A Reliable System for Quantitative G-Protein Activation Imaging in Cancer Cells.

Fluorescence resonance energy transfer (FRET) biosensors have proven to be an indispensable tool in cell biology and, more specifically, in the study of G-protein signalling. The best method of measuring the activation status or FRET state of a biosensor is often fluorescence lifetime imaging microscopy (FLIM), as it does away with many disadvantages inherent to fluorescence intensity-based methods and is easily quantitated. Despite the significant potential, there is a lack of reliable FLIM-FRET biosensors, and the data processing and analysis workflows reported previously face reproducibility challenges. Here, we established a system in live primary mouse pancreatic ductal adenocarcinoma cells, where we can detect the activation of an mNeonGreen-Gαi3-mCherry-Gγ2 biosensor through the lysophosphatidic acid receptor (LPAR) with 2-photon time-correlated single-photon counting (TCSPC) FLIM. This combination gave a superior signal to the commonly used mTurquoise2-mVenus G-protein biosensor. This system has potential as a platform for drug screening, or to answer basic cell biology questions in the field of G-protein signalling.

Single-sample image-fusion upsampling of fluorescence lifetime images.

Fluorescence lifetime imaging microscopy (FLIM) provides detailed information about molecular interactions and biological processes. A major bottleneck for FLIM is image resolution at high acquisition speeds due to the engineering and signal-processing limitations of time-resolved imaging technology. Here, we present single-sample image-fusion upsampling, a data-fusion approach to computational FLIM super-resolution that combines measurements from a low-resolution time-resolved detector (that measures photon arrival time) and a high-resolution camera (that measures intensity only). To solve this otherwise ill-posed inverse retrieval problem, we introduce statistically informed priors that encode local and global correlations between the two "single-sample" measurements. This bypasses the risk of out-of-distribution hallucination as in traditional data-driven approaches and delivers enhanced images compared, for example, to standard bilinear interpolation. The general approach laid out by single-sample image-fusion upsampling can be applied to other image super-resolution problems where two different datasets are available.

Sinking while you swim: A dual role for CCR7 in leukocyte migration.

The CCR7 receptor allows dendritic cells to sense the chemokine CCL19. It also depletes CCL19 from the environment by endocytosis, leading to local gradients that can steer cells accurately and robustly through tissues, even over long distances (see related Research Article by Alanko et al.).

Receptors, enzymes and self-attraction as autocrine generators and amplifiers of chemotaxis and cell steering.

Cells create their own steering cues, or modify cues from their outside, for a number of reasons. These include generating optimal, legible directional information; probing their environments for information to help decide an optimal route; symmetry breaking; generating new patterns and complexity; and bringing together collectives such as neutrophil swarms. Recent advances include more mechanisms of self-steering, in particular by using cell-generated mechanical cues, and gradients of respired oxygen. An increasing number of cell types are being found to use self-steering, in particular immune cells responding to chemokines and mesodermal cells during gastrulation. Finally, receptor modification has emerged as an important limit on the range of neutrophil swarming, allowing cells to monitor other areas as well as coming together. Self-steering is thus emerging as a dominant feature of cell motility.

Competition between chemoattractants causes unexpected complexity and can explain negative chemotaxis.

Negative chemotaxis, where eukaryotic cells migrate away from repellents, is important throughout biology, for example, in nervous system patterning and resolution of inflammation. However, the mechanisms by which molecules repel migrating cells are unknown. Here, we use predictive modeling and experiments with Dictyostelium cells to show that competition between different ligands that bind to the same receptor leads to effective chemorepulsion. 8-CPT-cAMP, widely described as a simple chemorepellent, is inactive on its own and only repels cells when it acts in combination with the attractant cAMP. If cells degrade either competing ligand, the pattern of migration becomes more complex; cells may be repelled in one part of a gradient but attracted elsewhere, leading to populations moving in different directions in the same assay or converging in an arbitrary place. More counterintuitively still, two chemicals that normally attract cells can become repellent when combined. Computational models of chemotaxis are now accurate enough to predict phenomena that have not been anticipated by experiments. We have used them to identify new mechanisms that drive reverse chemotaxis, which we have confirmed through experiments with real cells. These findings are important whenever multiple ligands compete for the same receptors.

AKT and SGK kinases regulate cell migration by altering Scar/WAVE complex activation and Arp2/3 complex recruitment.

Cell polarity and cell migration both depend on pseudopodia and lamellipodia formation. These are regulated by coordinated signaling acting through G-protein coupled receptors and kinases such as PKB/AKT and SGK, as well as the actin cytoskeletal machinery. Here we show that both Dictyostelium PKB and SGK kinases (encoded by pkbA and pkgB) are dispensable for chemotaxis towards folate. However, both are involved in the regulation of pseudopod formation and thus cell motility. Cells lacking pkbA and pkgB showed a substantial drop in cell speed. Actin polymerization is perturbed in pkbA- and reduced in pkgB- and pkbA-/pkgB- mutants. The Scar/WAVE complex, key catalyst of pseudopod formation, is recruited normally to the fronts of all mutant cells (pkbA-, pkgB- and pkbA-/pkgB-), but is unexpectedly unable to recruit the Arp2/3 complex in cells lacking SGK. Consequently, loss of SGK causes a near-complete loss of normal actin pseudopodia, though this can be rescued by overexpression of PKB. Hence both PKB and SGK are required for correct assembly of F-actin and recruitment of the Arp2/3 complex by the Scar/WAVE complex during pseudopodia formation.

Steering yourself by the bootstraps: how cells create their own gradients for chemotaxis.

Chemotaxis, where cell movement is steered by chemical gradients, is a widespread and essential way of organising cell behaviour. But where do the instructions come from - who makes gradients, and how are they controlled? We discuss the emerging concept that chemotactic cells often create attractant gradients at the same time as responding to them. This self-guidance is more robust, works across greater distances, and is more informative about the local environment than passive responses. Several mechanisms can establish autonomous gradients. Best known are self-generated gradients, in which the cells degrade a widespread attractant, but cells also produce repellents and 'relay' by secreting fresh attractant after stimulation. Understanding how cells make and interpret their own chemoattractant gradients is fundamental to understanding the spatial patterns seen in all organisms.

Under-Agarose Chemotaxis and Migration Assays for Dictyostelium.

Chemotaxis-directional cell movement steered by chemical gradients-involved in many biological processes including embryonic morphogenesis and immune cell function. Eukaryotic cells, in response to external gradients of attractants, use conserved mechanisms to achieve chemotaxis by regulating the actin cytoskeleton at their fronts and myosin II at their rears. Dictyostelium discoideum, an amoeba that is widely used to study chemotaxis, uses chemotaxis to move up gradients of folate to identify and locate its bacterial prey. Similarly, when starved, Dictyostelium cells synthesize and secrete cyclic AMP (cAMP) while simultaneously expressing cAMP receptors. This allows them to chemotax toward their neighbors and aggregate together. The chemotactic behavior of cells can be studied using several techniques. One such, under-agarose chemotaxis, is a robust, easy, and inexpensive assay that allows direct quantification of chemotactic parameters such as speed and directionality. With the use of high-resolution imaging, for example confocal microscopy, detailed examination of the distribution of actin and membrane proteins in migrating wild type and mutant cells can be performed. In this chapter, we describe simple and optimized methods for studying folate and cAMP chemotaxis in Dictyostelium cells under agarose.

Extracellular Signalling Modulates Scar/WAVE Complex Activity through Abi Phosphorylation.

The lamellipodia and pseudopodia of migrating cells are produced and maintained by the Scar/WAVE complex. Thus, actin-based cell migration is largely controlled through regulation of Scar/WAVE. Here, we report that the Abi subunit-but not Scar-is phosphorylated in response to extracellular signalling in Dictyostelium cells. Like Scar, Abi is phosphorylated after the complex has been activated, implying that Abi phosphorylation modulates pseudopodia, rather than causing new ones to be made. Consistent with this, Scar complex mutants that cannot bind Rac are also not phosphorylated. Several environmental cues also affect Abi phosphorylation-cell-substrate adhesion promotes it and increased extracellular osmolarity diminishes it. Both unphosphorylatable and phosphomimetic Abi efficiently rescue the chemotaxis of Abi KO cells and pseudopodia formation, confirming that Abi phosphorylation is not required for activation or inactivation of the Scar/WAVE complex. However, pseudopodia and Scar patches in the cells with unphosphorylatable Abi protrude for longer, altering pseudopod dynamics and cell speed. Dictyostelium, in which Scar and Abi are both unphosphorylatable, can still form pseudopods, but migrate substantially faster. We conclude that extracellular signals and environmental responses modulate cell migration by tuning the behaviour of the Scar/WAVE complex after it has been activated.

Moving the Research Forward: The Best of British Biology Using the Tractable Model System Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum provides an excellent model for research across a broad range of disciplines within biology. The organism diverged from the plant, yeast, fungi and animal kingdoms around 1 billion years ago but retains common aspects found in these kingdoms. Dictyostelium has a low level of genetic complexity and provides a range of molecular, cellular, biochemical and developmental biology experimental techniques, enabling multidisciplinary studies to be carried out in a wide range of areas, leading to research breakthroughs. Numerous laboratories within the United Kingdom employ Dictyostelium as their core research model. This review introduces Dictyostelium and then highlights research from several leading British research laboratories, covering their distinct areas of research, the benefits of using the model, and the breakthroughs that have arisen due to the use of Dictyostelium as a tractable model system.

Río-Hortega's drawings revisited with fluorescent protein defines a cytoplasm-filled channel system of CNS myelin.

A century ago this year, Pío del Río-Hortega (1921) coined the term 'oligodendroglia' for the 'interfascicular glia' with very few processes, launching an extensive discovery effort on his new cell type. One hundred years later, we review his original contributions to our understanding of the system of cytoplasmic channels within myelin in the context of what we observe today using light and electron microscopy of genetically encoded fluorescent reporters and immunostaining. We use the term myelinic channel system to describe the cytoplasm-delimited spaces associated with myelin; being the paranodal loops, inner and outer tongues, cytoplasm-filled spaces through compact myelin and further complex motifs associated to the sheath. Using a central nervous system myelinating cell culture model that contains all major neural cell types and produces compact myelin, we find that td-tomato fluorescent protein delineates the myelinic channel system in a manner reminiscent of the drawings of adult white matter by Río-Hortega, despite that he questioned whether some cytoplasmic figures he observed represented artefact. Together, these data lead us to propose a slightly revised model of the 'unrolled' sheath. Further, we show that the myelinic channel system, while relatively stable, can undergo subtle dynamic shape changes over days. Importantly, we capture an under-appreciated complexity of the myelinic channel system in mature myelin sheaths.

Actin in 2021.

Robert Insall introduces the cytoskeleton special issue and summarises some recent changes in our view of actin function and regulation.

Leep1 interacts with PIP3 and the Scar/WAVE complex to regulate cell migration and macropinocytosis.

Polarity is essential for diverse functions in many cell types. Establishing polarity requires targeting a network of specific signaling and cytoskeleton molecules to different subregions of the cell, yet the full complement of polarity regulators and how their activities are integrated over space and time to form morphologically and functionally distinct domains remain to be uncovered. Here, by using the model system Dictyostelium and exploiting the characteristic chemoattractant-stimulated translocation of polarly distributed molecules, we developed a proteomic screening approach, through which we identified a leucine-rich repeat domain-containing protein we named Leep1 as a novel polarity regulator. We combined imaging, biochemical, and phenotypic analyses to demonstrate that Leep1 localizes selectively at the leading edge of cells by binding to PIP3, where it modulates pseudopod and macropinocytic cup dynamics by negatively regulating the Scar/WAVE complex. The spatiotemporal coordination of PIP3 signaling, Leep1, and the Scar/WAVE complex provides a cellular mechanism for organizing protrusive structures at the leading edge.

Structural Basis of CYRI-B Direct Competition with Scar/WAVE Complex for Rac1.

Rac1 is a major regulator of actin dynamics, with GTP-bound Rac1 promoting actin assembly via the Scar/WAVE complex. CYRI competes with Scar/WAVE for interaction with Rac1 in a feedback loop regulating actin dynamics. Here, we reveal the nature of the CYRI-Rac1 interaction, through crystal structures of CYRI-B lacking the N-terminal helix (CYRI-BΔN) and the CYRI-BΔN:Rac1Q61L complex, providing the molecular basis for CYRI-B regulation of the Scar/WAVE complex. We reveal CYRI-B as having two subdomains - an N-terminal Rac1 binding subdomain with a unique Rac1-effector interface and a C-terminal Ratchet subdomain that undergoes conformational changes induced by Rac1 binding. Finally, we show that the CYRI protein family, CYRI-A and CYRI-B can produce an autoinhibited hetero- or homodimers, adding an additional layer of regulation to Rac1 signaling.

Fluorescence lifetime imaging with a megapixel SPAD camera and neural network lifetime estimation.

Fluorescence lifetime imaging microscopy (FLIM) is a key technology that provides direct insight into cell metabolism, cell dynamics and protein activity. However, determining the lifetimes of different fluorescent proteins requires the detection of a relatively large number of photons, hence slowing down total acquisition times. Moreover, there are many cases, for example in studies of cell collectives, where wide-field imaging is desired. We report scan-less wide-field FLIM based on a 0.5 MP resolution, time-gated Single Photon Avalanche Diode (SPAD) camera, with acquisition rates up to 1 Hz. Fluorescence lifetime estimation is performed via a pre-trained artificial neural network with 1000-fold improvement in processing times compared to standard least squares fitting techniques. We utilised our system to image HT1080-human fibrosarcoma cell line as well as Convallaria. The results show promise for real-time FLIM and a viable route towards multi-megapixel fluorescence lifetime images, with a proof-of-principle mosaic image shown with 3.6 MP.

Seeing around corners: Cells solve mazes and respond at a distance using attractant breakdown.

During development and metastasis, cells migrate large distances through complex environments. Migration is often guided by chemotaxis, but simple chemoattractant gradients between a source and sink cannot direct cells over such ranges. We describe how self-generated gradients, created by cells locally degrading attractant, allow single cells to navigate long, tortuous paths and make accurate choices between live channels and dead ends. This allows cells to solve complex mazes efficiently. Cells' accuracy at finding live channels was determined by attractant diffusivity, cell speed, and path complexity. Manipulating these parameters directed cells in mathematically predictable ways; specific combinations can even actively misdirect them. We propose that the length and complexity of many long-range migratory processes, including inflammation and germ cell migration, means that self-generated gradients are needed for successful navigation.

Melanoblasts Populate the Mouse Choroid Earlier in Development Than Previously Described.

Purpose: Human choroidal melanocytes become evident in the last trimester of development, but very little is known about them. To better understand normal and diseased choroidal melanocyte biology we examined their precursors, melanoblasts (MB), in mouse eyes during development, particularly their relation to the developing vasculature and immune cells. Methods: Naïve B6(Cg)-Tyrc-2J/J albino mice were used between embryonic (E) day 15.5 and postnatal (P) day 8, with adult controls. Whole eyes, posterior segments, or dissected choroidal wholemounts were stained with antibodies against tyrosinase-related protein 2, ionized calcium binding adaptor molecule-1 or isolectin B4, and examined by confocal microscopy. Immunoreactive cell numbers in the choroid were quantified with Imaris. One-way ANOVA with Tukey's post hoc test assessed statistical significance. Results: Small numbers of MB were present in the presumptive choroid at E15.5 and E18.5. The density significantly increased between E18.5 (381.4 ± 45.8 cells/mm2) and P0 (695.2 ± 87.1 cells/mm2; P = 0.032). In postnatal eyes MB increased in density and formed multiple layers beneath the choriocapillaris. MB in the periocular mesenchyme preceded the appearance of vascular structures at E15.5. Myeloid cells (Ionized calcium binding adaptor molecule-1-positive) were also present at high densities from this time, and attained adult-equivalent densities by P8 (556.4 ± 73.6 cells/mm2). Conclusions: We demonstrate that choroidal MB and myeloid cells are both present at very early stages of mouse eye development (E15.5). Although MB and vascularization seemed to be unlinked early in choroidal development, they were closely associated at later stages. MB did not migrate into the choroid in waves, nor did they have a consistent relationship with nerves.