. Significance StatementForward genetic screens in Drosophila have played an integral role in elucidating the cellular and molecular pathways that govern almost every facet of biology. However, current screening methods in Drosophila are either fast, but limited in their specificity for particular pathways or processes, or rely on imaging, which requires substantial expertise, time, and cost. We have developed a rapid GFP-based ELISA screening method that, when paired with the wealth of GFP-based genetic tools already available in Drosophila, can be used to screen for regulators of many subpopulations of cells, transcriptional programs and levels of thousands of different proteins. Using this assay, we have identified a novel family of genes required for astrocytes to mediate developmental synapse elimination. This technique provides a screening platform that is fast, accessible, and broadly applicable to many pathways and processes, making Drosophila an even more powerful screening tool.

neuroscience↗

bioRxiv2020

Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation

Calcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease, RHBDL2. We show that RHBDL2 prevents stochastic signalling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved, disease-linked proline residue is responsible for RHBDL2 recognising only the active conformation of Orai1, and cleavage by RHBDL2 is required to sharpen switch-like signalling triggered by store-operated calcium entry. Loss of RHBDL2 control of Orai1 causes severe dysregulation of CRAC channel effectors including transcription factor activation, inflammatory cytokine expression and T cell activation. We propose that this seek-and-destroy function may represent an ancient activity of rhomboid proteases in degrading unwanted signalling proteins.

biochemistry↗

bioRxiv2020

ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet mediated degeneration

Animals have evolved multiple mechanisms to protect themselves from the cumulative effects of age-related cellular damage. Here we reveal an unexpected link between the TNF (tumour necrosis factor) inflammatory pathway, triggered by the metalloprotease ADAM17/TACE, and a lipid droplet (LD)-mediated mechanism of protecting retinal cells from age related degeneration. Loss of ADAM17, TNF and the TNF receptor Grindelwald in pigmented glial cells of the Drosophila retina leads to age related degeneration of both glia and neurons, preceded by an abnormal accumulation of glial LDs. We show that the glial LDs initially buffer the cells against damage caused by neuronally generated reactive oxygen species (ROS), but that in later life the LDs dissipate, leading to the release of toxic peroxidated lipids. Finally, we demonstrate the existence of a conserved pathway in human iPS-derived microglia-like cells, which are central players in neurodegeneration. Overall, we have discovered a pathway mediated by TNF signalling acting not as a trigger of inflammation, but as a cytoprotective factor in the retina.

neuroscience↗

bioRxiv2019

Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells

Living cells function through the spatial compartmentalization of thousands of distinct proteins serving a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) has emerged as a pathway to directly view nanoscale protein relationships to the underlying global ultrastructure, but has traditionally suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional correlative cryogenic SR and focused ion beam milled block-face EM across entire vitreously frozen cells that addresses these issues by preserving native ultrastructure and enabling independent SR and EM workflow optimization. Application to a variety of biological systems revealed a number of unexpected protein-ultrastructure relationships and underscored the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

cell biology↗

bioRxiv2019

Spastin tethers lipid droplets to peroxisomes and directs fatty acid trafficking through ESCRT-III

Lipid droplets (LDs) are neutral lipid storage organelles that transfer lipids to various organelles including peroxisomes. Here, we show that the hereditary spastic paraplegia protein M1 Spastin, a membrane-bound AAA ATPase found on LDs, coordinates fatty acid (FA) trafficking from LDs to peroxisomes through two inter-related mechanisms. First, M1 Spastin forms a tethering complex with peroxisomal ABCD1 to promote LD-peroxisome contact formation. Second, M1 Spastin recruits the membrane-shaping ESCRT-III proteins IST1 and CHMP1B to LDs via its MIT domain to facilitate LD-to-peroxisome FA trafficking, possibly through IST1 and CHMP1B modifying LD membrane morphology. Furthermore, M1 Spastin, IST1 and CHMP1B are all required to relieve LDs of lipid peroxidation. M1 Spastins dual roles in tethering LDs to peroxisomes and in recruiting ESCRT-components to LD-peroxisome contact sites for FA trafficking may help explain the pathogenesis of diseases associated with defective FA metabolism in LDs and peroxisomes.

cell biology↗

bioRxiv2018

Modeling cellular response in large-scale radiogenomic databases to advance precision radiotherapy

Radiotherapy is integral to the care of a majority of cancer patients. Despite differences in tumor responses to radiation (radioresponse), dose prescriptions are not currently tailored to individual patients. Recent large-scale cancer cell line databases hold the promise of unravelling the complex molecular arrangements underlying cellular response to radiation, which is critical to novel predictive biomarker discovery. Here, we present RadioGx, a computational platform for integrative analyses of radioresponse using radiogenomic databases. We first used RadioGx to investigate the robustness of radioresponse assays and indicators. We then combined radioresponse and genome-wide molecular data with established radiobiological models to predict molecular pathways that are relevant for individual tissue types and conditions. We also applied RadioGx to pharmacogenomic data to identify several classes of drugs whose effects correlate with radioresponse. RadioGx provides a unique computational toolbox to advance preclinical research for radiation oncology and precision medicine.

bioinformatics↗

bioRxiv2018

Quantitative Proteomic Analysis of Prostate Tissue Specimens Identifies Deregulated ProteinComplexes in Primary Prostate Cancer

Prostate cancer (PCa) is the most frequently diagnosed non-skin cancer and a leading cause of mortality among males in developed countries. However, our understanding of the global changes of protein complexes within PCa tissue specimens remains very limited, although it has been well recognized that protein complexes carry out essentially all major processes in living organisms and that their deregulation drives the pathogenesis and progression of various diseases. By coupling tandem mass tagging-synchronous precursor selection-mass spectrometry/mass spectrometry/mass spectrometry (TMT-SPSMS3) with differential expression and co-regulation analyses, the present study compared the differences between protein complexes in normal prostate, low-grade PCa, and high-grade PCa tissue specimens. Globally, a large downregulated putative protein-protein interaction (PPI) network was detected in both low-grade and high-grade PCa, yet a large upregulated putative PPI network was only detected in high-grade but not low-grade PCa, compared with normal controls. To identify specific protein complexes that are deregulated in PCa, quantified proteins were mapped to protein complexes in CORUM, a collection of experimentally verified mammalian protein complexes. Differential expression analysis suggested that mitochondrial ribosomes and the fibrillin-associated protein complex were significantly overexpressed, whereas the ITGA6-ITGB4-Laminin10/12 and the P2X7 receptor signaling complexes were significantly downregulated, in PCa compared with normal prostate. Moreover, differential co-regulation analysis indicated that the assembly levels of some nuclear protein complexes involved in RNA synthesis and processing were significantly increased in low-grade PCa, and those of mitochondrial complex I and its subcomplexes were significantly increased in high-grade PCa, compared with normal prostate. In summary, the study represents the first global and quantitative comparison of protein complexes in prostate tissue specimens. It is expected to enhance our understanding of the molecular mechanisms underlying PCa development and progression in human patients, as well as lead to the discovery of novel biomarkers and therapeutic targets for precision management of PCa.

cancer biology↗

bioRxiv2018

MeCP2-E1 isoform is a dynamically expressed, weakly DNA-bound protein with different protein and DNA interactions compared to MeCP2-E2

MeCP2 - a chromatin-binding protein associated with Rett syndrome - has two main isoforms, MeCP2-E1 and MeCP2-E2, with 96% amino acid identity differing in a few N-terminal amino acid residues. Previous studies have shown brain region-specific expression of these isoforms which, in addition to their different cellular localization and differential expression during brain development, suggest they may also have non-overlapping molecular mechanisms. However, differential functions of MeCP2-E1 and E2 remain largely unexplored. Here, we show that the N-terminal domains (NTD) of MeCP2-E1 and E2 modulate the ability of the methyl binding domain (MBD) to interact with DNA as well as influencing the turnover rates, binding dynamics, response to nuclear depolarization, and circadian oscillations of the two isoforms. Our proteomics data indicate that both isoforms exhibit unique interacting protein partners. Moreover, genome-wide analysis using ChIP-seq provide evidence for a shared as well as a specific regulation of different sets of genes. Our findings provide insight into the functional complexity of MeCP2 by dissecting differential aspects of its two isoforms.\n\nSignificanceWhether the two E1 and E2 isoforms of MeCP2 have different structural and/or functional implications has been highly controversial and is not well known. Here we show that the relatively short N-terminal sequence variation between the two isoforms impinges them with an important DNA binding difference. Moreover, MeCP2-E1 and E2 exhibit a different cellular dynamic behavior and have some distinctive interacting partners. In addition, while sharing genome occupancy they specifically bind to several distinctive genes.

biochemistry↗

bioRxiv2018

Necroptosis and apoptosis contribute to cisplatin and aminoglycoside ototoxicity

Ototoxic side effects of cisplatin and aminoglycosides have been extensively studied, but no therapy is available to date. Sensory hair cells, upon exposure to cisplatin or aminoglycosides, undergo apoptotic and necrotic cell death. Blocking these cell death pathways has therapeutic potential in theory, but incomplete protection and lack of therapeutic targets in the case of necrosis, has hampered the development of clinically applicable drugs. Over the past decade, a novel form of necrosis, termed necroptosis, was established as an alternative cell death pathway. Necroptosis is distinguished from passive necrotic cell death, in that it follows a cellular program, involving the receptor-interacting protein kinases 1 and 3 (RIPK1 and 3). In this study, we used pharmacological and genetic intervention to test the relative contributions of necroptosis and caspase-8-mediated apoptosis towards cisplatin and aminoglycoside ototoxicity. We find that ex vivo, only apoptosis contributes to cisplatin and aminoglycoside ototoxicity, while in vivo, both necroptosis and apoptosis are involved. Inhibition of necroptosis and apoptosis using pharmacological compounds is thus a viable strategy to ameliorate aminoglycoside and cisplatin ototoxicity.\n\nSignificance statementThe clinical application of cisplatin and aminoglycosides is limited due to ototoxic side effects. Here, using pharmaceutical and genetic intervention, we present evidence that two types of programmed cell death, apoptosis and necroptosis, contribute to aminoglycoside and cisplatin ototoxicity. Key molecular factors mediating necroptosis are well characterized and druggable, presenting new avenues for pharmaceutical intervention.

neuroscience↗

bioRxiv2018

FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

Many intercellular signals are synthesised as transmembrane precursors that are released by proteolytic cleavage ( shedding) from the cell surface. ADAM17, a membrane-tethered metalloprotease, is the primary shedding enzyme responsible for the release of the inflammatory cytokine TNF and several EGF receptor ligands. ADAM17 exists in complex with the rhomboid-like iRhom proteins, which act as cofactors that regulate ADAM17 substrate shedding. Here we report that the poorly characterised FERM domain-containing protein FRMD8 is a new component of iRhom2/ADAM17 sheddase complex. FRMD8 binds to the cytoplasmic N-terminus of iRhoms, and is necessary to stabilise the iRhoms and ADAM17 beyond the Golgi. In the absence of FRMD8, iRhom2 and ADAM17 are degraded via the endolysosomal pathway, resulting in the reduction of ADAM17-mediated shedding. We have confirmed the pathophysiological significance of FRMD8 in iPSC-derived human macrophages and mouse tissues, thus demonstrating its role in the regulated release of multiple cytokine and growth factor signals.

biochemistry↗

bioRxiv2017

In vivo clonal analysis reveals spatiotemporal regulation of thalamic nucleogenesis

The thalamus, a crucial regulator of cortical functions, is composed of many nuclei arranged in a spatially complex pattern. Thalamic neurogenesis occurs over a short period during mammalian embryonic development. These features have hampered the effort to understand how regionalization, cell divisions and fate specification are coordinated and produce a wide array of nuclei that exhibit distinct patterns of gene expression and functions. Here, we performed in vivo clonal analysis to track the divisions of individual progenitor cells and spatial allocation of their progeny in the developing mouse thalamus. Quantitative analysis of clone compositions revealed evidence for sequential generation of distinct sets of thalamic nuclei based on the location of the founder progenitor cells. Furthermore, we identified intermediate progenitor cells that produced neurons populating more than one thalamic nuclei, indicating a prolonged specification of nuclear fate. Our study reveals an organizational principle that governs the spatial and temporal progression of cell divisions and fate specification, and provides a framework for studying cellular heterogeneity and connectivity in the mammalian thalamus.

neuroscience↗

bioRxiv2017

PharmacoDB: an integrative database for mining in vitro anticancer drug screening studies

Recent pharmacogenomic studies profiled large panels of cancer cell lines against hundreds of approved drugs and experimental chemical compounds. The overarching goal of these screens is to measure sensitivity of cell lines to chemical perturbation, correlate these measures to genomic features, and thereby develop novel predictors of drug response. However, leveraging this valuable data is challenging due to the lack of standards for annotating cell lines and chemical compounds, and quantifying drug response. Moreover, it has been recently shown that the complexity and complementarity of the experimental protocols used in the field result in high levels of technical and biological variation in the in vitro pharmacological profiles. There is therefore a need for new tools to facilitate rigorous comparison and integrative analysis of large-scale drug screening datasets. To address this issue, we have developed PharmacoDB (pharmacodb.pmgenomics.ca), a database integrating the largest pharmacogenomic studies published to date. Here, we describe how the curation of cell line and chemical compound identifiers maximizes the overlap between datasets and how users can leverage such data to compare and extract robust drug phenotypes. PharmacoDB provides a unique resource to mine a compendium of curated pharmacogenomic datasets that are otherwise disparate and difficult to integrate.\n\nKey pointsO_LICuration of cell line and drug identifiers in the largest pharmacogenomic studies published to date\nC_LIO_LIUniform processing of drug sensitivity data to reduce heterogeneity across studies\nC_LIO_LIMultiple drug response summary metrics enabling visual comparison and integrative analysis\nC_LI

bioinformatics↗

bioRxiv2016

Genome-wide association study in Collaborative Cross mice revealed a skeletal role for Rhbdf2

Osteoporosis, the most common bone disease, is characterized by a low bone mass and increased risk of fractures. Importantly, individuals with the same bone mineral density (BMD), as measured on two dimensional (2D) radiographs, have different risks for fracture, suggesting that microstructural architecture is an important determinant of skeletal strength. Here we took advantage of the rich phenotypic and genetic diversity of the Collaborative Cross (CC) mice. Using microcomputed tomography, we examined key structural parameters in the femoral cortical and trabecular compartments of male and female mice from 34 CC lines. These traits included the trabecular bone volume fraction, number, thickness, connectivity, and spacing, as well as structural morphometric index. In the mid-diaphyseal cortex, we recorded cortical thickness and volumetric BMD.\n\nThe broad-sense heritability of these traits ranged between 50 to 60%. We conducted a genome-wide association study to unravel 5 quantitative trait loci (QTL) significantly associated with 6 of the traits. We refined each locus by combining information obtained from the known ancestry of the mice and RNA-Seq data from publicly available sources, to shortlist potential candidate genes. We found strong evidence for new candidate genes, including Rhbdf2, which association to trabecular bone volume fraction and number was strongly suggested by our analyses. We then examined knockout mice, and validated the causal action of Rhbdf2 on bone mass accrual and microarchitecture.\n\nOur approach revealed new genome-wide QTLs and a series of genes that have never been associated with bone microarchitecture. This study demonstrates for the first time the skeletal role of Rhbdf2 on the physiological remodeling of both the cortical and trabecular bone. This newly assigned function for Rhbdf2 can prove useful in deciphering the predisposing factors of osteoporosis and propose new investigative avenues toward targeted therapeutic solutions.\n\nAuthor summaryIn this study, we used the novel mouse reference population, the Collaborative Cross (CC), to identify new causal genes in the regulation of bone microarchitecture, a critical determinant of bone strength. This approach provides a clear advantage in terms of resolution and dimensionality of the morphometric features (versus humans) and rich allelic diversity (versus classical mouse populations), over current practices of bone-related genome-wide association studies.\n\nOur genome-wide study revealed 5 loci significantly associated with microstructural traits in the cortical and trabecular bone. We found strong evidence for new candidate genes, in particular, Rhbdf2. We then validated the specific role of Rhbdf2 on bone mass accrual and microarchitecture using knockout mice. Importantly, this study is the first demonstration of a physiological role for Rhbdf2.

genetics↗

bioRxiv2016

Tissue specificity of in vitro drug sensitivity

Research in oncology traditionally focuses on specific tissue type from which the cancer develops. However, advances in high-throughput molecular profiling technologies have enabled the comprehensive characterization of molecular aberrations in multiple cancer types. It was hoped that these large-scale datasets would provide the foundation for a paradigm shift in oncology which would see tumors being classified by their molecular profiles rather than tissue types, but tumors with similar genomic aberrations may respond differently to targeted therapies depending on their tissue of origin. There is therefore a need to reassess the potential association between pharmacological response and tissue of origin for therapeutic drugs, and to test how these associations translate from preclinical to clinical settings.\n\nIn this paper, we investigate the tissue specificity of drug sensitivities in large-scale pharmacological studies and compare these associations to those found in clinical trial descriptions. Our meta-analysis of the four largest in vitro drug screening datasets indicates that tissue of origin is strongly associated with drug response. We identify novel tissue-drug associations, which may present exciting new avenues for drug repurposing. One caveat is that the vast majority of the significant associations found in preclinical settings do not concur with clinical observations. Accordingly, our results call for more testing to find the root cause of the discrepancies between preclinical and clinical observations.

bioinformatics↗