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Cancers are driven by multiple genetic mutations but evolve to evade treatments targeting specific mutations. Nonetheless, cancers cannot evade a treatment that targets mitochondria, which are essential for tumor progression. Iridium complexes have shown anticancer properties, but they lack specificity for their intracellular targets, leading to undesirable side effects. Herein we present a systematic study on structure-activity relationships of eight arylbenzazole-based Iridium(III) complexes of type [IrCl(Cp*)], that have revealed the role of each atom of the ancillary ligand in the physical chemistry properties, cytotoxicity and mechanism of biological action. Neutral complexes, especially those bearing phenylbenzimidazole (HL1 and HL2), restrict the binding to DNA and albumin. One of them, complex 1[C,NH-Cl], is the most selective one, does not bind DNA, targets exclusively the mitochondria, disturbs the mitochondria membrane permeability inducing proton leak and increases ROS levels, triggering the molecular machinery of regulated cell death. In mice with orthotopic lung tumors, the administration of complex 1[C,NH-Cl] reduced the tumor burden. Cancers are more vulnerable than normal tissues to a treatment that harnesses mitochondrial dysfunction. Thus, complex 1[C,NH-Cl] characterization opens the way to the development of new compounds to exploit this vulnerability, We acknowledge the “la Caixa” Foundation (LCF/PR/PR12/11070003), Ministerio de Ciencia Innovación y Universidades-FEDER (RTI2018-102040-B-100) and Junta de Castilla y León-FEDER (BU305P18) for financial support. Networking support by COST Action CA18202 (NECTAR) is also acknowledged.

The dinuclear Ru

With the aim of developing photostable near-infrared cell imaging probes, a convenient route to the synthesis of heteroleptic OsII complexes containing the Os(TAP)2 fragment is reported. This method was used to synthesize the dinuclear OsII complex, [{Os(TAP)2}2tpphz]4+ (where tpphz = tetrapyrido[3,2-a:2',3'-c:3″,2''-h:2‴,3'''-j]phenazine and TAP = 1,4,5,8- tetraazaphenanthrene). Using a combination of resonance Raman and time-resolved absorption spectroscopy, as well as computational studies, the excited state dynamics of the new complex were dissected. These studies revealed that, although the complex has several close lying excited states, its near-infrared, NIR, emission (λmax = 780 nm) is due to a low-lying Os → TAP based 3MCLT state. Cell-based studies revealed that unlike its RuII analogue, the new complex is neither cytotoxic nor photocytotoxic. However, as it is highly photostable as well as live-cell permeant and displays NIR luminescence within the biological optical window, its properties make it an ideal probe for optical microscopy, demonstrated by its use as a super-resolution NIR STED probe for nuclear DNA.

The synthesis of new dinuclear complexes containing linked RuII(dppz) and ReI(dppz) moieties is reported. The photophysical and biological properties of the new complex, which incorporates a N,N'-bis(4-pyridylmethyl)-1,6-hexanediamine tether ligand, are compared to a previously reported RuII/ReI complex linked by a simple dipyridyl alkane ligand. Although both complexes bind to DNA with similar affinities, steady-state and time-resolved photophysical studies reveal that the nature of the linker affects the excited state dynamics of the complexes and their DNA photocleavage properties. Quantum-based DFT calculations on these systems offer insights into these effects. While both complexes are live cells permeant, their intracellular localizations are significantly affected by the nature of the linker. Notably, one of the complexes displayed concentration-dependent localization and possesses photophysical properties that are compatible with SIM and STED nanoscopy. This allowed the dynamics of its intracellular localization to be tracked at super resolutions.

Osteoporosis is a leading world health problem that results from an imbalance between bone formation and bone resorption. &beta, glucans has been extensively reported to exhibit a wide range of biological activities, including antiosteoporosis both in vitro and in vivo. However, the molecular mechanisms responsible for &beta, glucan-mediated bone formation in osteoblasts have not yet been investigated. The oyster mushroom Pleurotus sajor-caju produces abundant amounts of an insoluble &beta, glucan, which is rendered soluble by enzymatic degradation using Hevea glucanase to generate low-molecular-weight glucanoligosaccharide (Ps-GOS). This study aimed to investigate the osteogenic enhancing activity and underlining molecular mechanism of Ps-GOS on osteoblastogenesis of pre-osteoblastic MC3T3-E1 cells. In this study, it was demonstrated for the first time that low concentrations of Ps-GOS could promote cell proliferation and division after 48 h of treatment. In addition, Ps-GOS upregulated the mRNA and protein expression level of bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor-2 (Runx2), which are both involved in BMP signaling pathway, accompanied by increased alkaline phosphatase (ALP) activity and mineralization. Ps-GOS also upregulated the expression of osteogenesis related genes including ALP, collagen type 1 (COL1), and osteocalcin (OCN). Moreover, our novel findings suggest that Ps-GOS may exert its effects through the mitogen-activated protein kinase (MAPK) and wingless-type MMTV integration site (Wnt)/&beta, catenin signaling pathways.

Nucleoside polyphosphates (NPPs) are mainly produced in mitochondria and used as a universal energy source for various cellular events. Although numerous fluorescent probes for adenosine triphosphate (ATP) have been reported, they are not ideally suited for live monitoring of the subtle variation of the mitochondrial ATP level. A new coumarin-based fluorescent probe is synthesized, and this reagent is utilized for specific recognition of NPPs in mitochondria by super-resolution microscopy in physiological condition. Detailed 31P NMR studies reveal that the probe, L·Zn(II), binds to NPPs through their pendent phosphate functionalities. Such binding leads to a substantial enhancement in the luminescence intensity of L·Zn(II) + ADP or L·Zn(II) + ATP as compared to L·Zn(II). This—as well as 1H NMR spectroscopy—has enabled us to evaluate the probe’s binding affinities to these NPPs. Structured illumination and wide field fluorescence microscopy confirmed that this physiologically benign reagent is localized wi...

BODIPY derivative, SF-1, exclusively shows a fluorescence ON response to HOCl and images endogenously generated HOCl in RAW 264.7 macrophages. Widefield and super resolution structured illumination microscopy images confirm localization in the Golgi complex and lysosomes, and hence specifically detects HOCl generated in these organelles. SF-1 is compatible with 3D-SIM imaging of individual cells.

Selective detection of nitroxyl (HNO), which has recently been identified as a reactive nitrogen species, is a challenging task. We report a BODIPY-based luminescence ON reagent for detection of HNO in aqueous solution and in live RAW 264.7 cells, based on the soft nucleophilicity of the phosphine oxide functionality toward HNO. The probe shows high selectivity to HNO over other reactive oxygen/nitrogen and sulfur species. Luminescence properties of the BODIPY-based chemodosimetric reagent make it an ideal candidate for use as a reagent for super-resolution structured illumination microscopy. The viability of the reagent for biological in vivo imaging application was also confirmed using Artemia as a model.

β-NaYF4:Yb,Gd up-conversion nanoparticles, UCNPs, surface functionalized with suitable targetting peptides function as nontoxic lysosome-specific imaging probes.\ud \ud

Medicinal leads that are also compatible with imaging technologies are attractive, as they facilitate the development of therapeutics through direct mechanistic observations at the molecular level. In this context, the uptake and antimicrobial activities of several luminescent dinuclear RuII complexes against E. coli were assessed and compared to results obtained for another ESKAPE pathogen, the Gram-positive major opportunistic pathogen Enterococcus faecalis, V583. The most promising lead displays potent activity, particularly against the Gram-negative bacteria, and potency is retained in the uropathogenic multidrug resistant EC958 ST131 strain. Exploiting the inherent luminescent properties of this complex, super-resolution STED nanoscopy was used to image its initial localization at/in cellular membranes and its subsequent transfer to the cell poles. Membrane damage assays confirm that the complex disrupts the bacterial membrane structure before internalization. Mammalian cell culture and animal model studies indicate that the complex is not toxic to eukaryotes, even at concentrations that are several orders of magnitude higher than its minimum inhibitory concentration (MIC). Taken together, these results have identified a lead molecular architecture for hard-to-treat, multiresistant, Gram-negative bacteria, which displays activities that are already comparable to optimized natural product-based leads.

Drug resistance to platinum chemotherapeutics targeting DNA often involves abrogation of apoptosis and has emerged as a significant challenge in modern, non-targeted chemotherapy. Consequently, there is great interest in the anti-cancer properties of metal complexes-particularly those that interact with DNA-and mechanisms of consequent cell death. Herein we compare a parent cytotoxic complex, [Ru(phen)2(tpphz)]2+ [phen = 1,10-phenanthroline, tpphz = tetrapyridyl[3,2- a:2',3'- c:3″,2″- h:2‴,3‴- j]phenazine], with a mononuclear analogue with a modified intercalating ligand, [Ru(phen)2(taptp)]2+ [taptp = 4,5,9,18-tetraazaphenanthreno[9,10- b] triphenylene], and two structurally related dinuclear, tpphz-bridged, heterometallic complexes, RuRe and RuPt. All three of these structural changes result in a switch from intercalation to groove-binding DNA interaction and concomitant reduction in cytotoxic potency, but no significant change in relative cytotoxicity toward platinum-resistant A2780CIS cancer cells, indicating that the DNA interaction mode is not critical for the mechanism of platinum resistance. All variants exhibited a light-switch effect, which for the first time was exploited to investigate timing of cell death by live-cell microscopy. Surprisingly, cell death occurred rapidly as a consequence of oncosis, characterized by loss of cytoplasmic volume control, absence of significant mitochondrial membrane potential loss, and lack of activation of apoptotic cell death markers. Importantly, a novel, quantitative proteomic analysis of the A2780 cell genome following exposure of the cells to either mononuclear complex reveals changes in protein expression associated with global cell responses to oxidative stress and DNA replication/repair cellular pathways. This combination of multiple targeting modalities and induction of a non-apoptotic death mechanism makes these complexes highly promising chemotherapeutic cytotoxicity leads.

Two-photon active Graphene Quantum Dots (GQDs) are obtained from extracts of the neem root. These biocompatible GQDs are found to be suitable for structured illumination microscopy. Two-photon microscopy ensured lysosome specificity of GQDs in live cells and confocal luminescence microscopic studies showed tissue-dependent localization of GQDs in Zebrafish

Although metal-ion-directed self-assembly has been widely used to construct a vast number of macrocycles and cages, it is only recently that the biological properties of these systems have begun to be explored. However, up until now, none of these studies have involved intrinsically photoexcitable self-assembled structures. Herein we report the first metallomacrocycle that functions as an intracellular singlet oxygen sensitizer. Not only does this Ru2 Re2 system possess potent photocytotoxicity at light fluences below those used for current medically employed systems, it offers an entirely new paradigm for the construction of sensitizers for photodynamic therapy.

Osteoporosis is widely recognized as a major health problem caused by an inappropriate rate of bone resorption compared to bone formation. Previously we showed that d-pinitol inhibits osteoclastogenesis but has no effect on osteoblastogenesis. However, the effect on osteoblast differentiation of its isomer, l-quebrachitol, has not yet been reported. The purpose of this study was, therefore, to investigate whether l-quebrachitol promotes the osteoblastogenesis of pre-osteoblastic MC3T3-E1 cells. Moreover, the molecular mechanism of action of l-quebrachitol was further explored. Here, it is shown for the first time that l-quebrachitol significantly promotes proliferation and cell DNA synthesis. It also enhances mineralization accompanied by increases in mRNA expression of bone matrix proteins including alkaline phosphatase (ALP), collagen type I (ColI), osteocalcin (OCN), and osteopontin (OPN). In addition, l-quebrachitol upregulates the mRNA and protein expression of bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor-2 (Runx2), while down-regulating the receptor activator of the nuclear factor-&kappa, B ligand (RANKL) mRNA level. Moreover, the expression of regulatory genes associated with the mitogen-activated protein kinase (MAPK) and wingless-type MMTV integration site (Wnt)/&beta, catenin signaling pathways are also upregulated. These findings indicate that l-quebrachitol may promote osteoblastogenesis by triggering the BMP-2-response as well as the Runx2, MAPK, and Wnt/&beta, catenin signaling pathway.

Surface engineering of nanocarriers allows fine-tuning of their interactions with biological organisms, potentially forming the basis of devices for the monitoring of intracellular events or for intracellular drug delivery. In this context, biodegradable nanocarriers or nanocapsules capable of carrying bioactive molecules or drugs into the mitochondrial matrix could offer new capabilities in treating mitochondrial diseases. Nanocapsules with a polymeric backbone that undergoes programmed rupture in response to a specific chemical or enzymatic stimulus with subsequent release of the bioactive molecule or drug at mitochondria would be particularly attractive for this function. With this goal in mind, we have developed biologically benign nanocapsules using polyurethane-based, polymeric backbone that incorporates repetitive ester functionalities. The resulting nanocapsules are found to be highly stable and monodispersed in size. Importantly, a new non-isocyanate route is adapted for the synthesis of these non-isocyanate polyurethane nanocapsules (NIPU). The embedded ester linkages of these capsules' shells have facilitated complete degradation of the polymeric backbone in response to a stimulus provided by an esterase enzyme. Hydrophilic payloads like rhodamine or doxorubicin can be loaded inside these nanocarriers during their synthesis by an interfacial polymerization reaction. The postgrafting of the nanocapsules with phosphonium ion, a mitochondria-targeting receptor functionality, has helped us achieve the site-specific release of the drug. Co-localization experiments with commercial mitotracker green as well as mitotracker deep red confirmed localization of the cargo in mitochondria. Our in vitro studies confirm that specific release of doxorubicin within mitochondria causes higher cytotoxicity and cell death compared to free doxorubicin. Endogenous enzyme triggered nanocapsule rupture and release of the encapsulated dye is also demonstrated in a zebrafish model. The results of this proof-of-concept study illustrate that NIPU nanocarriers can provide a site-specific delivery vehicle and improve the therapeutic efficacy of a drug or be used to produce organelle-specific imaging studies.

A new physiologically benign and cell membrane permeable BODIPY based molecular probe, MB-Sn, specifically senses intracellular hydrogen polysulfides (H2Sn, n > 1) localized in the endoplasmic reticulum. This reagent is suitable for mapping the intracellular distribution of H2Sn by wide-field as well as super-resolution Structured Illumination Microscopy (SIM).

The DNA binding and cellular localization properties of a new luminescent heterobimetallic Ir(III) Ru(II) tetrapyridophenazine complex are reported. Surprisingly, in standard cell media, in which its tetracationic, isostructural Ru(II) Ru(II) analogue is localized in the nucleus, the new tricationic complex is poorly taken up by live cells and demonstrates no nuclear staining. Consequent cell-free studies reveal that the Ir(III) Ru(II) complex binds bovine serum albumin, BSA, in Sudlow's Site I with a similar increase in emission and binding affinity to that observed with DNA. Contrastingly, in serum-free conditions the complex is rapidly internalized by live cells, where it localizes in cell nuclei and functions as a DNA imaging agent. The absence of serum proteins also greatly alters the cytotoxicity of the complex, where high levels of oncosis/necrosis are observed due to this enhanced uptake. This suggests that simply increasing the lipophilicity of a DNA imaging probe to enhance cellular uptake can be counterproductive as, due to increased binding to serum albumin protein, this strategy can actually disrupt nuclear targeting.

[Ru(phen)2(tpphz)]2+ simultaneously inhibits DNA replication, blocks mitosis and enhances DNA-damaging ionising radiation in oesophageal cancer cells., Substitutionally inert ruthenium(ii) polypyridyl complexes have been developed as DNA intercalating agents yet cellular DNA damage responses to this binding modality are largely unexplored. Here, we show the nuclear-targeting complex [Ru(phen)2(tpphz)]2+ (phen = 1,10-phenanthroline, tpphz = tetrapyridophenazine) generates rapid and pronounced stalling of replication fork progression in p53-deficient human oesophageal cancer cells. In response, replication stress and double-strand break (DSB) DNA damage response (DDR) pathways are activated and cell proliferation is inhibited by growth arrest. Moreover, mitotic progression is compromised by [Ru(phen)2(tpphz)]2+, where the generation of metaphase chromosome spindle attachment failure results in spindle assembly checkpoint (SAC) activation. This dual mechanism of action results in preferential growth inhibition of rapidly-proliferating oesophageal cancer cells with elevated mitotic indices. In addition to these single-agent effects, [Ru(phen)2(tpphz)]2+ functions as a radiosensitizer with efficiency comparable to cisplatin, which occurs through a synergistic enhancement of DNA damage. These results establish that DNA replication is the target for [Ru(phen)2(tpphz)]2+ and provide the first experimental evidence that ruthenium-based intercalation targets multiple genome integrity pathways in cancer cells, thereby achieving enhanced selectivity compared to existing DNA-damaging agents such as cisplatin.

Using a new mononuclear “building block,” for the first time, a dinuclear RuII(dppn) complex and a heteroleptic system containing both RuII(dppz) and RuII(dppn) moieties are reported. The complexes, including the mixed dppz/dppn system, are 1O2 sensitizers. However, unlike the homoleptic dppn systems, the mixed dppz/dppn complex also displays a luminescence “switch on” DNA light-switch effect. In both cisplatin sensitive and resistant human ovarian carcinoma lines the dinuclear complexes show enhanced uptake compared to their mononuclear analogue. Thanks to a favorable combination of singlet oxygen generation and cellular uptake properties all three of the new complexes are phototoxic and display potent activity against chemotherapeutically resistant cells.

Detailed studies on the live cell uptake properties of a dinuclear membrane-permeable RuII cell probe show that, at low concentrations, the complex localizes and images mitochondria. At concentrations above ∼20 μM, the complex images nuclear DNA. Because the complex is extremely photostable, has a large Stokes shift, and displays intrinsic subcellular targeting, its compatibility with super-resolution techniques was investigated. It was found to be very well suited to image mitochondria and nuclear chromatin in two color, 2C-SIM, and STED and 3D-STED, both in fixed and live cells. In particular, due to its vastly improved photostability compared to that of conventional SR probes, it can provide images of nuclear DNA at unprecedented resolution.

A new route to a tetracyclic lactam was developed and the product, called VG1, was found to inhibit nonsense-mediated mRNA decay at μM concentrations., During efforts to prepare the known compound NMDI1, a new tetracyclic compound, called VG1, was prepared in six steps. This compound was found to have good activity as an inhibitor of nonsense-mediated mRNA decay.

In vertebrates, the nuclear envelope (NE) assembles and disassembles during mitosis. As the NE is a complex structure consisting of inner and outer membranes, nuclear pore complexes (NPCs) and the nuclear lamina, NE assembly must be a controlled and systematic process. In Xenopus egg extracts, NE assembly is mediated by two distinct membrane vesicle populations, termed NEP-A and NEP-B. Here, we re-investigate how these two membrane populations contribute to NPC assembly. In growing stage III Xenopus oocytes, NPC assembly intermediates are frequently observed. High concentrations of NPC assembly intermediates always correlate with fusion of vesicles into preformed membranes. In Xenopus egg extracts, two integral membrane proteins essential for NPC assembly, POM121 and NDC1, are exclusively associated with NEP-B membranes. By contrast, a third integral membrane protein associated with the NPCs, gp210, associates only with NEP-A membranes. During NE assembly, fusion between NEP-A and NEP-B led to the formation of fusion junctions at which >65% of assembling NPCs were located. To investigate how each membrane type contributes to NPC assembly, we preferentially limited NEP-A in NE assembly assays. We found that, by limiting the NEP-A contribution to the NE, partially formed NPCs were assembled in which protein components of the nucleoplasmic face were depleted or absent. Our data suggest that fusion between NEP-A and NEP-B membranes is essential for NPC assembly and that, in contrast to previous reports, both membranes contribute to NPC assembly.

Under a Creative Commons license.-- Review.-- et al., Targeted therapies and the consequent adoption of >personalized> oncology have achieved notablesuccesses in some cancers; however, significant problems remain with this approach. Many targetedtherapies are highly toxic, costs are extremely high, and most patients experience relapse after a fewdisease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistantimmortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are notreliant upon the same mechanisms as those which have been targeted). To address these limitations, aninternational task force of 180 scientists was assembled to explore the concept of a low-toxicity >broad-spectrum> therapeutic approach that could simultaneously target many key pathways and mechanisms. Using cancer hallmark phenotypes and the tumor microenvironment to account for the various aspectsof relevant cancer biology, interdisciplinary teams reviewed each hallmark area and nominated a widerange of high-priority targets (74 in total) that could be modified to improve patient outcomes. For thesetargets, corresponding low-toxicity therapeutic approaches were then suggested, many of which werephytochemicals. Proposed actions on each target and all of the approaches were further reviewed forknown effects on other hallmark areas and the tumor microenvironment. Potential contrary or procar-cinogenic effects were found for 3.9% of the relationships between targets and hallmarks, and mixedevidence of complementary and contrary relationships was found for 7.1%. Approximately 67% of therelationships revealed potentially complementary effects, and the remainder had no known relationship. Among the approaches, 1.1% had contrary, 2.8% had mixed and 62.1% had complementary relationships. These results suggest that a broad-spectrum approach should be feasible from a safety standpoint. Thisnovel approach has potential to be relatively inexpensive, it should help us address stages and types ofcancer that lack conventional treatment, and it may reduce relapse risks. A proposed agenda for futureresearch is offered., Amr Amin was funded by Terry Fox Foundation Grant # TF-13-20 and UAEU Program for Advanced Research (UPAR) #31S118; Jack Arbiser was funded by NIHAR47901; Alexandra Arreola was funded by NIH NRSA Grant F31CA154080; Alla Arzumanyan was funded by NIH (NIAID) R01: Combination therapies for chronic HBV, liver disease, and cancer (AI076535); Work in the lab of Asfar S. Azmi is supported by NIH R21CA188818 as well as from Sky Foundation Inc. Michigan; Fabian Benencia was supported by NIH Grant R15 CA137499-01; Alan Bilsland was supported by the University of Glasgow, Beatson Oncology Centre Fund, CRUK (www.cancerresearchuk.org) Grant C301/A14762; Amancio Carnero was supported by grants from the Spanish Ministry of Economy and Competitivity, ISCIII (Fis: PI12/00137, RTICC: RD12/0036/0028) co-funded by FEDER from Regional Development European Funds (European Union), Consejeria de Ciencia e Innovacion (CTS-6844 and CTS-1848) and Consejeria de Salud of the Junta de Andalucia (PI-0135-2010 and PI-0306-2012). His work on this project has also been made possible thanks to the Grant PIE13/0004 co-funded by the ISCIII and FEDER funds; Stephanie C. Casey was supported by NIH Grant F32CA177139; Mrinmay Chakrabarti was supported by the United Soybean Board; Rupesh Chaturvedi was supported by an NIH NCCAM Grant (K01AT007324); Georgia Zhuo Chen was supported by an NIH NCI Grant (R33 CA161873-02); Helen Chen acknowledges financial support from the Michael Cuccione Childhood Cancer Foundation Graduate Studentship; Sophie Chen acknowledges financial support from the Ovarian and Prostate Cancer Research Trust, UK; Yi Charlie Chen acknowledges financial support from the West Virginia Higher Education Policy Commission/Division of Science Research, his research was also supported by NIH grants (P20RR016477 and P20GM103434) from the National Institutes of Health awarded to the West Virginia IDeA Network of Biomedical Research Excellence; Maria Rosa Ciriolo was partially supported by the Italian Association for Cancer Research (AIRC) Grants #IG10636 and #15403; Helen M. Coley acknowledges financial support from the GRACE Charity, UK and the Breast Cancer Campaign, UK; Marisa Connell was supported by a Michael Cuccione Childhood Cancer Foundation Postdoctoral Fellowship; Sarah Crawford was supported by a research grant from Connecticut State University; Charlotta Dabrosin acknowledges financial support from the Swedish Research Council and the Swedish Research Society; Giovanna Damia gratefully acknowledges the generous contributions of The Italian Association for Cancer Research (IG14536 to G.D.), Santanu Dasgupta gratefully acknowledges the support of the University of Texas Health Science Centre at Tyler, Elsa U. Pardee Foundation; William K. Decker was supported in part by CPRIT, the Cancer Prevention and Research Institute of Texas; Anna Mae E. Diehl was supported by NIH National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the NIH National Institute on Alcohol Abuse and Alcoholism (NIAAA), Gilead and Shire Pharmaceuticals; Q. Ping Dou was partially supported by NIH/NCI (1R01CA20009, 5R01CA127258-05 and R21CA184788), and NIH P30 CA22453 (to Karmanos Cancer Institute); Janice E. Drew was supported by the Scottish Government's Rural and Environment Science and Analytical Services Division; Eyad Elkord thanks the National Research Foundation, United Arab Emirates University and the Terry Fox Foundation for supporting research projects in his lab; Bassel El-Rayes was supported by Novartis Pharmaceutical, Aveo Pharmaceutical, Roche, Bristol Myers Squibb, Bayer Pharmaceutical, Pfizer, and Kyowa Kirin; Mark A. Feitelson was supported by NIH/NIAID Grant AI076535, Dean W. Felsher was supported by NIH grants (R01CA170378, U54CA149145, and U54CA143907); Lynnette R Ferguson was financially supported by the Auckland Cancer Society and the Cancer Society of New Zealand; Gary L. Firestone was supported by NIH Public Service Grant CA164095 awarded from the National Cancer Institute; Christian Frezza “would like to acknowledge funding from a Medical Research Council CCU-Program Grant on cancer metabolism, and a unique applicant AICR project grant”; Mark M. Fuster was supported by NIH Grant R01-HL107652; Alexandros G. Georgakilas was supported by an EU Marie Curie Reintegration Grant MC-CIG-303514, Greek National funds through the Operational Program ‘Educational and Lifelong Learning of the National Strategic Reference Framework (NSRF)-Research Funding Program THALES (Grant number MIS 379346) and COST Action CM1201 ‘Biomimetic Radical Chemistry’; Michelle F. Green was supported by a Duke University Molecular Cancer Biology T32 Training Grant; Brendan Grue was supported by a National Sciences Engineering and Research Council Undergraduate Student Research Award in Canada; Dorota Halicka was supported by by NIH NCI grant NCI RO1 28704; Petr Heneberg was supported by the Charles University in Prague projects UNCE 204015 and PRVOUK P31/2012, by the Czech Science Foundation projects 15-03834Y and P301/12/1686, by the Czech Health Research Council AZV project 15-32432A, and by the Internal Grant Agency of the Ministry of Health of the Czech Republic project NT13663-3/2012; Matthew D. Hirschey wishes to acknowledge Duke University Institutional Support, the Duke Pepper Older Americans Independence Center (OAIC) Program in Aging Research supported by the National Institute of Aging (P30AG028716-01) and NIH/NCI training grants to Duke University (T32-CA059365-19 and 5T32-CA059365), Lorne J. Hofseth was supported by NIH grants (1R01CA151304, 1R03CA1711326, and 1P01AT003961); Kanya Honoki was supported in part by the grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 24590493); Hsue-Yin Hsu was supported in part by grants from the Ministry of Health and Welfare (CCMP101-RD-031 and CCMP102-RD-112) and Tzu-Chi University (61040055-10) of Taiwan; Lasse D. Jensen was supported by Svenska Sallskapet for Medicinsk Forskning, Gosta Fraenkels Stiftelse, Ak.e Wibergs Stiftelse, Ollie och Elof Ericssons Stiftelse, Linkopings Universitet and the Karolinska Institute, Sweden; Wen G. Jiang wishes to acknowledge the support by Cancer Research Wales, the Albert Hung Foundation, the Fong Family Foundation, and Welsh Government A4B scheme; Lee W. Jones was supported in part by grants from the NIH NCI; W Nicol Keith was supported by the University of Glasgow, Beatson Oncology Centre Fund, CRUK (www.cancerresearchuk.org) Grant C301/A14762; Sid P. Kerkar was supported by the NIH Intramural Research Program; Rob J. Kulathinal was supported by the National Science Foundation, and the American Cancer Society; Byoung S. Kwon was supported in part by National Cancer Center (NCC-1310430-2) and National Research Foundation (NRF-2005-0093837); Anne Le was supported by Sol Goldman Pancreatic Cancer Research Fund Grant 80028595, a Lustgarten Fund Grant 90049125 and Grant NIHR21CA169757 (to Anne Le); Michael A. Lea was funded by the The Alma Toorock Memorial for Cancer Research; Ho-Young Lee., This work was supported by grants from the National Research Foundation of Korea (NRF), the Ministry of Science, ICT & Future Planning (MSIP), Republic of Korea (Nos. 2011-0017639 and 2011-0030001) and by a NIH Grant R01 CA100816; Liang-Tzung Lin was supported in part by a grant from the Ministry of Education of Taiwan (TMUTOP103005-4); Jason W. Locasale acknowledges support from NIH awards (CA168997 and AI110613) and the International Life Sciences Institute; Bal L. Lokeshwar was supported in part by United States’ Public Health Services Grants: NIH R01CA156776 and VA-BLR&D Merit Review Grant No. 5I01-BX001517-02; Valter D. Longo acknowledges support from NIH awards (P01AG034906 and R01AG020642) and from the V Foundation; Costas A. Lyssiotis was funded in part by the Pancreatic Cancer Action Network as a Pathway to Leadership Fellow and through a Dale F. Frey Breakthrough award from the Damon Runyon Cancer Research Foundation; Karen L. MacKenzie wishes to acknowledge the support from the Children's Cancer Institute Australia (affiliated with the University of New South Wales, Australia and the Sydney Children's Hospital Network); Maria Marino was supported by grant from University Roma Tre to M.M. (CLA 2013) and by the Italian Association for Cancer Research (AIRC-Grant #IG15221), Ander Matheu is funded by Carlos III Health Institute (AM: CP10/00539), Basque Foundation for Science (IKERBASQUE) and Marie Curie CIG Grant (AM: 2012/712404); Christopher Maxwell was supported by funding from the Canadian Institutes of Health Research, in partnership with the Avon Foundation for Women (OBC-134038) and the Canadian Institutes of Health Research New Investigator Salary Award (MSH-136647); Eoin McDonnell received Duke University Institutional Support; Kapil Mehta was supported by Bayer Healthcare System G4T (Grants4Targets); Gregory A. Michelotti received support from NIH NIDDK, NIH NIAAA, and Shire Pharmaceuticals; Vinayak Muralidhar was supported by the Harvard-MIT Health Sciences and Technology Research Assistantship Award; Elena Niccolai was supported by the Italian Ministry of University and the University of Italy; Virginia R. Parslow gratefully acknowledges the financial support of the Auckland Cancer Society Research Centre (ACSRC); Graham Pawelec was supported by the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF) Grant number 16SV5536K, and by the European Commission (FP7 259679 “IDEAL”); Peter L. Pedersen was supported by NIH Grant CA-10951; Brad Poore was supported by Sol Goldman Pancreatic Cancer Research Fund Grant 80028595, the Lustgarten Fund Grant 90049125, and Grant NIHR21CA169757 (to Anne Le); Satya Prakash was supported by a Canadian Institutes of Health Research Grant (MOP 64308); Lizzia Raffaghello was supported by an NIH Grant (P01AG034906-01A1) and Cinque per Mille dell’IRPEF–Finanziamento della Ricerca Sanitaria; Jeffrey C. Rathmell was supported by an NIH Grant (R01HL108006); Swapan K. Ray was supported by the United Soybean Board; Domenico Ribatti received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under Grant agreement n°278570; Luigi Ricciardiello was supported by the AIRC Investigator Grants 10216 and 13837, and the European Community's Seventh Framework Program FP7/2007–2013 under Grant agreement 311876; Francis Rodier acknowledges the support of the Canadian Institute for Health Research (FR: MOP114962, MOP125857), Fonds de Recherche Québec Santé (FR: 22624), and the Terry Fox Research Institute (FR: 1030), Gian Luigi Russo contributed to this effort while participating in the Fulbright Research Scholar Program 2013–14; Isidro Sanchez-Garcia is partially supported by FEDER and by MICINN (SAF2012-32810), by NIH Grant (R01 CA109335-04A1), by Junta de Castilla y León (BIO/SA06/13) and by the ARIMMORA project (FP7-ENV-2011, European Union Seventh Framework Program). Isidro Sanchez-Garcia's lab is also a member of the EuroSyStem and the DECIDE Network funded by the European Union under the FP7 program; Andrew J. Sanders wishes to acknowledge the support by Cancer Research Wales, the Albert Hung Foundation, the Fong Family Foundation, and Welsh Government A4B scheme; Neeraj K. Saxena was supported by grant funding from NIH NIDDK (K01DK077137, R03DK089130); Dipali Sharma was partially funded by NIH NCI grants (R01CA131294, R21 CA155686), the Avon Foundation and a Breast Cancer Research Foundation Grant (90047965); Markus David Siegelin received funding from National Institute of Health, NINDS Grant K08NS083732, and the 2013 AACR-National Brain Tumor Society Career Development Award for Translational Brain Tumor Research, Grant Number 13-20-23-SIEG; Neetu Singh was supported by funds from the Department of Science and Technology (SR/FT/LS-063/2008), New Delhi, India; Carl Smythe was supported by Yorkshire Cancer Research and The Wellcome Trust, UK; Carmela Spagnuolo was supported by funding from Project C.I.S.I.A., act n. 191/2009 from the Italian Ministry of Economy and Finance Project CAMPUS-QUARC, within program FESR Campania Region 2007/2013, objectives 2.1, 2.2; Diana M. Stafforini was supported by grants from the National Cancer Institute (5P01CA073992), IDEA Award W81XWH-12-1-0515 from the Department of Defense, and by the Huntsman Cancer Foundation; John Stagg was supported by the Canadian Institutes of Health Research; Pochi R. Subbarayan was supported by the University of Miami Clinical and Translational Science Institute (CTSI) Pilot Research Grant (CTSI-2013-P03) and SEEDS You Choose Awards; Phuoc T. Tran was funded by the DoD (W81XWH-11-1-0272 and W81XWH-13-1-0182), a Kimmel Translational Science Award (SKF-13-021), an ACS Scholar award (122688-RSG-12-196-01-TBG) and the NIH (R01CA166348); Kathryn E. Wellen receives funding from the National Cancer Institute, Pancreatic Cancer Action Network, Pew Charitable Trusts, American Diabetes Association, and Elsa U. Pardee Foundation; Huanjie Yang was partially supported by the Scientific Research Foundation for the Returned Oversea Scholars, State Education Ministry and Scientific and Technological Innovation Project, Harbin (2012RFLXS011), Paul Yaswen was supported by funding from the United States National Institutes of Health (ES019458) and the California Breast Cancer Research Program (17UB-8708); Clement Yedjou was supported by a grant from the National Institutes of Health (Grant # G1200MD007581), through the RCMI-Center for Environmental Health; Xin Yin was supported by NIH/National Heart, Lung, and Blood Institute Training Grant T32HL098062.; Jiyue Zhu was supported by NIH Grant R01GM071725; Massimo Zollo was supported by the European FP7-TuMIC HEALTH-F2-2008-201662, the Italian Association for Cancer research (AIRC) Grant IG # 11963 and the Regione Campania L.R:N.5, the European National Funds PON01-02388/1 2007-2013.

Introduction and objectivesThe zinc-finger transcription factor Krϋppel-like factor 2 (KLF2) transduces blood flow into molecular signals responsible for a wide range of responses within the vasculature. KLF2 maintains a healthy, quiescent endothelial phenotype. Previous studies report a range of phenotypes following morpholino antisense oligonucleotide-induced klf2a knockdown in zebrafish. Targeted genome editing is an increasingly applied method for functional assessment of candidate genes. We therefore generated a stable klf2a mutant zebrafish and characterised its cardiovascular and haematopoietic development.Methods and resultsUsing Transcription Activator-Like Effector Nucleases (TALEN) we generated a klf2a mutant (klf2ash317) with a 14bp deletion leading to a premature stop codon in exon 2. Western blotting confirmed loss of wild type Klf2a protein and the presence of a truncated protein in klf2ash317 mutants. Homozygous klf2ash317 mutants exhibit no defects in vascular patterning, survive to adulthood and are fertile, without displaying previously described morphant phenotypes such as high-output cardiac failure, reduced haematopoetic stem cell (HSC) development or impaired formation of the 5th accessory aortic arch. Homozygous klf2ash317 mutation did not reduce angiogenesis in zebrafish with homozygous mutations in von Hippel Lindau (vhl), a form of angiogenesis that is dependent on blood flow. We examined expression of three klf family members in wildtype and klf2ash317 zebrafish. We detected vascular expression of klf2b (but not klf4a or biklf/klf4b/klf17) in wildtypes but found no differences in expression that might account for the lack of phenotype in klf2ash317 mutants. klf2b morpholino knockdown did not affect heart rate or impair formation of the 5th accessory aortic arch in either wildtypes or klf2ash317 mutants.ConclusionsThe klf2ash317 mutation produces a truncated Klf2a protein but, unlike morpholino induced klf2a knockdown, does not affect cardiovascular development.

Cytostatic agents that interfere with specific cellular components to prevent cancer cell growth offer an attractive alternative, or complement, to traditional cytotoxic chemotherapy. Here, we describe the synthesis and characterization of a new binuclear RuII-PtII complex [Ru(tpy)(tpypma)Pt(Cl)(DMSO)]3+ (tpy=2,2′:6′,2′′-terpyridine and tpypma=4-([2,2′:6′,2′′-terpyridine]-4′-yl)-N-(pyridin-2-ylmethyl)aniline), VR54, which employs the extended terpyridine tpypma ligand to link the two metal centres. In cell-free conditions, VR54 binds DNA by non-intercalative reversible mechanisms (K b =1.3×105 M-1) and does not irreversibly bind guanosine. Cellular studies reveal that VR54 suppresses proliferation of A2780 ovarian cancer cells with no cross-resistance in the A2780CIS cisplatin-resistant cell line. Through the preparation of mononuclear RuII and PtII structural derivatives it was determined that both metal centres are required for this anti-proliferative activity. In stark contrast to cisplatin, VR54 neither activates the DNA-damage response network nor induces significant levels of cell death. Instead, VR54 is cytostatic and inhibits cell proliferation by up-regulating the cyclin-dependent kinase inhibitor p27KIP1 and inhibiting retinoblastoma protein phosphorylation, which blocks entry into S phase and results in G1 cell cycle arrest. Thus, VR54 inhibits cancer cell growth by a gain of function at the G1 restriction point. This is the first metal-coordination compound to demonstrate such activity. Arrested development: A binuclear ruthenium(II)-platinum(II) terpyridyl complex, VR54 (see structure), inhibits cancer cell proliferation. In contrast to the vast majority of metal anticancer agents, VR54 is cytostatic and acts by up-regulating p27KIP1, inhibiting retinoblastoma protein (Rb) phosphorylation and inducing G1 cell cycle arrest.

Genetically distinct checkpoints, activated as a consequence of either DNA replication arrest or ionizing radiation-induced DNA damage, integrate DNA repair responses into the cell cycle programme. The ataxia-telangiectasia mutated (ATM) protein kinase blocks cell cycle progression in response to DNA double strand breaks, whereas the related ATR is important in maintaining the integrity of the DNA replication apparatus. Here, we show that thymidine, which slows the progression of replication forks by depleting cellular pools of dCTP, induces a novel DNA damage response that, uniquely, depends on both ATM and ATR. Thymidine induces ATM-mediated phosphorylation of Chk2 and NBS1 and an ATM-independent phosphorylation of Chk1 and SMC1. AT cells exposed to thymidine showed decreased viability and failed to induce homologous recombination repair (HRR). Taken together, our results implicate ATM in the HRR-mediated rescue of replication forks impaired by thymidine treatment.

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo–AP2 interactions occur via the μ2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for μ2 phosphorylation is in cargo recruitment because μ2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of μ2 phosphorylation. We identify clathrin as a specific activator of the μ2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of μ2 phosphorylation. Furthermore, we show that AP2 containing phospho-μ2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-μ2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-μ2 levels.

Two dinuclear osmium polypyridyl complexes function as convenient, easy to handle TEM contrast agents and facilitate the high-resolution visualisation of intracellular structure, particularly sub-nuclear detail.

The synthesis of two new luminescent dinuclear Ir(III)-Ru(II) complexes containing tetrapyrido[3,2-a:2',3'-c:3'',2''-h:2''',3'''-j]phenazine (tpphz) as the bridging ligand is reported. Unlike many other complexes incorporating cyclometalated Ir(III) moieties, these complexes display good water solubility, allowing the first cell-based study on Ir(III)-Ru(II) bioprobes to be carried out. Photophysical studies indicate that emission from each complex is from a Ru(II) excited state and both complexes display significant in vitro DNA-binding affinities. Cellular studies show that each complex is rapidly internalised by HeLa cells, in which they function as luminescent nuclear DNA-imaging agents for confocal microscopy. Furthermore, the uptake and nuclear targeting properties of the complex incorporating cyclometalating 2-(4-fluorophenyl)pyridine ligands around its Ir(III) centre is enhanced in comparison to the non-fluorinated analogue, indicating that fluorination may provide a route to promote cell uptake of transition-metal bioprobes.

The first transition-metal complex-based two-photon absorbing luminescence lifetime probes for cellular DNA are presented. This allows cell imaging of DNA free from endogenous fluorophores and potentially facilitates deep tissue imaging. In this initial study, ruthenium(II) luminophores are used as phosphorescent lifetime imaging microscopy (PLIM) probes for nuclear DNA in both live and fixed cells. The DNA-bound probes display characteristic emission lifetimes of more than 160 ns, while shorter-lived cytoplasmic emission is also observed. These timescales are orders of magnitude longer than conventional FLIM, leading to previously unattainable levels of sensitivity, and autofluorescence-free imaging.

Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block–induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.

Cytoskeletal rearrangements during mitosis must be co-ordinated with chromosome movements. The ‘chromosomal passenger’ proteins [1], which include the inner centromere protein (INCENP [2]), the Aurora-related serine-threonine protein kinase AIRK2 [3,4] and the unidentified human autoantigen TD-60 [5], have been suggested to integrate mitotic events. These proteins are chromosomal until metaphase but subsequently transfer to the midzone microtubule array and the equatorial cortex during anaphase. Disruption of INCENP function affects both chromosome segregation and completion of cytokinesis [6,7], whereas interference with AIRK2 function primarily affects cytokinesis [3,8]. Here, we report that INCENP is stockpiled in Xenopus eggs in a complex with Xenopus AIRK2 (XAIRK2), and that INCENP and AIRK2 kinase bind one another in vitro. This association was found to be evolutionarily conserved. Sli15p, the binding partner of yeast Aurora kinase Ipl1p, can be recognized as an INCENP family member because of the presence of a conserved carboxy-terminal sequence region, which we term the IN box. This interaction between INCENP and Aurora kinase was found to be biologically relevant. INCENP and AIRK2 colocalized exactly in human cells, and INCENP was required to target AIRK2 correctly to centromeres and the central spindle.

In cell-free extracts of Xenopus eggs that support the assembly of replication-competent nuclei, we found that lamin B(3) specifically associates with four polypeptides (termed SLAPs, soluble lamin associated proteins). Here, one SLAP is identified as the nuclear pore complex protein Nup153, one member of the F/GXFG motif-containing nucleoporins. In vitro translated Nup153 and lamin B(3) co-immunoprecipitate, and lamin B(3) interacts specifically with the C-terminal domain of Nup153. During nuclear envelope assembly, other F/GXFG-containing nucleoporins are incorporated into the nuclear envelope preceding lamina assembly. Incorporation of Nup153 occurs at the same time as lamina assembly. When lamina assembly is prevented using the dominant-negative mutant XlaminB delta 2+, Nup153 does not appear at the nuclear envelope, while other F/GXFG-containing nucleoporins and Nup93 are recruited normally. When the lamina of pre-assembled nuclei is disrupted using the same dominant-negative mutant, the distribution of other nucleoporins is unaffected. However, Nup153 recruitment at the nuclear envelope is lost. Our results indicate that both the recruitment and maintenance of Nup153 at the pore are dependent upon the integrity of the lamina.

In this work, we have used novel mAbs against two proteins of the endoplasmic reticulum and outer nuclear membrane, termed NEP-B78 and p65, in addition to a polyclonal antibody against the inner nuclear membrane protein LBR (lamin B receptor), to study the order and dynamics of NE reassembly in the Xenopus cell-free system. Using these reagents, we demonstrate differences in the timing of recruitment of their cognate membrane proteins to the surface of decondensing chromatin in both the cell-free system and XLK-2 cells. We show unequivocally that, in the cell-free system, two functionally and biochemically distinct vesicle types are necessary for NE assembly. We find that the process of distinct vesicle recruitment to chromatin is an ordered one and that NEP-B78 defines a vesicle population involved in the earliest events of reassembly in this system. Finally, we present evidence that NEP-B78 may be required for the targeting of these vesicles to the surface of decondensing chromatin in this system. The results have important implications for the understanding of the mechanisms of nuclear envelope disassembly and reassembly during mitosis and for the development of systems to identify novel molecules that control these processes.

The characterization and bioactivity of the dinuclear ruthenium(ii) complex [(Ru(DIP)2)2(tpphz)]4+ (DIP = 4,7-diphenyl-1,10-phenanthroline and tpphz = tetrapyrido[3,2-a:2′,3′-c:3′′,2′′-h:2′′′,3′′′-j]phenazine) is reported., The characterization and bioactivity of the dinuclear ruthenium(ii) complex [(Ru(DIP)2)2(tpphz)]4+ (DIP = 4,7-diphenyl-1,10-phenanthroline and tpphz = tetrapyrido[3,2-a:2′,3′-c:3′′,2′′-h:2′′′,3′′′-j]phenazine) is reported. This new complex is found to be luminescent in acetonitrile, where excitation into MLCT (metal-to-ligand charge-transfer) bands in the visible area of the spectrum (λ ex = 450 nm, ε = 45 000 M–1 cm–1) result in red emission (λ em,max = 620 nm, Φ MLCT = 0.017). Aqueous in vitro binding studies indicate that this complex binds to duplex DNA with an affinity of 1.8 × 106 M–1 through a non-classical groove-binding interaction, however, unlike the parent complex [(Ru(phen)2)2(tpphz)]4+ (phen = 1,10-phenanthroline), it also displays an increase in MLCT luminescence on addition of liposomes. Confocal microscopy and TEM studies show that this lipophilic complex targets the endoplasmic reticulum of eukaryotic cells, where it functions as an imaging agent for this organelle, and cytotoxicity studies in human cancer cell lines indicate a comparable potency to the anti-cancer drug cisplatin.

The entry into mitosis is dependent on the activation of mitotic forms of cdc2 kinase. In many cell types, cyclin A-associated kinase activity peaks just prior to that of cyclin B, although the precise role of cyclin A-associated kinase in the entry into mitosis is still unclear. Previous work has suggested that while cyclin B is capable of triggering cyclin destruction in Xenopus cell-free systems, cyclin A-associated kinase is not able to support this function. Here we have expressed a full-length human cyclin A in Escherichia coli and purified the protein to homogeneity by virtue of an N-terminal histidine tag. We have found that when added to Xenopus cell-free extracts free of cyclin B and incapable of protein synthesis, the temporal pattern of cyclin A-associated cdc2 kinase activity showed distinct differences that were dependent on the concentration of cyclin A added. When cyclin A was added to a concentration that generated levels of cdc2 kinase activity capable of inducing nuclear envelope breakdown, the histone H1 kinase activity profile was bi-phasic, consisting of an activation phase followed by an inactivation phase. Inactivation was found to be due to cyclin destruction, which was prevented by mos protein. Cyclin destruction was followed by nuclear reassembly and an additional round of DNA replication, indicating that there is no protein synthesis requirement for DNA replication in this embryonic system. It has been suggested that the evolutionary recruitment of cyclin A into an S phase function may have necessitated the loss of an original mitotic ability to activate the cyclin destruction pathway. The results presented here indicate that cyclin A has not lost the ability to activate its own destruction and that cyclin A-mediated activation of the cyclin destruction pathway permitted destruction of cyclin B1 as well as cyclin A, indicating that there are not distinct cyclin A and cyclin B destruction pathways. Thus the ordered progression of the cell cycle requires the careful titration of cyclin. A concentration in order to avoid activation of the cyclin destruction pathway before sufficient active cyclin B/cdc2 kinase has accumulated.

Polarized growth is a fundamental property of cell growth and development. It requires the delivery of post-Golgi secretory vesicles to the site of polarized growth. This process is mediated by Rab GTPases activated by their guanine exchange factors (GEFs). The human fungal pathogen, Candida albicans, can grow in a budded yeast form or in a highly polarized hyphal form, and thus provides a model to study this phenomenon. During hyphal, but not yeast growth, secretory vesicles accumulate in an apical body called a Spitzenkorper, which acts to focus delivery of the vesicles to the tip. Post-Golgi transport of secretory vesicles is mediated by the Rab GTPase Sec4, activated by its GEF Sec2. Using a combination of deletion mapping, in vitro mutagenesis, an analogue-sensitive allele of Cdc28 and an in vitro kinase assay, we show that localization of Sec2 to the Spitzenkorper and normal hyphal development requires phosphorylation of Serine 584 by the cyclin-dependent kinase Cdc28. Thus, as well as controlling passage through the cell cycle, Cdc28 has an important function in controlling polarized secretion.

In the search for new biological imaging agents, metal coordination compounds able to emit from triplet metal-to-ligand charge transfer (MLCT) states offer many advantages as luminescent probes of DNA structure. However, poor cellular uptake restricts their use in live cells. Here, we present a dinuclear ruthenium(II) polypyridyl system that works as a multifunctional biological imaging agent staining the DNA of eukaryotic and prokaryotic cells for both luminescence and transition electron microscopy. This MLCT 'light switch' complex directly images nuclear DNA of living cells without requiring prior membrane permeabilization. Furthermore, inhibition and transmission electron microscopy studies show this to be via a non-endocytotic, but temperature-dependent, mechanism of cellular uptake in MCF-7 cells, and confocal microscopy reveals multiple emission peaks that function as markers for cellular DNA structure.

The biogenesis of glycogen in skeletal muscle requires a priming mechanism that has recently been elucidated. The first step is catalysed by a protein tyrosine glucosyltransferase and involves the formation of a novel glycosidic linkage, namely the covalent attachment of glucose to a single tyrosine residue (Tyr194) on a priming protein, termed glycogenin. The next stage is the extension of the glucan chain from Tyr194 and involves the sequential addition of up to seven further glucosyl residues. This reaction is brought about autocatalytically by glycogenin itself, which is a Mn2+/Mg(2+)-dependent UDP-Glc-requiring glucosyltransferase. The glucan primer is elongated by glycogen synthase, but only when glycogenin and glycogen synthase are complexed together. Glycogen synthase dissociates from glycogenin during the synthesis of a glycogen molecule, enabling glycogen molecules to reach their maximum theoretical size. Each mature glycogen beta particle in muscle contains one molecule of glycogenin attached covalently, and an average one glycogen synthase catalytic subunit bound non-covalently. As evidence accumulates that a priming protein may be a fundamental property of polysaccharide synthesis in general, the molecular details of mammalian glycogen biogenesis may serve as a useful model for other systems.

Dissociation and association of membranes with chromatin at the beginning and end of mitosis are critical in controlling nuclear dynamics during these stages of the cell cycle. Employing purified membrane and cytosolic fractions from Xenopus eggs, a simple assay was developed for the reversible binding of nuclear membrane vesicles to chromatin. We have shown, using phosphatase and kinase inhibitors, that membrane-chromatin association is regulated by a phosphatase/kinase system. In interphase, the balance in this system favors dephosphorylation, possibly of a membrane receptor, which then mediates chromatin binding. At mitosis the membrane receptor is phosphorylated, causing release of chromatin-bound membrane. Purified MPF kinase does not directly cause membranes to dissociate from chromatin. Rather, binding of membranes to chromatin at mitosis appears to be regulated indirectly by MPF through its action on a phosphatase/kinase system that directly modulates the phosphorylation state of a nuclear membrane component.

About 90% of the glycogenin in skeleta muscle extracts prepared from fed, 24-h starved or alloxan-diabetic rabbits sedimented at 140 000xg with the glycogen/sarcovesicular fraction, from which it was released by glycogenolysis, but not by 1% SDS. Glycogenin in the glycogen/sarcovesicular fraction is therefore bound covalently to glycogen, and not associated (covalently or non-covalently) with the sarcoplasmic reticulum. The same proportion of glycogen synthase was also recovered in the glycogen/sarcovesicle fraction, was solubilised by glycogenolysis, and copurified with glycogenin to yield a heterodimer composed of a 1:1 complex between these proteins. Glycogen synthase and glycogenin are therefore present in equimolar amounts in skeletal muscle and there is an average of one glycogen synthase catalytic subunit associated with each molecule of glycogen in vivo. Glycogenin and glycogen synthase released into the muscle cytosol by degradation of glycogen did not form a complex initially, and only 50% reassociation took place after storage for several hours or overnight dialysis. This suggests that the muscle cytosol may contain a factor(s) which regulates glycogen biogenesis by modulating the association of glycogenin and glycogen synthase. Only glycogen synthase that was complexed to glycogenin was capable of elongating the primer formed by incubation of glycogenin with Mn2+and micromolar concentration of UDP-glucose, demonstrating the critical importance of this complex for glycogen biogenesis.

In metazoans, accurate replication of chromosomes is ensured by the coupling of DNA synthesis to the synthesis of histone proteins. Expression of replication-dependent histone genes is restricted to S-phase by a combination of cell cycle-regulated transcriptional and post-transcriptional control mechanisms and is linked to DNA replication by a poorly understood mechanism involving checkpoint kinases [Su, Gao, Schneider, Helt, Weiss, O'Reilly, Bohmann and Zhao (2004) EMBO J. 23, 1133–1143; Kaygun and Marzluff (2005) Nat. Struct. Mol. Biol. 12, 794–800]. Here we propose a model for the molecular mechanisms that link these two important processes within S-phase, and propose roles for multiple checkpoints in this mechanism.

Somatic cells possess control mechanisms which monitor DNA replication and assure that it is complete before mitosis is initiated. We have been investigating these mechanisms in Xenopus egg extracts. Using in vitro cycling extracts, which spontaneously alternate between interphase and mitosis, we found that the onset of mitosis is inhibited by the presence of unreplicated DNA, demonstrating that the completion of DNA replication and the initiation of mitosis are coupled in these extracts. As in somatic cells, this coupling is sensitive to caffeine and to okadaic acid. In Xenopus extracts unreplicated DNA increases the tyrosine phosphorylation of p34cdc2, thereby maintaining MPF (mitosis-promoting factor) in an inactive state and preventing the onset of mitosis. The block to mitosis in the presence of unreplicated DNA can be reversed by the addition of bacterially expressed cdc25 protein. The extent of MPF activation by cdc25 protein under these conditions depends on the number of nuclei present. We have developed an assay to examine the rate of tyrosine phosphorylation on p34cdc2. It is increased by unreplicated DNA, in a manner consistent with unreplicated DNA up-regulating the kinase that phosphorylates p34cdc2. We have begun to examine how unreplicated DNA generates the signal that inhibits MPF activation by testing the ability of naked single- and double-stranded DNA templates to inhibit mitosis, and by investigating the role of RCC1, a chromatin-associated protein required for the coupling of DNA replication and mitosis.