A role for local calcium signaling in rapid synaptic partner selection by dendritic filopodia

Synapse elimination is an important process underlying the establishment of functional neuronal networks during development. Here, we tested the idea that neurons select among potential synaptic partners already during initial contact formation between dendritic filopodia and axons-well before mature synapses are established. We show that filopodia frequently make contact with axons, and while some contacts are selectively stabilized, many are short-lived. More specifically, we demonstrate that contacts with a certain population of GABAergic axons never get stabilized, indicating that filopodia already early on select between different types of axons. Local dendritic calcium transients that are independent of glutamate occur within seconds after contact formation, and their frequency is high where contacts become stabilized and low at short-lived contacts. Thus, filopodia are capable of choosing between potential synaptic partners well before a mature synapse is established.     

MIG-10/lamellipodin and AGE-1/PI3K promote axon guidance and outgrowth in response to slit and netrin

BACKGROUND: The cytoplasmic C. elegans protein MIG-10 affects cell migrations and is related to mammalian proteins that bind phospholipids and Ena/VASP actin regulators. In cultured cells, mammalian MIG-10 promotes lamellipodial growth and Ena/VASP proteins induce filopodia. RESULTS: We show here that during neuronal development, mig-10 and the C. elegans Ena/VASP homolog unc-34 cooperate to guide axons toward UNC-6 (netrin) and away from SLT-1 (Slit). The single mutants have relatively mild phenotypes, but mig-10; unc-34 double mutants arrest early in development with severe axon guidance defects. In axons that are guided toward ventral netrin, unc-34 is required for the formation of filopodia and mig-10 increases the number of filopodia. In unc-34 mutants, developing axons that lack filopodia are still guided to netrin through lamellipodial growth. In addition to its role in axon guidance, mig-10 stimulates netrin-dependent axon outgrowth in a process that requires the age-1 phosphoinositide-3 lipid kinase but not unc-34. CONCLUSIONS: mig-10 and unc-34 organize intracellular responses to both attractive and repulsive axon guidance cues. mig-10 and age-1 lipid signaling promote axon outgrowth; unc-34 and to a lesser extent mig-10 promote filopodia formation. Surprisingly, filopodia are largely dispensable for accurate axon guidance.     

Activation of the neurokinin 3 receptor promotes filopodia growth and sprouting in rat embryonic hypothalamic cells

Members of the tachykinin family have trophic effects on developing neurons. The tachykinin neurokinin 3 receptor (NK3R) appears early in embryonic development; during the peak birthdates of hypothalamic neurons, but its involvement in neural development has not been examined. To address its possible role, immortalized embryonic hypothalamic neurons (CLU209) were treated with CellMask, a plasma membrane stain, or the membranes were imaged in CLU209 cells that were transfected with a pEGFP‐NK3R expression vector. Nontransfected cells and transfected cells were then treated with senktide, a NK3R agonist, or Dulbecco's Modified Eagle's Medium (DMEM) and time‐lapse confocal images were captured for the following 30 min. Compared to DMEM, senktide treatment led to filopodia initiation from the soma of both nontransfected and transfected CLU209 cells. These filopodia had diameters and lengths of approximately 200 nm and 3 µm, respectively. Pretreatment with an IP3 receptor blocker, 2‐aminoethoxydiphenyl borate (2‐APB), prevented the senktide‐induced growth in filopodia; demonstrating that NK3R‐induced outgrowth of filopodia likely involves the release of intracellular calcium. Exposure of transfected CLU209 cells to senktide for 24 h led to further growth of filopodia and processes that extended 10–20 µm. A mathematical model, composed of a linear and population model was developed to account for the dynamics of filopodia growth during a timescale of minutes. The results suggest that the ligand‐induced activation of NK3R affects early developmental processes by initiating filopodia formation that are a prerequisite for neuritogenesis. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 12–22, 2015     

Sleep contributes to dendritic spine formation and elimination in the developing mouse somatosensory cortex

Sleep is maximal during early postnatal life when rapid and extensive synapse remodeling occurs. It remains unknown whether and how sleep affects synapse development and plasticity. Using transcranial two‐photon microscopy, we examined the formation and elimination of fluorescently labeled dendritic spines and filopodia of Layer 5 pyramidal neurons in the barrel cortex of 3‐week‐old mice during wakefulness and sleep. We observed high turnover of dendritic protrusions over 2 h in both wake and sleep states. The formation rate of dendritic spines or filopodia over 2 h was comparable between the two states. The elimination rate of dendritic spines or filopodia was lower during 2‐h wakefulness than during 2‐h sleep. Similar results were observed on dendritic protrusion dynamics over 12‐h light/dark cycle when mice spent more time asleep or awake. The substantial remodeling of dendritic protrusions during the sleep state supports the notion that sleep plays an important role in the development and plasticity of synaptic connections in the mouse cortex. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Focal adhesion kinase regulates actin nucleation and neuronal filopodia formation during axonal growth

The establishment of neural circuits depends on the ability of axonal growth cones to sense their surrounding environment en route to their target. To achieve this, a coordinated rearrangement of cytoskeleton in response to extracellular cues is essential. Although previous studies have identified different chemotropic and adhesion molecules that influence axonal development, the molecular mechanism by which these signals control the cytoskeleton remains poorly understood. Here, we show that in vivo conditional ablation of the focal adhesion kinase gene (Fak) from mouse hippocampal pyramidal cells impairs axon outgrowth and growth cone morphology during development, which leads to functional defects in neuronal connectivity. Time-lapse recordings and in vitro FRAP analysis indicate that filopodia motility is altered in growth cones lacking FAK, probably owing to deficient actin turnover. We reveal the intracellular pathway that underlies this process and describe how phosphorylation of the actin nucleation-promoting factor N-WASP is required for FAK-dependent filopodia formation. Our study reveals a novel mechanism through which FAK controls filopodia formation and actin nucleation during axonal development.     

Eps8 Regulates Axonal Filopodia in Hippocampal Neurons in Response to Brain-Derived Neurotrophic Factor (BDNF)

The regulation of filopodia plays a crucial role during neuronal development and synaptogenesis. Axonal filopodia, which are known to originate presynaptic specializations, are regulated in response to neurotrophic factors. The structural components of filopodia are actin filaments, whose dynamics and organization are controlled by ensembles of actin-binding proteins. How neurotrophic factors regulate these latter proteins remains, however, poorly defined. Here, using a combination of mouse genetic, biochemical, and cell biological assays, we show that genetic removal of Eps8, an actin-binding and regulatory protein enriched in the growth cones and developing processes of neurons, significantly augments the number and density of vasodilator-stimulated phosphoprotein (VASP)-dependent axonal filopodia. The reintroduction of Eps8 wild type (WT), but not an Eps8 capping-defective mutant, into primary hippocampal neurons restored axonal filopodia to WT levels. We further show that the actin barbed-end capping activity of Eps8 is inhibited by brain-derived neurotrophic factor (BDNF) treatment through MAPK-dependent phosphorylation of Eps8 residues S624 and T628. Additionally, an Eps8 mutant, impaired in the MAPK target sites (S624A/T628A), displays increased association to actin-rich structures, is resistant to BDNF-mediated release from microfilaments, and inhibits BDNF-induced filopodia. The opposite is observed for a phosphomimetic Eps8 (S624E/T628E) mutant. Thus, collectively, our data identify Eps8 as a critical capping protein in the regulation of axonal filopodia and delineate a molecular pathway by which BDNF, through MAPK-dependent phosphorylation of Eps8, stimulates axonal filopodia formation, a process with crucial impacts on neuronal development and synapse formation.     

Cytoneme-mediated cell-to-cell signaling during development

Cell-to-cell communication is vital for animal tissues and organs to develop and function as organized units. Throughout development, intercellular communication is crucial for the generation of structural diversity, mainly by the regulation of differentiation and growth. During these processes, several signaling molecules function as messengers between cells and are transported from producing to receptor cells. Thus, a tight spatial and temporal regulation of signaling transport is likely to be critical during morphogenesis. Despite much experimental and theoretical work, the question as to how these signals move between cells remains. Cell-to-cell contact is probably the most precise spatial and temporal mechanism for the transference of signaling molecules from the producing to the receiving cells. However, most of these molecules can also function at a distance between cells that are not juxtaposed. Recent research has shown the way in which cells may achieve direct physical contact and communication through actin-based filopodia. In addition, increasing evidence is revealing the role of such filopodia in regulating spatial patterning during development; in this context, the filopodia are referred to as cytonemes. In this review, we highlight recent work concerning the roles of these filopodia in cell signaling during development. The processes that initiate and regulate the formation, orientation and dynamics of cytonemes are poorly understood but are potentially extremely important areas for our knowledge of intercellular communication.     

Developmental relocation of presynaptic terminals along distinct types of dendritic filopodia

Dendritic filopodia are long thin protrusions occurring predominantly on developing neurons. Data from different systems suggest a range of crucial functions for filopodia in central circuit formation, including steering of dendritic growth, branch formation, synaptogenesis, and spinogenesis. Are the same filopodia competent to mediate all these processes, do filopodia acquire different functions through development, or do different filopodial types with distinct functions exist? In this study, 3-dimensional reconstructions from confocal image stacks demonstrate the existence of two morphologically and functionally distinct types of filopodia located on the dendritic tips versus the dendritic shafts of the same developing motoneuron. During dendritic growth, both filopodial types undergo a process of stage-specific morphogenesis. Using novel quantification strategies of 3-dimensional co-localization analysis for immunocytochemically labeled presynaptic specializations along postsynaptic filopodia, we find that presynaptic terminals accumulate along filopodia towards the dendrites at both stable dendritic shafts and on growing dendritic tips. On tips, this is likely to reflect synaptotrophic growth of the dendrite. At stable shafts, however, presynaptic sites become relocated along filopodia towards dendritic branches. This indicates the interactive growth of both pre- and postsynaptic partner towards one another during synaptogenesis, using filopodia as guides.     

Drebrin coordinates the actin and microtubule cytoskeleton during the initiation of axon collateral branches

Drebrin is a cytoskeleton‐associated protein which can interact with both actin filaments and the tips of microtubules. Its roles have been studied mostly in dendrites, and the functions of drebrin in axons are less well understood. In this study, we analyzed the role of drebrin, through shRNA‐mediated depletion and overexpression, in the collateral branching of chicken embryonic sensory axons. We report that drebrin promotes the formation of axonal filopodia and collateral branches in vivo and in vitro. Live imaging of cytoskeletal dynamics revealed that drebrin promotes the formation of filopodia from precursor structures termed axonal actin patches. Endogenous drebrin localizes to actin patches and depletion studies indicate that drebrin contributes to the development of patches. In filopodia, endogenous drebrin localizes to the proximal portion of the filopodium. Drebrin was found to promote the stability of axonal filopodia and the entry of microtubule plus tips into axonal filopodia. The effects of drebrin on the stabilization of filopodia are independent of its effects on promoting microtubule targeting to filopodia. Inhibition of myosin II induces a redistribution of endogenous drebrin distally into filopodia, and further increases branching in drebrin overexpressing neurons. Finally, a 30 min treatment with the branch‐inducing signal nerve growth factor increases the levels of axonal drebrin. This study determines the specific roles of drebrin in the regulation of the axonal cytoskeleton, and provides evidence that drebrin contributes to the coordination of the actin and microtubule cytoskeleton during the initial stages of axon branching. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1092–1110, 2016     

Myosin-X Induces Filopodia by Multiple Elongation Mechanism

Filopodia are actin-rich finger-like cytoplasmic projections extending from the leading edge of cells. Unconventional myosin-X is involved in the protrusion of filopodia. However, the underlying mechanism of myosin-X-induced filopodia formation is obscure. Here, we studied the movements of myosin-X during filopodia protrusion using a total internal reflection microscope to clarify the mechanism of myosin-X-induced filopodia formation. Myosin-X was recruited to the discrete site at the leading edge where it assembles with exponential kinetics before the filopodia extension. The myosin-X-induced filopodia showed repeated extension-retraction cycles with each extension of 2.4 μm, which was critical to produce long filopodia. Myosin-X, lacking the FERM domain, could move to the tip as does the wild type. However, it was transported toward the cell body during filopodia retraction, did not undergo multiple extension-retraction cycles, and failed to produce long filopodia. During the filopodia protrusion, the single molecules of full-length myosin-X moved within filopodia. The majority of the fluorescence spots showed two-step photobleaching, suggesting that the moving myosin-X is a dimer. Deletion of the FERM domain did not change the movement at the single molecule level with the same velocity of ∼600 nm/s as wild-type, suggesting that the myosin-X in filopodia moves without interaction with the attached membrane via the FERM domain. Based upon these results, we have proposed a model of myosin-X-induced filopodia protrusion.     

Developmental regulation of spine and filopodial motility in primary visual cortex: reduced effects of activity and sensory deprivation

Dendritic protrusions are highly motile during postnatal development. Although spine morphological plasticity could be associated with synaptic plasticity, the function of rapid spine/filopodial motility is still unknown. To investigate the role of spine motility in the development of the visual cortex and its relation with critical periods, we used two-photon imaging of neurons from layers receiving visual input in developing mouse primary visual cortex and compared motility between control and visually deprived animals. Spine and filopodia motility was prominent during early synaptogenesis (P11-P13) but greatly decreased after P15. This "switch" was coincident with a 2.5-fold increase in protrusion density and spine formation. Spine motility was not regulated during the critical period for monocular deprivation (P19-P34). Moreover, delaying the critical period by dark rearing did not delay the normal developmental decrease of spine motility, but caused a modest further reduction in motility at P28-P35. Dark rearing and enucleation also mildly reduced spine motility before eye opening and dark rearing reduced the proportion of filopodia. We conclude that (1) rapid spine motility is not related to critical period plasticity, but is likely to play a role in early synaptogenesis, and (2) neuronal activity stimulates spine motility during synaptogenesis and promotes the appearance of dendritic filopodia.     

Paralemmin-1, a modulator of filopodia induction is required for spine maturation

Dendritic filopodia are thought to participate in neuronal contact formation and development of dendritic spines; however, molecules that regulate filopodia extension and their maturation to spines remain largely unknown. Here we identify paralemmin-1 as a regulator of filopodia induction and spine maturation. Paralemmin-1 localizes to dendritic membranes, and its ability to induce filopodia and recruit synaptic elements to contact sites requires protein acylation. Effects of paralemmin-1 on synapse maturation are modulated by alternative splicing that regulates spine formation and recruitment of AMPA-type glutamate receptors. Paralemmin-1 enrichment at the plasma membrane is subject to rapid changes in neuronal excitability, and this process controls neuronal activity-driven effects on protrusion expansion. Knockdown of paralemmin-1 in developing neurons reduces the number of filopodia and spines formed and diminishes the effects of Shank1b on the transformation of existing filopodia into spines. Our study identifies a key role for paralemmin-1 in spine maturation through modulation of filopodia induction.     

Septin-driven coordination of actin and microtubule remodeling regulates the collateral branching of axons

Axon branching is fundamental to the development of the peripheral and central nervous system. Branches that sprout from the axon shaft are termed collateral or interstitial branches. Collateral branching of axons requires the formation of filopodia from actin microfilaments (F-actin) and their engorgement with microtubules (MTs) that splay from the axon shaft. The mechanisms that drive and coordinate the remodeling of actin and MTs during branch morphogenesis are poorly understood. Septins comprise a family of GTP-binding proteins that oligomerize into higher-order structures, which associate with membranes and the actin and microtubule cytoskeleton. Here, we show that collateral branching of axons requires SEPT6 and SEPT7, two interacting septins. In the axons of sensory neurons, both SEPT6 and SEPT7 accumulate at incipient sites of filopodia formation. We show that SEPT6 localizes to axonal patches of F-actin and increases the recruitment of cortactin, a regulator of Arp2/3-mediated actin polymerization, triggering the emergence of filopodia. Conversely, SEPT7 promotes the entry of axonal MTs into filopodia, enabling the formation of collateral branches. Surprisingly, septins provide a novel mechanism for the collateral branching of axons by coordinating the remodeling of the actin and microtubule cytoskeleton.     

Regulation of neuronal growth cone filopodia by nitric oxide

Nitric oxide (NO) has been proposed to play an important role during neuronal development. Since many of its effects occur during the time of growth cone pathfinding and target interaction, we here test the hypothesis that part of NO's effects might be exerted at the growth cone. We found that low concentrations of the NO-donors DEA/NO, SIN-1, and SNP caused a rapid and transient elongation of filopodia as well as a reduction in filopodial number. These effects resulted from distinct changes in filopodial extension and retraction rates. Our novel findings suggest that NO could play a physiological role by temporarily changing a growth cone's morphology and switching its behavior from a close-range to a long-range exploratory mode. We subsequently dissected the pathway by which NO acted on growth cones. The effect of NO donors on filopodial length could be blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase (sGC), indicating that NO acted via sGC. Supporting this idea, injection of cyclic GMP (cGMP) mimicked the effect of NO donors on growth cone filopodia. Moreover, application of NO-donors as well as injection of cGMP elicited a rapid and transient rise in intracellular calcium in growth cones, indicating that NO acted via cGMP to elevate calcium. This calcium rise, as well as the morphological effects of SIN-1 on filopodia, were blocked by preventing calcium entry. Given the role of filopodia in axonal guidance, our new data suggest that NO could function at the neuronal growth cone as an intracellular and/or intercellular signaling molecule by affecting steering decisions during neuronal pathfinding.     

Fascin1-Dependent Filopodia are Required for Directional Migration of a Subset of Neural Crest Cells

Directional migration of neural crest (NC) cells is essential for patterning the vertebrate embryo, including the craniofacial skeleton. Extensive filopodial protrusions in NC cells are thought to sense chemo-attractive/repulsive signals that provide directionality. To test this hypothesis, we generated null mutations in zebrafish fascin1a (fscn1a), which encodes an actin-bundling protein required for filopodia formation. Homozygous fscn1a zygotic null mutants have normal NC filopodia due to unexpected stability of maternal Fscn1a protein throughout NC development and into juvenile stages. In contrast, maternal/zygotic fscn1a null mutant embryos (fscn1a MZ) have severe loss of NC filopodia. However, only a subset of NC streams display migration defects, associated with selective loss of craniofacial elements and peripheral neurons. We also show that fscn1a-dependent NC migration functions through cxcr4a/cxcl12b chemokine signaling to ensure the fidelity of directional cell migration. These data show that fscn1a-dependent filopodia are required in a subset of NC cells to promote cell migration and NC derivative formation, and that perdurance of long-lived maternal proteins can mask essential zygotic gene functions during NC development.     

Plasticity-related Gene 5 (PRG5) Induces Filopodia and Neurite Growth and Impedes Lysophosphatidic Acid– and Nogo-A–mediated Axonal Retraction

Members of the plasticity-related gene (PRG1-4) family are brain-specific integral membrane proteins and implicated in neuronal plasticity, such as filopodia formation and axon growth after brain lesion. Here we report on the cloning of a novel member of the PRG family, PRG5, with high homologies to PRG3. PRG5 is regulated during brain and spinal cord development and is exclusively allocated within the nervous system. When introduced in neurons, PRG5 is distributed in the plasma membrane and induces filopodia as well as axon elongation and growth. Conversely, siRNA mediated knockdown of PRG5 impedes axon growth and disturbs filopodia formation. Here we show that PRG5 induces filopodia growth independently of Cdc42. Moreover, axon collapse and RhoA activation induced by LPA and myelin-associated neurite inhibitor Nogo-A is attenuated in the presence of PRG5, although direct activation of the RhoA-Rho-PIP5K kinase pathway abolishes PRG5 -formed neurites. Thus, we describe here the identification of a novel member of the PRG family that induces filopodia and axon elongation in a Cdc42-independent manner. In addition, PRG5 impedes brain injury-associated growth inhibitory signals upstream of the RhoA-Rho kinase pathway.     

Filopodia: molecular architecture and cellular functions

Filopodia are thin, actin-rich plasma-membrane protrusions that function as antennae for cells to probe their environment. Consequently, filopodia have an important role in cell migration, neurite outgrowth and wound healing and serve as precursors for dendritic spines in neurons. The initiation and elongation of filopodia depend on the precisely regulated polymerization, convergence and crosslinking of actin filaments. The increased understanding of the functions of various actin-associated proteins during the initiation and elongation of filopodia has provided new information on the mechanisms of filopodia formation in distinct cell types.     

c-Abl phosphorylates Dok1 to promote filopodia during cell spreading

Filopodia are dynamic F-actin structures that cells use to explore their environment. c-Abl tyrosine kinase promotes filopodia during cell spreading through an unknown mechanism that does not require Cdc42 activity. Using an unbiased approach, we identified Dok1 as a specific c-Abl substrate in spreading fibroblasts. When activated by cell adhesion, c-Abl phosphorylates Y361 of Dok1, promoting its association with the Src homology 2 domain (SH2)/SH3 adaptor protein Nck. Each signaling component was critical for filopodia formation during cell spreading, as evidenced by the finding that mouse fibroblasts lacking c-Abl, Dok1, or Nck had fewer filopodia than cells reexpressing the product of the disrupted gene. Dok1 and c-Abl stimulated filopodia in a mutually interdependent manner, indicating that they function in the same signaling pathway. Dok1 and c-Abl were both detected in filopodia of spreading cells, and therefore may act locally to modulate actin. Our data suggest a novel pathway by which c-Abl transduces signals to the actin cytoskeleton through phosphorylating Dok1 Y361 and recruiting Nck.     

Regulation of neuronal growth cone filopodia by nitric oxide depends on soluble guanylyl cyclase

Nitric oxide has been proposed to play an important role in neuronal development. We have previously shown that growth cones from an identified neuron, B5, in the snail Helisoma trivolvis, respond to nitric oxide (NO) donors by increasing the length of their filopodia within minutes of application (Van Wagenen and Rehder, 1999). This effect was mediated through a cGMP-induced increase in [Ca2+]i and resulted in an enlargement of the growth cone's action radius, suggesting that NO could function as a signaling molecule during neuronal pathfinding. We show here that NO functions as a specific rather than a general regulator of growth cone filopodia, because another identified neuron from the same ganglion, B19, failed to respond to NO with an increase in filopodial length. We found that, contrary to B5 neurons, B19 growth cones contained little or no soluble guanylyl cyclase (sGC) immunoreactivity, presumably preventing their response to NO. This hypothesis was supported by the finding that the sGC activator YC-1 (10 microM) had no effect on B19 filopodia but induced elongation of B5 filopodia. These results indicate that the effects of NO can be quite specific, and raise the interesting possibility that neurons could selectively tune in to NO by differentially expressing the target enzyme sGC in the appropriate cellular location during critical developmental stages. In addition, our NADPH-diaphorase staining and anti-NOS immunohistochemisty suggest that growth cones of B5 neurons, but not of B19 neurons, could be a source of NO, making NO a potential intra- and transcellular messenger.     

An Ultrastructural Investigation on Vitellophage Invasion of the Yolk Mass during and after Germ Band Formation in Embryos of the Stick Insect Carausius morosus Br.

Developing embryos of the stick insect Carausius morosus were examined ultrastructurally with a view to studying vitellophage invasion of the yolk mass during and after germ band formation. Newly laid eggs in C.morosus have a unique yolk fluid compartment surrounded by a narrow fringe of cytoplasm comprising several small yolk granules. Vitellophages originate mainly from a thin layer of stem cells, the so-called yolk cell membrane, interposed between the germ band and the yolk mass. Throughout development, a thin basal lamina separates the yolk cell membrane from the overlying embryo.
Vitellophages extend from the yolk cell membrane with long cytoplasmic processes or filopodia to invade the central yolk mass. Along their route of entrance, filopodia engulf portions of the yolk mass and sequester it into membrane-bounded granules. As this process continues, the yolk mass is gradually partitioned into a number of yolk granules inside the vitellophages.
Later in development, the yolk cell membrane is gradually replaced by the endodermal cells that emerge from the anterior and posterior embryonic rudiments. From this stage of development onwards, vitellophages remain attached to the basal lamina through long filopodia extending between the endodermal cells. Yolk confined in different vitellophagic cells appears heterogeneous both in density and texture, suggesting that yolk degradation may be spatially differentiated.