Discrimination of Kinetic Models by a Combination of Microirradiation and Fluorescence Photobleaching.

Fluorescence recovery after photobleaching (FRAP) is an excellent tool to measure the chemical rate constants of fluorescently labeled proteins in living cells. Usually FRAP experiments are conducted with the protein concentrations being in a steady state, i.e., when the association and dissociation of the proteins are equilibrated. This is a strong limitation because situations in which rate constants change with time are of great scientific interest. In this study, we present an approach in which FRAP is used shortly after DNA damage introducing laser microirradiation, which results in the recruitment of the DNA clamp protein proliferating cell nuclear antigen (PCNA) to DNA lesions. We establish different kinetic models that are compatible with the observed PCNA recruitment data if FRAP is not used. By using FRAP at different time points during protein accumulation, we can not only exclude two out of three models, but we can also determine the rate constants with increased reliability. This study thus demonstrates the feasibility of using FRAP during protein recruitment and its application in the discrimination of possible kinetic models.

Histone hypoacetylation is required to maintain late replication timing of constitutive heterochromatin.

The replication of the genome is a spatio-temporally highly organized process. Yet, its flexibility throughout development suggests that this process is not genetically regulated. However, the mechanisms and chromatin modifications controlling replication timing are still unclear. We made use of the prominent structure and defined heterochromatic landscape of pericentric regions as an example of late replicating constitutive heterochromatin. We manipulated the major chromatin markers of these regions, namely histone acetylation, DNA and histone methylation, as well as chromatin condensation and determined the effects of these altered chromatin states on replication timing. Here, we show that manipulation of DNA and histone methylation as well as acetylation levels caused large-scale heterochromatin decondensation. Histone demethylation and the concomitant decondensation, however, did not affect replication timing. In contrast, immuno-FISH and time-lapse analyses showed that lowering DNA methylation, as well as increasing histone acetylation, advanced the onset of heterochromatin replication. While dnmt1(-)(/)(-) cells showed increased histone acetylation at chromocenters, histone hyperacetylation did not induce DNA demethylation. Hence, we propose that histone hypoacetylation is required to maintain normal heterochromatin duplication dynamics. We speculate that a high histone acetylation level might increase the firing efficiency of origins and, concomitantly, advances the replication timing of distinct genomic regions.

Cell entry of arginine-rich peptides is independent of endocytosis.

Arginine-rich peptides are a subclass of cell-penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytic mode of uptake and a subsequent release from vesicles or to direct membrane penetration (transduction). To distinguish between both possibilities, we have blocked endocytic pathways suggested to be involved in uptake of cell-penetrating peptides. We have then monitored by confocal microscopy the uptake and distribution of the cell-penetrating transactivator of transcription (TAT) peptide into living mammalian cells over time. To prevent side effects of chemical inhibitors, we used genetically engineered cells as well as different temperature. We found that a knockdown of clathrin-mediated endocytosis and a knock-out of caveolin-mediated endocytosis did not affect the ability of TAT to enter cells. In addition, the TAT peptide showed the same intracellular distribution throughout the cytoplasm and nucleus as in control cells. Even incubation of cells at 4 degrees C did not abrogate TAT uptake nor change its intracellular distribution. We therefore conclude that this distribution results from TAT peptide that directly penetrated (transduced) the plasma membrane. The formation of nonselective pores is unlikely, because simultaneously added fluorophores were not taken up together with the TAT peptide. In summary, although the frequency and kinetics of TAT transduction varied between cell types, it was independent of endocytosis.

Generation and characterization of a rat monoclonal antibody specific for multiple red fluorescent proteins.

Abstract Fluorescent proteins (FP) are widely used as in vivo reporter molecules and are available in multiple colors spanning almost the entire visible light spectrum. Genetically fused to any protein target, FPs offer a powerful tool to study protein localization and dynamics. After the isolation of the prototypical green fluorescent protein (GFP) from the jellyfish Aequorea victoria, a red fluorescent protein (DsRed) was discovered in the coral Discosoma sp. that provided a better spectral separation from cellular autofluorescence and allowed multicolor tracking of fusion proteins. However, the obligate tetramerization of DsRed caused serious problems for its use in live-cell imaging. Subsequent mutageneses of the red progenitor have resulted in several monomeric red FPs (mRFP1, mCherry, mOrange, mPlum, etc). These improved red FPs are characterized by higher brightness and photostability, complete chromophore maturation, and promise a wide variety of features for biological imaging and multicolor labeling. Here we report the generation and characterization of the first rat monoclonal antibody (MAb) against multiple red FPs, designated as multi-red 5F8. We demonstrate that multi-red 5F8 is a MAb with high affinity and specificity against the DsRed derivatives and corresponding fusion proteins, and that it is suitable for ELISA, immunoblotting, immunoprecipitation, and immunofluorescence assays. Applying our versatile antibody, one and the same red fluorescent protein tag can be used to perform not only microscopic studies, but also multiple biochemical assays.

Uncoupling the replication machinery: replication fork progression in the absence of processive DNA synthesis.

The precise coordination of the different steps of DNA replication is critical for the maintenance of genome stability. We have probed the mechanisms coupling various components of the replication machinery and their response to polymerase stalling by inhibition of the DNA polymerases in living mammalian cells with aphidicolin. We observed little change in the behaviour of proteins involved in the initiation of DNA replication. In contrast, we detected a marked accumulation of the single stranded DNA binding factor RPA34 at sites of DNA replication. Finally, we demonstrate that proteins involved in the elongation step of DNA synthesis dissociate from replication foci in the presence of aphidicolin. Taken together, these data indicate that inhibition of processive DNA polymerases uncouples the initiation of DNA replication from subsequent elongation steps. We, therefore, propose that the replication machinery is made up of distinct functional sub-modules that allow a flexible and dynamic response to challenges during DNA replication.

Generation and characterization of a rat monoclonal antibody specific for PCNA.

Proliferating cell nuclear antigen (PCNA) is a homotrimeric ring-shaped protein that encircles the DNA and acts as a stationary loading platform for multiple, transiently interacting partners participating in various DNA transactions. This essential cellular component, originally characterized as a nuclear antigen of dividing cells, is evolutionary highly conserved from yeast to human. Within the eukaryotic cell, PCNA serves as a processivity factor for DNA polymerase delta and plays a key role in DNA replication, repair, cell cycle regulation, and post-replicative transactions like DNA methylation and chromatin remodelling. All these cellular processes are regulated by a complex network comprising cell cycle dependent changes in expression levels, dynamics, interactions, and localization of PCNA. Here we report the generation and characterization of the first rat monoclonal antibody (MAb) against human PCNA, designated as PCNA 16D10. We demonstrated that PCNA 16D10 MAb has high affinity and specificity and is suited for ELISA, immunoblotting, immunoprecipitation, and immunofluorescence. The characteristic punctate staining of S phase cells allows the identification of proliferating cells and the monitoring of cell cycle progression.

MeCP2 interacts with HP1 and modulates its heterochromatin association during myogenic differentiation.

There is increasing evidence of crosstalk between epigenetic modifications such as histone and DNA methylation, recognized by HP1 and methyl CpG-binding proteins, respectively. We have previously shown that the level of methyl CpG-binding proteins increased dramatically during myogenesis leading to large-scale heterochromatin reorganization. In this work, we show that the level of HP1 isoforms did not change significantly throughout myogenic differentiation but their localization did. In particular, HP1gamma relocalization to heterochromatin correlated with MeCP2 presence. Using co-immunoprecipitation assays, we found that these heterochromatic factors interact in vivo via the chromo shadow domain of HP1 and the first 55 amino acids of MeCP2. We propose that this dynamic interaction of HP1 and MeCP2 increases their concentration at heterochromatin linking two major gene silencing pathways to stabilize transcriptional repression during differentiation.

Targeting and tracing antigens in live cells with fluorescent nanobodies.

We fused the epitope-recognizing fragment of heavy-chain antibodies from Camelidae sp. with fluorescent proteins to generate fluorescent, antigen-binding nanobodies (chromobodies) that can be expressed in living cells. We demonstrate that chromobodies can recognize and trace antigens in different subcellular compartments throughout S phase and mitosis. Chromobodies should enable new functional studies, as potentially any antigenic structure can be targeted and traced in living cells in this fashion.

Methyl CpG-binding proteins induce large-scale chromatin reorganization during terminal differentiation.

Pericentric heterochromatin plays an important role in epigenetic gene regulation. We show that pericentric heterochromatin aggregates during myogenic differentiation. This clustering leads to the formation of large chromocenters and correlates with increased levels of the methyl CpG-binding protein MeCP2 and pericentric DNA methylation. Ectopic expression of fluorescently tagged MeCP2 mimicked this effect, causing a dose-dependent clustering of chromocenters in the absence of differentiation. MeCP2-induced rearrangement of heterochromatin occurred throughout interphase, did not depend on the H3K9 histone methylation pathway, and required the methyl CpG-binding domain (MBD) only. Similar to MeCP2, another methyl CpG-binding protein, MBD2, also increased during myogenic differentiation and could induce clustering of pericentric regions, arguing for functional redundancy. This MeCP2- and MBD2-mediated chromatin reorganization may thus represent a molecular link between nuclear genome topology and the epigenetic maintenance of cellular differentiation.