Stem-like cells may be integral to the development and maintenance of human cancers. Direct proof is still lacking, mainly because of our poor understanding of the biological differences between normal and cancer stem cells (SCs). Using the ErbB2 transgenic model of breast cancer, we found that self-renewing divisions of cancer SCs are more frequent than their normal counterparts, unlimited and symmetric, thus contributing to increasing numbers of SCs in tumoral tissues. SCs with targeted mutation of the tumor suppressor p53 possess the same self-renewal properties as cancer SCs, and their number increases progressively in the p53 null premalignant mammary gland. Pharmacological reactivation of p53 correlates with restoration of asymmetric divisions in cancer SCs and tumor growth reduction, without significant effects on additional cancer cells. These data demonstrate that p53 regulates polarity of cell division in mammary SCs and suggest that loss of p53 favors symmetric divisions of cancer SCs, contributing to tumor growth.

Ubiquitination is a post-translation modification in which ubiquitin chains or single ubiquitin molecules are appended to target proteins, giving rise to poly- or monoubiquitination, respectively. Polyubiquitination targets proteins for destruction by the proteasome. The role of monoubiquitination is less understood, although a function in membrane trafficking is emerging, at least in yeast. Here we report that a short amino-acid stretch at the carboxy-termini of the monoubiquitinated endocytic proteins Eps15 and eps15R is indispensable for their monoubiquitination. A similar sequence, also required for this modification, is found in other cytosolic endocytic proteins, such as epsins and Hrs. These sequences comprise a protein motif, UIM (ref. 6), which has been proposed to bind to ubiquitin. We confirm this for the UIMs of eps15, eps15R, epsins and Hrs. Thus, the same motif in several endocytic proteins is responsible for ubiquitin recognition and monoubiquitination. Our results predict the existence of a UIM:ubiquitin-based intracellular network. Eps15/eps15R, epsins and Hrs may function as adaptors between ubiquitinated membrane cargo and either the clathrin coat or other endocytic scaffolds. In addition, through their own ubiquitination, they may further contribute to the amplification of this network in the endocytic pathway.

The yeast Sir2 protein mediates chromatin silencing through an intrinsic NAD-dependent histone deacetylase activity. Sir2 is a conserved protein and was recently shown to regulate lifespan extension both in budding yeast and worms. Here, we show that SIRT1, the human Sir2 homolog, is recruited to the promyelocytic leukemia protein (PML) nuclear bodies of mammalian cells upon overexpression of either PML or oncogenic Ras (Ha-rasV12). SIRT1 binds and deacetylates p53, a component of PML nuclear bodies, and it can repress p53-mediated transactivation. Moreover, we show that SIRT1 and p53 co-localize in nuclear bodies upon PML upregulation. When overexpressed in primary mouse embryo ®broblasts (MEFs), SIRT1 antagonizes PML-induced acetylation of p53 and rescues PML-mediated premature cellular senescence. Taken together, our data establish the SIRT1 deacetylase as a novel negative regulator of p53 function capable of modulating cellular senescence.

We propose that closed Mad2 bound to Mad1 represents a template for the conversion of open Mad2 into closed Mad2 bound to Cdc20. This simple model, which we have named the "Mad2 template" model, predicts a mechanism for cytosolic propagation of the spindle checkpoint signal away from kinetochores.

DNA methylation of tumor suppressor genes is a frequent mechanism of transcriptional silencing in cancer. The molecular mechanisms underlying the specificity of methylation are unknown. We report here that the leukemia-promoting PML-RAR fusion protein induces gene hypermethylation and silencing by recruiting DNA methyltransferases to target promoters and that hypermethylation contributes to its leukemogenic potential. Retinoic acid treatment induces promoter demethylation, gene reexpression, and reversion of the transformed phenotype. These results establish a mechanistic link between genetic and epigenetic changes during transformation and suggest that hypermethylation contributes to the early steps of carcinogenesis.

The small GTPases, Rab5 and Rac, are essential for endocytosis and actin remodeling, respectively. Coordination of these processes is critical to achieve spatial restriction of intracellular signaling, which is essential for a variety of polarized functions. Here, we show that clathrin- and Rab5-mediated endocytosis are required for the activation of Rac induced by motogenic stimuli. Rac activation occurs on early endosomes, where the RacGEF Tiam1 is also recruited. Subsequent recycling of Rac to the plasma membrane ensures localized signaling, leading to the formation of actin-based migratory protrusions. Thus, membrane trafficking of Rac is required for the spatial resolution of Rac-dependent motogenic signals. We further demonstrate that a Rab5-to-Rac circuitry controls the morphology of motile mammalian tumor cells and primordial germinal cells during zebrafish development, suggesting that this circuitry is relevant for the regulation of migratory programs in various cells, in both in vitro settings and whole organisms.

We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50-150 μm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids.

Signaling from receptor tyrosine kinases (RTKs)* requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1–Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. Here, we demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1–Grb2 (S/G) or a Sos-1–E3b1–Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1–Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell.