SciCombinator

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Concept: Filopodia

168

Intracellular Ca(2+) signals control the development and regeneration of spinal axons downstream of chemical guidance cues, but little is known about the roles of mechanical cues in axon guidance. Here we show that transient receptor potential canonical 1 (TRPC1) subunits assemble mechanosensitive (MS) channels on Xenopus neuronal growth cones that regulate the extension and direction of axon outgrowth on rigid, but not compliant, substrata. Reducing expression of TRPC1 by antisense morpholinos inhibits the effects of MS channel blockers on axon outgrowth and local Ca(2+) transients. Ca(2+) influx through MS TRPC1 activates the protease calpain, which cleaves the integrin adaptor protein talin to reduce Src-dependent axon outgrowth, likely through altered adhesion turnover. We found that talin accumulates at the tips of dynamic filopodia, which is lost upon cleavage of talin by active calpain. This pathway may also be important in axon guidance decisions since asymmetric inhibition of MS TRPC1 is sufficient to induce growth cone turning. Together our results suggest that Ca(2+) influx through MS TRPC1 on filopodia activates calpain to control growth cone turning during development.

Concepts: Nervous system, Neuron, Developmental biology, Action potential, Axon, Axon guidance, Growth cone, Filopodia

26

Cell-microstructure surface interactions play a significant role in tissue engineering to guide cell spreading and migration. However, the mechanisms underlying cell-topography interactions are complex and remain elusive. To address this topic, microsphere array patterns were prepared on silk fibroin films through polystyrene microsphere self-assembly, followed by culturing rat bone marrow derived mesenchymal stem cells on the films to study cell-substrate interactions. Filopodia sensed and anchored to the microspheres to form initial attachments prior to spreading. Importantly, the anchored filopodia converted into lamellipodia, and this conversion initiated the directional formation of lamellipodia. Therefore, the conversion of exploratory filopodia into lamellipodia was the main driving force for directional extension of the lamellipodia. Correspondingly, cell spreading, morphology and migration were modulated by pseudopodial recognition and conversion. This finding demonstrated that filopodia not only act as an antenna to detect microenvironment, but also serve as skeleton to guide lamellipodial extension for directing cell motions. The micropatterned films promoted cell adhesion and proliferation due to accelerated lamellipodia formation and cell spreading, with recognition and conversion of filopodia into lamellipodia as a critical role in cell response to surface topography.

Concepts: Cell, Stem cell, Mesenchymal stem cell, Bone marrow, Cell biology, Cellular differentiation, Lamellipodia, Filopodia

25

Growth cone filopodia are actin-based mechanosensory structures essential for chemoreception and generation of contractile forces necessary for directional motility. However, little is known about the influence of filopodial actin structures on substrate adhesion and filopodial contractility.Formin-2 (Fmn2) localizes along filopodial actin bundles and its depletion does not affect filopodia initiation or elongation. However, Fmn2 activity is required for filopodial tip adhesion maturation and the ability of filopodia to generate traction forces. Dysregulation of filopodia in Fmn2 depleted neurons leads to compromised growth cone motility. Additionally, in fibroblasts, Fmn2 regulates ventral stress fiber assembly and affects the stability of focal adhesions. In the developing spinal cord, Fmn2 activity is required cell autonomously for the outgrowth and pathfinding of spinal commissural neurons.Our results reveal an unanticipated function for Fmn2 in neural development. Fmn2 regulates structurally diverse bundled actin structures, parallel filopodial bundles in growth cones and anti-parallel stress fibers in fibroblasts, in turn modulating the stability of substrate adhesions. We propose Fmn2 as a mediator of actin bundle integrity enabling efficient force transmission to the adhesion sites.

Concepts: Nervous system, Neuron, Axon, Axon guidance, Force, Growth cone, Filopodia, Actin based structures

10

Chemical cues presented on the adhesive substrate direct cell migration, a process termed haptotaxis. To migrate, cells must generate traction forces upon the substrate. However, how cells probe substrate-bound cues and generate directional forces for migration remains unclear. Here, we show that the cell adhesion molecule (CAM) L1-CAM is involved in laminin-induced haptotaxis of axonal growth cones. L1-CAM underwent grip and slip on the substrate. The ratio of the grip state was higher on laminin than on the control substrate polylysine; this was accompanied by an increase in the traction force upon laminin. Our data suggest that the directional force for laminin-induced growth cone haptotaxis is generated by the grip and slip of L1-CAM on the substrates, which occur asymmetrically under the growth cone. This mechanism is distinct from the conventional cell signaling models for directional cell migration. We further show that this mechanism is disrupted in a human patient with L1-CAM syndrome, suffering corpus callosum agenesis and corticospinal tract hypoplasia.

Concepts: Enzyme, Cell biology, Axon, Corpus callosum, Agenesis of the corpus callosum, Filopodia

6

Filopodia have important sensory and mechanical roles in motile cells. The recruitment of actin regulators, such as ENA/VASP proteins, to sites of protrusion underlies diverse molecular mechanisms of filopodia formation and extension. We developed Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia and at their tips and bases concurrently with their morphological and dynamic properties. Filopodyan supports high-throughput phenotype characterization as well as detailed interactive editing of filopodia reconstructions through an intuitive graphical user interface. Our highly customizable pipeline is widely applicable, capable of detecting filopodia in four different cell types in vitro and in vivo. We use Filopodyan to quantify the recruitment of ENA and VASP preceding filopodia formation in neuronal growth cones, and uncover a molecular heterogeneity whereby different filopodia display markedly different responses to changes in the accumulation of ENA and VASP fluorescence in their tips over time.

Concepts: Protein, Cell, In vivo, In vitro, User interface, Growth cone, Graphical user interface, Filopodia

6

Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors.

Concepts: Signal transduction, Adenosine triphosphate, Cell membrane, Cell biology, Förster resonance energy transfer, Growth cone, Fluorescence in the life sciences, Filopodia

4

Cells can interact with their surroundings via filopodia, which are membrane protrusions that extend beyond the cell body. Filopodia are essential during dynamic cellular processes like motility, invasion, and cell-cell communication. Filopodia contain cross-linked actin filaments, attached to the surrounding cell membrane via protein linkers such as integrins. These actin filaments are thought to play a pivotal role in force transduction, bending, and rotation. We investigated whether, and how, actin within filopodia is responsible for filopodia dynamics by conducting simultaneous force spectroscopy and confocal imaging of F-actin in membrane protrusions. The actin shaft was observed to periodically undergo helical coiling and rotational motion, which occurred simultaneously with retrograde movement of actin inside the filopodium. The cells were found to retract beads attached to the filopodial tip, and retraction was found to correlate with rotation and coiling of the actin shaft. These results suggest a previously unidentified mechanism by which a cell can use rotation of the filopodial actin shaft to induce coiling and hence axial shortening of the filopodial actin bundle.

Concepts: Protein, Gene, Cell, Cell membrane, Cell biology, Cytoplasm, Cytoskeleton, Filopodia

3

Filopodia have a key role in sensing both chemical and mechanical cues in surrounding extracellular matrix (ECM). However, quantitative understanding is still missing in the filopodial mechanosensing of local ECM stiffness, resulting from dynamic interactions between filopodia and the surrounding 3D ECM fibers. Here we present a method for characterizing the stiffness of ECM that is sensed by filopodia based on the theory of elasticity and discrete ECM fiber. We have applied this method to a filopodial mechanosensing model for predicting directed cell migration toward stiffer ECM. This model provides us with a distribution of force and displacement as well as their time rate of changes near the tip of a filopodium when it is bound to the surrounding ECM fibers. Aggregating these effects in each local region of 3D ECM, we express the local ECM stiffness sensed by the cell and explain polarity in the cellular durotaxis mechanism.

Concepts: Time, Scientific method, Cell, Stiffness, Hooke's law, American films, Filopodia, Movable cellular automaton

1

Single-headed myosin 1 has been identified in neurons, but its function in these cells is still unclear. We demonstrate that depletion of myosin 1b (Myo1b), inhibition of its motor activity, or its binding to phosphoinositides impairs the formation of the axon, whereas overexpression of Myo1b increases the number of axon-like structures. Myo1b is associated with growth cones and actin waves, two major contributors to neuronal symmetry breaking. We show that Myo1b controls the dynamics of the growth cones and the anterograde propagation of the actin waves. By coupling the membrane to the actin cytoskeleton, Myo1b regulates the size of the actin network as well as the stability and size of filopodia in the growth cones. Our data provide the first evidence that a myosin 1 plays a major role in neuronal symmetry breaking and argue for a mechanical control of the actin cytoskeleton both in actin waves and in the growth cones by this myosin.

Concepts: Protein, Neuron, Actin, Retina, Axon, Cytoskeleton, Growth cone, Filopodia

1

Sprouting blood vessels are led by filopodia-studded endothelial tip cells that respond to angiogenic signals. Mosaic lineage tracing previously revealed that NRP1 is essential for tip cell function, although its mechanistic role in tip cells remains poorly defined. Here, we show that NRP1 is dispensable for genetic tip cell identity. Instead, we find that NRP1 is essential to form the filopodial bursts that distinguish tip cells morphologically from neighboring stalk cells, because it enables the extracellular matrix (ECM)-induced activation of CDC42, a key regulator of filopodia formation. Accordingly, NRP1 knockdown and pharmacological CDC42 inhibition similarly impaired filopodia formation in vitro and in developing zebrafish in vivo. During mouse retinal angiogenesis, CDC42 inhibition impaired tip cell and vascular network formation, causing defects that resembled those due to loss of ECM-induced, but not VEGF-induced, NRP1 signaling. We conclude that NRP1 enables ECM-induced filopodia formation for tip cell function during sprouting angiogenesis.

Concepts: Inflammation, Gene, Extracellular matrix, Angiogenesis, Cell membrane, Blood vessel, Endothelium, Filopodia