Costello et al. identify ACBD5 and VAPB as key components of a peroxisome–ER tether in mammalian cells. Disruption of this tethering complex leads to reduced peroxisomal membrane expansion and increased peroxisomal movement.

BackgroundClonal microbial populations often harbor rare phenotypic variants that are typically hidden within the majority of the remaining cells, but are crucial for the population’s resilience to external perturbations. Persister and viable but non-culturable (VBNC) cells are two important clonal bacterial subpopulations that can survive antibiotic treatment. Both persister and VBNC cells pose a serious threat to human health. However, unlike persister cells, which quickly resume growth following drug removal, VBNC cells can remain non-growing for prolonged periods of time, thus eluding detection via traditional microbiological assays. Therefore, understanding the molecular mechanisms underlying the formation of VBNC cells requires the characterization of the clonal population with single-cell resolution. A combination of microfluidics, time-lapse microscopy, and fluorescent reporter strains offers the perfect platform for investigating individual cells while manipulating their environment.MethodsHere, we report a novel single-cell approach to investigate VBNC cells. We perform drug treatment, bacterial culturing, and live/dead staining in series by using transcriptional reporter strains and novel adaptations to the mother machine technology. Since we track each cell throughout the experiment, we are able to quantify the size, morphology and fluorescence that each VBNC cell displayed before, during and after drug treatment.ResultsWe show that VBNC cells are not dead or dying cells but share similar phenotypic features with persister cells, suggesting a link between these two subpopulations, at least in the Escherichia coli strain under investigation. We strengthen this link by demonstrating that, before drug treatment, both persister and VBNC cells can be distinguished from the remainder of the population by their lower fluorescence when using a reporter strain for tnaC, encoding the leader peptide of the tnaCAB operon responsible for tryptophan metabolism.ConclusionOur data demonstrates the suitability of our approach for studying the physiology of non-growing cells in response to external perturbations. Our approach will allow the identification of novel biomarkers for the isolation of VBNC and persister cells and will open new opportunities to map the detailed biochemical makeup of these clonal subpopulations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-017-0465-4) contains supplementary material, which is available to authorized users.

Peroxisomes are dynamic organelles which fulfil essential roles in lipid and ROS metabolism. Peroxisome movement and positioning allows interaction with other organelles and is crucial for their cellular function. In mammalian cells, such movement is microtubule‐dependent and mediated by kinesin and dynein motors. The mechanisms of motor recruitment to peroxisomes are largely unknown, as well as the role this plays in peroxisome membrane dynamics and proliferation. Here, using a combination of microscopy, live‐cell imaging analysis and mathematical modelling, we identify a role for Mitochondrial Rho GTPase 1 (MIRO1) as an adaptor for microtubule‐dependent peroxisome motility in mammalian cells. We show that MIRO1 is targeted to peroxisomes and alters their distribution and motility. Using a peroxisome‐targeted MIRO1 fusion protein, we demonstrate that MIRO1‐mediated pulling forces contribute to peroxisome membrane elongation and proliferation in cellular models of peroxisome disease. Our findings reveal a molecular mechanism for establishing peroxisome‐motor protein associations in mammalian cells and provide new insights into peroxisome membrane dynamics in health and disease.

Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes.

SummaryThe mitotic spindle is defined by its organized, bipolar mass of microtubules, which drive chromosome alignment and segregation. Although different cells have been shown to use different molecular pathways to generate the microtubules required for spindle formation, how these pathways are coordinated within a single cell is poorly understood. We have tested the limits within which the Drosophila embryonic spindle forms, disrupting the inherent temporal control that overlays mitotic microtubule generation, interfering with the molecular mechanism that generates new microtubules from preexisting ones, and disrupting the spatial relationship between microtubule nucleation and the usually dominant centrosome. Our work uncovers the possible routes to spindle formation in embryos and establishes the central role of Augmin in all microtubule-generating pathways. It also demonstrates that the contributions of each pathway to spindle formation are integrated, highlighting the remarkable flexibility with which cells can respond to perturbations that limit their capacity to generate microtubules.