CDC14 was originally identified by L. Hartwell in his famous screen for genes that regulate the budding yeast cell cycle. Subsequent work showed that Cdc14 belongs to a family of highly conserved dual-specificity phosphatases that are present in a wide range of organisms from yeast to human. Human CDC14B is even able to fulfill the essential functions of budding yeast Cdc14. In budding yeast, Cdc14 counteracts the activity of cyclin dependent kinase (Cdk1) at the end of mitosis and thus has important roles in the regulation of anaphase, mitotic exit and cytokinesis. On the basis of the functional conservation of other cell-cycle genes it seemed obvious to assume that Cdc14 phosphatases also have roles in late mitosis in mammalian cells and regulate similar targets to those found in yeast. However, analysis of the human Cdc14 proteins (CDC14A, CDC14B and CDC14C) by overexpression or by depletion using small interfering RNA (siRNA) has suggested functions that are quite different from those of ScCdc14. Recent studies in avian and human somatic cell lines in which the gene encoding either Cdc14A or Cdc14B had been deleted, have shown – surprisingly – that neither of the two phosphatases on its own is essential for viability, cell-cycle progression and checkpoint control. In this Commentary, we critically review the available data on the functions of yeast and vertebrate Cdc14 phosphatases, and discuss whether they indeed share common functions as generally assumed.

Cdc14A and Cdc14B knockout cells with double-strand breaks still arrest in G2, but they fail to repair the damage.

Metazoan development requires robust proliferation of progenitor cells, whose identities are established by tightly controlled transcriptional networks 1 . As gene expression is globally inhibited during mitosis, the transcriptional programs defining cell identity must be restarted in each cell cycle 2 - 5 , yet how this is accomplished is poorly understood. Here, we identified a ubiquitin-dependent mechanism that integrates gene expression with cell division to preserve cell identity. We found that WDR5 and TBP, which bind active interphase promoters 6 , 7 , recruit the anaphase-promoting complex (APC/C) to specific transcription start sites (TSS) during mitosis. This allows APC/C to decorate histones with K11/K48-branched ubiquitin chains that recruit p97/VCP and the proteasome and ensure rapid expression of pluripotency genes in the next cell cycle. Mitotic exit and transcription re-initiation are thus controlled by the same regulator, APC/C, which provides a robust mechanism to maintain cell identity through cell division.

Commentary 255Introduction Accurate cell division is no trivial task: cells need to duplicate their genomic material, correct mistakes made by sloppy DNA polymerases, repair damage caused by harsh environments and yet still distribute their chromosomes into identical daughter cells. Errors in this program can be deadly for the cell, or, if they result in transformation, have detrimental effects on the organism. To prevent this from happening, the cell division machinery is subject to multiple layers of regulation, with ubiquitylation being of central importance.The post-translational modification with ubiquitin controls the stability, activity or localization of numerous proteins, including multiple cell cycle regulators. It is catalyzed by an enzymatic cascade composed of E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases (Deshaies and Joazeiro, 2009;Rotin and Kumar, 2009;Schulman and Harper, 2009;Ye and Rape, 2009). Together, these enzymes promote the formation of an isopeptide bond between a lysine residue within the substrate and the C-terminus of ubiquitin. The covalent addition of a single ubiquitin, referred to as monoubiquitylation, can alter protein localization or its interactions (Mukhopadhyay and Riezman, 2007). Attachment of further ubiquitin molecules to one of the seven lysine residues or the N-terminus of a substrate-linked ubiquitin results in formation of polymeric chains ( Fig. 1) (Ye and Rape, 2009). When connected through lysine 48 (K48) of ubiquitin, these chains trigger degradation of the substrate by the proteasome (Chau et al., 1989), but when linked through K63, they act as a molecular scaffold that orchestrates kinase activation or DNA repair (Mukhopadhyay and Riezman, 2007). K48-and K63-linked ubiquitin chains have long been recognized as essential regulators of cell division, as they provide a signal for the degradation of inhibitors of cell cycle progression or the activation of cell cycle checkpoints, respectively (Fig. 1).Among the ~600 human E3s, two enzymes -the SCF (Skp1-cullin1-F-box) and APC/C (anaphase-promoting complex/ cyclosome) -are well known for their roles in cell cycle control. These E3s share similar domain architectures, as they are composed of a cullin (in the case of SCF) or cullin-related (in the case of the APC/C) scaffold, a RING domain for binding the ubiquitin-charged E2 and a module for substrate recruitment (Box 1) (Petroski and Deshaies, 2005a;Schreiber et al., 2011). The SCF and APC/C regulate cell division by triggering the degradation of cyclins, Aurora or Polo-like kinases, Cdc25 phosphatases and cyclin-dependent kinase (CDK) inhibitors (Petroski and Deshaies, 2005a;Sullivan and Morgan, 2007). Despite similarities in structure and function, the regulatory mechanisms that ensure proper activation of the SCF and the APC/C are strikingly different: in the case of SCF, the substrate usually needs to be phosphorylated to be recognized by the E3, and mutations in the phosphorylation sites of SCF substrates can result...

Despite improvements in the CRISPR molecular toolbox, identifying and purifying properly edited clones remains slow, laborious, and low-yield. Here, we establish a method to enable clonal isolation, selection, and expansion of properly edited cells, using OptoElectroPositioning technology for single-cell manipulation on a nanofluidic device. Briefly, after electroporation of primary T cells with CXCR4-targeting Cas9 ribonucleoproteins, single T cells are isolated on a chip and expanded into colonies. Phenotypic consequences of editing are rapidly assessed on-chip with cell-surface staining for CXCR4. Furthermore, individual colonies are identified based on their specific genotype. Each colony is split and sequentially exported for on-target sequencing and further off-chip clonal expansion of the validated clones. Using this method, single-clone editing efficiencies, including the rate of mono- and bi-allelic indels or precise nucleotide replacements, can be assessed within 10 days from Cas9 ribonucleoprotein introduction in cells.

CRISPR-Cas9 gene editing has revolutionized cell engineering and promises to open new doors in gene and cell therapies. Despite improvements in the CRISPR-editing molecular toolbox in cell lines and primary cells, identifying and purifying properly edited clones remains slow, laborious and low-yield. Here, we establish a new method that uses cell manipulation on a chip with Opto-Electronic Positioning (OEP) technology to enable clonal isolation and selection of edited cells. We focused on editing CXCR4 in primary human T cells, a gene that encodes a co-receptor for HIV entry. T cells hold significant potential for cell-based therapy, but the gene-editing efficiency and expansion potential of these cells is limited. We describe here a method to obviate these limitations. Briefly, after electroporation of cells with CXCR4-targeting Cas9 ribonucleoproteins (RNPs), single T cells were isolated on a chip, where they proliferated over time into well-resolved colonies. Phenotypic consequences of genome editing could be rapidly assessed on-chip with cell-surface staining for CXCR4. Furthermore, independent of phenotype, individual colonies could be identified based on their specific genotype at the 5-10 cell stage. Each colony was split and sequentially exported for immediate "ontarget" sequencing and validation, and further off-chip clonal expansion of the validated clones. We were able to assess single-clone editing efficiencies, including the rate of monoallelic and biallelic indels or precise nucleotide replacements. This new method will enable identification and selection of perfectly edited clones within 10 days from Cas9-RNP introduction in cells based on the phenotype and/or genotype.