Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression

EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo–electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α- and β-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser144 and Ser146 in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression. In addition, constitutive activation of NEK6 or NEK7 reduced the association of EML4 with interphase microtubules. Together, these data support a model in which NEK6- and NEK7-dependent phosphorylation promotes the dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression

CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence

Summary. Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2− breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients

Septicaemia models using Streptococcus pneumoniae and Listeria monocytogenes: understanding the role of complement properdin

Streptococcus pneumoniae and Listeria monocytogenes, pathogens which can cause severe infectious disease in human, were used to infect properdin-deficient and wildtype mice. The aim was to deduce a role for properdin, positive regulator of the alternative pathway of complement activation, by comparing and contrasting the immune response of the two genotypes in vivo. We show that properdin-deficient and wildtype mice mounted antipneumococcal serotype-specific IgM antibodies, which were protective. Properdin-deficient mice, however, had increased survival in the model of streptococcal pneumonia and sepsis. Low activity of the classical pathway of complement and modulation of FcγR2b expression appear to be pathogenically involved. In listeriosis, however, properdin-deficient mice had reduced survival and a dendritic cell population that was impaired in maturation and activity. In vitro analyses of splenocytes and bone marrow-derived myeloid cells support the view that the opposing outcomes of properdin-deficient and wildtype mice in these two infection models is likely to be due to a skewing of macrophage activity to an M2 phenotype in the properdin-deficient mice. The phenotypes observed thus appear to reflect the extent to which M2- or M1-polarised macrophages are involved in the immune responses to S. pneumoniae and L. monocytogenes. We conclude that properdin controls the strength of immune responses by affecting humoral as well as cellular phenotypes during acute bacterial infection and ensuing inflammation

EML4: Cell Cycle-Dependent Regulation And Function

EMLs are a highly conserved family of microtubule-associated proteins. However, whether and how they regulate microtubule dynamics remains far from clear. Human cells express six members of this family, EML1 to EML6. EML1 to 4 have a similar canonical structure, consisting of an N-terminal region containing a coiled-coil trimerization motif and C-terminal tandem b-propeller (TAPE) domain, whereas EML5 and EML6 lack the coiled-coil but have three repeats of the TAPE domain. Structure-function studies have revealed that the TAPE domain allows interaction with soluble tubulin dimers, whereas the N-terminal region of EML1 to 4 including the coiled-coil promotes microtubule binding. In this project, we explored the microtubule binding of endogenous EML4 and its regulation by Nek mitotic kinases. Using localization and depletion approaches, we found that EML4 decorates the interphase microtubule lattice as punctate foci, while it exhibits reduced binding affinity to spindle microtubules. We also showed that endogenous EML4 stabilises microtubules, promotes microtubule acetylation and detyrosination, and protects microtubules from nocodazoleinduced depolymerisation. Depletion of EML4 disturbs mitotic spindle organisation and results in loss of K-fibres suggesting that it also stabilises microtubules in mitosis. We discovered that phosphorylation of EML4 within its N-terminal region at S144 and S146 by the Nek6 and Nek7 mitotic kinases weakens the affinity of EML4 for microtubules. We propose that this phosphorylation of EML4 by Nek kinases disturbs electrostatic interactions between the basic EML4 N-terminal region and the acidic C-terminal tubulin tails exposed on the surface of microtubules. This leads to reduced association of EML4 with microtubules in mitosis enabling the increase in microtubule dynamics that is required for chromosome capture and congression. Consistent with this model, overexpression of an EML4 phosphonull mutant, S144/146A, led to inappropriate retention of EML4 on spindle microtubules disrupting spindle organization and chromosome congresssion

EML Proteins in Microtubule Regulation and Human Disease

The EMLs are a conserved family of microtubule-associated proteins (MAPs). The founding member was discovered in sea urchins as a 77-kDa polypeptide that co-purified with microtubules. This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified microtubule preparations made from unfertilized sea urchin eggs [J. Cell Sci. (1993) 104, 445–450; J. Cell Sci. (1987) 87(Pt 1), 71–84]. Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like protein) has been adopted [BMC Dev. Biol. (2008) 8, 110; Dev. Genes Evol. (2000) 210, 2–10]. The common property of these proteins is their ability to decorate microtubules. However, whether they are associated with particular microtubule populations or exercise specific functions in different microtubule-dependent processes remains unknown. Furthermore, although there is limited evidence that they regulate microtubule dynamics, the biochemical mechanisms of their molecular activity have yet to be explored. Nevertheless, interest in these proteins has grown substantially because of the identification of EML mutations in neuronal disorders and oncogenic fusions in human cancers. Here, we summarize our current knowledge of the expression, localization and structure of what is proving to be an interesting and important class of MAPs. We also speculate about their function in microtubule regulation and highlight how the studies of EMLs in human diseases may open up novel avenues for patient therapy

CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence

Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2− breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients

Images Of Research 2016

Images Of Research 2016 Winners: Damian Roland – ‘Spotting the Sick Child – Development of the ‘POPS’ tool‘ - Winner of the Best Image from the College of Medicine, Biological Sciences and Psychology Sarah Hainsworth – ‘Fly Pupae’ - Winner of the Best Image from the College of Science and Engineering Stevie-Jade Hardy – ‘A Human Right’ - Winner of the Best Image from the College of Social Sciences, Arts and Humanities Chris Nixon – ‘Star Eaters’ - Winner of the Peoples Choice Award Mark Williams – ‘Creature From the Black Lagoon’ - Winner of the Best Postgraduate Researcher Image Josephina Sampson – ‘Clustered centrosomes in cancer’ - Second Place for the Best Postgraduate Researcher Image Images Of Research 2016 successful submissions: Aarti Patel – ‘Untitled’ Alex Sutton – ‘Visualisations to assist the analysis of “Which treatment is best?”: a collaboration between medical statisticians and computer scientists from academia and industry’ Andrew Fry – ‘Understanding the mechanics of cancer cell division’ Andrew Hopper – ‘Leicester historians with the wheelchair of Sir Thomas Fairfax at the National Civil War Centre’ Benjamin Hall – ‘The Martian Space Plasma Environment’ Chee Kay Cheung – ‘Shining a light into the kidney’ Christine Pulla – ‘The web of life’ Clare Gunby – ‘The Pocket‘ Dan Stewart – ‘Geophysical Survey of Roman Knossos’ David Siveter – ‘Spectacular 430 MILLION-YEAR-OLD ‘VIRTUAL FOSSILS’ help interpret the evolution of life’ Dawn Watkins – ‘Law in Children’s Lives ‘ Duncan Murdock – ‘Fossils Are Rotten’ Elizabeth Jones – ‘Small Town Urbanity in Nineteenth-Century Wales.’ Emma Jones – ‘An invitation to imagine a world where complete accounts of research are always published’ Emma Raven – ‘Iron Heart of the Crystal – Neutron crystal structure of ascorbate peroxidase compound II’ Geoff Belknap – ‘Citizen Science, and the Uncovering of History of Female Scientists’ Giannis Koukkidis – ‘Salads and Salmonellas’ Giovanna Puppin – ‘Advertising Cultures‘ Janet Nale – ‘Clostridium difficile bacteriophages are effective anti-biofilm agents’ John Goodwin – ‘Pearl Jephcott (1900-1980): The ‘Czechoslovakia’ Notebook’ Jun Li – ‘Untitled’ Kristina Wright – ‘Kenyan artist Michael Soi painting at an exhibition of his work in Seoul, South Korea.’ Laura Gray – ‘Are activity trackers telling us the truth about our physical activity level?’ Loveday Hodgeson – ‘Feminist International Judgments Project: Women’s Voices in International Law’ Luciano Ost – ‘Embedding smart and runtime techniques to improve multi-core systems’ reliability’ Maria Theresia Walach – ‘The Auroral Heart’ Mesut Erzurumluoglu – ‘Breathtaking genes’ Michael Barer – ‘SURVIVAL OF THE FATTEST – a TB bacterium (approximately 0.003 mm in length)’ Nicholas Vass – ‘Visual Community Organising’ Paul Dickinson – ‘A Brightspot on a glass darkly’ Emmanuel Georgoulis, Dr Andrew Norozov and Andrea Cangiani – ‘Chaotic Ice Cream Cones’ Ravi Purohit, Dr Zhanhan Tu, Helen Kuht – ‘Infants’ eye scan’ Rob Hirst – ‘Transmission Electron Microscope image of the unusual case of swollen human respiratory cilia’ Rona Aldo – ‘Supersonic flow modelling thrusts forward airframe-engine design integration of large twin aircraft’ Rozita Adib – ‘The microtubule cytoskeleton‘ Ruslan Davidchack – ‘Tadpole’ Sarah Johnson – ‘Persistence of Flood Waters - Vale of York - Autumn 2015’ Sarah Thornton – ‘Senyum‘ Tu Zhanhan – ‘Hope’ Wendy Fitzgibbon – ‘Supervisible’ Yewande Okuleye – ‘Sense about Cannabis’ </p