Understanding the role of the TOPBP1 and BLM interaction in promoting genome stability

The ability to sense, respond to and repair DNA damage is essential in normal development and survival of an organism. A number of human congenital syndromes are associated with mutations in pathways involved in the DNA damage response, including Seckel and Bloom syndromes. Such diseases are often characterised by developmental abnormalities and cancer, emphasising the importance of gaining a deeper understanding of the mechanisms underlying these pathways. The protein kinase ATR, mutated in Seckel syndrome, is a critical mediator of the intra S-phase checkpoint in response to replicative stress. ATR activation is a multistep process that requires its interaction with TOPBP1. Recently published data identified an interaction between TOPBP1 and the BLM helicase. This interaction is dependent on the phosphorylation of the conserved serine 304 of BLM and mutation of this residue results in genome instability. However, exactly how TOPBP1-BLM interaction protects the genome remains unclear. The findings presented in this thesis demonstrate that DT40 cells expressing the BLM S251A mutant are defective in activation of the ATR kinase upon DNA damage. In line with this, these cells display reduced CHK1 phosphorylation, increased origin firing and unreliable replication fork restart. Importantly, these phenotypes could be rescued by fusing the ATR activating domain of TOPBP1 to the mutated BLM. Furthermore, results from BLM-deficient human cell lines demonstrate similar phenotypes. Thus, this data establishes a non-enzymatic role for the BLM helicase in promoting genome stability via activation of the ATR kinase. This may help in explaining why many Bloom syndrome patients display many of the same symptoms as ATR-Seckel patients, including short stature and microcephaly

Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal.

The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient

Galaxy Zoo: quantitative visual morphological classifications for 48 000 galaxies from CANDELS

We present quantified visual morphologies of approximately 48 000 galaxies observed in three Hubble Space Telescope legacy fields by the Cosmic Assembly Near-infrared Deep Extragalactic Legacy Survey (CANDELS) and classified by participants in the Galaxy Zoo project. 90 per cent of galaxies have z ≤ 3 and are observed in rest-frame optical wavelengths by CANDELS. Each galaxy received an average of 40 independent classifications, which we combine into detailed morphological information on galaxy features such as clumpiness, bar instabilities, spiral structure, and merger and tidal signatures. We apply a consensus-based classifier weighting method that preserves classifier independence while effectively down-weighting significantly outlying classifications. After analysing the effect of varying image depth on reported classifications, we also provide depth-corrected classifications which both preserve the information in the deepest observations and also enable the use of classifications at comparable depths across the full survey. Comparing the Galaxy Zoo classifications to previous classifications of the same galaxies shows very good agreement; for some applications, the high number of independent classifications provided by Galaxy Zoo provides an advantage in selecting galaxies with a particular morphological profile, while in others the combination of Galaxy Zoo with other classifications is a more promising approach than using any one method alone. We combine the Galaxy Zoo classifications of ‘smooth’ galaxies with parametric morphologies to select a sample of featureless discs at 1 ≤ z ≤ 3, which may represent a dynamically warmer progenitor population to the settled disc galaxies seen at later epochs

EXD2 Protects Stressed Replication Forks and Is Required for Cell Viability in the Absence of BRCA1/2.

Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells' ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process. EXD2 recruitment to stressed forks suppresses their degradation by restraining excessive fork regression. Accordingly, EXD2 deficiency leads to fork collapse, hypersensitivity to replication inhibitors, and genomic instability. Impeding fork regression by inactivation of SMARCAL1 or removal of RECQ1's inhibition in EXD2-/- cells restores efficient fork restart and genome stability. Moreover, purified EXD2 efficiently processes substrates mimicking regressed forks. Thus, this work identifies a mechanism underpinned by EXD2's nuclease activity, by which cells balance fork regression with fork restoration to maintain genome stability. Interestingly, from a clinical perspective, we discover that EXD2's depletion is synthetic lethal with mutations in BRCA1/2, implying a non-redundant role in replication fork protection.Work in W.N.’s laboratory is funded by ICR Intramural Grant and Cancer Research UK Programme (A24881). R.A.S. and L.S. were supported by WIMM Senior Non-Clinical Fellowship awarded to W.N. M.M.S. and M.A.B. were supported by the Intramural Research Program of the NIH, National Institute on Aging, United States (Z01-AG000746-08). Work in S.G.’s laboratory is supported by BRFAA Intramural Funds. V.C. was supported by John S. Latsis Public Benefit Foundation and Alexander S. Onassis Public Benefit Foundation. Work in the P.P.’s laboratory is supported by grants from the Agence Nationale pour la Recherche (ANR), the Ligue Contre le Cancer (équipe labellisée), SIRIC Montpellier Cancer (INCa Inserm DGOS 12553), and the MSDAvenir fund

A dynamic actin cytoskeleton is required to prevent constitutive VDAC-dependent MAPK-signalling and aberrant lipid homeostasis.

The dynamic nature of the actin cytoskeleton is required to coordinate many cellular processes and a loss of its plasticity has been linked to accelerated cell ageing and attenuation of adaptive response mechanisms. Cofilin is an actin-binding protein that controls actin dynamics and has been linked to mitochondrial signalling pathways that control drug resistance and cell death. Here we show that cofilin-driven chronic depolarisation of the actin cytoskeleton activates cell wall integrity MAPK-signalling and disrupts lipid homeostasis in a VDAC-dependent manner. Expression of the cof1-5 mutation, which reduces the dynamic nature of actin, triggers loss of cell wall integrity, vacuole fragmentation, disruption of lipid homeostasis, lipid droplet (LD) accumulation and the promotion of cell death. The integrity of the actin cytoskeleton is therefore essential to maintain the fidelity of MAPK signalling, lipid homeostasis and cell health in S. cerevisiae. Graphical abstrac

Contains full lipidomic data covering two separate experiments where the relative amount of PA species has been analysed in different brain regions.

For each experiment, brain regions are described using the following code: Mouse gender (Male, M, or Female, F), Genotype, brain region: CRB, Cerebellum (green); CTX, Cortex (blue); OLF, Olfactory bulbs (purple); TH, Thalamus-Hypothalamus and Striatum (white); HP, Hippocampus; Rest, Midbrain-Hindbrain and Medulla (pink). Univariate Analysis of Variance is presented for each condition. (XLSX)</p

In situ hybridisation reveals widespread expression of PLD2.

LNA in situ hybridisation detects PLD2 mRNA in a variety of neuronal but not glial structures in the mouse adult brain. A, B: reconstruction of a sagittal section stained with PLD2 LNA (A) and counterstained with Hoechst to show overall histology (B). Scale bar = 2mm. C-J: different regions of the brain magnified from the boxes in (A) with PLD2 LNA (red) and Hoechst counterstain (blue). C: Hippocampus; PLD2 is expressed a high levels in the CA pyramidal layers, specially the CA2 field (CA2, defined by dashed lines) and at lower levels in the dentate gyrus (DG) while it’s absent from the fimbria (fi). The lateral dorsal (LD) and lateral posterior (LP) nuclei of the thalamus and the subiculum (sub) also express PLD2. D: Cerebellum; high levels of PLD2 are detected in the Purkinje cell layer (PJc, arrow) and the Cerebellar Nuclei (CBN) but not in the glia of the arbor vitae (arb), the molecular layer (mol) and the granular layer (gra). E: Caudoputamen (CP), PLD2 is detected at low level in all neurons but not in the surrounding glial-rich fiber tracks of the corpus callosum (cc), fimbria (fi) and internal capsule (int, dashed lines). F: Isocortex; layers II-VI of the cortex (CTX) express PLD2 but not the corpus callosum (cc) or the outermost glia-rich layer I (I). G: Pons; PLD2 is expressed at high levels in the facial motor nucleus (VII), at lower levels in the superior olivary complex (SOC) and intermediate reticular nucleus (IRN) and not at all in the rubrospinal track (rust). H: Medulla; high levels of PLD2 are detected in the spinal vestibular nucleus (SPIV), the spinal nucleus of the trigeminal (SPV) and the lateral vestibular nucleus (LAV) but not in the parvicellular reticular nucleus (PARN). I: Hypothalamus; PLD2 positive nuclei of the hypothalamus such as the subthalamic nucleus (STN) and lateral hypothalamic area (LHA) are separated by signal-free glial-rich fiber tracks of the optic nerve (opt) and cerebral peduncle (cpd). J: Midbrain; most neurons express PLD2 such as the inferior colliculus (IC) or the cuneiform nucleus (CUN). The parabrachial nucleus also expresses high levels of PLD2. K: Olfactory bulb: PLD2 is detected at high levels in the mitral cell layer (Mi) and interspersed in a few interneurons in the outer plexiform layer (opl) but not in the glomerular layer (Gl) or the granular layer (Gr). L-N: higher magnification of the CA fields of the hippocampus showing that there is more PLD2 detected in the CA2 field relative to cell content defined by DAPI staining. Scale bars A, B = 2mm C-M = 500 μm</p

Lipidomic analysis indicates lipid imbalance in PLD2KO mouse brain.

<p><b>A-F</b> Lipidomic analysis of six regions of the brain indicate that PLD2KO mice have significant changes in their pools of PA. Percentage of PA species are calculated compared to the total sum of PA species identified. A) 32: species, B) 34: species, C) 36: species, D) 38: species, E) Polyunsaturated species, F) Saturated- and monounsaturated species. <i>CRB</i>, Cerebellum; <i>CTX</i>, Cortex; <i>OLF</i>, Olfactory bulbs; <i>TH</i>, Thalamus-Hypothalamus and Striatum; <i>HP</i>, Hippocampus; <i>Rest</i>, Midbrain-Hindbrain and Medulla. <b>G</b> Comparison of difference between percentage of species in WT and PLD2KO indicate that the overall proportion of 32:, 34: and 36: species have significantly changed. A-F significance is calculated by Univariate Analysis of Variance test over two different experiments. G significance is calculated as paired t-test comparing the profile of all brain regions. When p<0.05, *; p<0.01, **; p<0.005, ***</p