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During cell division, chromosomes are maintained as individual units; this process is shown to be mediated by the cell proliferation marker Ki-67, which has biophysical properties similar to those of surfactants. Surfactant activity of Ki-67 protein As chromosomes prepare for division by the process of mitosis, they undergo reorganization into separate bodies. Through an RNAi screen, Daniel Gerlich and colleagues demonstrate that this process is mediated by Ki-67 — a protein known as a cell proliferation marker and used in cancer diagnostics. After nuclear envelope disassembly, Ki-67 prevents chromosomes from collapsing into a single cluster by acting as a biological surfactant. In fact, many biophysical properties of this protein match those of surfactants, and it appears to be through these properties, rather than though a specific region of the protein, that Ki-67 ensures chromosome separation. Eukaryotic genomes are partitioned into chromosomes that form compact and spatially well-separated mechanical bodies during mitosis 1 , 2 , 3 . This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes 4 and the discovery of proteins at the chromosome surface 3 , 5 , 6 , the molecular and biophysical bases of mitotic chromosome structural individuality have remained unclear. Here we report that the proliferation marker protein Ki-67 (encoded by the MKI67 gene), a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of human Ki-67 is not confined within a specific protein domain, but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrostatic charge barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-colour labelling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic of polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization.

Centromeres are defined by a self-propagating chromatin structure based on stable inheritance of CENP-A containing nucleosomes. Here, we present a genetic screen coupled to pulse-chase labeling that allow us to identify proteins selectively involved in deposition of nascent CENP-A or in long-term transmission of chromatin-bound CENP-A. These include factors with known roles in DNA replication, repair, chromatin modification, and transcription, revealing a broad set of chromatin regulators that impact on CENP-A dynamics. We further identify the SUMO-protease SENP6 as a key factor, not only controlling CENP-A stability but virtually the entire centromere and kinetochore. Loss of SENP6 results in hyper-SUMOylation of CENP-C and CENP-I but not CENP-A itself. SENP6 activity is required throughout the cell cycle, suggesting that a dynamic SUMO cycle underlies a continuous surveillance of the centromere complex that in turn ensures stable transmission of CENP-A chromatin. Centromeres are a self-propagating chromatin structure that feature nucleosomes containing histone H3 variant CENP-A. Here, the authors screen for factors that play a role in CENP-A chromatin maintenance, finding that SUMO-protease SENP6 controls inheritance of chromatin bound CENP-A and is required for the maintenance of the centromere and kinetochore complex.

Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane’s lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy. It was unknown how the most upstream Atg protein transits from the cytosol to autophagosome formation sites. Here, the authors show that ULK1 palmitoylation by ZDHHC13 recruits the complex to the formation site and enhances ATG14L phosphorylation.

Drug conjugates using toxic payloads are a promising approach for selectively combating cancer while sparing healthy tissue. The lack of highly cytotoxic and at the same time selective therapeutics against cancer is an ongoing challenge. Cryptophycins are a class of cyclic depsipeptides renowned for their high cytotoxicity in the picomolar range often combined with efficacy against multidrug-resistant tumour cell lines. However, cryptophycins failed as stand-alone drugs in cancer treatment, and their naturally occurring derivatives lack a covalent attachment handle. By making use of drug conjugates, toxic payloads such as cryptophycins can be selectively delivered to the target site. We present the synthesis of two conjugable cryptophycins with amino groups in unit B, representing potential payloads for drug conjugates particularly effective against multidrug-resistant cancers.

Incorporation of time information into the annotation of distinct biological states in automated fluorescence time-lapse live-cell imaging of complex cellular dynamics reduces both classification noise and confusion between cell states with similar morphology. A computational framework for achieving this is implemented in the open-source software package CellCognition. Fluorescence time-lapse imaging has become a powerful tool to investigate complex dynamic processes such as cell division or intracellular trafficking. Automated microscopes generate time-resolved imaging data at high throughput, yet tools for quantification of large-scale movie data are largely missing. Here we present CellCognition, a computational framework to annotate complex cellular dynamics. We developed a machine-learning method that combines state-of-the-art classification with hidden Markov modeling for annotation of the progression through morphologically distinct biological states. Incorporation of time information into the annotation scheme was essential to suppress classification noise at state transitions and confusion between different functional states with similar morphology. We demonstrate generic applicability in different assays and perturbation conditions, including a candidate-based RNA interference screen for regulators of mitotic exit in human cells. CellCognition is published as open source software, enabling live-cell imaging–based screening with assays that directly score cellular dynamics.

Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the ∼21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community. Cell division genes revealed Jan Ellenberg and colleagues have used RNA interference to silence each of the approximately 21,000 protein-coding genes in a human cell line, then used high-throughput time-lapse imaging of live dividing cells to record the results. Phenotypes were scored quantitatively by computational image processing of at least six two-day movies per gene. Hundreds of genes were found to function in mitosis and other cellular processes including cell survival and migration. The entire data set is available as a public functional genomics resource at www.mitocheck.org. In the new News & View Forum, Jason Swedlow, Cecilia Cotta-Ramusino and Stephen Elledge consider the contribution of this remarkable data set to cell biology, and the challenge of drawing meaningful conclusions from future genome-wide screens. High-throughput microscopy combined with gene silencing by RNA interference is a powerful method for studying gene function. Here, a genome-wide method is presented for phenotypic screening of each of the ∼21,000 human protein-coding genes, using two-day imaging of dividing cells with fluorescently labelled chromosomes. The method enabled the identification of hundreds of genes involved in biological functions such as cell division, migration and survival.

RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.

Decarbonylation of a cyclic bis‐phosphaethynolatostannylene [(ADC)Sn(PCO)]2 based on an anionic dicarbene framework (ADC = PhCN(Dipp)C2; Dipp = 2,6‐iPr2C6H3) under UV light results in the formation of a Sn2P2 cluster compound [(ADC)SnP]2 as a green crystalline solid. The electronic structure of [(ADC)SnP]2 is analyzed by quantum‐chemical calculations. At room temperature, [(ADC)SnP]2 reversibly binds with CO2 and forms [(ADC)2SnOC(O)PSnP]. [(ADC)SnP]2 enables catalytic hydroboration of CO2 and reacts with elemental selenium and Fe2(CO)9 to afford [(ADC)2Sn(Se)P2SnSe] and [(ADC)SnFe(CO)4P]2, respectively. All compounds are characterized by multinuclear NMR spectroscopy and their solid‐state molecular structures are determined by single‐crystal X‐ray diffraction. The Sn2P2 cluster compound [(ADC)SnP]2 based on an anionic dicarbene (ADC) is isolated as a green crystalline solid. [(ADC)SnP]2 reversibly binds with CO2 at room temperature to form [(ADC)2SnOC(O)PSnP] and enables catalytic hydroboration of CO2.

Genome integrity relies on precise coordination between DNA replication and chromosome segregation. Whereas replication stress attracted much attention, the consequences of mitotic perturbations for genome integrity are less understood. Here, we knockdown 47 validated mitotic regulators to show that a broad spectrum of mitotic errors correlates with increased DNA breakage in daughter cells. Unexpectedly, we find that only a subset of these correlations are functionally linked. We identify the genuine mitosis-born DNA damage events and sub-classify them according to penetrance of the observed phenotypes. To demonstrate the potential of this resource, we show that DNA breakage after cytokinesis failure is preceded by replication stress, which mounts during consecutive cell cycles and coincides with decreased proliferation. Together, our results provide a resource to gauge the magnitude and dynamics of DNA breakage associated with mitotic aberrations and suggest that replication stress might limit propagation of cells with abnormal karyotypes. DNA damage arising from replication stress is well studied, but the effect of mitotic errors on genome integrity is less understood. Here the authors knock down 47 mitotic regulators and record how they impact on DNA breakage events, providing a resource for future studies on the relation between cell division and genome integrity.