Publications

Weatherdon L, Stuart K, Cassidy MA, de la Gándara AM, Okkenhaug H, Muellener M, Mckenzie G, Cook SJ, Gilley R Signalling, Imaging

The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is activated in cancer due to mutations in RAS proteins (especially KRAS), BRAF, CRAF, MEK1 and MEK2. Whilst inhibitors of KRASG12C (lung adenocarcinoma) and BRAF and MEK1/2 (melanoma and colorectal cancer) are clinically approved, acquired resistance remains a problem. Consequently, the search for new inhibitors (especially of RAS proteins), new inhibitor modalities and regulators of this pathway, which may be new drug targets, continues and increasingly involves cell-based screens with small molecules or genetic screens such as RNAi, CRISPR or Protein Interference. Here we describe cell lines that exhibit doxycycline-dependent expression KRASG12V or BRAFV600E and harbour a stably integrated EGR1:EmGFP reporter gene that can be detected by flow cytometry, high-content microscopy or immunoblotting. KRASG12V or BRAFV600E-driven EmGFP expression is inhibited by MEK1/2 or ERK1/2 inhibitors (MEKi and ERKi). BRAFi inhibit BRAFV600E-driven EmGFP expression but enhance the response to KRASG12V, recapitulating paradoxical activation of wild type RAF proteins. In addition to small molecules, expression of iDab6, encoding a RAS-specific antibody fragment inhibited KRASG12V- but not BRAFV600E-driven EmGFP expression. Finally, substitution of EmGFP for a bacterial nitroreductase gene allowed KRASG12V or BRAFV600E to drive cell death in the presence of a pro-drug, which may allow selection of pathway inhibitors that promote survival. These cell lines should prove useful for cell-based screens to identify new regulators of KRAS- or BRAF-dependent ERK1/2 signalling (drug target discovery) as well as screening or triaging 'hits' from drug discovery screens.

+view abstract The Biochemical journal, PMID: 38381045 21 Feb 2024

Hornigold K, Baker MJ, Machin PA, Chetwynd SA, Johnsson AK, Pantarelli C, Islam P, Stammers M, Crossland L, Oxley D, Okkenhaug H, Walker S, Walker R, Segonds-Pichon A, Fukui Y, Malliri A, Welch HCE Signalling, Imaging, Mass Spectrometry, Bioinformatics, Flow Cytometry

Rac GTPases are required for neutrophil adhesion and migration, and for the neutrophil effector responses that kill pathogens. These Rac-dependent functions are impaired when neutrophils lack the activators of Rac, Rac-GEFs from the Prex, Vav, and Dock families. In this study, we demonstrate that Tiam1 is also expressed in neutrophils, governing focal complexes, actin cytoskeletal dynamics, polarisation, and migration, in a manner depending on the integrin ligand to which the cells adhere. Tiam1 is dispensable for the generation of reactive oxygen species but mediates degranulation and NETs release in adherent neutrophils, as well as the killing of bacteria. , Tiam1 is required for neutrophil recruitment during aseptic peritonitis and for the clearance of during pulmonary infection. However, Tiam1 functions differently to other Rac-GEFs. Instead of promoting neutrophil adhesion to ICAM1 and stimulating β2 integrin activity as could be expected, Tiam1 restricts these processes. In accordance with these paradoxical inhibitory roles, Tiam1 limits the fMLP-stimulated activation of Rac1 and Rac2 in adherent neutrophils, rather than activating Rac as expected. Tiam1 promotes the expression of several regulators of small GTPases and cytoskeletal dynamics, including αPix, Psd4, Rasa3, and Tiam2. It also controls the association of Rasa3, and potentially αPix, Git2, Psd4, and 14-3-3ζ/δ, with Rac. We propose these latter roles of Tiam1 underlie its effects on Rac and β2 integrin activity and on cell responses. Hence, Tiam1 is a novel regulator of Rac-dependent neutrophil responses that functions differently to other known neutrophil Rac-GEFs.

+view abstract Frontiers in immunology, PMID: 38077328 2023

Johansen VBI, Hampson E, Tsonou E, Pantarelli C, Chu JY, Crossland L, Okkenhaug H, Massey AJ, Hornigold DC, Welch HCE, Chetwynd SA Signalling, Imaging

Norbin is an adaptor protein that binds numerous G protein-coupled receptors (GPCRs), is highly expressed in neurons, and is essential for a functioning nervous system in rodent models. Yet, beyond its control of neurite outgrowth and synaptic plasticity, few cellular roles of Norbin have been investigated to date. Furthermore, while Norbin is known to regulate the steady-state cell surface levels of several GPCRs, only in one case has the protein been shown to control the agonist-induced receptor internalisation which serves to attenuate GPCR signalling. Here, we generated a Norbin-deficient PC12 cell line which enabled us to study both the cellular functions of Norbin and its roles in GPCR trafficking and signalling. We show that Norbin limits cell size and spreading, and is required for the growth, viability and cell cycle progression of PC12 cells. We also found that Norbin regulates both the steady-state surface level and agonist-induced internalisation of the GPCR sphingosine-1-phosphate receptor 1 (S1PR1) in these cells, suggesting that its role in agonist-dependent GPCR trafficking is more widespread than previously appreciated. Finally, we show that Norbin limits the S1P-stimulated activation of Akt and p38 Mapk, and is required for the activation of Erk in PC12 cells. Together, our findings provide a better understanding of the cellular functions of Norbin and its control of GPCR trafficking.

+view abstract Scientific reports, PMID: 37880240 25 Oct 2023

Chessa TAM, Jung P, Anwar A, Suire S, Anderson KE, Barneda D, Kielkowska A, Sadiq BA, Lai IW, Felisbino S, Turnham DJ, Pearson HB, Phillips WA, Sasaki J, Sasaki T, Oxley D, Spensberger D, Segonds-Pichon A, Wilson M, Walker S, Okkenhaug H, Cosulich S, Hawkins PT, Stephens LR Signalling, Imaging, Mass Spectrometry, Bioinformatics, Gene Targeting

The PIP/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP/PI(3,4)P phosphatase, PTEN. Despite huge research investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signaling and constrained by pathway feedback. In the absence of PTEN, the network is dramatically remodeled. A poorly understood YXXM- and PIP/PI(3,4)P-binding PH domain-containing adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP, AKT phosphorylation, and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K- and Src-family kinase-dependent phosphorylation of YXXM, eliciting PI3K activation. hPLEKHS1 mRNA and activating Y phosphorylation of hSrc correlated with PI3K pathway activity in human prostate cancers. We propose that in PTEN-null cells receptor-independent, Src-dependent tyrosine phosphorylation of PLEKHS1 creates positive feedback that escapes homeostasis, drives PIP signaling, and supports tumor progression.

+view abstract Molecular cell, PMID: 37567175 17 Aug 2023

Machin PA, Johnsson AE, Massey EJ, Pantarelli C, Chetwynd SA, Chu JY, Okkenhaug H, Segonds-Pichon A, Walker S, Malliri A, Fukui Y, Welch HCE Signalling, Bioinformatics, Imaging

Rac-GTPases and their Rac-GEF activators play important roles in neutrophil-mediated host defence. These proteins control the adhesion molecules and cytoskeletal dynamics required for neutrophil recruitment to inflamed and infected organs, and the neutrophil effector responses that kill pathogens.

+view abstract Frontiers in immunology, PMID: 37383235 2023

Maskalenka K, Alagöz G, Krueger F, Wright J, Rostovskaya M, Nakhuda A, Bendall A, Krueger C, Walker S, Scally A, Rugg-Gunn PJ Epigenetics, Gene Targeting, Bioinformatics, Imaging

Gene duplication events can drive evolution by providing genetic material for new gene functions, and create opportunities for diverse developmental strategies to emerge between species. To study the contribution of duplicated genes to human early development, we examined the evolution and function of NANOGP1, a tandem duplicate of the transcription factor NANOG. We found that NANOGP1 and NANOG have overlapping but distinct expression profiles, with high NANOGP1 expression restricted to early epiblast cells and naïve-state pluripotent stem cells. Sequence analysis and epitope-tagging revealed that NANOGP1 is protein-coding with an intact homeobox domain. The duplication that created NANOGP1 occurred earlier in primate evolution than previously thought and has been retained only in great apes, whereas Old World monkeys have disabled the gene in different ways including homeodomain point mutations. NANOGP1 is a strong inducer of naïve pluripotency; however, unlike NANOG, it is not required to maintain the undifferentiated status of human naïve pluripotent cells. By retaining expression, sequence and partial functional conservation with its ancestral copy, NANOGP1 exemplifies how gene duplication and subfunctionalisation can contribute to transcription factor activity in human pluripotency and development.

+view abstract Development (Cambridge, England), PMID: 36621005 09 Jan 2023

Gill D, Parry A, Santos F, Okkenhaug H, Todd CD, Hernando-Herraez I, Stubbs TM, Milagre I, Reik W Epigenetics, Imaging

Ageing is the gradual decline in organismal fitness that occurs over time leading to tissue dysfunction and disease. At the cellular level, ageing is associated with reduced function, altered gene expression and a perturbed epigenome. Recent work has demonstrated that the epigenome is already rejuvenated by the maturation phase of somatic cell reprogramming, which suggests full reprogramming is not required to reverse ageing of somatic cells. Here we have developed the first "maturation phase transient reprogramming" (MPTR) method, where reprogramming factors are selectively expressed until this rejuvenation point then withdrawn. Applying MPTR to dermal fibroblasts from middle-aged donors, we found that cells temporarily lose and then reacquire their fibroblast identity, possibly as a result of epigenetic memory at enhancers and/or persistent expression of some fibroblast genes. Excitingly, our method substantially rejuvenated multiple cellular attributes including the transcriptome, which was rejuvenated by around 30 years as measured by a novel transcriptome clock. The epigenome was rejuvenated to a similar extent, including H3K9me3 levels and the DNA methylation ageing clock. The magnitude of rejuvenation instigated by MPTR appears substantially greater than that achieved in previous transient reprogramming protocols. In addition, MPTR fibroblasts produced youthful levels of collagen proteins, and showed partial functional rejuvenation of their migration speed. Finally, our work suggests that optimal time windows exist for rejuvenating the transcriptome and the epigenome. Overall, we demonstrate that it is possible to separate rejuvenation from complete pluripotency reprogramming, which should facilitate the discovery of novel anti-ageing genes and therapies.

+view abstract eLife, PMID: 35390271 08 Apr 2022

Robinson EL, Drawnel FM, Mehdi S, Archer CR, Liu W, Okkenhaug H, Alkass K, Aronsen JM, Nagaraju CK, Sjaastad I, Sipido KR, Bergmann O, Arthur JSC, Wang X, Roderick HL Signalling, Imaging

Heart failure is a leading cause of death that develops subsequent to deleterious hypertrophic cardiac remodelling. MAPK pathways play a key role in coordinating the induction of gene expression during hypertrophy. Induction of the immediate early gene (IEG) response including activator protein 1 (AP-1) complex factors is a necessary and early event in this process. How MAPK and IEG expression are coupled during cardiac hypertrophy is not resolved. Here, in vitro, in rodent models and in human samples, we demonstrate that MAPK-stimulated IEG induction depends on the mitogen and stress-activated protein kinase (MSK) and its phosphorylation of histone H3 at serine 28 (pH3S28). pH3S28 in IEG promoters in turn recruits Brg1, a BAF60 ATP-dependent chromatin remodelling complex component, initiating gene expression. Without MSK activity and IEG induction, the hypertrophic response is suppressed. These studies provide new mechanistic insights into the role of MAPK pathways in signalling to the epigenome and regulation of gene expression during cardiac hypertrophy.

+view abstract Cells, PMID: 35203255 09 Feb 2022

Prescott JA, Balmanno K, Mitchell JP, Okkenhaug H, Cook SJ Signalling, Imaging

Inhibitor of kappa B (IκB) kinase β (IKKβ) has long been viewed as the dominant IKK in the canonical nuclear factor-κB (NF-κB) signalling pathway, with IKKα being more important in non-canonical NF-κB activation. Here we have investigated the role of IKKα and IKKβ in canonical NF-κB activation in colorectal cells using CRISPR-Cas9 knock-out cell lines, siRNA and selective IKKβ inhibitors. IKKα and IKKβ were redundant for IκBα phosphorylation and turnover since loss of IKKα or IKKβ alone had little (SW620 cells) or no (HCT116 cells) effect. However, in HCT116 cells IKKα was the dominant IKK required for basal phosphorylation of p65 at S536, stimulated phosphorylation of p65 at S468, nuclear translocation of p65 and the NF-κB-dependent transcriptional response to both TNFα and IL-1α. In these cells IKKβ was far less efficient at compensating for the loss of IKKα than IKKα was able to compensate for the loss of IKKβ. This was confirmed when siRNA was used to knock-down the non-targeted kinase in single KO cells. Critically, the selective IKKβ inhibitor BIX02514 confirmed these observations in WT cells and similar results were seen in SW620 cells. Notably, whilst IKKα loss strongly inhibited TNFα-dependent p65 nuclear translocation, IKKα and IKKβ contributed equally to c-Rel nuclear translocation indicating that different NF-κB subunits exhibit different dependencies on these IKKs. These results demonstrate a major role for IKKα in canonical NF-κB signalling in colorectal cells and may be relevant to efforts to design IKK inhibitors, which have focused largely on IKKβ to date.

+view abstract The Biochemical journal, PMID: 35029639 14 Jan 2022

Fra-Bido S, Walker SA, Innocentin S, Linterman MA Immunology, Imaging

Location of immune cells that form the germinal center reaction within secondary lymphoid tissues can be characterized using confocal microscopy. Here, we present an optimized immunofluorescence staining protocol to image germinal center structures in fixed/frozen spleen sections from ChAdOx1 nCoV-19 immunized mice. This protocol can be adapted to identify other cell types within secondary lymphoid tissues. For complete information on the generation and use of this protocol to examine immune responses to the COVID vaccine ChAdOx1 nCoV-19, please refer to Silva-Cayetano et al. (2020).

+view abstract STAR protocols, PMID: 34195671 17 Sep 2021

Lehmann LC, Bacic L, Hewitt G, Brackmann K, Sabantsev A, Gaullier G, Pytharopoulou S, Degliesposti G, Okkenhaug H, Tan S, Costa A, Skehel JM, Boulton SJ, Deindl S Imaging

Upon DNA damage, the ALC1/CHD1L nucleosome remodeling enzyme (remodeler) is activated by binding to poly(ADP-ribose). How activated ALC1 recognizes the nucleosome, as well as how this recognition is coupled to remodeling, is unknown. Here, we show that remodeling by ALC1 requires a wild-type acidic patch on the entry side of the nucleosome. The cryo-electron microscopy structure of a nucleosome-ALC1 linker complex reveals a regulatory linker segment that binds to the acidic patch. Mutations within this interface alter the dynamics of ALC1 recruitment to DNA damage and impede the ATPase and remodeling activities of ALC1. Full activation requires acidic patch-linker segment interactions that tether the remodeler to the nucleosome and couple ATP hydrolysis to nucleosome mobilization. Upon DNA damage, such a requirement may be used to modulate ALC1 activity via changes in the nucleosome acidic patches.

+view abstract Cell reports, PMID: 33357431 22 Dec 2020

Vardaka P, Lozano T, Bot C, Ellery J, Whiteside SK, Imianowski CJ, Farrow S, Walker S, Okkenhaug H, Yang J, Okkenhaug K, Kuo P, Roychoudhuri R Immunology, Imaging

Whereas effector CD4 and CD8 T cells promote immune activation and can drive clearance of infections and cancer, CD4 regulatory T (T) cells suppress their function, contributing to both immune homeostasis and cancer immunosuppression. The transcription factor BACH2 functions as a pervasive regulator of T cell differentiation, promoting development of CD4 T cells and suppressing the effector functions of multiple effector T cell (T) lineages. Here, we report the development of a stable cell-based bioluminescence assay of the transcription factor activity of BACH2. Tetracycline-inducible BACH2 expression resulted in suppression of phorbol 12-myristate 13-acetate (PMA)/ionomycin-driven activation of a luciferase reporter containing BACH2/AP-1 target sequences from the mouse Ifng + 18k enhancer. BACH2 expression repressed the luciferase signal in a dose-dependent manner but this activity was abolished at high levels of AP-1 signalling, suggesting contextual regulation of AP-1 driven gene expression by BACH2. Finally, using the reporter assay developed, we find that the histone deacetylase 3 (HDAC3)-selective inhibitor, RGFP966, inhibits BACH2-mediated repression of signal-driven luciferase expression. In addition to enabling mechanistic studies, this cell-based reporter may enable identification of small molecule agonists or antagonists of BACH2 function for drug development.

+view abstract Scientific reports, PMID: 33144667 03 Nov 2020

Okkenhaug H, Chauve L, Masoudzadeh F, Okkenhaug L, Casanueva O Epigenetics, Imaging

An issue often encountered when acquiring image data from fixed or anesthetized C. elegans is that worms cross and cluster with their neighbors. This problem is aggravated with increasing density of worms and creates challenges for imaging and quantification. We developed a FIJI-based workflow, Worm-align, that can be used to generate single- or multi-channel montages of user-selected, straightened and aligned worms from raw image data of C. elegans. Worm-align is a simple and user-friendly workflow that does not require prior training of either the user or the analysis algorithm. Montages generated with Worm-align can aid the visual inspection of worms, their classification and representation. In addition, the output of Worm-align can be used for subsequent quantification of fluorescence intensity in single worms, either in FIJI directly, or in other image analysis software platforms. We demonstrate this by importing the Worm-align output into Worm_CP, a pipeline that uses the open-source CellProfiler software. CellProfiler's flexibility enables the incorporation of additional modules for high-content screening. As a practical example, we have used the pipeline on two datasets: the first dataset are images of heat shock reporter worms that express green fluorescent protein (GFP) under the control of the promoter of a heat shock inducible gene hsp-70, and the second dataset are images obtained from fixed worms, stained for fat-stores with a fluorescent dye.

+view abstract Journal of visualized experiments, PMID: 32538914 28 May 2020

Dalle Pezze P, Karanasios E, Kandia V, Manifava M, Walker SA, Gambardella Le Novère N, Ktistakis NT Signalling, Imaging

During macroautophagy/autophagy, the ULK complex nucleates autophagic precursors, which give rise to autophagosomes. We analyzed, by live imaging and mathematical modeling, the translocation of ATG13 (part of the ULK complex) to the autophagic puncta in starvation-induced autophagy and ivermectin-induced mitophagy. In nonselective autophagy, the intensity and duration of ATG13 translocation approximated a normal distribution, whereas wortmannin reduced this effect and shifted to a log-normal distribution. During mitophagy, multiple translocations of ATG13 with increasing time between peaks were observed. We hypothesized that these multiple translocations arise because the engulfment of mitochondrial fragments required successive nucleation of phagophores on the same target, and a mathematical model based on this idea reproduced the oscillatory behavior. Significantly, model and experimental data were also in agreement that the number of ATG13 translocations is directly proportional to the diameter of the targeted mitochondrial fragments. Thus, our data provide novel insights into the early dynamics of selective and nonselective autophagy. ATG: autophagy related 13; CFP: cyan fluorescent protein; dsRED: red fluorescent protein; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; IVM: ivermectin; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: PtdIns-3-phosphate; ULK: unc-51 like autophagy activating kinase.

+view abstract Autophagy, PMID: 32320309 22 Apr 2020

Kazakevych J, Denizot J, Liebert A, Portovedo M, Mosavie M, Jain P, Stellato C, Fraser C, Corrêa RO, Célestine M, Mattiuz R, Okkenhaug H, Miller JR, Vinolo MAR, Veldhoen M, Varga-Weisz P Immunology, Imaging

How intestinal epithelial cells interact with the microbiota and how this is regulated at the gene expression level are critical questions. Smarcad1 is a conserved chromatin remodeling factor with a poorly understood tissue function. As this factor is highly expressed in the stem and proliferative zones of the intestinal epithelium, we explore its role in this tissue.

+view abstract Genome biology, PMID: 32160911 11 Mar 2020

Walker SA, Ktistakis NT Signalling, Imaging

We review current knowledge of the process of autophagosome formation with special emphasis on the very early steps: turning on the autophagy pathway, assembling the autophagy machinery, and building the autophagosome. The pathway is remarkably well coordinated spatially and temporally, and it shows broad conservation across species and cell types, including neurons. In addition, although much current knowledge derives mostly from settings of nonselective autophagy, recent work also indicates that selective autophagy, and more specifically mitophagy, shows similar dynamics. Having an understanding of this remarkable process may help the design of novel therapeutics for neurodegeneration and other pathologies.

+view abstract Journal of molecular biology, PMID: 31705882 03 04 2020

Zachari M, Gudmundsson SR, Li Z, Manifava M, Shah R, Smith M, Stronge J, Karanasios E, Piunti C, Kishi-Itakura C, Vihinen H, Jokitalo E, Guan JL, Buss F, Smith AM, Walker SA, Eskelinen EL, Ktistakis NT Signalling, Imaging

The dynamics and coordination between autophagy machinery and selective receptors during mitophagy are unknown. Also unknown is whether mitophagy depends on pre-existing membranes or is triggered on the surface of damaged mitochondria. Using a ubiquitin-dependent mitophagy inducer, the lactone ivermectin, we have combined genetic and imaging experiments to address these questions. Ubiquitination of mitochondrial fragments is required the earliest, followed by auto-phosphorylation of TBK1. Next, early essential autophagy proteins FIP200 and ATG13 act at different steps, whereas ULK1 and ULK2 are dispensable. Receptors act temporally and mechanistically upstream of ATG13 but downstream of FIP200. The VPS34 complex functions at the omegasome step. ATG13 and optineurin target mitochondria in a discontinuous oscillatory way, suggesting multiple initiation events. Targeted ubiquitinated mitochondria are cradled by endoplasmic reticulum (ER) strands even without functional autophagy machinery and mitophagy adaptors. We propose that damaged mitochondria are ubiquitinated and dynamically encased in ER strands, providing platforms for formation of the mitophagosomes.

+view abstract Developmental cell, PMID: 31353311 2019

Dautova Y, Kapustin AN, Pappert K, Epple M, Okkenhaug H, Cook SJ, Shanahan CM, Bootman MD, Proudfoot D Signalling, Imaging

Calcium phosphate (CaP) particle deposits are found in several inflammatory diseases including atherosclerosis and osteoarthritis. CaP, and other forms of crystals and particles, can promote inflammasome formation in macrophages leading to caspase-1 activation and secretion of mature interleukin-1β (IL-1β). Given the close association of small CaP particles with vascular smooth muscle cells (VSMCs) in atherosclerotic fibrous caps, we aimed to determine if CaP particles affected pro-inflammatory signalling in human VSMCs.

+view abstract Journal of molecular and cellular cardiology, PMID: 29274344 2017

Sipthorp J, Lebraud H, Gilley R, Kidger A, Okkenhaug H, Saba-El-Leil MK, Meloche S, Caunt CJ, Cook S, Heightman TD Signalling, Imaging

The RAS-RAF-MEK-ERK pathway has been intensively studied in oncology with RAS known to be mutated in ~30% of all human cancers. The recent emergence of ERK1/2 inhibitors and their ongoing clinical investigation demands a better understanding of ERK1/2 behaviour following small molecule inhibition. Although fluorescent fusion proteins and fluorescent antibodies are well-established methods to visualise proteins, we show that ERK1/2 can be visualised via a less invasive approach based on a two-step process using Inverse Electron Demand Diels-Alder cycloaddition. Our previously reported TCO-tagged covalent ERK1/2 inhibitor was used in a series of imaging experiments following a click reaction with a tetrazine-tagged fluorescent dye. Although limitations were encountered with this approach, endogenous ERK1/2 was successfully imaged in cells and 'on target' staining was confirmed by overexpressing DUSP5, a nuclear ERK1/2 phosphatase which anchors ERK1/2 in the nucleus.

+view abstract Bioconjugate chemistry, PMID: 28449575 2017

Thienpont B, Aronsen JM, Robinson EL, Okkenhaug H, Loche E, Ferrini A, Brien P, Alkass K, Tomasso A, Agrawal A, Bergmann O, Sjaastad I, Reik W, Roderick HL Epigenetics, Imaging

Cardiac hypertrophic growth in response to pathological cues is associated with reexpression of fetal genes and decreased cardiac function and is often a precursor to heart failure. In contrast, physiologically induced hypertrophy is adaptive, resulting in improved cardiac function. The processes that selectively induce these hypertrophic states are poorly understood. Here, we have profiled 2 repressive epigenetic marks, H3K9me2 and H3K27me3, which are involved in stable cellular differentiation, specifically in cardiomyocytes from physiologically and pathologically hypertrophied rat hearts, and correlated these marks with their associated transcriptomes. This analysis revealed the pervasive loss of euchromatic H3K9me2 as a conserved feature of pathological hypertrophy that was associated with reexpression of fetal genes. In hypertrophy, H3K9me2 was reduced following a miR-217-mediated decrease in expression of the H3K9 dimethyltransferases EHMT1 and EHMT2 (EHMT1/2). miR-217-mediated, genetic, or pharmacological inactivation of EHMT1/2 was sufficient to promote pathological hypertrophy and fetal gene reexpression, while suppression of this pathway protected against pathological hypertrophy both in vitro and in mice. Thus, we have established a conserved mechanism involving a departure of the cardiomyocyte epigenome from its adult cellular identity to a reprogrammed state that is accompanied by reexpression of fetal genes and pathological hypertrophy. These results suggest that targeting miR-217 and EHMT1/2 to prevent H3K9 methylation loss is a viable therapeutic approach for the treatment of heart disease.

+view abstract The Journal of clinical investigation, PMID: 27893464 2016

Ktistakis NT, Walker SA, Karanasios E Signalling, Imaging

+view abstract Oncotarget, PMID: 27829241 2016

Manifava M, Smith M, Rotondo S, Walker S, Niewczas I, Zoncu R, Clark J, Ktistakis NT Signalling, Imaging

Amino acids are essential activators of mTORC1 via a complex containing RAG GTPases, RAGULATOR and the vacuolar ATPase. Sensing of amino acids causes translocation of mTORC1 to lysosomes, an obligate step for activation. To examine the spatial and temporal dynamics of this translocation, we used live imaging of the mTORC1 component RAPTOR and a cell permeant fluorescent analogue of di-leucine methyl ester. Translocation to lysosomes is a transient event, occurring within 2 min of aa addition and peaking within 5 min. It is temporally coupled with fluorescent leucine appearance in lysosomes and is sustained in comparison to aa stimulation. Sestrin2 and the vacuolar ATPase are negative and positive regulators of mTORC1 activity in our experimental system. Of note, phosphorylation of canonical mTORC1 targets is delayed compared to lysosomal translocation suggesting a dynamic and transient passage of mTORC1 from the lysosomal surface before targetting its substrates elsewhere.

+view abstract eLife, PMID: 27725083 2016

Karanasios E, Walker SA, Okkenhaug H, Manifava M, Hummel E, Zimmermann H, Ahmed Q, Domart MC, Collinson L, Ktistakis NT Signalling, Imaging

Autophagosome formation requires sequential translocation of autophagy-specific proteins to membranes enriched in PI3P and connected to the ER. Preceding this, the earliest autophagy-specific structure forming de novo is a small punctum of the ULK1 complex. The provenance of this structure and its mode of formation are unknown. We show that the ULK1 structure emerges from regions, where ATG9 vesicles align with the ER and its formation requires ER exit and coatomer function. Super-resolution microscopy reveals that the ULK1 compartment consists of regularly assembled punctate elements that cluster in progressively larger spherical structures and associates uniquely with the early autophagy machinery. Correlative electron microscopy after live imaging shows tubulovesicular membranes present at the locus of this structure. We propose that the nucleation of autophagosomes occurs in regions, where the ULK1 complex coalesces with ER and the ATG9 compartment.

+view abstract Nature communications, PMID: 27510922 2016

Petrini E, Baillet V, Cridge J, Hogan CJ, Guillaume C, Ke H, Brandetti E, Walker S, Koohy H, Spivakov M, Varga-Weisz P Imaging

Endocytosis is essential for uptake of many substances into the cell, but how it links to nutritional signalling is poorly understood. Here we show a novel role for endocytosis in regulating the response to low phosphate in Schizosaccharomyces pombe. Loss of function of Myo1, Sla2/End4 or Arp2, proteins involved in the early steps of endocytosis, led to increased proliferation in low phosphate media compared to controls. We show that once cells are deprived of phosphate they undergo a quiescence response that is dependent on the endocytic function of Myo1. Transcriptomic analysis revealed a wide perturbation of gene expression with induction of stress-regulated genes upon phosphate starvation in wildtype but not Δmyo1 cells. Thus, endocytosis plays a pivotal role in mediating the cellular response to nutrients, bridging the external environment and internal molecular functions of the cell.

+view abstract Journal of cell science, PMID: 26345368 2015

Karanasios E, Stapleton E, Manifava M, Kaizuka T, Mizushima N, Walker SA, Ktistakis NT Signalling, Imaging

Induction of autophagy requires the ULK1 protein kinase complex and the Vps34 lipid kinase complex. PtdIns3P synthesised by Vps34 accumulates in omegasomes, membrane extensions of the ER within which some autophagosomes form. The ULK1 complex is thought to target autophagosomes independently of PtdIns3P, and its functional relationship to omegasomes is unclear. Here we show that the ULK1 complex colocalises with omegasomes in a PtdIns3P-dependent way. Live-cell imaging of Atg13 (a ULK1 complex component), omegasomes and LC3 establishes and annotates for the first time a complete sequence of steps leading to autophagosome formation, as follows. Upon starvation, the ULK1 complex forms puncta associated with the ER and sporadically with mitochondria. If PtdIns3P is available, these puncta become omegasomes. Subsequently, the ULK1 complex exits omegasomes and autophagosomes bud off. If PtdIns3P is unavailable, ULK1 puncta are greatly reduced in number and duration. Atg13 contains a region with affinity for acidic phospholipids, required for translocation to punctate structures and autophagy progression.

+view abstract Journal of cell science, PMID: 24013547 2013

Musiwaro P, Smith M, Manifava M, Walker SA, Ktistakis NT Signalling, Imaging

Basal autophagy-here defined as macroautophagic activity during cellular growth in normal medium containing amino acids and serum-appears to be highly active in many cell types and in animal tissues. Here we characterized this pathway in mammalian HEK 293 cells. First, we examined, side by side, three compounds that are widely used to reveal basal autophagy by blocking maturation of autophagosomes: bafilomycin A 1 (BafA1), chloroquine and vinblastine. Only BafA1 appeared to be without complicating side effects. Chloroquine partially inhibited mechanistic target of rapamycin (MTOR) activity, which would induce autophagy induction as well as block autophagosome maturation. Vinblastine caused the distribution of early omegasome components into punctate phagophore assembly sites, and therefore it would also induce autophagy, complicating interpretation. Basal autophagy was significantly sensitive to inhibition by wortmannin, and therefore required formation of phosphatidylinositol 3-phosphate (PtdIns3P), but it was twice as resistant to wortmannin as starvation-induced autophagy. We also determined that basal autophagy was significantly suppressed by MTOR activation brought about by overexpression of RHEB or activated RAGs. Finally we investigated the spatial relationship of nascent autophagosomes to the endoplasmic reticulum (ER) or to mitochondria by live imaging experiments under conditions that reveal basal autophagy (with BafA1 treatment), or upon MTOR inactivation (which would result in autophagy induction). Side-by-side comparison showed that under both basal and induced autophagy, 100% of autophagosomes first appeared in close proximity to ER strands. In parallel measurements, 40% were in close proximity to mitochondria under both conditions. We concluded that in HEK 293 cells, basal autophagy is mechanistically similar to that induced by MTOR inactivation in all aspects examined.

+view abstract Autophagy, PMID: 23800949 2013

R Adalbert, G Morreale, M Paizs, L Conforti, SA Walker, HL Roderick, MD Bootman, L Siklós, MP Coleman Signalling, Imaging

Calcium accumulation induces the breakdown of cytoskeleton and axonal fragmentation in the late stages of Wallerian degeneration. In the early stages there is no evidence for any long-lasting, extensive increase in intra-axonal calcium but there does appear to be some redistribution. We hypothesized that changes in calcium distribution could have an early regulatory role in axonal degeneration in addition to the late executionary role of calcium. Schmidt-Lanterman clefts (SLCs), which allow exchange of metabolites and ions between the periaxonal and extracellular space, are likely to have an increased role when axon segments are separated from the cell body, so we used the oxalate-pyroantimonate method to study calcium at SLCs in distal stumps of transected wild-type and slow Wallerian degeneration (Wld(S)) mutant sciatic nerves, in which Wallerian degeneration is greatly delayed. In wild-type nerves most SLCs show a step gradient of calcium distribution, which is lost at around 20% of SLCs within 3mm of the lesion site by 4-24h after nerve transection. To investigate further the association with Wallerian degeneration, we studied nerves from Wld(S) rats. The step gradient of calcium distribution in Wld(S) is absent in around 20% of the intact nerves beneath SLCs but 4-24h following injury, calcium distribution in transected axons remained similar to that in uninjured nerves. We then used calcium indicators to study influx and buffering of calcium in injured neurites in primary culture. Calcium penetration and the early calcium increase in this system were indistinguishable between Wld(S) and wild-type axons. However, a significant difference was observed during the following hours, when calcium increased in wild-type neurites but not in Wld(S) neurites. We conclude that there is little relationship between calcium distribution and the early stages of Wallerian degeneration at the time points studied in vivo or in vitro but that Wld(S) neurites fail to show a later calcium rise that could be a cause or consequence of the later stages of Wallerian degeneration.

+view abstract Neuroscience, PMID: 22960623 2012

S Walker, N Cunniffe, M Bootman, HL Roderick , Imaging

+view abstract BioTechniques, PMID: 18778263 2008