The spatial organization of replicons into clusters is believed to be of critical importance for genome duplication in higher eukaryotes, but its functional organization still remains to be fully clarified. The coordinated activation of origins is insufficient on its own to account for a timely completion of genome duplication when interorigin distances vary significantly and fork velocities are constant. Mechanisms coordinating origin distribution with fork progression are still poorly elucidated, because of technical difficulties of visualizing the process. Taking advantage of a single molecule approach, we delineated and compared the DNA replication kinetics at the genome level in human normal primary and malignant cells. Our results show that replication forks moving from one origin, as well as from neighboring origins, tend to exhibit the same velocity, although the plasticity of the replication program allows for their adaptation to variable interorigin distances. We also found that forks that emanated from closely spaced origins tended to move slower than those associated with long replicons. Taken together, our results indicate a functional role for origin clustering in the dynamic regulation of genome duplication. INTRODUCTIONThe complete and correct duplication of the genome once and only once per cell cycle is one of the most challenging cellular tasks. In metazoan cells, DNA replication initiates at multiple sites, called origins of replication, from which two forks emanate and progress bidirectionally. The efficient duplication of the eukaryotic genome depends on the orderly activation of those origins, estimated to be several tens of thousands, and on the proper progression of their forks. In spite of its importance in the transmission of genetic information, the regulation of origin distribution and fork progression and the coordination of these two processes still remain to be fully elucidated in human cells. In metazoans, origins of replication are generally not encoded by specific sequences (DePamphilis, 1999;Todorovic et al., 1999;Gilbert, 2001;Mechali, 2001). Origins are dynamically regulated by a number of cis-acting, metabolic and epigenetic factors depending on the transcriptional and developmental programs (Maric et al., 1999;Sasaki et al., 1999;Aladjem and Fanning, 2004;Danis et al., 2004).In early Xenopus embryos, transcription is repressed and DNA replication starts at nonspecific sites every 5-15 kb. The exit from midblastula and the beginning of transcription reorganizes the spatio-temporal pattern of origin firing, with replication occurring at specific sites every 150 -300 kb (Hyrien et al., 1995). In spite of this flexibility in the distribution of active origins along the genome, the total number of origins is a crucial parameter for the efficient duplication of the genome (Machida et al., 2005;Shechter and Gautier, 2005). Initiation of DNA replication from a reduced number of origins is associated with gross chromosomal abnormalities in Saccharomyces cerevisiae Sic1 mutants (Len...

The ubiquitin proteasome system (UPS) regulates many cellular functions by degrading key proteins. Notably, the role of UPS in regulating mitochondrial metabolic functions is unclear. Here, we show that ubiquitination occurs in different mitochondrial compartments, including the inner mitochondrial membrane, and that turnover of several metabolic proteins is UPS dependent. We specifically detailed mitochondrial ubiquitination and subsequent UPS-dependent degradation of succinate dehydrogenase subunit A (SDHA), which occurred when SDHA was minimally involved in mitochondrial energy metabolism. We demonstrate that SDHA ubiquitination occurs inside the organelle. In addition, we show that the specific inhibition of SDHA degradation by UPS promotes SDHA-dependent oxygen consumption and increases ATP, malate, and citrate levels. These findings suggest that the mitochondrial metabolic machinery is also regulated by the UPS.

BackgroundThe aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties. Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells.Methodology/Principal FindingsWe analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity.Conclusion/SignificanceThe association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells. Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability.