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The mammary epithelium is indispensable for the continued survival of more than 5,000 mammalian species. For some, the volume of milk ejected in a single day exceeds their entire blood volume. Here, we unveil the spatiotemporal properties of physiological signals that orchestrate the ejection of milk from alveolar units and its passage along the mammary ductal network. Using quantitative, multidimensional imaging of mammary cell ensembles from GCaMP6 transgenic mice, we reveal how stimulus evoked Ca2+ oscillations couple to contractions in basal epithelial cells. Moreover, we show that Ca2+-dependent contractions generate the requisite force to physically deform the innermost layer of luminal cells, compelling them to discharge the fluid that they produced and housed. Through the collective action of thousands of these biological positive-displacement pumps, each linked to a contractile ductal network, milk begins its passage toward the dependent neonate, seconds after the command.

Background With recent advances in microscopy, recordings of cell behaviour can result in terabyte-size datasets. The lattice light sheet microscope (LLSM) images cells at high speed and high 3D resolution, accumulating data at 100 frames/second over hours, presenting a major challenge for interrogating these datasets. The surfaces of vertebrate cells can rapidly deform to create projections that interact with the microenvironment. Such surface projections include spike-like filopodia and wave-like ruffles on the surface of macrophages as they engage in immune surveillance. LLSM imaging has provided new insights into the complex surface behaviours of immune cells, including revealing new types of ruffles. However, full use of these data requires systematic and quantitative analysis of thousands of projections over hundreds of time steps, and an effective system for analysis of individual structures at this scale requires efficient and robust methods with minimal user intervention. Results We present LLAMA, a platform to enable systematic analysis of terabyte-scale 4D microscopy datasets. We use a machine learning method for semantic segmentation, followed by a robust and configurable object separation and tracking algorithm, generating detailed object level statistics. Our system is designed to run on high-performance computing to achieve high throughput, with outputs suitable for visualisation and statistical analysis. Advanced visualisation is a key element of LLAMA: we provide a specialised tool which supports interactive quality control, optimisation, and output visualisation processes to complement the processing pipeline. LLAMA is demonstrated in an analysis of macrophage surface projections, in which it is used to i) discriminate ruffles induced by lipopolysaccharide (LPS) and macrophage colony stimulating factor (CSF-1) and ii) determine the autonomy of ruffle morphologies. Conclusions LLAMA provides an effective open source tool for running a cell microscopy analysis pipeline based on semantic segmentation, object analysis and tracking. Detailed numerical and visual outputs enable effective statistical analysis, identifying distinct patterns of increased activity under the two interventions considered in our example analysis. Our system provides the capacity to screen large datasets for specific structural configurations. LLAMA identified distinct features of LPS and CSF-1 induced ruffles and it identified a continuity of behaviour between tent pole ruffling, wave-like ruffling and filopodia deployment.

Toll-like receptor (TLR)-inducible zinc toxicity is a recently described macrophage antimicrobial response used against bacterial pathogens. Here we investigated deployment of this pathway against uropathogenic (UPEC), the major cause of urinary tract infections. Primary human macrophages subjected EC958, a representative strain of the globally disseminated multidrug-resistant UPEC ST131 clone, to zinc stress. We therefore used transposon-directed insertion site sequencing to identify the complete set of UPEC genes conferring protection against zinc toxicity. Surprisingly, zinc-susceptible EC958 mutants were not compromised for intramacrophage survival, whereas corresponding mutants in the nonpathogenic K-12 strain MG1655 displayed significantly reduced intracellular bacterial loads within human macrophages. To investigate whether the intramacrophage zinc stress response of EC958 reflected the response of only a subpopulation of bacteria, we generated and validated reporter systems as highly specific sensors of zinc stress. Using these tools we show that, in contrast to MG1655, the majority of intramacrophage EC958 evades the zinc toxicity response, enabling survival within these cells. In addition, EC958 has a higher tolerance to zinc than MG1655, with this likely being important for survival of the minor subset of UPEC cells exposed to innate immune-mediated zinc stress. Indeed, analysis of zinc stress reporter strains and zinc-sensitive mutants in an intraperitoneal challenge model in mice revealed that EC958 employs both evasion and resistance against zinc toxicity, enabling its dissemination to the liver and spleen. We thus demonstrate that a pathogen of global significance uses multiple mechanisms to effectively subvert innate immune-mediated zinc poisoning for systemic spread.

The formation of new blood vessel networks occurs via angiogenesis during development, tissue repair, and disease. Angiogenesis is regulated by intracellular endothelial signalling pathways, induced downstream of vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). A major challenge in understanding angiogenesis is interpreting how signalling events occur dynamically within endothelial cell populations during sprouting, proliferation, and migration. Extracellular signal-regulated kinase (Erk) is a central downstream effector of Vegf-signalling and reports the signalling that drives angiogenesis. We generated a vascular Erk biosensor transgenic line in zebrafish using a kinase translocation reporter that allows live-imaging of Erk-signalling dynamics. We demonstrate the utility of this line to live-image Erk activity during physiologically relevant angiogenic events. Further, we reveal dynamic and sequential endothelial cell Erk-signalling events following blood vessel wounding. Initial signalling is dependent upon Ca 2+ in the earliest responding endothelial cells, but is independent of Vegfr-signalling and local inflammation. The sustained regenerative response, however, involves a Vegfr-dependent mechanism that initiates concomitantly with the wound inflammatory response. This work reveals a highly dynamic sequence of signalling events in regenerative angiogenesis and validates a new resource for the study of vascular Erk-signalling in real-time.

Nuclear hormone receptors have important roles in the regulation of metabolic and inflammatory pathways. The retinoid-related orphan receptor alpha (Rorα)-deficient staggerer (sg/sg) mice display several phenotypes indicative of aberrant lipid metabolism, including dyslipidemia, and increased susceptibility to atherosclerosis. In this study we demonstrate that macrophages from sg/sg mice have increased ability to accumulate lipids and accordingly exhibit larger lipid droplets (LD). We have previously shown that BMMs from sg/sg mice have significantly decreased expression of cholesterol 25-hydroxylase (Ch25h) mRNA, the enzyme that produces the oxysterol, 25-hydroxycholesterol (25HC), and now confirm this at the protein level. 25HC functions as an inverse agonist for RORα. siRNA knockdown of Ch25h in macrophages up-regulates Vldlr mRNA expression and causes increased accumulation of LDs. Treatment with physiological concentrations of 25HC in sg/sg macrophages restored lipid accumulation back to normal levels. Thus, 25HC and RORα signify a new pathway involved in the regulation of lipid homeostasis in macrophages, potentially via increased uptake of lipid which is suggested by mRNA expression changes in Vldlr and other related genes.

The surface of mammalian cells offers an interface between the cell interior and its surrounding milieu. As part of the innate immune system, macrophages have cell surface features optimised for probing and sampling as they patrol our tissues for pathogens, debris or dead cells. Their highly dynamic and constantly moving cell surface has extensions such as lamellipodia, filopodia and dorsal ruffles that help detect pathogens. Dorsal ruffles give rise to macropinosomes for rapid, high volume non‐selective fluid sampling, receptor internalisation and plasma membrane turnover. Ruffles can also generate phagocytic cups for the receptor‐mediated uptake of pathogens or particles. The membrane lipids, actin cytoskeleton, receptors and signalling proteins that constitute these cell surface domains are discussed. Although the cell surface is designed to counteract pathogens, many bacteria, viruses and other pathogens have evolved to circumvent or hijack these cell structures and their underlying machinery for entry and survival. Nevertheless, these features offer important potential for developing vaccines, drugs and preventative measures to help fight infection.

While apoptosis dismantles the cell to enforce immunological silence, pyroptotic cell death provokes inflammation. Little is known of the structural architecture of cells undergoing pyroptosis, and whether pyroptotic corpses are immunogenic. Here we report that inflammasomes trigger the Gasdermin-D- and calcium-dependent eruption of filopodia from the plasma membrane minutes before pyroptotic cell rupture, to crown the resultant corpse with filopodia. As a rich store of F-actin, pyroptotic filopodia are recognized by dendritic cells through the F-actin receptor, CLEC9A (DNGR1). We propose that cells assemble filopodia before cell rupture to serve as a posthumous mark for a cell that has died by gasdermin-induced pyroptosis, or MLKL-induced necroptosis, for recognition by dendritic cells. This study reveals the spectacular morphology of pyroptosis and identifies a mechanism by which inflammasomes induce pyroptotic cells to construct a de novo alarmin that activates dendritic cells via CLEC9A, which coordinates the transition from innate to adaptive immunity 1 , 2 . Pyroptotic cell death results in inflammation. Here the authors find that F-actin-rich structures formed during macrophage pyroptosis persist after cell death to activate dendritic cells.

Macropinocytosis is a prevalent and essential pathway in macrophages where it contributes to anti-microbial responses and innate immune cell functions. Cell surface ruffles give rise to phagosomes and to macropinosomes as multi-functional compartments that contribute to environmental sampling, pathogen entry, plasma membrane turnover and receptor signalling. Rapid, high resolution, lattice light sheet imaging demonstrates the dynamic nature of macrophage ruffling. Pathogen-mediated activation of surface and endosomal Toll-like receptors (TLRs) in macrophages upregulates macropinocytosis. Here, using multiple forms of imaging and microscopy, we track membrane-associated, fluorescently-tagged Rab8a expressed in live macrophages, using a variety of cell markers to demonstrate Rab8a localization and its enrichment on early macropinosomes. Production of a novel biosensor and its use for quantitative FRET analysis in live cells, pinpoints macropinosomes as the site for TLR-induced activation of Rab8a. We have previously shown that TLR signalling, cytokine outputs and macrophage programming are regulated by the GTPase Rab8a with PI3 Kγ as its effector. Finally, we highlight another effector, the phosphatase OCRL, which is located on macropinosomes and interacts with Rab8a, suggesting that Rab8a may operate on multiple levels to modulate phosphoinositides in macropinosomes. These findings extend our understanding of macropinosomes as regulatory compartments for innate immune function in macrophages. This article is part of the Theo Murphy meeting issue ‘Macropinocytosis’.

Animal and plant colouration presents a striking dimension of phenotypic variation, the study of which has driven general advances in ecology, evolution, and animal behaviour. Quantitative Colour Pattern Analysis (QCPA) is a dynamic framework for analysing colour patterns through the eyes of non-human observers. However, its extensive array of user-defined image processing and analysis tools means image analysis is often time-consuming. This hinders the full use of analytical power provided by QCPA and its application to large datasets. Here, we offer a robust and comprehensive batch script, allowing users to automate many QCPA workflows. We also provide a complimentary set of useful R scripts for downstream data extraction and analysis. The presented batch processing extension will empower users to further utilise the analytical power of QCPA and facilitate the development of customised semi-automated workflows. Such quantitatively scaled workflows are crucial for exploring colour pattern spaces and developing ever-richer frameworks for analysing organismal colouration accounting for visual perception in animals other than humans. These advances will, in turn, facilitate testing hypotheses on the function and evolution of vision and signals at quantitative and qualitative scales, which are otherwise computationally unfeasible.

Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS) to mount innate immune responses. The TLR4-induced release of pro- and anti-inflammatory cytokines generates robust inflammatory responses, which must then be restrained to avoid disease. New mechanisms for the critical regulation of TLR-induced cytokine responses are still emerging. Here we find TLR4 complexes localized in LPS-induced dorsal ruffles on the surface of macrophages. We discover that the small GTPase Rab8a is enriched in these ruffles and recruits phosphatidylinositol 3-kinase (PI3Kγ) as an effector by interacting directly through its Ras-binding domain. Rab8a and PI3Kγ function to regulate Akt signalling generated by surface TLR4. Rab8a and PI3Kγ do not affect TLR4 endocytosis, but instead regulate mammalian target of rapamycin signalling as a mechanism for biasing the cytokine profile to constrain inflammation in innate immunity. Dorsal membrane ruffles in macrophages accompany pathogen contact and appear before macropinocytosis and phagocytosis. Here, the authors show that these structures are the sites of the accumulation of Toll-like receptor 4 complexes involved in the signalling cascade activated by bacterial lipopolysaccharide.