Fluorescent labelling is used to show that in E. coli, outer membrane protein (OMP) turnover is passive and binary in nature, and OMPs cluster to form islands in which diffusion of individual proteins is restricted owing to lateral interactions with other OMPs; new OMPs are inserted mostly at mid-cell, meaning that old OMP islands are displaced to the poles of growing cells. Gram-negative bacteria must ensure that they have the appropriate complement of outer membrane proteins (OMPs) to cope with fluctuating environmental conditions. However, the mechanism by which OMP turnover occurs has been unclear. Here Colin Kleanthous and colleagues use fluorescent labelling to show that in Escherichia coli this process is passive and binary in nature. OMPs form islands in which diffusion of individual proteins is restricted by lateral interactions with other OMPs. New proteins are inserted mostly at mid-cell, so that older OMP islands are displaced to, and preserved at, the poles of growing cells. Gram-negative bacteria inhabit a broad range of ecological niches. For Escherichia coli, this includes river water as well as humans and animals, where it can be both a commensal and a pathogen1,2,3. Intricate regulatory mechanisms ensure that bacteria have the right complement of β-barrel outer membrane proteins (OMPs) to enable adaptation to a particular habitat4,5. Yet no mechanism is known for replacing OMPs in the outer membrane, an issue that is further confounded by the lack of an energy source and the high stability6 and abundance of OMPs5. Here we uncover the process underpinning OMP turnover in E. coli and show it to be passive and binary in nature, in which old OMPs are displaced to the poles of growing cells as new OMPs take their place. Using fluorescent colicins as OMP-specific probes, in combination with ensemble and single-molecule fluorescence microscopy in vivo and in vitro, as well as molecular dynamics simulations, we established the mechanism for binary OMP partitioning. OMPs clustered to form ∼0.5-μm diameter islands, where their diffusion is restricted by promiscuous interactions with other OMPs. OMP islands were distributed throughout the cell and contained the Bam complex, which catalyses the insertion of OMPs in the outer membrane7,8. However, OMP biogenesis occurred as a gradient that was highest at mid-cell but largely absent at cell poles. The cumulative effect is to push old OMP islands towards the poles of growing cells, leading to a binary distribution when cells divide. Hence, the outer membrane of a Gram-negative bacterium is a spatially and temporally organized structure, and this organization lies at the heart of how OMPs are turned over in the membrane.

Light-responsive biomaterials can be used for the delivery of therapeutic drugs and nucleic acids, where the tunable/precise delivery of payload highlights the potential of such biomaterials for treating a variety of conditions. The translucency of eyes and advances of laser technology in ophthalmology make light-responsive delivery of drugs feasible. Importantly, light can be applied in a non-invasive fashion; therefore, light-triggered drug delivery systems have great potential for clinical impact. This review will examine various types of light-responsive polymers and the chemistry that underpins their application as ophthalmic drug delivery systems. Graphical abstract

Cellular homeostasis requires adaption to environmental stress. In response to various environmental and genotoxic stresses, all cells produce dinucleoside polyphosphates (Np_nNs), the best studied of which is diadenosine tetraphosphate (Ap₄A). Despite intensive investigation, the precise biological roles of these molecules have remained elusive. However, recent studies have elucidated distinct and specific signaling mechanisms for these nucleotides in prokaryotes and eukaryotes. This review summarizes these key discoveries and describes the mechanisms of Ap₄A and Ap₄N synthesis, the mediators of the cellular responses to increased intracellular levels of these molecules and the hydrolytic mechanisms required to maintain low levels in the absence of stress. The intracellular responses to dinucleotide accumulation are evaluated in the context of the “friend” and “foe” scenarios. The “friend (or alarmone) hypothesis” suggests that Ap_nN act as bona fide secondary messengers mediating responses to stress. In contrast, the “foe” hypothesis proposes that Ap_nN and other Np_nN are produced by non-canonical enzymatic synthesis as a result of physiological and environmental stress in critically damaged cells but do not actively regulate mitigating signaling pathways. In addition, we will discuss potential target proteins, and critically assess new evidence supporting roles for Ap_nN in the regulation of gene expression, immune responses, DNA replication and DNA repair. The recent advances in the field have generated great interest as they have for the first time revealed some of the molecular mechanisms that mediate cellular responses to Ap_nN. Finally, areas for future research are discussed with possible but unproven roles for intracellular Ap_nN to encourage further research into the signaling networks that are regulated by these nucleotides.

Initiation of mammalian DNA replication can be reconstituted from isolated G1-phase nuclei and cell extracts, supplemented with cyclin-dependent protein kinases (CDKs). Under these conditions, cyclin E supports pre-replication complex assembly, whereas cyclin-A-associated kinase acts later to terminate assembly and activate DNA replication. The mechanism by which these events are coordinated is unknown. Here, we show that the replication factor Ciz1 interacts with cyclins E and A sequentially through distinct cyclin-binding motifs. Cyclin A displaces cyclin E from Ciz1 in a manner that is dependent on functional domains that are essential for its role in DNA replication. Furthermore, in cell-free assays, recombinant cyclin-A–CDK2 complexes and recombinant Ciz1 cooperate to promote initiation of DNA replication in late G1-phase nuclei. In addition, Ciz1 supports immobilization of cyclin A in isolated nuclei and depletion of Ciz1 by RNAi impairs immobilization, suggesting that Ciz1 promotes initiation by helping to target the kinase to a specific subnuclear compartment. We propose that Ciz1 acts to coordinate the functions of cyclins E and A in the nucleus, by delivering cyclin-A-associated kinase to sites that are specified by cyclin E, helping to ensure that they execute their functions in the same place and in the correct order.

Regulation of gene expression is one of several roles proposed for the stress-induced nucleotide diadenosine tetraphosphate (Ap₄A). We have examined this directly by a comparative RNA-Seq analysis of KBM-7 chronic myelogenous leukemia cells and KBM-7 cells in which the NUDT2 Ap₄A hydrolase gene had been disrupted (NuKO cells), causing a 175-fold increase in intracellular Ap₄A. 6,288 differentially expressed genes were identified with P < 0.05. Of these, 980 were up-regulated and 705 down-regulated in NuKO cells with a fold-change ≥ 2. Ingenuity^® Pathway Analysis (IPA^®) was used to assign these genes to known canonical pathways and functional networks. Pathways associated with interferon responses, pattern recognition receptors and inflammation scored highly in the down-regulated set of genes while functions associated with MHC class II antigens were prominent among the up-regulated genes, which otherwise showed little organization into major functional gene sets. Tryptophan catabolism was also strongly down-regulated as were numerous genes known to be involved in tumor promotion in other systems, with roles in the epithelial-mesenchymal transition, proliferation, invasion and metastasis. Conversely, some pro-apoptotic genes were up-regulated. Major upstream factors predicted by IPA^® for gene down-regulation included NFκB, STAT1/2, IRF3/4 and SP1 but no major factors controlling gene up-regulation were identified. Potential mechanisms for gene regulation mediated by Ap₄A and/or NUDT2 disruption include binding of Ap₄A to the HINT1 co-repressor, autocrine activation of purinoceptors by Ap₄A, chromatin remodeling, effects of NUDT2 loss on transcript stability, and inhibition of ATP-dependent regulatory factors such as protein kinases by Ap₄A. Existing evidence favors the last of these as the most probable mechanism. Regardless, our results suggest that the NUDT2 protein could be a novel cancer chemotherapeutic target, with its inhibition potentially exerting strong anti-tumor effects via multiple pathways involving metastasis, invasion, immunosuppression and apoptosis.