Talin and kindlin cooperate to control the density of integrin clusters.

Focal adhesions are composed of transmembrane integrins, linking the extracellular matrix to the actomyosin cytoskeleton, via cytoplasmic proteins. Adhesion depends on the activation of integrins. Talin and kindlin proteins are intracellular activators of integrins that bind to ß-integrin cytoplasmic tails. Integrin activation and clustering through extracellular ligands guide the organization of adhesion complexes. However, the roles of talin and kindlin in this process are poorly understood. To determine the contribution of talin, kindlin, lipids and actomyosin in integrin clustering, we used a biomimetic in vitro system, made of giant unilamellar vesicles, containing transmembrane integrins (herein αIIbß3), with purified talin (talin-1), kindlin (kindlin-2, also known as FERMT2) and actomyosin. Here, we show that talin and kindlin individually have the ability to cluster integrins. Talin and kindlin synergize to induce the formation of larger integrin clusters containing the three proteins. Comparison of protein density reveals that kindlin increases talin and integrin density, whereas talin does not affect kindlin and integrin density. Finally, kindlin increases integrin-talin-actomyosin coupling. Our study unambiguously demonstrates how kindlin and talin cooperate to induce integrin clustering, which is a major parameter for cell adhesion.

Polo-like kinase 1 (Plk1) regulates DNA replication origin firing and interacts with Rif1 in Xenopus.

The activation of eukaryotic DNA replication origins needs to be strictly controlled at multiple steps in order to faithfully duplicate the genome and to maintain its stability. How the checkpoint recovery and adaptation protein Polo-like kinase 1 (Plk1) regulates the firing of replication origins during non-challenged S phase remained an open question. Using DNA fiber analysis, we show that immunodepletion of Plk1 in the Xenopus in vitro system decreases replication fork density and initiation frequency. Numerical analyses suggest that Plk1 reduces the overall probability and synchrony of origin firing. We used quantitative chromatin proteomics and co-immunoprecipitations to demonstrate that Plk1 interacts with firing factors MTBP/Treslin/TopBP1 as well as with Rif1, a known regulator of replication timing. Phosphopeptide analysis by LC/MS/MS shows that the C-terminal domain of Rif1, which is necessary for its repressive action on origins through protein phosphatase 1 (PP1), can be phosphorylated in vitro by Plk1 on S2058 in its PP1 binding site. The phosphomimetic S2058D mutant interrupts the Rif1-PP1 interaction and modulates DNA replication. Collectively, our study provides molecular insights into how Plk1 regulates the spatio-temporal replication program and suggests that Plk1 controls origin activation at the level of large chromatin domains in vertebrates.

A non-transcriptional function of Yap regulates the DNA replication program in Xenopus laevis.

In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signalling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. Furthermore, we show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that either Yap or Rif1 depletion triggers an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as brakes to control the DNA replication program in early embryos and post-embryonic stem cells.

Organization of DNA Replication Origin Firing in Xenopus Egg Extracts: The Role of Intra-S Checkpoint.

During cell division, the duplication of the genome starts at multiple positions called replication origins. Origin firing requires the interaction of rate-limiting factors with potential origins during the S(ynthesis)-phase of the cell cycle. Origins fire as synchronous clusters which is proposed to be regulated by the intra-S checkpoint. By modelling the unchallenged, the checkpoint-inhibited and the checkpoint protein Chk1 over-expressed replication pattern of single DNA molecules from Xenopus sperm chromatin replicated in egg extracts, we demonstrate that the quantitative modelling of data requires: (1) a segmentation of the genome into regions of low and high probability of origin firing; (2) that regions with high probability of origin firing escape intra-S checkpoint regulation and (3) the variability of the rate of DNA synthesis close to replication forks is a necessary ingredient that should be taken in to account in order to describe the dynamic of replication origin firing. This model implies that the observed origin clustering emerges from the apparent synchrony of origin firing in regions with high probability of origin firing and challenge the assumption that the intra-S checkpoint is the main regulator of origin clustering.

Polo-like kinase 1 (Plk1) is a positive regulator of DNA replication in the Xenopus in vitro system.

Polo-like kinase 1 (Plk1) is a cell cycle kinase essential for mitosis progression, but also important for checkpoint recovery and adaptation in response to DNA damage and replication stress. However, although Plk1 is expressed in S phase, little is known about its function during unperturbed DNA replication. Using Xenopus laevis egg extracts, mimicking early embryonic replication, we demonstrate that Plk1 is simultaneously recruited to chromatin with pre-replication proteins where it accumulates throughout S phase. Further, we found that chromatin-bound Plk1 is phosphorylated on its activating site T201, which appears to be sensitive to dephosphorylation by protein phosphatase 2A. Extracts immunodepleted of Plk1 showed a decrease in DNA replication, rescued by wild type recombinant Plk1. Inversely, modest Plk1 overexpression accelerated DNA replication. Plk1 depletion led to an increase in Chk1 phosphorylation and to a decrease in Cdk2 activity, which strongly suggests that Plk1 could inhibit the ATR/Chk1-dependent intra-S phase checkpoint during normal S phase. In addition, we observed that phosphorylated Plk1 levels are high during the rapid, early cell cycles of Xenopus development but decrease after the mid-blastula transition when the cell cycle and the replication program slow down along with more active checkpoints. These data shed new light on the role of Plk1 as a positive regulating factor for DNA replication in early, rapidly dividing embryos.

Genome wide decrease of DNA replication eye density at the midblastula transition of Xenopus laevis.

During the first rapid divisions of early development in many species, the DNA:cytoplasm ratio increases until the midblastula transition (MBT) when transcription resumes and cell cycles lengthen. S phase is very rapid in early embryos, about 20-30 times faster than in differentiated cells. Using a combination of DNA fiber studies and a Xenopus laevis embryonic in vitro replication system, we show that S phase slows down shortly after the MBT owing to a genome wide decrease of replication eye density. Increasing the dNTP pool did not accelerate S phase or increase replication eye density implying that dNTPs are not rate limiting for DNA replication at the Xenopus MBT. Increasing the ratio of DNA:cytoplasm in egg extracts faithfully recapitulates changes in the spatial replication program in embryos, supporting the hypothesis that titration of soluble limiting factors could explain the observed changes in the DNA replication program at the MBT in Xenopus laevis.

Tight Chk1 Levels Control Replication Cluster Activation in Xenopus.

DNA replication in higher eukaryotes initiates at thousands of origins according to a spatio-temporal program. The ATR/Chk1 dependent replication checkpoint inhibits the activation of later firing origins. In the Xenopus in vitro system initiations are not sequence dependent and 2-5 origins are grouped in clusters that fire at different times despite a very short S phase. We have shown that the temporal program is stochastic at the level of single origins and replication clusters. It is unclear how the replication checkpoint inhibits late origins but permits origin activation in early clusters. Here, we analyze the role of Chk1 in the replication program in sperm nuclei replicating in Xenopus egg extracts by a combination of experimental and modelling approaches. After Chk1 inhibition or immunodepletion, we observed an increase of the replication extent and fork density in the presence or absence of external stress. However, overexpression of Chk1 in the absence of external replication stress inhibited DNA replication by decreasing fork densities due to lower Cdk2 kinase activity. Thus, Chk1 levels need to be tightly controlled in order to properly regulate the replication program even during normal S phase. DNA combing experiments showed that Chk1 inhibits origins outside, but not inside, already active clusters. Numerical simulations of initiation frequencies in the absence and presence of Chk1 activity are consistent with a global inhibition of origins by Chk1 at the level of clusters but need to be combined with a local repression of Chk1 action close to activated origins to fit our data.