Cell adhesion defines the topology of endocytosis and signaling.

Preferred sites of endocytosis have been observed in various cell types, but whether they occur randomly or are linked to cellular cues is debated. Here, we quantified the sites of endocytosis of transferrin (Tfn) and epidermal growth factor (EGF) in cells whose adhesion geometry was defined by micropatterns. 3D probabilistic density maps revealed that Tfn was enriched in adhesive sites during uptake, whereas EGF endocytosis was restricted to the dorsal cellular surface. This spatial separation was not due to distributions of corresponding receptors but was regulated by uptake mechanisms. Asymmetric uptake of Tfn resulted from the enrichment of clathrin and adaptor protein 2 at adhesive areas. Asymmetry in EGF uptake was strongly dependent on the actin cytoskeleton and led to asymmetry in EGF receptor activation. Mild alteration of actin dynamics abolished asymmetry in EGF uptake and decreased EGF-induced downstream signaling, suggesting that cellular adhesion cues influence signal propagation. We propose that restriction of endocytosis at distinct sites allows cells to sense their environment in an "outside-in" mechanism.

Cell cycle-dependent subcellular translocation of the human DNA licensing inhibitor geminin.

Once per cell cycle replication is crucial for maintaining genome integrity. Geminin interacts with the licensing factor Cdt1 to prevent untimely replication and is controlled by APC/C-dependent cell cycle specific proteolysis during mitosis and in G1. We show here that human geminin, when expressed in human cells in culture under a constitutive promoter, is excluded from the nucleus during part of the G1 phase and at the transition from G0 to G1. The N-terminal 30 amino acids of geminin, which contain its destruction box, are essential for nuclear exclusion. In addition, 30 amino acids within the central domain of geminin are required for both nuclear exclusion and nuclear accumulation. Cdt1 overexpression targets geminin to the nucleus, while reducing Cdt1 levels by RNAi leads to the appearance of endogenous geminin in the cytoplasm. Our data propose a novel means of regulating the balance of Cdt1/geminin in human cells, at the level of the subcellular localization of geminin.

Origin and function of tumor stroma fibroblasts.

Tumor development is critically dependent on the formation of a supporting stroma consisting of neovasculature, inflammatory cells and activated fibroblasts. Activated fibroblasts present a heterogeneous cell population not only in regard to the expression of marker molecules but also to their origin and molecular signaling properties. The plasticity of this cell type is pointed out by the multiple transdifferentiation events that lead to the generation of activated fibroblasts which can arise from resting fibroblasts, epithelial and endothelial cells as well as from mesenchymal stem cells. Cellular in vitro and in vivo experiments have changed the perspective of fibroblasts from passive "bystanders" in the tumor microenvironment to that of important drivers of tumor progression. Here, we describe the multiple origins of fibroblast recruitment to the tumor tissue as well as the function of activated fibroblasts during tumor initiation, progression, metastasis and anti-VEGF resistance. The identification of markers present in activated fibroblasts as well as a better understanding how these cells influence other tumor compartments has led to the clinical development of anti-tumor therapies.

Ubiquitination switches EphA2 vesicular traffic from a continuous safeguard to a finite signalling mode.

Autocatalytic phosphorylation of receptor tyrosine kinases (RTKs) enables diverse, context-dependent responses to extracellular signals but comes at the price of autonomous, ligand-independent activation. Using a conformational biosensor that reports on the kinase activity of the cell guidance ephrin receptor type-A (EphA2) in living cells, we observe that autonomous EphA2 activation is suppressed by vesicular recycling and dephosphorylation by protein tyrosine phosphatases 1B (PTP1B) near the pericentriolar recycling endosome. This spatial segregation of catalytically superior PTPs from RTKs at the plasma membrane is essential to preserve ligand responsiveness. Ligand-induced clustering, on the other hand, promotes phosphorylation of a c-Cbl docking site and ubiquitination of the receptor, thereby redirecting it to the late endosome/lysosome. We show that this switch from cyclic to unidirectional receptor trafficking converts a continuous suppressive safeguard mechanism into a transient ligand-responsive signalling mode.

EGF-dependent re-routing of vesicular recycling switches spontaneous phosphorylation suppression to EGFR signaling.

Autocatalytic activation of epidermal growth factor receptor (EGFR) coupled to dephosphorylating activity of protein tyrosine phosphatases (PTPs) ensures robust yet diverse responses to extracellular stimuli. The inevitable tradeoff of this plasticity is spontaneous receptor activation and spurious signaling. We show that a ligand-mediated switch in EGFR trafficking enables suppression of spontaneous activation while maintaining EGFR's capacity to transduce extracellular signals. Autocatalytic phosphorylation of tyrosine 845 on unliganded EGFR monomers is suppressed by vesicular recycling through perinuclear areas with high PTP1B activity. Ligand-binding results in phosphorylation of the c-Cbl docking tyrosine and ubiquitination of the receptor. This secondary signal relies on EGF-induced EGFR self-association and switches suppressive recycling to directional trafficking. The re-routing regulates EGFR signaling response by the transit-time to late endosomes where it is switched-off by high PTP1B activity. This ubiquitin-mediated switch in EGFR trafficking is a uniquely suited solution to suppress spontaneous activation while maintaining responsiveness to EGF.

Cdt1 interactions in the licensing process: a model for dynamic spatiotemporal control of licensing.

Within each cell cycle, a cell must ensure that the processes of selection of replication origins (licensing) and initiation of DNA replication are well coordinated to prevent reinitiation of DNA replication from the same DNA segment during the same cell cycle. This is achieved by restricting the licensing process to G1 phase when the prereplicative complexes (preRCs) are assembled onto the origin DNA, while DNA replication is initiated only during S phase when de novo preRC assembly is blocked. Cdt1 is an important member of the preRC complex and its tight regulation through ubiquitin-dependent proteolysis and binding to its inhibitor Geminin ensure that Cdt1 will only be present in G1 phase, preventing relicensing of replication origins. We have recently reported that Cdt1 associates with chromatin in a dynamic way and recruits its inhibitor Geminin onto chromatin in vivo. Here we discuss how these dynamic Cdt1-chromatin interactions and the local recruitment of Geminin onto origins of replication by Cdt1 may provide a tight control of the licensing process in time and in space.

Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin.

To maintain genome integrity, eukaryotic cells initiate DNA replication once per cell cycle after assembling prereplicative complexes (preRCs) on chromatin at the end of mitosis and during G1. In S phase, preRCs are disassembled, precluding initiation of another round of replication. Cdt1 is a key member of the preRC and its correct regulation via proteolysis and by its inhibitor Geminin is essential to prevent premature re-replication. Using quantitative fluorescence microscopy, we study the interactions of Cdt1 with chromatin and Geminin in living cells. We find that Cdt1 exhibits dynamic interactions with chromatin throughout G1 phase and that the protein domains responsible for chromatin and Geminin interactions are separable. Contrary to existing in vitro data, we show that Cdt1 simultaneously binds Geminin and chromatin in vivo, thereby recruiting Geminin onto chromatin. We propose that dynamic Cdt1-chromatin associations and the recruitment of Geminin to chromatin provide spatio-temporal control of the licensing process.

DNA replication in the fission yeast: robustness in the face of uncertainty.

DNA replication, the process of duplication of a cell's genetic content, must be carried out with great precision every time the cell divides, so that genetic information is preserved. Control mechanisms must ensure that every base of the genome is replicated within the allocated time (S-phase) and only once per cell cycle, thereby safeguarding genomic integrity. In eukaryotes, replication starts from many points along the chromosome, termed origins of replication, and then proceeds continuously bidirectionally until an opposing moving fork is encountered. In contrast to bacteria, where a specific site on the genome serves as an origin in every cell division, in most eukaryotes origin selection appears highly stochastic: many potential origins exist, of which only a subset is selected to fire in any given cell, giving rise to an apparently random distribution of initiation events across the genome. Origin states change throughout the cell cycle, through the ordered formation and modification of origin-associated multisubunit protein complexes. State transitions are governed by fluctuations of cyclin-dependent kinase (CDK) activity and guards in these transitions ensure system memory. We present here DNA replication dynamics, emphasizing recent data from the fission yeast Schizosaccharomyces pombe, and discuss how robustness may be ensured in spite of (or even assisted by) system randomness.

Cdt1 and geminin are down-regulated upon cell cycle exit and are over-expressed in cancer-derived cell lines.

Licensing origins for replication upon completion of mitosis ensures genomic stability in cycling cells. Cdt1 was recently discovered as an essential licensing factor, which is inhibited by geminin. Over-expression of Cdt1 was shown to predispose cells for malignant transformation. We show here that Cdt1 is down-regulated at both the protein and RNA level when primary human fibroblasts exit the cell cycle into G0, and its expression is induced as cells re-enter the cell cycle, prior to S phase onset. Cdt1's inhibitor, geminin, is similarly down-regulated upon cell cycle exit at both the protein and RNA level, and geminin protein accumulates with a 3-6 h delay over Cdt1, following serum re-addition. Similarly, mouse NIH3T3 cells down-regulate Cdt1 and geminin mRNA and protein when serum starved. Our data suggest a transcriptional control over Cdt1 and geminin at the transition from quiescence to proliferation. In situ hybridization and immunohistochemistry localize Cdt1 as well as geminin to the proliferative compartment of the developing mouse gut epithelium. Cdt1 and geminin levels were compared in primary cells vs. cancer-derived human cell lines. We show that Cdt1 is consistently over-expressed in cancer cell lines at both the protein and RNA level, and that the Cdt1 protein accumulates to higher levels in individual cancer cells. Geminin is similarly over-expressed in the majority of cancer cell lines tested. The relative ratios of Cdt1 and geminin differ significantly amongst cell lines. Our data establish that Cdt1 and geminin are regulated at cell cycle exit, and suggest that the mechanisms controlling Cdt1 and geminin levels may be altered in cancer cells.