8 research outputs found
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 cryoâelectron 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 ÎČ-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+/HER2â 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 FcÎłR2b 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, 445â450; J. Cell Sci. (1987) 87(Pt 1), 71â84]. 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, 2â10]. 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+/HER2â 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 â âSpotting the Sick Child â Development of the âPOPSâ
toolâ - Winner of the Best Image
from the College of Medicine, Biological Sciences and Psychology
Sarah Hainsworth â âFly Pupaeâ - Winner of the Best Image from the College of Science and Engineering
Stevie-Jade Hardy â âA Human Rightâ - Winner of the Best Image from the College of
Social Sciences, Arts and Humanities
Chris Nixon â âStar Eatersâ - Winner of the Peoples Choice Award
Mark Williams â âCreature From the Black Lagoonâ - Winner of the Best Postgraduate Researcher
Image
Josephina Sampson â âClustered centrosomes in cancerâ
- Second Place for the Best
Postgraduate Researcher Image
Images Of Research 2016 successful submissions:
Aarti Patel â âUntitledâ
Alex Sutton â âVisualisations to assist the analysis of âWhich
treatment is best?â: a collaboration between medical statisticians and computer
scientists from academia and industryâ
Andrew Fry â âUnderstanding the mechanics of cancer cell
divisionâ
Andrew Hopper â âLeicester historians with the wheelchair of Sir
Thomas Fairfax at the National Civil War Centreâ
Benjamin Hall â âThe Martian Space Plasma Environmentâ
Chee Kay Cheung â âShining a light into the kidneyâ
Christine Pulla â âThe web of lifeâ
Clare Gunby â âThe Pocketâ
Dan Stewart â âGeophysical Survey of Roman Knossosâ
David Siveter â âSpectacular 430 MILLION-YEAR-OLD âVIRTUAL FOSSILSâ
help interpret the evolution of lifeâ
Dawn Watkins â âLaw in Childrenâs Lives â
Duncan Murdock â âFossils Are Rottenâ
Elizabeth Jones â âSmall Town Urbanity in
Nineteenth-Century Wales.â
Emma Jones â âAn invitation to imagine a world where complete
accounts of research are always publishedâ
Emma Raven â âIron Heart of the Crystal â Neutron crystal structure
of ascorbate peroxidase compound IIâ
Geoff Belknap â âCitizen Science, and the Uncovering of History of
Female Scientistsâ
Giannis Koukkidis â âSalads and Salmonellasâ
Giovanna Puppin â âAdvertising Culturesâ
Janet Nale â âClostridium difficile bacteriophages
are effective anti-biofilm agentsâ
John Goodwin â âPearl Jephcott (1900-1980): The âCzechoslovakiaâ
Notebookâ
Jun Li â âUntitledâ
Kristina Wright â âKenyan artist Michael Soi
painting at an exhibition of his work in Seoul, South Korea.â
Laura Gray â âAre activity trackers telling us the truth about
our physical activity level?â
Loveday Hodgeson â âFeminist International Judgments
Project: Womenâs Voices in International
Lawâ
Luciano Ost â âEmbedding smart and runtime techniques to improve
multi-core systemsâ reliabilityâ
Maria Theresia Walach â âThe Auroral Heartâ
Mesut Erzurumluoglu â âBreathtaking genesâ
Michael Barer â âSURVIVAL OF THE FATTEST â a TB bacterium (approximately
0.003 mm in length)â
Nicholas Vass â âVisual Community Organisingâ
Paul Dickinson â âA Brightspot on a glass darklyâ
Emmanuel Georgoulis, Dr Andrew Norozov and Andrea Cangiani â
âChaotic Ice Cream Conesâ
Ravi Purohit, Dr Zhanhan Tu, Helen Kuht â âInfantsâ eye
scanâ
Rob Hirst â âTransmission Electron Microscope image of the
unusual case of swollen human respiratory ciliaâ
Rona Aldo â âSupersonic flow modelling thrusts forward
airframe-engine design integration of large twin aircraftâ
Rozita Adib â âThe microtubule cytoskeletonâ
Ruslan Davidchack â âTadpoleâ
Sarah Johnson â âPersistence of Flood Waters - Vale of York - Autumn
2015â
Sarah Thornton â âSenyumâ
Tu Zhanhan â âHopeâ
Wendy Fitzgibbon â âSupervisibleâ
Yewande Okuleye â âSense about Cannabisâ
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