PCNA appears in two populations of slow and fast diffusion with a constant ratio throughout S-phase in replicating mammalian cells

DNA replication is a fundamental cellular process that precedes cell division. Proliferating cell nuclear antigen (PCNA) is a central scaffold protein that orchestrates DNA replication by recruiting many factors essential for the replication machinery. We studied the mobility of PCNA in live mammalian cells using single-particle tracking in combination with photoactivated-localization microscopy (sptPALM) and found two populations. The first population which is only present in cells with active DNA replication, showed slow diffusion and was found to be located in replication foci. The second population showed fast diffusion, and represents the nucleoplasmic pool of unbound PCNA not involved in DNA replication. The ratio of these two populations remained constant throughout different stages of S-phase. A fraction of molecules in both populations showed spatially constrained mobility. We determined an exploration radius of ~100 nm for 13% of the slow-diffusing PCNA molecules, and of ~600 nm for 46% of the fast-diffusing PCNA molecules

PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins

In DNA replication, the leading strand is synthesized continuously, but lagging strand synthesis requires the complex, discontinuous synthesis of Okazaki fragments, and their subsequent joining. We have used a combination of in situ extraction and dual color photobleaching to compare the dynamic properties of three proteins essential for lagging strand synthesis: the polymerase clamp proliferating cell nuclear antigen (PCNA) and two proteins that bind to it, DNA Ligase I and Fen1. All three proteins are localized at replication foci (RF), but in contrast to PCNA, Ligase and Fen1 were readily extracted. Dual photobleaching combined with time overlays revealed a rapid exchange of Ligase and Fen1 at RF, which is consistent with de novo loading at every Okazaki fragment, while the slow recovery of PCNA mostly occurred at adjacent, newly assembled RF. These data indicate that PCNA works as a stationary loading platform that is reused for multiple Okazaki fragments, while PCNA binding proteins only transiently associate and are not stable components of the replication machinery

Simple method for sub-diffraction resolution imaging of cellular structures on standard confocal microscopes by three-photon absorption of quantum dots

This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts

TSG101 associates with PARP1 and is essential for PARylation and DNA damage-induced NF-κB activation

In a genome-wide screening for components of the dsDNA-break-induced IKK-NF-κB pathway, we identified scores of regulators, including tumor susceptibility gene TSG101. TSG101 is essential for DNA damage-induced formation of cellular poly(ADP-ribose) (PAR). TSG101 binds to PARP1 and is required for PARP1 activation. This function of TSG101 is independent of its role in the ESCRT-I endosomal sorting complex. In the absence of TSG101, the PAR-dependent formation of a nuclear PARP1-IKKγ signalosome, which triggers IKK activation, is impaired. According to its requirement for PARP1 and NF-κB activation, TSG101-deficient cells are defective in DNA repair and apoptosis protection. Loss of TSG101 results in PARP1 trapping at damage sites and mimics the effect of pharmacological PARP inhibition. We also show that the loss of TSG101 in connection with inactivated tumor suppressors BRCA1/2 in breast cancer cells is lethal. Our results imply TSG101 as a therapeutic target to achieve synthetic lethality in cancer treatment

PCNA stimulates catalysis by structure-specific nucleases using two distinct mechanisms: substrate targeting and catalytic step

The sliding clamp Proliferating Cell Nuclear Antigen (PCNA) functions as a recruiter and organizer of a wide variety of DNA modifying enzymes including nucleases, helicases, polymerases and glycosylases. The 5′-flap endonuclease Fen-1 is essential for Okazaki fragment processing in eukaryotes and archaea, and is targeted to the replication fork by PCNA. Crenarchaeal XPF, a 3′-flap endonuclease, is also stimulated by PCNA in vitro. Using a novel continuous fluorimetric assay, we demonstrate that PCNA activates these two nucleases by fundamentally different mechanisms. PCNA stimulates Fen-1 by increasing the enzyme's binding affinity for substrates, as suggested previously. However, PCNA activates XPF by increasing the catalytic rate constant by four orders of magnitude without affecting the KM. PCNA may function as a platform upon which XPF exerts force to distort DNA substrates, destabilizing the substrate and/or stabilizing the transition state structure. This suggests that PCNA can function directly in supporting catalysis as an essential cofactor in some circumstances, a new role for a protein that is generally assumed to perform a passive targeting and organizing function in molecular biology. This could provide a mechanism for the exquisite control of nuclease activity targeted to specific circumstances, such as replication forks or damaged DNA with pre-loaded PCNA

IFT88 transports Gucy2d, a guanylyl cyclase, to maintain sensory cilia function in Drosophila

Cilia are involved in a plethora of motility and sensory-related functions. Ciliary defects cause several ciliopathies, some of which with late-onset, suggesting cilia are actively maintained. While much is known about cilia assembly, little is understood about the mechanisms of their maintenance. Given that intraflagellar transport (IFT) is essential for cilium assembly, we investigated the role of one of its main players, IFT88, in ciliary maintenance. We show that DmIFT88, the Drosophila melanogaster orthologue of IFT88, continues to move along fully formed sensory cilia, and that its acute knockdown in the ciliated neurons of the adult affects sensory behaviour. We further identify DmGucy2d, the Drosophila guanylyl cyclase 2d, as a DmIFT88 cargo, whose loss also leads to defects in sensory behaviour maintenance. DmIFT88 binds to the intracellular part of DmGucy2d, a highly, evolutionarily conserved and mutated in several degenerative retina diseases, taking the cyclase into the cilia. Our results offer a novel mechanism for the maintenance of sensory cilia function and its potential role in human diseases

Functional traits influence patterns in vegetative and reproductive plant phenology - a multi-botanical garden study

Phenology has emerged as key indicator of the biological impacts of climate change, yet the role of functional traits constraining variation in herbaceous species' phenology has received little attention. Botanical gardens are ideal places in which to investigate large numbers of species growing under common climate conditions. We ask whether interspecific variation in plant phenology is influenced by differences in functional traits. We recorded onset, end, duration and intensity of initial growth, leafing out, leaf senescence, flowering and fruiting for 212 species across five botanical gardens in Germany. We measured functional traits, including plant height, absolute and specific leaf area, leaf dry matter content, leaf carbon and nitrogen content and seed mass and accounted for species' relatedness. Closely related species showed greater similarities in timing of phenological events than expected by chance, but species' traits had a high degree of explanatory power, pointing to paramount importance of species' life-history strategies. Taller plants showed later timing of initial growth, and flowered, fruited and underwent leaf senescence later. Large-leaved species had shorter flowering and fruiting durations. Taller, large-leaved species differ in their phenology and are more competitive than smaller, small-leaved species. We assume climate warming will change plant communities' competitive hierarchies with consequences for biodiversity

Photocaged Hoechst enables subnuclear visualization and cell selective staining of DNA in vivo

Selective targeting of DNA by means of fluorescent labelling has become a mainstay in the life sciences. While genetic engineering serves as a powerful technique and allows for the visualization of nucleic acid by using DNA-targeting fluorescent fusion proteins in a cell-type and subcellular specific manner, it relies on the introduction of foreign genes. On the other hand, DNA-binding small fluorescent molecules can be used without genetic engineering but they are not spatially restricted. Here, we report a photocaged version of the DNA dye Hoechst33342 (pcHoechst), which can be uncaged using UV to blue light for the selective staining of chromosomal DNA in subnuclear regions of live cells. Expanding its application to a vertebrate model organism, we demonstrate uncaging in epithelial cells and short-term cell tracking  in vivo  in zebrafish. We envision pcHoechst as a valuable tool for targeting and interrogating DNA with precise spatiotemporal resolution in living cells and wild-type organisms

Dynamics of Dnmt1 interaction with the replication machinery and its role in postreplicative maintenance of DNA methylation

Postreplicative maintenance of genomic methylation patterns was proposed to depend largely on the binding of DNA methyltransferase 1 (Dnmt1) to PCNA, a core component of the replication machinery. We investigated how the slow and discontinuous DNA methylation could be mechanistically linked with fast and processive DNA replication. Using photobleaching and quantitative live cell imaging we show that Dnmt1 binding to PCNA is highly dynamic. Activity measurements of a PCNA-binding-deficient mutant with an enzyme-trapping assay in living cells showed that this interaction accounts for a 2-fold increase in methylation efficiency. Expression of this mutant in mouse dnmt1−/− embryonic stem (ES) cells restored CpG island methylation. Thus association of Dnmt1 with the replication machinery enhances methylation efficiency, but is not strictly required for maintaining global methylation. The transient nature of this interaction accommodates the different kinetics of DNA replication and methylation while contributing to faithful propagation of epigenetic information

Microglia sense neuronal activity via GABA in the early postnatal hippocampus

Microglia, the resident macrophages in the central nervous system, express receptors for classical neurotransmitters, such as γ-aminobutyric acid (GABA) and glutamate, suggesting that they sense synaptic activity. To detect microglial Ca(2+) responses to neuronal activity, we generate transgenic mouse lines expressing the fluorescent Ca(2+) indicator GCaMP6m, specifically in microglia and demonstrate that electrical stimulation of the Schaffer collateral pathway results in microglial Ca(2+) responses in early postnatal but not adult hippocampus. Preceding the microglial responses, we also observe similar Ca(2+) responses in astrocytes, and both are sensitive to tetrodotoxin. Blocking astrocytic glutamate uptake or GABA transport abolishes stimulation-induced microglial responses as well as antagonizing the microglial GABA(B) receptor. Our data, therefore, suggest that the neuronal activity-induced glutamate uptake and the release of GABA by astrocytes trigger the activation of GABA(B) receptors in microglia. This neuron, astrocyte, and microglia communication pathway might modulate microglial activity in developing neuronal networks