Cortical Development and Brain Malformations: Insights From the Differential Regulation of Early Events of DNA Replication

During the development of the cortex distinct populations of Neural Stem Cells (NSCs) are defined by differences in their cell cycle duration, self-renewal capacity and transcriptional profile. A key difference across the distinct populations of NSCs is the length of G1 phase, where the licensing of the DNA replication origins takes place by the assembly of a pre-replicative complex. Licensing of DNA replication is a process that is adapted accordingly to the cell cycle length of NSCs to secure the timed duplication of the genome. Moreover, DNA replication should be efficiently coordinated with ongoing transcription for the prevention of conflicts that would impede the progression of both processes, compromising the normal course of development. In the present review we discuss how the differential regulation of the licensing and initiation of DNA replication in different cortical NSCs populations is integrated with the properties of these stem cells populations. Moreover, we examine the implication of the initial steps of DNA replication in the pathogenetic mechanisms of neurodevelopmental defects and Zika virus-related microcephaly, highlighting the significance of the differential regulation of DNA replication during brain development

Inference of protein kinetics by stochastic modeling and simulation of fluorescence recovery after photobleaching experiments

Motivation: Fluorescence recovery after photobleaching (FRAP) is a functional live cell imaging technique that permits the exploration of protein dynamics in living cells. To extract kinetic parameters from FRAP data, a number of analytical models have been developed. Simplifications are inherent in these models, which may lead to inexhaustive or inaccurate exploitation of the experimental data. An appealing alternative is offered by the simulation of biological processes in realistic environments at a particle level. However, inference of kinetic parameters using simulation-based models is still limited. Results: We introduce and demonstrate a new method for the inference of kinetic parameter values from FRAP data. A small number of in silico FRAP experiments is used to construct a mapping from FRAP recovery curves to the parameters of the underlying protein kinetics. Parameter estimates from experimental data can then be computed by applying the mapping to the observed recovery curves. A bootstrap process is used to investigate identifiability of the physical parameters and determine confidence regions for their estimates. Our method circumvents the computational burden of seeking the best-fitting parameters via iterative simulation. After validation on synthetic data, the method is applied to the analysis of the nuclear proteins Cdt1, PCNA and GFPnls. Parameter estimation results from several experimental samples are in accordance with previous findings, but also allow us to discuss identifiability issues as well as cell-to-cell variability of the protein kinetics. Implementation: All methods were implemented in MATLAB R2011b. Monte Carlo simulations were run on the HPC cluster Brutus of ETH Zurich. Contact: [email protected] or [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

Running title: Maximal loading of MCM2/4 in late G1

Once-per-cell cycle replication is regulated through the assembly onto chromatin of multisubunit protein complexes that license DNA for a further round of replication. Licensing consists of the loading of the hexameric MCM2-7 complex onto chromatin during G1 phase and is dependent on the licensing factor Cdt1. In vitro experiments have suggested a two-step binding mode for minichromosome maintenance (MCM) proteins, with transient initial interactions converted to stable chromatin loading. Here, we assess MCM loading in live human cells using an in vivo licensing assay on the basis of fluorescence recovery after photobleaching of GFP-tagged MCM protein subunits through the cell cycle. We show that, in telophase, MCM2 and MCM4 maintain transient interactions with chromatin, exhibiting kinetics similar to Cdt1. These are converted to stable interactions from early G1 phase. The immobile fraction of MCM2 and MCM4 increases during G1 phase, suggestive of reiterative licensing. In late G1 phase, a large fraction of MCM proteins are loaded onto chromatin, with maximal licensing observed just prior to S phase onset. Fluorescence loss in photobleaching experiments show subnuclear concentrations of MCM-chromatin interactions that differ as G1 phase progresses and do not colocalize with sites of DNA synthesis in S phase.Fil: Symeonidou, Ioanna Eleni. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Kotsantis, Panagiotis. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Roukos, Vassilis. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Rapsomaniki, Maria Anna. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Grecco, Hernan Edgardo. Max Planck Institute of Molecular Physiology. Department of Systemic Cell Biology; Alemania; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires; Argentina;Fil: Bastiaens, Philippe. Max Planck Institute of Molecular Physiology. Department of Systemic Cell Biology; Alemania;Fil: Taraviras, Stavros. University of Patras. School of Medicine. Laboratory of Physiology; Grecia;Fil: Lygerou, Zoi. University of Patras. School of Medicine. Laboratory of General Biology; Grecia

CK1δ restrains lipin-1 induction, lipid droplet formation and cell proliferation under hypoxia by reducing HIF-1α/ARNT complex formation.

Proliferation of cells under hypoxia is facilitated by metabolic adaptation, mediated by the transcriptional activator Hypoxia Inducible Factor-1 (HIF-1). HIF-1α, the inducible subunit of HIF-1 is regulated by oxygen as well as by oxygen-independent mechanisms involving phosphorylation. We have previously shown that CK1δ phosphorylates HIF-1α in its N-terminus and reduces its affinity for its heterodimerization partner ARNT. To investigate the importance of this mechanism for cell proliferation under hypoxia, we visually monitored HIF-1α interactions within the cell nucleus using the in situ proximity ligation assay (PLA) and fluorescence recovery after photobleaching (FRAP). Both methods show that CK1δ-dependent modification of HIF-1α impairs the formation of a chromatin binding HIF-1 complex. This is confirmed by analyzing expression of lipin-1, a direct target of HIF-1 that mediates hypoxic neutral lipid accumulation. Inhibition of CK1δ increases lipid droplet formation and proliferation of both cancer and normal cells specifically under hypoxia and in an HIF-1α- and lipin-1-dependent manner. These data reveal a novel role for CK1δ in regulating lipid metabolism and, through it, cell adaptation to low oxygen conditions.This work was supported by the “ARISTEIA ΙΙ” Action of the “OPERATIONAL PROGRAMME EDUCATION AND LIFELONG LEARNING” and was co-funded by the European Social Fund (ESF) and National Resources. Partial support was provided by the Proof of Concept Studies for the ESFRI project Euro-BioImaging (Greek BioImaging Facility, PCS facility Nr. 9, Unit 2). N.-N.G., M.A.R. and Z.L. were supported by a grant from the European Research Council and S.S. was supported by a Medical Research Council Senior Fellowship (grant number G0701446).This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S0898656815000637

DNA replication control: Liquid-liquid phase separation comes into play

Liquid-liquid phase separation (LLPS) has been recently suggested as a new potential mechanism underpinning various organizational aspects of the cell, from the formation of sub-cellular, biomolecule enrichments to the assembly of organelles. In eukaryotes, DNA replication follows a strict temporal and spatial program that is majorly affected by the chromatin structure, the nuclear organization and the availability of limiting initiation factors, however the regulatory mechanisms driving the process have not been fully elucidated. Original data published lately revealed for the first time that the components of the pre-replicative complex, ORC, Cdc6 and Cdt1, are able to phase separate indicating a possible connection between LLPS and DNA replication. Here, we critically present these preliminary data and propose mechanistic models that could support this regulatory link and lead to new future research directions

Visualizing the dynamics of histone variants in the S-phase nucleus

Abstract Histone variants constitute a fundamental feature of the epigenome. However, their dynamics during normal and challenged DNA replication and their distribution in the three-dimensional space of the nucleus remain poorly characterized. A recent study employed stochastic optical reconstruction microscopy (STORM) to obtain a high-resolution view of the spatial distribution of H3 histone variants in the nucleus and related this to the timing of DNA replication

Expression of Cdc18/Cdc6 and Cdt1 during G(2) phase induces initiation of DNA replication

Cdc18/Cdc6 and Cdt1 are essential initiation factors for DNA replication. In this paper we show that expression of Cdc18 in fission yeast G(2) cells is sufficient to override the controls that ensure one S phase per cell cycle. Cdc18 expression in G(2) induces DNA synthesis by re-firing replication origins and recruiting the MCM Cdc21 to chromatin in the presence of low levels of Cdt1. However, when Cdt1 is expressed together with Cdc18 in G(2), cells undergo very rapid, uncontrolled DNA synthesis, accumulating DNA contents of 64C or more. Our data suggest that Cdt1 may potentiate re-replication by inducing origins to fire more persistently, possibly by stabilizing Cdc18 on chromatin. In addition, low level expression of a mutant form of Cdc18 that cannot be phosphorylated by cyclin-dependent kinases is not sufficient to induce replication in G(2), but does so only when co-expressed with Cdt1. Thus, regulation of both Cdc18 and Cdt1 in G(2) plays a crucial role in preventing the re-initiation of DNA synthesis until the next cell cycle

IPP Complex Reinforces Adhesion by Relaying Tension-Dependent Signals to Inhibit Integrin Turnover

SummaryCytoskeleton-mediated forces regulate the assembly and function of integrin adhesions; however, the underlying mechanisms remain unclear. The tripartite IPP complex, comprising ILK, Parvin, and PINCH, mediates the integrin-actin link at Drosophila embryo muscle attachment sites (MASs). Here, we demonstrate a developmentally earlier function for the IPP complex: to reinforce integrin-extracellular matrix (ECM) adhesion in response to tension. In IPP-complex mutants, the integrin-ECM linkage at MASs breaks in response to intense muscle contractility. Mechanistically, the IPP complex is required to relay force-elicited signals that decelerate integrin turnover at the plasma membrane so that the integrin immobile fraction is adequate to withstand tension. Epistasis analysis shows that alleviation of muscle contractility, downregulation of endocytosis, and enhanced integrin binding to the ECM are sufficient to restore integrin-ECM adhesion and maintain integrin-adhesome organization in IPP-complex mutants. Our findings reveal a role for the IPP complex as an essential mechanosensitive regulatory switch of integrin turnover in vivo