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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., Using fluorescent reporter protein and immunostaining we revisit Pío del Río‐Hortega’s exquisite drawings of the cytoplasm of CNS myelin. Together, these data highlight an under‐appreciated complexity of the myelinic channel system. We employ 3D reconstructions to illustrate some key features.

Cell migration requires the constant modification of cellular shape by reorganization of the actin cytoskeleton. The pentameric Scar/WAVE regulatory complex (WRC) is the main catalyst of pseudopod and lamellipodium formation. Its actin nucleation activity has been attributed to its ability to combine monomeric actin and Arp2/3 complex through the VCA domain of Scar/WAVE, while other regions of the complex are typically thought to mediate spatial and temporal regulation and have no direct role in actin polymerization.Here we show that the Scar/WAVE with its VCA domain deleted can still induce the formation of morphologically normal actin protrusions. Equivalent results are seen in B16-F1 mouse melanoma cells and Dictyostelium discoideum cells. This actin polymerization occurs independently of the Arp2/3 complex, whose recruitment to the leading edge is greatly reduced by the loss of the VCA domain. We also expressed Scar/WAVE with VCA and polyproline domains both deleted. In Dictyostelium cells, these were only active if WASP (which contains its own proline-rich domain) was available. Similarly, in B16-F1 cells both Abi and WAVE proline-rich domains needed to be deleted before the function of the WRC was lost. Thus we conclude that proline-rich domains play a central role in actin nucleation.Our data demonstrate a new actin nucleation mechanism of the WRC that is independent of its VCA domain and the Arp2/3 complex. We also show that proline-rich domains are more fundamental than has been thought. Together, these findings suggest a new mechanism for WRC action.

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.

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

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 - not Scar/WAVE – is phosphorylated in response to extracellular signalling. 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/WAVE 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/WAVE patches in the cells with unphosphorylatable Abi protrude for longer, altering pseudopod dynamics and cell speed. Cells 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.

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.

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.

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.

Mass-conservative reaction-diffusion systems have recently been proposed as a general framework to describe intracellular pattern formation. These systems have been used to model the conformational switching of proteins as they cycle from an inactive state in the cell cytoplasm, to an active state at the cell membrane. The active state then acts as input to downstream effectors. The paradigm of activation by recruitment to the membrane underpins a range of biological pathways, including G-protein signalling, growth control through Ras and PI 3-kinase, and cell polarity through Rac and Rho; all activate their targets by recruiting them from the cytoplasm to the membrane. Global mass conservation lies at the heart of these models, reflecting the property that the total number of active and inactive forms and total number of targets remains constant. Here we present a conservative arbitrary Lagrangian Eulerian (ALE) finite element method for the approximate solution of systems of bulk-surface reaction-diffusion equations on an evolving two-dimensional domain. Fundamental to the success of the method is the robust generation of bulk and surface meshes. For this purpose, we use a moving mesh partial differential equation (MMPDE) approach. Global conservation of the fully discrete finite element solution is established independently of the ALE velocity field and the time step size. The developed method is applied to model problems with known analytical solutions; these experiments indicate that the method is second-order accurate and globally conservative. The method is further applied to a model of a single cell migrating in the presence of an external chemotactic signal.

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.

Introduction: \ud Cells migrate over long distances during normal embryonic development and in disease, and they navigate through complex, branched paths. Chemotaxis, cell migration steered by gradients of attractive chemicals, is a central regulator of this behavior. Its sensitivity is limited by the responsiveness of attractant receptors. Simple chemotaxis is unable to guide cells over long distances and cannot resolve complexities and branches.\ud \ud Self-generated chemotaxis, a process in which cells create their own local, dynamic gradients by breaking down an attractant in their environment, is different. Gradients can be steeper because they are only made in the vicinity of the cells. They also work over long distances because they are remade locally as the cells migrate. Furthermore, self-generated gradients can follow complex paths because they are constantly generated by interactions between the cells and their environment.\ud Rationale: \ud The migration of cells through complex structures such as developing embryos or vascularized tumors involves both long distances and numerous decisions about which path to follow. Therefore, we examined chemotaxis in artificial complex environments, which are represented here by mazes. We predicted the pathfinding of cells in mazes using computational and mathematical modeling of self-generated gradients and tested the outcomes with real cells in microfluidic mazes.\ud Results: \ud We modeled cells as they navigated through various junctions between a connection to an attractant well and a dead end. We then tested the model’s predictions with cells that typically use long-range navigation: Dictyostelium discoideum cells (which find each other over large distances in the environment) and metastatic cancer cells (which spread around the human body). Both successfully solved a range of mazes, even remarkably complex ones (see figure), and could identify optimum paths. The results confirm that a model of self-generated chemotaxis captures all essential decision-making in these mazes: Cells broke down attractants locally, which were replenished at different, predictable rates by each possible path. The resulting local attractant gradients allowed cells to sense upcoming maze junctions before reaching them, even around corners, and to make correct decisions about paths they had not yet encountered. Attractant breakdown thus allowed cells to create a detailed picture of their surroundings.\ud \ud Real cell behavior closely agreed with the models for both rapidly moving D. discoideum and slower-moving pancreatic cancer cells, implying that the underlying conceptual model is an effective explanation of general cell behavior. In particular, the diffusivity of attractant, which is ignored by simple models, is crucial to cells’ responses. We simulated and then constructed “easy” and “hard” mazes, which appear similar but caused radically different pathfinding by cells. Finally, we designed maze geometries that passively created a temporary peak of attractant flux, deliberately misdirecting cells into a dead end and creating a chemotactic “mirage.”\ud Conclusion: \ud Self-generated chemoattractant gradients allow cells to navigate complex paths with great efficiency. Diffusion and attractant breakdown allow cells to obtain detailed information about their surroundings that could not be provided by simple attractant gradients. Cell migration in vivo cannot be understood without considering the interplay among cells, attractants, and the structure of the local environment. Microfluidic mazes are an excellent experimental system for studying these interactions.

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 Megapixel 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 megapixels.

We report a technique for high-resolution fluorescence lifetime imaging that is based on data-fusion of fluorescence lifetime images at low spatial resolution with fluorescence intensity images at high-resolution, via deep learning, for wide-field, real-time FLIM.

Cell biology and imaging technology have vastly improved over the past decades, enabling scientists to dissect the inner workings of a cell. In addition to technical limits on spatial and temporal resolution, which obscure the picture at the molecular level, the sheer density and complexity of information impede clear understanding. 3D molecular visualisation has therefore blossomed as a way to translate molecular data in a more tangible form. Whilst the molecular machinery involved in cell locomotion has been extensively studied, existing narratives describing how cells generate the forces that drive movement remain unclear. Polymerisation of a protein called actin is clearly essential. The general belief in the cell migration field is that actin polymerisation’s main role is to push the leading edge of the cell forwards, while the rest of the cell follows passively. The cell migration & chemotaxis group at the CRUK Beatson Institute propose an alternative hypothesis, in which actin filaments constitute cables. Motor proteins pull on these cables, causing them to behave like the treads of a tank and drive cell movement. This article describes the development of a 3D animation that uses analogical reasoning to contrast the ‘tank’ hypothesis for cell locomotion with the current dogma.

The picosecond temporal resolution of a SPAD array is computationally extended to the higher spatial resolution of a conventional CMOS camera and is applied to high resolution fluorescence lifetime imaging of cancer cells.

Among the amoebozoan species capable of forming fruiting bodies, the dictyostelid social amoebae stand out since they form true multicellular organisms by means of single cell aggregation. Upon food depletion, cells migrate across gradients of extracellular signals initiated by cells in aggregation centers. The model species that is widely used to study multicellular development of social amoebae, Dictyostelium discoideum, uses cyclic adenosine monophosphate (cAMP) as a chemoattractant to coordinate aggregation. Molecular phylogeny studies suggested that social amoebae evolved in four major groups, of which groups 1 and 2 are paraphyletic to groups 3 and 4. During early development, intercellular communication with cAMP appears to be restricted to group 4 species. Cells of group 1 and 2 taxa do not respond chemotactically to extracellular cAMP and likely use a dipeptide chemoattractant known as glorin ( N-propionyl-γ-L-glutamyl-L-ornithin-δ-lactam-ethylester) to regulate aggregation. Directional migration of glorin-responsive cells requires the periodic breakdown of the chemoattractant. Here, we identified an extracellular enzymatic activity (glorinase) in the glorin-responsive group 2 taxon Polysphondylium pallidum leading to the inactivation of glorin. We determined the inactivation mechanism to proceed via hydrolytic ethyl ester cleavage of the γ-glutamyl moiety of glorin. Synthetic glorinamide, in which the ethyl ester group was substituted by an ethyl amide group, had glorin-like biological activity but was resistant to degradation by glorinase. Our observations pave the way for future investigations toward an ancient eukaryotic chemotaxis system.

Summary Efficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP’s Cdc42 and Rac interacting binding (“CRIB”) motif has been thought to be essential for its activation. However, we show that the CRIB motif’s biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought—Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif., Graphical Abstract, Highlights • WASP exploits its CRIB motif to remove active Rac from the membrane via endocytosis • WASP is recruited to clathrin-coated pits (CCPs) independently of small GTPases • WASP triggers actin polymerization at CCPs independently of small GTPase activation • WASP maintains homeostasis in Dictyostelium by controlling the level of active Rac, Amato et al. have discovered a mechanism that contributes to front-rear polarization in migrating cells. During clathrin-mediated endocytosis, Dictyostelium WASP interacts with active Rac via its CRIB motif. This interaction leads to incorporation of active Rac within endocytic vesicles, which facilitates its removal from inappropriate locations.

Pancreatic ductal adenocarcinoma is one of the most invasive and metastatic cancers and has a dismal 5-year survival rate. We show that N-WASP drives pancreatic cancer metastasis, with roles in both chemotaxis and matrix remodeling. lysophosphatidic acid, a signaling lipid abundant in blood and ascites fluid, is both a mitogen and chemoattractant for cancer cells. Pancreatic cancer cells break lysophosphatidic acid down as they respond to it, setting up a self-generated gradient driving tumor egress. N-WASP-depleted cells do not recognize lysophosphatidic acid gradients, leading to altered RhoA activation, decreased contractility and traction forces, and reduced metastasis. We describe a signaling loop whereby N-WASP and the endocytic adapter SNX18 promote lysophosphatidic acid-induced RhoA-mediated contractility and force generation by controlling lysophosphatidic acid receptor recycling and preventing degradation. This chemotactic loop drives collagen remodeling, tumor invasion, and metastasis and could be an important target against pancreatic cancer spread.

Actin polymerization is essential for cells to migrate, as well as for various cell biological processes such as cytokinesis and vesicle traffic. This brief review describes the mechanisms underlying its different roles and recent advances in our understanding. Actin usually requires “nuclei”—preformed actin filaments—to start polymerizing, but, once initiated, polymerization continues constitutively. The field therefore has a strong focus on nucleators, in particular the Arp2/3 complex and formins. These have different functions, are controlled by contrasting mechanisms, and generate alternate geometries of actin networks. The Arp2/3 complex functions only when activated by nucleation-promoting factors such as WASP, Scar/WAVE, WASH, and WHAMM and when binding to a pre-existing filament. Formins can be individually active but are usually autoinhibited. Each is controlled by different mechanisms and is involved in different biological roles. We also describe the processes leading to actin disassembly and their regulation and conclude with four questions whose answers are important for understanding actin dynamics but are currently unanswered.

Chemotaxis, in which cells steer using chemical gradients, drives fundamental biological processes like embryogenesis, metastasis and immune responses. Self-generated chemotaxis, where cells break down abundant attractants to create gradients, is an important but under-studied aspect of physiological navigation. Here we show that self-generated gradients allow cells to navigate arbitrarily complex paths and, remarkably, make accurate choices about pathways they have not yet encountered. This enables cells to solve microfluidic mazes, even with initially homogeneous environments and distant correct destinations. We combine computational models and experiments to understand how cells anticipate environmental features, and how decision accuracy is determined by path complexity, attractant diffusibility and cell speed. This permits mazes that are easy or hard for cells to resolve, despite similar appearances. Counterintuitively, slowly-diffusing attractants can generate a “mirage”, making cells prefer dead ends over correct paths.In vivoenvironments resemble complex mazes, and only self-generated gradients realistically explain cell behaviour.

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE’s proline-rich domain is polyphosphorylated after the complex is activated. Treatments that stop activation block phosphorylation in bothDictyosteliumand mammalian cells. This implies that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether a protrusion is initiated. Unexpectedly, activation-dependent phosphorylation is not promoted by chemotactic signalling, or by signal-dependent kinases such as ERKs, but is greatly stimulated by cell:substrate adhesion. Scar/WAVE that has been mutated to be either unphosphorylatable or phosphomimetic is activated normally, and rescues the phenotype ofscar−cells, demonstrating that phosphorylation is dispensible for activation and actin regulation. However, pseudopods and patches of Scar/WAVE complex recruitment last substantially longer in unphosphorylatable mutants, altering cell polarisation and the efficiency of migration. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE’s activity, and that phosphorylation acts as a timer, restricting pseudopod lifetime by promoting Scar/WAVE turnover.

Stereotyped behaviors are series of postures that show very little variability between repeats. They have been used to classify the dynamics of individuals, groups and species without reference to the lower-level mechanisms that drive them. Stereotypes are easily identified in animals due to strong constraints on the number, shape, and relative positions of anatomical features, such as limbs, that may be used as landmarks for posture identification. In contrast, the identification of stereotypes in single cells poses a significant challenge as the cell lacks these landmark features, and finding constraints on cell shape is a non-trivial task. Here, we use the maximum caliber variational method to build a minimal model of cell behavior during migration. Without reference to biochemical details, we are able to make behavioral predictions over timescales of minutes using only changes in cell shape over timescales of seconds. We use drug treatment and genetics to demonstrate that maximum caliber descriptors can discriminate between healthy and aberrant migration, thereby showing potential applications for maximum caliber methods in automated disease screening, for example in the identification of behaviors associated with cancer metastasis., 5 figures plus SI

SummaryEfficient motility requires polarised cells, with anterior pseudopods and a retracting rear. This polarisation requires that the pseudopod catalyst Rac is restricted to the front. Here we show that the Arp2/3 complex regulator WASP is important for maintaining front–rear polarity, using a mechanism that limits where active Rac localises. Dictyostelium cells lacking WASP inappropriately activate Rac and SCAR/WAVE at their rears, leading to reduced cell speed. WASP facilitates the internalisation of clathrin-coated pits, and its Rac-binding CRIB motif is considered essential for its localisation and activity. However, WASP mutants with deleted CRIB domains, or harbouring a new mutation that prevents Rac binding, localise normally, recruit Arp2/3 complex, and drive actin polymerisation. Similarly, Rac inhibitors do not block WASP localisation or activation. Despite this, WASP CRIB mutants cannot restore polarisation of active Rac. Thus, WASP’s interaction with Rac regulates Rac activity and cell polarity, but is dispensable for activating actin polymerization.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.