Cellular quiescence is a dormant but reversible cellular state in which cell-cycle entry and proliferation are prevented. Recent studies both in vivo and in vitro demonstrate that quiescence is actively maintained through synergistic interactions between intrinsic and extrinsic signals. Subtypes of adult mammalian stem cells can be maintained in this poised, quiescent state, and subsequently reactivated upon tissue injury to restore homeostasis. However, quiescence can become deregulated in pathological settings. In this review, we discuss the recent advances uncovering intracellular signaling pathways, transcriptional changes, and extracellular cues within the stem cell niche that control induction and exit from quiescence in tissue stem cells. We discuss the implications of quiescence as well as the pharmacological and genetic approaches that are being explored to either induce or prevent quiescence as a therapeutic strategy.

Number-average molecular weight; b) Weight-average molecular weight; c) Molecular weight of highest peak; d) Dispersion index.

Members of the Flaviviridae family constitute a severe risk to human health. Whilst effective drugs have been developed against the hepacivirus HCV, no antiviral therapy is currently available for any other viruses, including the flaviviruses dengue (DENV), West Nile and Zika viruses. The RNA-dependent RNA polymerase (RdRp) is responsible for viral replication and represents an excellent therapeutic target with no homologue found in mammals. The identification of compounds targeting the RdRp of other flaviviruses is an active area of research. One of the main factors hampering further developments in the field is the difficulty in obtaining high-quality crystal information that could aid a structure-based drug discovery approach. To address this, we have developed a convenient and economical 96-well screening platform. We validated the screen by successfully obtaining crystals of both native DENV serotype 2 and 3 RdRps under several conditions included in the screen. In addition, we have obtained crystal structures of RdRp3 in complex with a previously identified fragment using both soaking and co-crystallization techniques. This work will streamline and accelerate the generation of crystal structures of viral RdRps and provide the community with a valuable tool to aid the development of structure-based antiviral design.

Dysregulated neuronal excitability is a hallmark of amyotrophic lateral sclerosis (ALS). We sought to investigate how functional changes to the axon initial segment (AIS), the site of action potential generation, could impact neuronal excitability in a human iPSC model of ALS. We found that early (6-week) ALS-related TDP-43G298S motor neurons showed an increase in the length of the AIS, relative to CRISPR-corrected controls. This was linked to neuronal hyperexcitability and increased spontaneous contractions of hiPSC-myofibers in compartmentalised neuromuscular co-cultures. In contrast late (10-week) TDP-43G298S motor neurons showed reduced AIS length and hypoexcitability. At a molecular level aberrant expression of the AIS master scaffolding protein Ankyrin-G, and the AIS-specific voltage-gated ion channels SCN1A (Nav1.1) and SCN8A (Nav1.6) mirrored these dynamic changes in excitability. Finally, at all stages, TDP-43G298S motor neurons showed compromised activity-dependent plasticity of the AIS, further contributing to abnormal excitability. Our results point toward the AIS as an important subcellular target driving changes to neuronal excitability in ALS.

To coordinate cellular physiology, eukaryotic cells rely on the inter-organelle transfer of molecules at specialized organelle-organelle contact sites. Endoplasmic reticulum-mitochondria contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signaling molecules, lipids, and metabolites. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation, a clear understanding of their nanoscale structure and regulation is still lacking. Here, we combine 3D electron microscopy with high-speed molecular tracking of a model organelle tether, VAPB, to map the structure and diffusion landscape of ERMCSs. From EM reconstructions, we identified subdomains within the contact site where ER membranes dramatically deform to match local mitochondrial curvature. In parallel live cell experiments, we observed that the VAPB tethers that mediate this interface were not immobile, but rather highly dynamic, entering and leaving the site in seconds. These subdomains enlarged during nutrient stress, indicating ERMCSs can readily remodel under different physiological conditions. An ALS-associated mutation in VAPB altered the normal fluidity of contact sites, likely perturbing effective communication across the contact site and preventing remodeling. These results establish high speed single molecule imaging as a new tool for mapping the structure of contact site interfaces and suggest that the diffusion landscape of VAPB is a crucial component of ERMCS homeostasis.

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V3 spinal interneurons are a key element of the spinal circuits, which control motor function. However, to date, there are no effective ways of deriving a pure V3 population from human pluripotent stem cells. Here, we report a method for differentiation and isolation of spinal V3 interneurons, combining extrinsic factor-mediated differentiation and magnetic activated cell sorting. We found that differentiation of V3 progenitors can be enhanced with a higher concentration of Sonic Hedgehog agonist, as well as culturing cells in 3D format. To enable V3 progenitor purification from mixed differentiation cultures, we developed a transgene reporter, with a part of the regulatory region of V3-specific gene Nkx2-2 driving the expression of a membrane marker CD14. We found that in human cells, NKX2-2 initially exhibited co-labelling with motor neuron progenitor marker, but V3 specificity emerged as the differentiation culture progressed. At these later differentiation timepoints, we were able to enrich V3 progenitors labelled with CD14 to ~ 95% purity, and mature them to postmitotic V3 interneurons. This purification tool for V3 interneurons will be useful for in vitro disease modeling, studies of normal human neural development and potential cell therapies for disorders of the spinal cord.