Papers by Bram Van Den Broek
Scientific Reports
Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative method to screen for protei... more Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative method to screen for protein–protein interactions and is frequently used to record the outcome of signal transduction events. With new highly sensitive and photon efficient FLIM instrumentation, the technique also becomes attractive to screen, with high temporal resolution, for fast changes in Förster Resonance Energy Transfer (FRET), such as those occurring upon activation of cell signaling. The second messenger cyclic adenosine monophosphate (cAMP) is rapidly formed following activation of certain cell surface receptors. cAMP is subsequently degraded by a set of phosphodiesterases (PDEs) which display cell-type specific expression and may also affect baseline levels of the messenger. To study which specific PDEs contribute most to cAMP regulation, we knocked down individual PDEs and recorded breakdown rates of cAMP levels following transient stimulation in HeLa cells stably expressing the FRET/FLIM sensor, Epac-...
Additional file 1: Fig. S1. G1 cell cycle synchronization and controls of ATM and CHK2 inhibitors... more Additional file 1: Fig. S1. G1 cell cycle synchronization and controls of ATM and CHK2 inhibitors. a RPE-1 cells were grown to confluency until they achieved contact inhibition; confluent cells were seeded, allowed to attach for 4 hours, washed three times with PBS and left cultures in starvation media for at least 36 hours for G0 establishment. Cells were re-stimulated with serum-containing medium for 4 hours to obtain a G1-enriched population. b G1 cells were collected by trypsinization, stained with propidium iodide (PI) and analyzed by flow cytometry. PI profile shows that 90% of RPE-1 cells are in G1 phase after the synchronization protocol. c RPE-1 cells irradiated with a dose of 4 Gy were left untreated or pre-treated with ATM inhibitor (ATMi) before IR; cells were harvested 1 hour after IR. Cells treated with ATMi show lower pCHK2 T68 levels, similar to the non-irradiated counterparts (0 Gy), indicating that pCHK2 T68 phosphosite is a good readout for ATM activity. d RPE-1 c...
Release accompanying the article "Resistance of Hypoxic Cells to Ionizing Radiation Is Media... more Release accompanying the article "Resistance of Hypoxic Cells to Ionizing Radiation Is Mediated in Part via Hypoxia-Induced Quiescence", Menegakis et al., Cells (2021)
<b>Copyright information:</b>Taken from "Real-time observation of DNA looping dy... more <b>Copyright information:</b>Taken from "Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI"Nucleic Acids Research 2006;34(1):167-174.Published online 10 Jan 2006PMCID:PMC1326248.© The Author 2006. Published by Oxford University Press. All rights reserved Template #1 is 960 bp in length and has two NaeI recognition sites (and one NarI site). Template #2 is slightly longer, 1296 bp and harbors two NarI sites. In the looped state template #1 is 505 bp and template #2 991 bp in length. () Schematic representation of the experiment. Small beads are tethered with the DNA molecule in question to the glass slide. By tracking the - and -positions of the bead the magnitude of the Brownian motion is monitored, which is a measure for the tether length. Upon DNA loop formation by a restriction enzyme the Brownian motion of the bead suddenly decreases. This allows following DNA looping kinetics in real-time.
<b>Copyright information:</b>Taken from "DNA-tension dependence of restriction e... more <b>Copyright information:</b>Taken from "DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway"Nucleic Acids Research 2005;33(8):2676-2684.Published online 10 May 2005PMCID:PMC1092278.© The Author 2005. Published by Oxford University Press. All rights reserved () Schematic representation of DNA bending by a restriction enzyme under an applied external force. The enzyme induces a sharp kink in the DNA at the center of the recognition sequence. α is the half bend angle θ/2. () Theoretical model curves representing the effect of tension on the induced-fit rate using a bend angle θ = 50°. Dashed curve: overcoming the enthalpic stretching of the recognition sequence. Dotted curve: local shortening of the DNA end-to-end distance due to a kink at the center of the recognition site. Dashed–dotted curve: additional bending and end-to-end shortening of the DNA protruding from the enzyme–DNA complex. Solid curve: all three effects added, providing the dependence of the induced-fit rate on DNA tension.
<b>Copyright information:</b>Taken from "DNA-tension dependence of restriction e... more <b>Copyright information:</b>Taken from "DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway"Nucleic Acids Research 2005;33(8):2676-2684.Published online 10 May 2005PMCID:PMC1092278.© The Author 2005. Published by Oxford University Press. All rights reserved () The effect of tension on cleavage of linearized by EcoRV (number of recognition sites = 1). EcoRV concentration was 25 nM in terms of dimers. The total amount of cutting events was 68. () EcoRV on Lambda phage DNA ( = 21), 32 cutting events. () Tension dependence on DNA cleavage by BamHI (300 nM) on the derivative containing a single recognition site (squares, 78 events) and on Lambda phage DNA with five BamHI sites (triangles, 28 events) using 2.5 nM BamHI. Each point consists of at least 6 cleavage events. Vertical error bars represent the standard error of the mean rate. Horizontal error bars are the standard deviation of the combined DNA tensions (a result of the binning). The data in (a and b) are fitted to the described model (normalized χ are 0.6 and 0.2, respectively). The BamHI rates in (c) do not significantly vary with DNA tension and were fitted with constant values [normalized χ are 0.8 (hydrolysis, green line) and 1.6 (diffusion, blue line)].
<b>Copyright information:</b>Taken from "DNA-tension dependence of restriction e... more <b>Copyright information:</b>Taken from "DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway"Nucleic Acids Research 2005;33(8):2676-2684.Published online 10 May 2005PMCID:PMC1092278.© The Author 2005. Published by Oxford University Press. All rights reserved The association rate is the only rate that depends on enzyme concentration. Under normal conditions, the induced-fit rate () is much faster than hydrolysis () and product dissociation (). The applied tension opposes DNA bending by the enzyme in the induced-fit process.
Many restriction enzymes require binding of two copies of a recognition sequence for DNA cleavage... more Many restriction enzymes require binding of two copies of a recognition sequence for DNA cleavage, thereby introducing a loop in the DNA. We investigated looping dynamics of Type IIE restriction enzymes NaeI and NarI by tracking the Brownian motion of single tethered DNA molecules. DNA containing two endonuclease recognition sites spaced a few 100 bp apart connect small polystyrene beads to a glass surface. The position of a bead is tracked through video microscopy. Protein-mediated looping and unlooping is then observed as a sudden specific change in Brownian motion of the bead. With this method we are able to directly follow DNA looping kinetics of single protein–DNA complexes to obtain loop stability and loop formation times. We show that, in the absence of divalent cations, NaeI induces DNA loops of specific size. In contrast, under these conditions NarI mainly creates nonspecific loops, resulting in effective DNA compaction for higher enzyme concentrations. Addition of Ca 21 in...
bioRxiv, 2021
Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative method to screen for protei... more Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative method to screen for protein-protein interactions and frequently used to record the outcome of signal transduction events. With new highly sensitive and photon efficient FLIM instrumentation, the technique also becomes attractive to screen, with high temporal resolution, for fast changes in Förster Resonance Energy Transfer (FRET), such as those occurring upon activation of cell signaling. We studied the effects of siRNA-mediated individual knockdown of an extensive set of 22 different phosphodiesterases (PDEs) on baseline levels and agonist-induced changes of the second messenger cAMP. Using HeLa cells stably expressing our FRET-FLIM sensor we imaged many hundreds of cells at 5 second intervals for each condition. Following segmentation of cells by the deep-learning implementation Cellpose, FLIM time traces were calculated and fitted for dynamic analysis with custom-made Python scripts. Taking advantage of the qu...
Cell Cycle, 2014
Cytoplasmic dynein is recruited to the cell cortex in early mitosis, where it can generate pullin... more Cytoplasmic dynein is recruited to the cell cortex in early mitosis, where it can generate pulling forces on astral microtubules to position the mitotic spindle. Recent work has shown that dynein displays a dynamic asymmetric cortical localization, and that dynein recruitment is negatively regulated by spindle pole-proximity. this results in oscillating dynein recruitment to opposite sides of the cortex to center the mitotic spindle. However, although the centrosome-derived signal that promotes displacement of dynein has been identified, it is currently unknown how dynein is re-recruited to the cortex once it has been displaced. Here we show that re-recruitment of cortical dynein requires astral microtubules. We find that microtubules are necessary for the sustained localized enrichment of dynein at the cortex. Furthermore, we show that stabilization of astral microtubules causes spindle misorientation, followed by mispositioning of dynein at the cortex. thus, our results demonstrate the importance of astral microtubules in the dynamic regulation of cortical dynein recruitment in mitosis.
<b>Copyright information:</b>Taken from "Real-time observation of DNA looping dy... more <b>Copyright information:</b>Taken from "Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI"Nucleic Acids Research 2006;34(1):167-174.Published online 10 Jan 2006PMCID:PMC1326248.© The Author 2006. Published by Oxford University Press. All rights reserved () Black trace: looping of DNA template #2 (two recognition sites) by NarI (2 U/ml). Grey trace: signal without protein. The histogram on the right clarifies even more that, in contrast to NaeI (), it is not possible to distinguish two discrete levels of Brownian motion. () Black trace: non-specific looping by NarI (2 U/ml) on DNA template #1 (one site). The signal is similar to the trace observed with DNA template #2. Grey trace: signal without protein. () Effect of higher NarI concentrations. Addition of 10 U/ml NarI to DNA substrate #2 tethers results in a drop of Brownian motion from 170 nm to ∼110 nm over several minutes, presumably due to non-specific looping of multiple enzymes. After this initial process the average RMS motion of the bead fluctuates around this last value and never recovers toward the initial value. Upon addition of a high NarI concentration (100 U/ml), the Brownian motion is quickly reduced to a very low level due to non-specific DNA looping of many enzymes. This happens regardless of whether the DNA substrate has one or two NarI sites.
Förster(Fluorescence) Resonance Energy Transfer (FRET) has become a powerful tool to study protei... more Förster(Fluorescence) Resonance Energy Transfer (FRET) has become a powerful tool to study protein-protein interactions and signal transduction in living cells. FRET is commonly read out either by detecting the ratio of the donor and acceptor intensities (sensitized emission) or by detecting the excited state lifetime of the donor, which decreases with increasing FRET (Fluorescence Lifetime IMaging or FLIM). FLIM is robust, immune to bleaching and inherently quantitative, but typically rather slow and photon-inefficient. Moreover, FLIM detection typically requires several images to be collected from the cells, leading to potential artifacts in lifetime when FRET changes rapidly, for example, during fast transients in metabolite concentrations, and when vesicles move within the cell. Using a new generation of cameras capable of collecting two phase images simultaneously, we developed a fast and artifact-immune technique to obtain lifetime images in just a single image. This approach,...
The glucocorticoid receptor (GR) regulates gene expression, governing aspects of homeostasis, but... more The glucocorticoid receptor (GR) regulates gene expression, governing aspects of homeostasis, but is also involved in cancer. Pharmacological GR activation is frequently used to alleviate therapy-related side-effects. While prior studies have shown GR activation might also have anti-proliferative action on tumours, the underpinnings of glucocorticoid action and its direct effectors in non-lymphoid solid cancers remain elusive. Here, we study the mechanisms of glucocorticoid response, focusing on lung cancer. We show that GR activation induces reversible cancer cell dormancy characterised by anticancer drug tolerance, and activation of growth factor survival signalling accompanied by vulnerability to inhibitors. GR-induced dormancy is dependent on a single GR-target gene, CDKN1C, regulated through chromatin looping of a GR-occupied upstream distal enhancer in a SWI/SNF-dependent fashion. These insights illustrate the importance of GR signalling in non-lymphoid solid cancer biology, p...
Current Biology
Summary The endosomal system constitutes a highly dynamic vesicle network used to relay materials... more Summary The endosomal system constitutes a highly dynamic vesicle network used to relay materials and signals between the cell and its environment.1 Once internalized, endosomes gradually mature into late acidic compartments and acquire a multivesicular body (MVB) organization through invagination of the limiting membrane (LM) to form intraluminal vesicles (ILVs).2 Cargoes sequestered into ILVs can either be delivered to lysosomes for degradation or secreted following fusion of the MVB with the plasma membrane.3 It has been speculated that commitment to ILVs is not a terminal event, and that a return pathway exists, allowing “back-fusion” or “retrofusion” of intraluminal membranes to the LM.4 The existence of retrofusion as a way to support membrane equilibrium within the MVB has been widely speculated in various cell biological contexts, including exosome uptake5 and major histocompatibility complex class II (MHC class II) antigen presentation.6, 7, 8, 9 Given the small physical scale, retrofusion of ILVs cannot be measured with conventional techniques. To circumvent this, we designed a chemically tunable cell-based system to monitor retrofusion in real time. Using this system, we demonstrate that retrofusion occurs as part of the natural MVB lifestyle, with attributes parallel to those of viral infection. Furthermore, we find that retrofusion and exocytosis coexist in an equilibrium, implying that ILVs inert to retrofusion comprise a significant fraction of exosomes destined for secretion. MVBs thus contain three types of ILVs: those committed to lysosomal degradation, those retrofusing ILVs, and those subject to secretion in the form of exosomes. Video abstract
Science
We investigated genome folding across the eukaryotic tree of life. We find two types of three-dim... more We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.
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Papers by Bram Van Den Broek