Cytoneme signaling provides essential contributions to mammalian tissue patterning.

During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function.

Load Adaptation of Lamellipodial Actin Networks.

Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load.

Cytoneme-mediated transport of active Wnt5b-Ror2 complexes in zebrafish.

Chemical signalling is the primary means by which cells communicate in the embryo. The underlying principle refers to a group of ligand-producing cells and a group of cells that respond to this signal because they express the appropriate receptors1,2. In the zebrafish embryo, Wnt5b binds to the receptor Ror2 to trigger the Wnt-planar cell polarity (PCP) signalling pathway to regulate tissue polarity and cell migration3,4. However, it remains unclear how this lipophilic ligand is transported from the source cells through the aqueous extracellular space to the target tissue. In this study, we provide evidence that Wnt5b, together with Ror2, is loaded on long protrusions called cytonemes. Our data further suggest that the active Wnt5b-Ror2 complexes form in the producing cell and are handed over from these cytonemes to the receiving cell. Then, the receiving cell has the capacity to initiate Wnt-PCP signalling, irrespective of its functional Ror2 receptor status. On the tissue level, we further show that cytoneme-dependent spreading of active Wnt5b-Ror2 affects convergence and extension in the zebrafish gastrula. We suggest that cytoneme-mediated transfer of ligand-receptor complexes is a vital mechanism for paracrine signalling. This may prompt a reevaluation of the conventional concept of characterizing responsive and non-responsive tissues solely on the basis of the expression of receptors.

Role of PFKFB3-driven glycolysis in vessel sprouting.

Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.

CellTracksColab is a platform that enables compilation, analysis, and exploration of cell tracking data.

In life sciences, tracking objects from movies enables researchers to quantify the behavior of single particles, organelles, bacteria, cells, and even whole animals. While numerous tools now allow automated tracking from video, a significant challenge persists in compiling, analyzing, and exploring the large datasets generated by these approaches. Here, we introduce CellTracksColab, a platform tailored to simplify the exploration and analysis of cell tracking data. CellTracksColab facilitates the compiling and analysis of results across multiple fields of view, conditions, and repeats, ensuring a holistic dataset overview. CellTracksColab also harnesses the power of high-dimensional data reduction and clustering, enabling researchers to identify distinct behavioral patterns and trends without bias. Finally, CellTracksColab also includes specialized analysis modules enabling spatial analyses (clustering, proximity to specific regions of interest). We demonstrate CellTracksColab capabilities with 3 use cases, including T cells and cancer cell migration, as well as filopodia dynamics. CellTracksColab is available for the broader scientific community at https://github.com/CellMigrationLab/CellTracksColab.

Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis.

Lamellipodia are sheet-like, leading edge protrusions in firmly adherent cells that contain Arp2/3-generated dendritic actin networks. Although lamellipodia are widely believed to be critical for directional cell motility, this notion has not been rigorously tested. Using fibroblasts derived from Ink4a/Arf-deficient mice, we generated a stable line depleted of Arp2/3 complex that lacks lamellipodia. This line shows defective random cell motility and relies on a filopodia-based protrusion system. Utilizing a microfluidic gradient generation system, we tested the role of Arp2/3 complex and lamellipodia in directional cell migration. Surprisingly, Arp2/3-depleted cells respond normally to shallow gradients of PDGF, indicating that lamellipodia are not required for fibroblast chemotaxis. Conversely, these cells cannot respond to a surface-bound gradient of extracellular matrix (haptotaxis). Consistent with this finding, cells depleted of Arp2/3 fail to globally align focal adhesions, suggesting that one principle function of lamellipodia is to organize cell-matrix adhesions in a spatially coherent manner.

Membrane tension maintains cell polarity by confining signals to the leading edge during neutrophil migration.

Little is known about how neutrophils and other cells establish a single zone of actin assembly during migration. A widespread assumption is that the leading edge prevents formation of additional fronts by generating long-range diffusible inhibitors or by sequestering essential polarity components. We use morphological perturbations, cell-severing experiments, and computational simulations to show that diffusion-based mechanisms are not sufficient for long-range inhibition by the pseudopod. Instead, plasma membrane tension could serve as a long-range inhibitor in neutrophils. We find that membrane tension doubles during leading-edge protrusion, and increasing tension is sufficient for long-range inhibition of actin assembly and Rac activation. Furthermore, reducing membrane tension causes uniform actin assembly. We suggest that tension, rather than diffusible molecules generated or sequestered at the leading edge, is the dominant source of long-range inhibition that constrains the spread of the existing front and prevents the formation of secondary fronts.

The scaffolding protein AKAP12 regulates mRNA localization and translation.

Regulation of subcellular messenger (m)RNA localization is a fundamental biological mechanism, which adds a spatial dimension to the diverse layers of post-transcriptional control of gene expression. The cellular compartment in which mRNAs are located may define distinct aspects of the encoded proteins, ranging from production rate and complex formation to localized activity. Despite the detailed roles of localized mRNAs that have emerged over the past decades, the identity of factors anchoring mRNAs to subcellular domains remains ill-defined. Here, we used an unbiased method to profile the RNA-bound proteome in migrating endothelial cells (ECs) and discovered that the plasma membrane (PM)-associated scaffolding protein A-kinase anchor protein (AKAP)12 interacts with various mRNAs, including transcripts encoding kinases with Actin remodeling activity. In particular, AKAP12 targets a transcript coding for the kinase Abelson Tyrosine-Protein Kinase 2 (ABL2), which we found to be necessary for adequate filopodia formation and angiogenic sprouting. Moreover, we demonstrate that AKAP12 is necessary for anchoring ABL2 mRNA to the PM and show that in the absence of AKAP12, the translation efficiency of ABL2 mRNA is reduced. Altogether, our work identified a unique post-transcriptional function for AKAP12 and sheds light into mechanisms of spatial control of gene expression.

Filopodial protrusion driven by density-dependent Ena-TOCA-1 interactions.

Filopodia are narrow actin-rich protrusions with important roles in neuronal development where membrane-binding adaptor proteins, such as I-BAR- and F-BAR-domain-containing proteins, have emerged as upstream regulators that link membrane interactions to actin regulators such as formins and proteins of the Ena/VASP family. Both the adaptors and their binding partners are part of diverse and redundant protein networks that can functionally compensate for each other. To explore the significance of the F-BAR domain-containing neuronal membrane adaptor TOCA-1 (also known as FNBP1L) in filopodia we performed a quantitative analysis of TOCA-1 and filopodial dynamics in Xenopus retinal ganglion cells, where Ena/VASP proteins have a native role in filopodial extension. Increasing the density of TOCA-1 enhances Ena/VASP protein binding in vitro, and an accumulation of TOCA-1, as well as its coincidence with Ena, correlates with filopodial protrusion in vivo. Two-colour single-molecule localisation microscopy of TOCA-1 and Ena supports their nanoscale association. TOCA-1 clusters promote filopodial protrusion and this depends on a functional TOCA-1 SH3 domain and activation of Cdc42, which we perturbed using the small-molecule inhibitor CASIN. We propose that TOCA-1 clusters act independently of membrane curvature to recruit and promote Ena activity for filopodial protrusion.

filoVision - using deep learning and tip markers to automate filopodia analysis.

Filopodia are slender, actin-filled membrane projections used by various cell types for environment exploration. Analyzing filopodia often involves visualizing them using actin, filopodia tip or membrane markers. Due to the diversity of cell types that extend filopodia, from amoeboid to mammalian, it can be challenging for some to find a reliable filopodia analysis workflow suited for their cell type and preferred visualization method. The lack of an automated workflow capable of analyzing amoeboid filopodia with only a filopodia tip label prompted the development of filoVision. filoVision is an adaptable deep learning platform featuring the tools filoTips and filoSkeleton. filoTips labels filopodia tips and the cytosol using a single tip marker, allowing information extraction without actin or membrane markers. In contrast, filoSkeleton combines tip marker signals with actin labeling for a more comprehensive analysis of filopodia shafts in addition to tip protein analysis. The ZeroCostDL4Mic deep learning framework facilitates accessibility and customization for different datasets and cell types, making filoVision a flexible tool for automated analysis of tip-marked filopodia across various cell types and user data.

Steering cell migration: lamellipodium dynamics and the regulation of directional persistence.

Membrane protrusions at the leading edge of cells, known as lamellipodia, drive cell migration in many normal and pathological situations. Lamellipodial protrusion is powered by actin polymerization, which is mediated by the actin-related protein 2/3 (ARP2/3)-induced nucleation of branched actin networks and the elongation of actin filaments. Recently, advances have been made in our understanding of positive and negative ARP2/3 regulators (such as the SCAR/WAVE (SCAR/WASP family verprolin-homologous protein) complex and Arpin, respectively) and of proteins that control actin branch stability (such as glial maturation factor (GMF)) or actin filament elongation (such as ENA/VASP proteins) in lamellipodium dynamics and cell migration. This Review highlights how the balance between actin filament branching and elongation, and between the positive and negative feedback loops that regulate these activities, determines lamellipodial persistence. Importantly, directional persistence, which results from lamellipodial persistence, emerges as a critical factor in steering cell migration.

Life at the leading edge.

Cell migration requires sustained forward movement of the plasma membrane at the cell's front or "leading edge." To date, researchers have uncovered four distinct ways of extending the membrane at the leading edge. In lamellipodia and filopodia, actin polymerization directly pushes the plasma membrane forward, whereas in invadopodia, actin polymerization couples with the extracellular delivery of matrix-degrading metalloproteases to clear a path for cells through the extracellular matrix. Membrane blebs drive the plasma membrane forward using a combination of actomyosin-based contractility and reversible detachment of the membrane from the cortical actin cytoskeleton. Each protrusion type requires the coordination of a wide spectrum of signaling molecules and regulators of cytoskeletal dynamics. In addition, these different protrusion methods likely act in concert to move cells through complex environments in vivo.

Ena/VASP clustering at microspike tips involves lamellipodin but not I-BAR proteins, and absolutely requires unconventional myosin-X.

Sheet-like membrane protrusions at the leading edge, termed lamellipodia, drive 2D-cell migration using active actin polymerization. Microspikes comprise actin-filament bundles embedded within lamellipodia, but the molecular mechanisms driving their formation and their potential functional relevance have remained elusive. Microspike formation requires the specific activity of clustered Ena/VASP proteins at their tips to enable processive actin assembly in the presence of capping protein, but the factors and mechanisms mediating Ena/VASP clustering are poorly understood. Systematic analyses of B16-F1 melanoma mutants lacking potential candidate proteins revealed that neither inverse BAR-domain proteins, nor lamellipodin or Abi is essential for clustering, although they differentially contribute to lamellipodial VASP accumulation. In contrast, unconventional myosin-X (MyoX) identified here as proximal to VASP was obligatory for Ena/VASP clustering and microspike formation. Interestingly, and despite the invariable distribution of other relevant marker proteins, the width of lamellipodia in MyoX-KO mutants was significantly reduced as compared with B16-F1 control, suggesting that microspikes contribute to lamellipodium stability. Consistently, MyoX removal caused marked defects in protrusion and random 2D-cell migration. Strikingly, Ena/VASP-deficiency also uncoupled MyoX cluster dynamics from actin assembly in lamellipodia, establishing their tight functional association in microspike formation.

Neuronal filopodia: From stochastic dynamics to robustness of brain morphogenesis.

Brain development relies on dynamic morphogenesis and interactions of neurons. Filopodia are thin and highly dynamic membrane protrusions that are critically required for neuronal development and neuronal interactions with the environment. Filopodial interactions are typically characterized by non-deterministic dynamics, yet their involvement in developmental processes leads to stereotypic and robust outcomes. Here, we discuss recent advances in our understanding of how filopodial dynamics contribute to neuronal differentiation, migration, axonal and dendritic growth and synapse formation. Many of these advances are brought about by improved methods of live observation in intact developing brains. Recent findings integrate known and novel roles ranging from exploratory sensors and decision-making agents to pools for selection and mechanical functions. Different types of filopodial dynamics thereby reveal non-deterministic subcellular decision-making processes as part of genetically encoded brain development.

Myosin XV is a negative regulator of signaling filopodia during long-range lateral inhibition.

The self-organization of cells during development is essential for the formation of healthy tissues and requires the coordination of cell activities at local scales. Cytonemes, or signaling filopodia, are dynamic actin-based cellular protrusions that allow cells to engage in contact mediated signaling at a distance. While signaling filopodia have been shown to support several signaling paradigms during development, less is understood about how these protrusions are regulated. We investigated the role of the plus-end directed, unconventional MyTH4-FERM myosins in regulating signaling filopodia during sensory bristle patterning on the dorsal thorax of the fruit fly Drosophila melanogaster. We found that Myosin XV is required for regulating signaling filopodia dynamics and, as a consequence, lateral inhibition more broadly throughout the patterning epithelium. We found that Myosin XV is required for limiting the length and number of signaling filopodia generated by bristle precursor cells. Cells with additional and longer signaling filopodia due to loss of Myosin XV are not signaling competent, due to altered levels of Delta ligand and Notch receptor along their lengths. We conclude that Myosin XV acts to negatively regulate signaling filopodia, as well as promote the ability of signaling filopodia to engage in long-range Notch signaling. Since Myosin XV isoforms are present across several vertebrate and invertebrate systems, this may have significance for other long-range signaling mechanisms.

Myo10 tail is crucial for promoting long filopodia.

Filopodia are slender cellular protrusions containing parallel actin bundles involved in environmental sensing and signaling, cell adhesion and migration, and growth cone guidance and extension. Myosin 10 (Myo10), an unconventional actin-based motor protein, was reported to induce filopodial initiation with its motor domain. However, the roles of the multifunctional tail domain of Myo10 in filopodial formation and elongation remain elusive. Herein, we generated several constructs of Myo10-full-length Myo10, Myo10 with a truncated tail (Myo10 HMM), and Myo10 containing four mutations to disrupt its coiled-coil domain (Myo10 CC mutant). We found that the truncation of the tail domain decreased filopodial formation and filopodial length, while four mutations in the coiled-coil domain disrupted the motion of Myo10 toward filopodial tips and the elongation of filopodia. Furthermore, we found that filopodia elongated through multiple elongation cycles, which was supported by the Myo10 tail. These findings suggest that Myo10 tail is crucial for promoting long filopodia.

PTENε suppresses tumor metastasis through regulation of filopodia formation.

PTEN is one of the most frequently mutated genes in malignancies and acts as a powerful tumor suppressor. Tumorigenesis is involved in multiple and complex processes including initiation, invasion, and metastasis. The complexity of PTEN function is partially attributed to PTEN family members such as PTENα and PTENß. Here, we report the identification of PTENε (also named as PTEN5), a novel N-terminal-extended PTEN isoform that suppresses tumor invasion and metastasis. We show that the translation of PTENε/PTEN5 is initiated from the CUG816 codon within the 5'UTR region of PTEN mRNA. PTENε/PTEN5 mainly localizes in the cell membrane and physically associates with and dephosphorylates VASP and ACTR2, which govern filopodia formation and cell motility. We found that endogenous depletion of PTENε/PTEN5 promotes filopodia formation and enhances the metastasis capacity of tumor cells. Overall, we identify a new isoform of PTEN with distinct subcellular localization and molecular function compared to the known members of the PTEN family. These findings advance our current understanding of the importance and diversity of PTEN functions.

Myosin-X recruits lamellipodin to filopodia tips.

Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. However, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped the RAPH1 interaction domain for adhesome components to its talin-binding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises a conserved helix located just after the RAPH1 pleckstrin homology domain with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not required to activate integrins at filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip.

Cell-based optimization and characterization of genetically encoded location-based biosensors for Cdc42 or Rac activity.

Rac (herein referring to the Rac family) and Cdc42 are Rho GTPases that regulate the formation of lamellipoda and filopodia, and are therefore crucial in processes such as cell migration. Relocation-based biosensors for Rac and Cdc42 have not been characterized well in terms of their specificity or affinity. In this study, we identify relocation sensor candidates for both Rac and Cdc42. We compared their (1) ability to bind the constitutively active Rho GTPases, (2) specificity for Rac and Cdc42, and (3) relocation efficiency in cell-based assays. Subsequently, the relocation efficiency was improved by a multi-domain approach. For Rac1, we found a sensor candidate with low relocation efficiency. For Cdc42, we found several sensors with sufficient relocation efficiency and specificity. These optimized sensors enable the wider application of Rho GTPase relocation sensors, which was showcased by the detection of local endogenous Cdc42 activity at assembling invadopodia. Moreover, we tested several fluorescent proteins and HaloTag for their influence on the recruitment efficiency of the Rho location sensor, to find optimal conditions for a multiplexing experiment. This characterization and optimization of relocation sensors will broaden their application and acceptance.

A minimal cell model for lamellipodia-based cellular dynamics and migration.

One ubiquitous cellular structure for performing various tasks, such as spreading and migration over external surfaces, is the sheet-like protrusion called a lamellipodium, which propels the leading edge of the cell. Despite the detailed knowledge about the many components of this cellular structure, it is not yet fully understood how these components self-organize spatiotemporally to form lamellipodia. We review here recent theoretical works where we have demonstrated that membrane-bound protein complexes that have intrinsic curvature and recruit the protrusive forces of the cytoskeleton result in a simple, yet highly robust, organizing feedback mechanism that organizes the cytoskeleton and the membrane. This self-organization mechanism accounts for the formation of flat lamellipodia at the leading edge of cells spreading over adhesive substrates, allowing for the emergence of a polarized, motile 'minimal cell' model. The same mechanism describes how lamellipodia organize to drive robust engulfment of particles during phagocytosis and explains in simple physical terms the spreading and migration of cells over fibers and other curved surfaces. This Review highlights that despite the complexity of cellular composition, there might be simple general physical principles that are utilized by the cell to drive cellular shape dynamics.