Filopodial Protrusions Research Articles

Interplay of geometry and mechanics in epithelial wound healing.

Wound healing is a complex biological process critical for maintaining an organism's structural integrity and tissue repair following an infection or injury. Recent studies have unveiled the mechanisms involving the coordination of biochemical and mechanical responses in the tissue in wound healing. In this article, we focus on the healing property of an epithelial tissue as a material while the effects of biological mechanisms such as cell proliferation, tissue intercalation, cellular migration, cell crawling, and filopodia protrusion is minimal. We present a mathematical framework that predicts the fate of a wounded tissue based on the wound's geometrical features and the tissue's mechanical properties. Precisely, adapting the vertex model of tissue mechanics, we predict whether a wound of a specific size in an epithelial monolayer characterized by certain levels of actomyosin contractility and cell-cell adhesion will heal (i.e., close), shrink in size, or rupture the tissue further. Moreover, we show how tissue-mediated mechanisms such as purse-string tension at the wound boundary facilitate wound healing. Finally, we validate the predictions of our model by designing an experimental setup that enables us to create wounds of specific sizes in kidney epithelial cells (MDCK) monolayers. Altogether, this work sets up a basis for interpreting the interplay of mechanical and geometrical features of a tissue in the process of wound healing.

WAVE1 and WAVE2 facilitate human papillomavirus-driven actin polymerization during cellular entry.

Human PapillomavirusType 16 (HPV16) is an etiological agent of human cancers that requires endocytosis to initiate infection. HPV16 entry into epithelial cells occurs through a non-canonical endocytic pathway that is actin-driven, but it is not well understood how HPV16-cell surface interactions trigger actin reorganization in a way that facilitates entry. This study provides evidence that Wiskott-Aldrich syndrome protein family verprolin-homologous proteins 1 and 2 (WAVE1 and WAVE2) are molecular mediators of the actin polymerization that facilitates HPV endocytosis and intracellular trafficking. We demonstrate through post-transcriptional gene silencing and genome editing that WAVE1 and WAVE2 are critical for efficient HPV16 infection, and that restoration of each in knockout cells rescues HPV16 infection. Cells lacking WAVE1, WAVE2, or both, internalize HPV16 at a significantly reduced rate. Analysis of fluorescently labeled cells exposed to HPV16 and acquired by confocal fluorescence microscopy revealed that HPV16, WAVE1, WAVE2, and actin are all colocalized at the cellular dorsal surface. We also found that HPV16 stimulates WAVE1 and WAVE2-mediated cellular dorsal surface filopodia formation during the viral endocytic process. Taken together, this study provides evidence that the HPV endocytic process needed for infection is controlled by actin reorganization into filopodial protrusions and that this process is mediated by WAVE1 and WAVE2.

Biophysical modeling of membrane curvature generation and curvature sensing by the glycocalyx.

Generation of membrane curvature is fundamental to cellular function. Recent studies have established that the glycocalyx, a sugar-rich polymer layer at the cell surface, can generate membrane curvature. While there have been some theoretical efforts to understand the interplay between the glycocalyx and membrane bending, there remain open questions about how the properties of the glycocalyx affect membrane bending. For example, the relationship between membrane curvature and the density of glycosylated proteins on its surface remains unclear. In this work, we use polymer brush theory to develop a detailed biophysical model of the energetic interactions of the glycocalyx with the membrane. Using this model, we identify the conditions under which the glycocalyx can both generate and sense curvature. Our model predicts that the extent of membrane curvature generated depends on the grafting density of the glycocalyx and the length of the polymers constituting the glycocalyx. Furthermore, when coupled with the intrinsic membrane properties such as spontaneous curvature and a line tension along the membrane, the curvature generation properties of the glycocalyx are enhanced. These predictions were tested experimentally by examining the propensity of glycosylated transmembrane proteins to drive the assembly of highly-curved filopodial protrusions at the plasma membrane of adherent mammalian cells. Our model also predicts that the glycocalyx has curvature-sensing capabilities, in agreement with the results of our experiments. Thus, our study develops a quantitative framework for mapping the properties of the glycocalyx to the curvature generation capability of the membrane.

SRC-1 controls growth cone polarity and protrusion with the UNC-6/Netrin receptor UNC-5 in Caenorhabditis elegans.

The Polarity/Protusion model of UNC-6/Netrin function in axon repulsion does not rely on a gradient of UNC-6/Netrin. Instead, the UNC-5 receptor polarizes the VD growth cone such that filopodial protrusions are biased to the dorsal leading edge. UNC-5 then inhibits growth cone protrusion ventrally based upon this polarity, resulting in dorsally-biased protrusion and dorsal migration away from UNC-6/Netrin. While previous studies have shown that UNC-5 inhibits growth cone protrusion by destabilizing actin, preventing microtubule + end entry, and preventing vesicle fusion, the signaling pathways involved are unclear. The SRC-1 tyrosine kinase has been previously shown to physically interact with and phosphorylate UNC-5, and to act with UNC-5 in axon guidance and cell migration. Here, the role of SRC-1 in VD growth cone polarity and protrusion is investigated. A precise deletion of src-1 was generated, and mutants displayed unpolarized growth cones with increased size, similar to unc-5 mutants. Transgenic expression of src-1(+) in VD/DD neurons resulted in smaller growth cones, and rescued growth cone polarity defects of src-1 mutants, indicating cell-autonomous function. Transgenic expression of a putative kinase-dead src-1(D831A) mutant caused a phenotype similar to src-1 loss-of-function, suggesting that this is a dominant negative mutation. The D381A mutation was introduced into the endogenous src-1 gene by genome editing, which also had a dominant-negative effect. Genetic interactions of src-1 and unc-5 suggest they act in the same pathway on growth cone polarity and protrusion, but might have overlapping, parallel functions in other aspects of axon guidance. src-1 function was not required for the effects of activated myr::unc-5, suggesting that SRC-1 might be involved in UNC-5 dimerization and activation by UNC-6, of which myr::unc-5 is independent. In sum, these results show that SRC-1 acts with UNC-5 in growth cone polarity and inhibition of protrusion.

Viscoelastic cues to induce stem cell migration and neuronal differentiation in cell-free hydrogel-assisted TBI recovery

The concept of tissue-inducing biomaterials, such as osteoinductive biomaterials, has inspired the design of regenerative material with biomimetic cues to manipulate cell/tissue responses but has been little applied for neuroinduction in traumatic brain injury (TBI) treatment. Meanwhile, material design has typically focused on elasticity without viscoelasticity taken into consideration. Here, guided by the intrinsic viscoelasticity of brain tissue and the decisive role of viscoelasticity in cell-matrix interactions, we developed a family of brain-mimicking hydrogels, in which the viscoelasticity can be tuned over a wide range under the premise that hydrogels have a low modulus comparable to that of brain tissue. Then, we revealed the promoted migration of stem cells on the viscoelastic hydrogel resulted from the increased number of motor − clutch pairs and filopodia protrusions, as well as their enhanced neuronal differentiation. In a rat TBI model, the cell-free viscoelastic hydrogel successfully induced endogenous stem cells to migrate into lesions and differentiate into neurons, contributing to brain tissue regeneration and neurological function restoration. This study reveals the great promise of biomimetic viscoelastic matrices for TBI treatment, and simultaneously, provides intriguing insights for the design of tissue-inducing biomaterials.

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.

First person – Thomas Blake

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Thomas Blake is first author on ‘ Filopodial protrusion driven by density-dependent Ena–TOCA-1 interactions’, published in JCS. Thomas is a Research Associate in the lab of Jennifer Gallop at the Wellcome/Cancer Research UK Gurdon Institute, Cambridge, UK, investigating the molecular mechanisms that drive cell motility using quantitative microscopy techniques.

Generative modeling of living cells with SO(3)-equivariant implicit neural representations

Data-driven cell tracking and segmentation methods in biomedical imaging require diverse and information-rich training data. In cases where the number of training samples is limited, synthetic computer-generated data sets can be used to improve these methods. This requires the synthesis of cell shapes as well as corresponding microscopy images using generative models. To synthesize realistic living cell shapes, the shape representation used by the generative model should be able to accurately represent fine details and changes in topology, which are common in cells. These requirements are not met by 3D voxel masks, which are restricted in resolution, and polygon meshes, which do not easily model processes like cell growth and mitosis. In this work, we propose to represent living cell shapes as level sets of signed distance functions (SDFs) which are estimated by neural networks. We optimize a fully-connected neural network to provide an implicit representation of the SDF value at any point in a 3D+time domain, conditioned on a learned latent code that is disentangled from the rotation of the cell shape. We demonstrate the effectiveness of this approach on cells that exhibit rapid deformations (Platynereis dumerilii), cells that grow and divide (C. elegans), and cells that have growing and branching filopodial protrusions (A549 human lung carcinoma cells). A quantitative evaluation using shape features and Dice similarity coefficients of real and synthetic cell shapes shows that our model can generate topologically plausible complex cell shapes in 3D+time with high similarity to real living cell shapes. Finally, we show how microscopy images of living cells that correspond to our generated cell shapes can be synthesized using an image-to-image model.

Differences and similarities in biophysical and biological characteristics between U87 MG glioblastoma and astrocyte cells.

Current cancer studies focus on molecular-targeting diagnostics and interactions with surroundings; however, there are still gaps in characterization based on topological differences and elemental composition. Glioblastoma (GBM cells; GBMCs) is an astrocytic aggressive brain tumor. At the molecular level, GBMCs and astrocytes may differ, and cell elemental/topological analysis is critical for identifying potential new cancer targets. Here, we used U87 MG cells for GBMCS. U87 MG cell lines, which are frequently used in glioblastoma research, are an important tool for studying the various features and underlying mechanisms of this aggressive brain tumor. For the first time, atomic force microscopy (AFM), scanning electron microscopy (SEM) accompanied by energy-dispersive X-ray spectroscopy (EDS), and X-ray photoelectron spectroscopy (XPS) are used to report the topology and chemistry of cancer (U87 MG) and healthy (SVG p12) cells. In addition, F-actin staining and cytoskeleton-based gene expression analyses were performed. The degree of gene expression for genes related to the cytoskeleton was similar; however, the intensity of F-actin, anisotropy values, and invasion-related genes were different. Morphologically, GBMCs were longer and narrower while astrocytes were shorter and more disseminated based on AFM. Furthermore, the roughness values of these cells differed slightly between the two call types. In contrast to the rougher astrocyte surfaces in the lamellipodial area, SEM-EDS analysis showed that elongated GBMCs displayed filopodial protrusions. Our investigation provides considerable further insight into rapid cancer cell characterization in terms of a combinatorial spectroscopic and microscopic approach.

A short isoform of the UNC-6/Netrin receptor UNC-5 is required for growth cone polarity and robust growth cone protrusion in Caenorhabditis elegans.

Introduction: UNC-6/Netrin is a conserved bi-functional guidance cue which regulates dorsal-ventral axon guidance in C. elegans. In the Polarity/Protrusion model of UNC-6/Netrin mediated dorsal growth away from UNC-6/Netrin, The UNC-5 receptor first polarizes the VD growth cone such that filopodial protrusions are biased dorsally. Based on this polarity, the UNC-40/DCC receptor stimulates growth cone lamellipodial and filopodial protrusion dorsally. The UNC-5 receptor maintains dorsal polarity of protrusion, and inhibits growth cone protrusion ventrally, resulting in net dorsal growth cone advance. Methods: Growth cone imaging in mutants, combined with Cas9 genome editing and genetic analysis, were used to analyze the role of a novel short isoform on unc-5 in growth cone polarity and protrusion. Results: Work presented here demonstrates a novel role of a previously undescribed, conserved short isoform of UNC-5 (UNC-5B). UNC-5B lacks the cytoplasmic domains of UNC-5 long, including the DEATH domain, the UPA/DB domain, and most of the ZU5 domain. Mutations that specifically affect only the unc-5 long isoforms were hypomorphic, suggesting a role of unc-5B short. A mutation specifically affecting unc-5B caused loss of dorsal polarity of protrusion and reduced growth cone filopodial protrusion, the opposite of unc-5 long mutations. Transgenic expression of unc-5B partially rescued unc-5 axon guidance defects, and resulted in large growth cones. Tyrosine 482 (Y482) in the cytoplasmic juxtamembrane region has been shown to be important for UNC-5 function, and is present in both UNC-5 long and UNC-5B short. Results reported here show that Y482 is required for the function of UNC-5 long and for some functions of UNC-5B short. Finally, genetic interactions with unc-40 and unc-6 suggest that UNC-5B short acts in parallel to UNC-6/Netrin to ensure robust growth cone lamellipodial protrusion. Discussion: These results demonstrate a previously-undescribed role for the UNC-5B short isoform, which is required for dorsal polarity of growth cone filopodial protrusion and to stimulate growth cone protrusion, in contrast to the previously-described role of UNC-5 long in inhibiting growth cone protrusion.

Optimized Fixation and Phalloidin Staining of Basally Localized F-Actin Networks in Collectively Migrating Follicle Cells.

The follicular epithelial cells of the Drosophila egg chamber have become a premier model to study how cells globally orient their actin-based machinery for collective migration. The basal surface of each follicle cell has lamellipodial and filopodial protrusions that extend from its leading edge and an array of stress fibers that mediate its adhesion to the extracellular matrix; these migratory structures are all globally aligned in the direction of tissue movement. To understand how this global alignment is achieved, one must be able to reliably visualize the underlying F-actin; however, dynamic F-actin networks can be difficult to preserve in fixed tissues. Here, we describe an optimized protocol for the fixation and phalloidin staining of the follicular epithelium. We also provide a brief primer on relevant aspects of the image acquisition process to ensure high quality data are collected.

Antimetastatic Properties of Prodigiosin and the BH3-Mimetic Obatoclax (GX15-070) in Melanoma

Metastasis is the primary cause of death in cancer patients. Many current chemotherapeutic agents only show cytotoxic, but not antimetastatic properties. This leads to a reduction in tumor size, but allows cancer cells to disseminate, which ultimately causes patient death. Therefore, novel anticancer compounds with both effects need to be developed. In this work, we analyze the antimetastatic properties of prodigiosin and obatoclax (GX15-070), anticancer drugs of the Prodiginines (PGs) family. We studied PGs' effects on cellular adhesion and morphology in the human primary and metastatic melanoma cell lines, SK-MEL-28 and SK-MEL-5, and in the murine melanoma cell line, B16F10A. Cell adhesion sharply decreased in the treated cells, and this was accompanied by a reduction in filopodia protrusions and a significant decrease in the number of focal-adhesion structures. Moreover, cell migration was assessed through the wound-healing assay and cell motility was severely inhibited after 24 h of treatment. To elucidate the molecular mechanisms involved, changes in metastasis-related genes were analyzed through a gene-expression array. Key genes related to cellular invasion, migration and chemoresistance were significantly down-regulated. Finally, an in vivo model of melanoma-induced lung metastasis was established and significant differences in lung tumors were observed in the obatoclax-treated mice. Altogether, these results describe, in depth, PGs' cellular antimetastatic effects and identify in vivo antimetastatic properties of Obatoclax.

Fucoidan from Sargassum hemiphyllum inhibits the stemness of cancer stem cells and epithelial-mesenchymal transitions in bladder cancer cells

A variety of anticancer activities have been established for fucoidan from brown algae, whereas whether cancer stem cells (CSCs) are inhibited by sulfated polysaccharides is unexplored. In this study, fucoidan extracted from Sargassum hemiphyllum was showed heat stable and might tolerate 140 °C treatment. Fucoidan did not exhibit cytotoxicity in 5637 and T24 bladder cancer cells. After fucoidan treatment, the stress fibers were aggregated into thick and abundant underneath the plasma membrane and getting around the cells, and the structure of F-actin showed a remarkable change in the filopodial protrusion in T24 and 5637 cells. Using culture inserts, transwell assays and time lapse recordings showed that fucoidan inhibited cell migration. In the epithelial-mesenchymal transition (EMT), fucoidan downregulated the expression of vimentin, a mesenchymal marker, and upregulated the expression of E-cadherin, an epithelial marker. Additionally, the transcription levels of Snail, Slug, Twist1, Twist2, MMP2 and MMP9 were significantly decreased by fucoidan, indicating EMT suppression. CSCs are implicated in tumor initiation, metastatic spread, drug resistance and tumor recurrence. Our results showed that fucoidan inhibited stemness gene expression and sphere formation in bladder CSCs. For the first time, our findings demonstrated that fucoidan inhibits CSC formation and provides evidence as potential anticancer therapy.

Effect of monetite reinforced into the chitosan-based lyophilized 3D scaffolds on physicochemical, mechanical, and osteogenic properties

Chitosan and monetite nanoparticles-based composite scaffold with tailored structures and properties possesses tremendous potential for bone restoration. Here, we aimed to enhance the physicochemical, mechanical, and biological properties of a three-dimensional composite scaffold by the addition of monetite nanoparticles into a chitosan matrix with varying content of monetite in the developed scaffolds. The scaffolds were fabricated from the viscous slurry containing chitosan and nano monetite particles using a free-drying process. Macro-porous scaffolds were fabricated by incorporation of nano-sized monetite particles up to 20 wt% of total chitosan concentration into pure chitosan solution. These are then characterized using an X-ray diffractometer (XRD), FTIR, and field emission electron microscope (FESEM). Results revealed that the chitosan/monetite composite scaffolds showed highly porous architecture with an average pore size varying between 90 and 390 µm, and high interconnected porosity varying from 77 to 88%, respectively. Scaffold containing 20 wt% monetite (CS-M20) revealed the highest compressive strength of 1.93 MPa, which is closely matching with that of the lower range of compressive strength in cancellous bone. The incorporation of monetite content into the chitosan matrix significantly diminished (p < 0.05) pore size, and porosity and retarded the rate of biodegradation of the monetite-containing scaffolds as evident from 20 wt% and 38 wt% of weight loss in CSM20 and CSM10 scaffolds after 28 days of incubation in a sterile PBS. Monetite-containing scaffolds showed a higher ability of mineralization as compared to the pristine chitosan (CS-M0) scaffold, depositing bone-like carbonated hydroxyapatite crystals onto their surface after 14 days of immersion in SBF at 37 °C. A higher extent of lamellipodia and filopodia protrusions and superior spreading behavior of osteoblasts were noticed in FESEM images of MG-63 cultured monetite containing scaffolds. MTT assay exhibited significantly higher (p < 0.05) MG-63 cell proliferation in monetite-containing CS-M20 scaffold than that in pristine chitosan scaffold suggesting the fact that monetite nanoparticles incorporation into the chitosan matrix stimulated superior osteoblast attachment and proliferation. Further, immunocytochemistry analysis of MG-63 cultured onto developed scaffolds revealed higher osteogenic activity and differentiation ability in CS-M20 scaffold as compared to pristine chitosan scaffold. The obtained results demonstrated that the monetite-containing scaffolds have a great potential for the regeneration of bone tissue defects.

Matrix Metalloproteinase‐9 Promotes Membrane Protrusive Activity in Ovarian Cancer Cells

Matrix Metalloproteinase‐9 (MMP9) is an extracellular endopeptidase associated with ovarian cancer development and progression. To further understand MMP9 contributions to metastatic behavior of an established ovarian cancer cell line (OVCA433), we utilized CRISPR to generate seven cell clones null for MMP9 expression. Analysis of the pooled clones revealed MMP9‐null cells exhibited identical phenotype and growth compared to parental OVCA 433 cells. Cell adhesion and transwell migration towards type I collagen was also not impacted by MMP9 loss. MMP9‐null cells were significantly delayed, however, in EGF‐driven non‐directional filamentous actin‐based membrane protrusions on a variety of matrix substrates. Further, over or re‐expression of MMP9 in OVCA433 or MMP9‐null cells, respectively, was sufficient to drive both lamellipodial and filopodial membrane protrusions on glass. These results indicate MMP9 directly contributes to ovarian cancer cell protrusive activity.

Aluminum hydroxide adjuvant diverts the uptake and trafficking of genetically detoxified pertussis toxin to lysosomes in macrophages.

Aluminum salts have been successfully utilized as adjuvants to enhance the immunogenicity of vaccine antigens since the 1930s. However, the cellular mechanisms behind the immune adjuvanticity effect of these materials in antigen‐presenting cells are poorly understood. In this study, we investigated the uptake and trafficking of aluminum oxy‐hydroxide (AlOOH), in RAW 264.7 murine and U‐937 human macrophages‐like cells. Furthermore, we determined the impact that the adsorption to AlOOH particulates has on the trafficking of a Bordetella pertussis vaccine candidate, the genetically detoxified pertussis toxin (gdPT). Our results indicate that macrophages internalize AlOOH by constitutive macropinocytosis assisted by the filopodial protrusions that capture the adjuvant particles. Moreover, we show that AlOOH has the capacity to nonspecifically adsorb IgG, engaging opsonic phagocytosis, which is a feature that may allow for more effective capture and uptake of adjuvant particles by antigen‐presenting cells (APCs) at the site of vaccine administration. We found that AlOOH traffics to endolysosomal compartments that hold degradative properties. Importantly, while we show that gdPT escapes degradative endolysosomes and traffics toward the retrograde pathway, as reported for the wild‐type pertussis toxin, the adsorption to AlOOH diverts gdPT to traffic to the adjuvant’s lysosome‐type compartments, which may be key for MHC‐II‐driven antigen presentation and activation of CD4+ T cell. Thus, our findings establish a direct link between antigen adsorption to AlOOH and the intracellular trafficking of antigens within antigen‐presenting cells and bring to light a new potential mechanism for aluminum adjuvancy. Moreover, the in‐vitro single‐cell approach described herein provides a general framework and tools for understanding critical attributes of other vaccine formulations.

The PH/MyTH4/FERM molecule MAX-1 inhibits UNC-5 activity in the regulation of VD growth cone protrusion in Caenorhabditis elegans.

UNC-6/Netrin is a secreted conserved guidance cue that regulates dorsal-ventral axon guidance of Caenorhabditis elegans and in the vertebral spinal cord. In the polarity/protrusion model of VD growth cone guidance away from ventrally expressed UNC-6 (repulsion), UNC-6 first polarizes the growth cone via the UNC-5 receptor such that filopodial protrusions are biased dorsally. UNC-6 then regulates a balance of protrusion in the growth cone based upon this polarity. UNC-5 inhibits protrusion ventrally, and the UNC-6 receptor UNC-40/DCC stimulates protrusion dorsally, resulting in net dorsal growth cone outgrowth. UNC-5 inhibits protrusion through the flavin monooxygenases FMO-1, 4, and 5 and possible actin destabilization, and inhibits pro-protrusive microtubule entry into the growth cone utilizing UNC-33/CRMP. The PH/MyTH4/FERM myosin-like protein was previously shown to act with UNC-5 in VD axon guidance utilizing axon guidance endpoint analysis. Here, we analyzed the effects of MAX-1 on VD growth cone morphology during outgrowth. We found that max-1 mutant growth cones were smaller and less protrusive than wild type, the opposite of the unc-5 mutant phenotype. Furthermore, genetic interactions suggest that MAX-1 might normally inhibit UNC-5 activity, such that in a max-1 mutant growth cone, UNC-5 is overactive. Our results, combined with previous studies suggesting that MAX-1 might regulate UNC-5 levels in the cell or plasma membrane localization, suggest that MAX-1 attenuates UNC-5 signaling by regulating UNC-5 stability or trafficking. Alternately, MAX-1 might inhibit UNC-5 independent of this known mechanism. We also show that the effects of MAX-1 in growth cone protrusion are independent of UNC-40/DCC, UNC-33/CRMP, and UNC-34/Enabled. In summary, in the context of growth cone protrusion, MAX-1 inhibits UNC-5, demonstrating the mechanistic insight that can be gained by analyzing growth cones during outgrowth in addition to axon guidance endpoint analysis.

Activating RAC1 variants in the switch II region cause a developmental syndrome and alter neuronal morphology.

RAC1 is a highly conserved Rho GTPase critical for many cellular and developmental processes. De novo missense RAC1 variants cause a highly variable neurodevelopmental disorder. Some of these variants have previously been shown to have a dominant negative effect. Most previously reported patients with this disorder have either severe microcephaly or severe macrocephaly. Here, we describe eight patients with pathogenic missense RAC1 variants affecting residues between Q61 and R68 within the switch II region of RAC1. These patients display variable combinations of developmental delay, intellectual disability, brain anomalies such as polymicrogyria and cardiovascular defects with normocephaly or relatively milder micro- or macrocephaly. Pulldown assays, NIH3T3 fibroblast spreading assays and staining for activated PAK1/2/3 and WAVE2 suggest that these variants increase RAC1 activity and over-activate downstream signalling targets. Axons of neurons isolated from Drosophila embryos expressing the most common of the activating variants are significantly shorter, with an increased density of filopodial protrusions. In vivo, these embryos exhibit frequent defects in axonal organization. Class IV dendritic arborization neurons expressing this variant exhibit a significant reduction in the total area of the dendritic arbour, increased branching and failure of self-avoidance. RNAi knock down of the WAVE regulatory complex component Cyfip significantly rescues these morphological defects. These results establish that activating substitutions affecting residues Q61-R68 within the switch II region of RAC1 cause a developmental syndrome. Our findings reveal that these variants cause altered downstream signalling, resulting in abnormal neuronal morphology and reveal the WAVE regulatory complex/Arp2/3 pathway as a possible therapeutic target for activating RAC1 variants. These insights also have the potential to inform the mechanism and therapy for other disorders caused by variants in genes encoding other Rho GTPases, their regulators and downstream effectors.

Cannabinoid Receptor Type 1 regulates growth cone filopodia and axon dispersion in the optic tract of Xenopus laevis tadpoles.

Previous studies show that the main cannabinoid receptor in the brain-cannabinoid type 1 receptor (CB1R)-is required for establishment of axonal projections in developing neurons but questions remain regarding the cellular and molecular mechanisms, especially in neurons developing in their native environment. We assessed the effects of CB1R signalling on growth cone filopodia and axonal projections of retinal ganglion cells (RGCs) in whole mount brains from Xenopus laevis tadpoles. Our results indicate that growth cones of RGC axons in brains from tadpoles exposed to a CB1R agonist had fewer filopodial protrusions, whereas growth cones from tadpoles exposed to a CB1R inverse agonist had more filopodia than growth cones of RGC axons in whole brains from control tadpoles. However, application of both the CB1R agonist and inverse agonist resulted in RGC axons that were overly dispersed and undulatory in the optic tract in situ. In addition, expression of a mutant for cadherin adhesive factor, β-catenin, that disrupts its binding to α-catenin, and application of an inhibitor for actin regulator non-muscle Myosin II, phenocopied the effects of the CB1R agonist and inverse agonist on growth cone filopodia, respectively. These findings suggest that both destablization and stabilization of growth cone filopodia are required for RGC axonal fasciculation/defasciculation in the optic tract and that CB1R regulates growth cone filopodia and axon dispersion of RGCs by oppositely modulating β-catenin adhesive and Myosin II actin regulatory functions. This study extends and confirms our understanding of cannabinoid mechanisms in sculpting developing neuronal circuits in vivo.

Designing Single-Component Optogenetic Membrane Recruitment Systems: The Rho-Family GTPase Signaling Toolbox.

We describe the efficient creation of single-component optogenetic tools for membrane recruitment-based signaling perturbation using BcLOV4 technology. The workflow requires two plasmids to create six different domain arrangements of the dynamic membrane binder BcLOV4, a fluorescent reporter, and the fused signaling protein of interest. Screening of this limited set of genetic constructs for expression characteristics and dynamic translocation in response to one pulse of light is sufficient to identify viable signaling control tools. The reliability of this streamlined approach is demonstrated by the creation of an optogenetic Cdc42 GTPase and Rac1-activating Tiam1 GEF protein, which together with our other recently reported technologies, completes a toolbox for spatiotemporally precise induction of Rho-family GTPase signaling at the GEF or GTPase level, for driving filopodial protrusions, lamellipodial protrusions, and cell contractility, respectively mediated by Cdc42, Rac1, and RhoA.