Two distinct filopodia populations at the growth cone allow to sense nanotopographical extracellular matrix cues to guide neurite outgrowth

Background

The process of neurite outgrowth is the initial step in producing the neuronal processes that wire the brain. Current models about neurite outgrowth have been derived from classic two-dimensional (2D) cell culture systems, which do not recapitulate the topographical cues that are present in the extracellular matrix (ECM) in vivo. Here, we explore how ECM nanotopography influences neurite outgrowth.

Methodology/Principal Findings

We show that, when the ECM protein laminin is presented on a line pattern with nanometric size features, it leads to orientation of neurite outgrowth along the line pattern. This is also coupled with a robust increase in neurite length. The sensing mechanism that allows neurite orientation occurs through a highly stereotypical growth cone behavior involving two filopodia populations. Non-aligned filopodia on the distal part of the growth cone scan the pattern in a lateral back and forth motion and are highly unstable. Filopodia at the growth cone tip align with the line substrate, are stabilized by an F-actin rich cytoskeleton and enable steady neurite extension. This stabilization event most likely occurs by integration of signals emanating from non-aligned and aligned filopodia which sense different extent of adhesion surface on the line pattern. In contrast, on the 2D substrate only unstable filopodia are observed at the growth cone, leading to frequent neurite collapse events and less efficient outgrowth.

Conclusions/Significance

We propose that a constant crosstalk between both filopodia populations allows stochastic sensing of nanotopographical ECM cues, leading to oriented and steady neurite outgrowth. Our work provides insight in how neuronal growth cones can sense geometric ECM cues. This has not been accessible previously using routine 2D culture systems.     

Filopodia initiation

Filopodia are long, slender, actin-rich cellular protrusions, which recently have become a focus of cell biology research because of their proposed roles as sensory and exploratory organelles that allow for “intelligent” cell behavior. Actin nucleation, elongation and bundling are believed to be essential for filopodia formation and functions. However, the identity of actin filament nucleators responsible for the initiation of filopodia remains controversial. Two alternative models, the convergent elongation and tip nucleation, emphasize two different actin filament nucleators, the Arp2/3 complex or formins, respectively, as key players during filopodia initiation. Although these two models in principle are not mutually exclusive, it is important to understand which of them is actually employed by cells. In this review, we discuss the existing evidence regarding the relative roles of the Arp2/3 complex and formins in filopodia initiation.     

Ena/VASP regulates mDia2-initiated filopodial length,dynamics, and function

Filopodia are long plasma membrane extensions involved in the formation of adhesive, contractile, and protrusive actin-based structures in spreading and migrating cells. Whether filopodia formed by different molecular mechanisms equally support these cellular functions is unresolved. We used Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP)–deficient MV^D7 fibroblasts, which are also devoid of endogenous mDia2, as a model system to investigate how these different actin regulatory proteins affect filopodia morphology and dynamics independently of one another. Filopodia initiated by either Ena/VASP or mDia2 contained similar molecular inventory but differed significantly in parameters such as number, length, F-actin organization, lifetime, and protrusive persistence. Moreover, in the absence of Ena/VASP, filopodia generated by mDia2 did not support initiation of integrin-dependent signaling cascades required for adhesion and subsequent lamellipodial extension, thereby causing a defect in early cell spreading. Coexpression of VASP with constitutively active mDia2^M/A rescued these early adhesion defects. We conclude that Ena/VASP and mDia2 support the formation of filopodia with significantly distinct properties and that Ena/VASP regulates mDia2-initiated filopodial morphology, dynamics, and function.     

Dissecting regulatory networks of filopodia formation in a Drosophila growth cone model

F-actin networks are important structural determinants of cell shape and morphogenesis. They are regulated through a number of actin-binding proteins. The function of many of these proteins is well understood, but very little is known about how they cooperate and integrate their activities in cellular contexts. Here, we have focussed on the cellular roles of actin regulators in controlling filopodial dynamics. Filopodia are needle-shaped, actin-driven cell protrusions with characteristic features that are well conserved amongst vertebrates and invertebrates. However, existing models of filopodia formation are still incomplete and controversial, pieced together from a wide range of different organisms and cell types. Therefore, we used embryonic Drosophila primary neurons as one consistent cellular model to study filopodia regulation. Our data for loss-of-function of capping proteins, enabled, different Arp2/3 complex components, the formin DAAM and profilin reveal characteristic changes in filopodia number and length, providing a promising starting point to study their functional relationships in the cellular context. Furthermore, the results are consistent with effects reported for the respective vertebrate homologues, demonstrating the conserved nature of our Drosophila model system. Using combinatorial genetics, we demonstrate that different classes of nucleators cooperate in filopodia formation. In the absence of Arp2/3 or DAAM filopodia numbers are reduced, in their combined absence filopodia are eliminated, and in genetic assays they display strong functional interactions with regard to filopodia formation. The two nucleators also genetically interact with enabled, but not with profilin. In contrast, enabled shows strong genetic interaction with profilin, although loss of profilin alone does not affect filopodia numbers. Our genetic data support a model in which Arp2/3 and DAAM cooperate in a common mechanism of filopodia formation that essentially depends on enabled, and is regulated through profilin activity at different steps.     

Filopodia and adhesion in cancer cell motility

Slender bundled actin containing plasma membrane protrusions, called filopodia, are important for many essential cellular processes like cell adhesion, migration, angiogenesis and the formation of cell-cell contacts. In migrating cells, filopodia are the pioneers at the leading edge which probe the environment for cues. Integrins are cell surface adhesion receptors critically implicated in cell migration and they are transported actively to filopodia tips by an unconventional myosin, myosin-X. Integrin mediated adhesion stabilizes filopodia and promotes cell migration even though integrins are not essential for filopodia initiation. Myosin-X binds also PtdIns(3,4,5)P₃ and this regulates its activation and localization to filopodia. Filopodia stimulate cell migration in many cell types and increased filopodia density has been described in cancer. Furthermore, several proteins implicated in filopodia formation, like fascin, are also relevant for cancer progression. To investigate this further, we performed a meta-analysis of the expression profiles of 10 filopodia-linked genes in human breast cancer. These data implicated that several different filopodia-inducing genes may contribute in a collective manner to cancer progression and the high metastasis rates associated with basal-type breast carcinomas.Key words: filopodia, integrins, migration, cancer     

A Highlights from MBoC Selection: Cell type–dependent mechanisms for formin-mediated assembly of filopodia

Filopodia are finger-like protrusions from the plasma membrane and are of fundamental importance to cellular physiology, but the mechanisms governing their assembly are still in question. One model, called convergent elongation, proposes that filopodia arise from Arp2/3 complex–nucleated dendritic actin networks, with factors such as formins elongating these filaments into filopodia. We test this model using constitutively active constructs of two formins, FMNL3 and mDia2. Surprisingly, filopodial assembly requirements differ between suspension and adherent cells. In suspension cells, Arp2/3 complex is required for filopodial assembly through either formin. In contrast, a subset of filopodia remains after Arp2/3 complex inhibition in adherent cells. In adherent cells only, mDia1 and VASP also contribute to filopodial assembly, and filopodia are disproportionately associated with focal adhesions. We propose an extension of the existing models for filopodial assembly in which any cluster of actin filament barbed ends in proximity to the plasma membrane, either Arp2/3 complex dependent or independent, can initiate filopodial assembly by specific formins.     

Arp2/3 complex activity in filopodia of spreading cells

Background  

Cells use filopodia to explore their environment and to form new adhesion contacts for motility and spreading. The Arp2/3 complex has been implicated in lamellipodial actin assembly as a major nucleator of new actin filaments in branched networks. The interplay between filopodial and lamellipodial protrusions is an area of much interest as it is thought to be a key determinant of how cells make motility choices.     

Maturation of Filopodia Shaft Adhesions Is Upregulated by Local Cycles of Lamellipodia Advancements and Retractions

While cell-substrate adhesions that form between the protruding edge of a spreading cell and flat surfaces have been studied extensively, processes that regulate the maturation of filopodia adhesions are far less characterized. Since little is known about how the kinetics of formation or disassembly of filopodia adhesions is regulated upon integration into the lamellum, a kinetic analysis of the formation and disassembly of filopodia adhesions was conducted at the leading edge of β3-integrin-EGFP-expressing rat embryonic fibroblasts spreading on fibronectin-coated glass or on soft polyacrylamide gels. Filopodia β3-integrin adhesions matured only if the lamellipodium in their immediate vicinity showed cyclic protrusions and retractions. Filopodia β3-integrin shaft adhesions elongated rapidly when they were overrun by the advancing lamellipodium. Subsequently and once the lamellipodium stopped its advancement at the distal end of the filopodia β3-integrin adhesion, these β3-integrin shaft adhesions started to grow sidewise and colocalize with the newly assembled circumferential actin stress fibers. In contrast, the suppression of the cyclic protrusions and retractions of the lamellipodium by blocking myosin light chain kinase suppressed the growth of filopodia adhesion and resulted in the premature disassembly of filopodia adhesions. The same failure to stabilize those adhesions was found for the advancing lamellipodium that rapidly overran filopodia shaft adhesions without pausing as seen often during fast cell spreading. In turn, plating cells on soft polyacrylamide gels resulted in a reduction of lamellipodia activity, which was partially restored locally by the presence of filopodia adhesions. Thus filopodia adhesions could also mature and be integrated into the lamellum for fibroblasts on soft polyacrylamide substrates.     

Islands of Conformational Stability for Filopodia

Filopodia are long, thin protrusions formed when bundles of fibers grow outwardly from a cell surface while remaining closed in a membrane tube. We study the subtle issue of the mechanical stability of such filopodia and how this depends on the deformation of the membrane that arises when the fiber bundle adopts a helical configuration. We calculate the ground state conformation of such filopodia, taking into account the steric interaction between the membrane and the enclosed semiflexible fiber bundle. For typical filopodia we find that a minimum number of fibers is required for filopodium stability. Our calculation elucidates how experimentally observed filopodia can obviate the classical Euler buckling condition and remain stable up to several tens of . We briefly discuss how experimental observation of the results obtained in this work for the helical-like deformations of enclosing membrane tubes in filopodia could possibly be observed in the acrosomal reactions of the sea cucumber Thyone, and the horseshoe crab Limulus. Any realistic future theories for filopodium stability are likely to rely on an accurate treatment of such steric effects, as analysed in this work.     

Asynchronous adaptive time step in quantitative cellular automata modeling

Background  

The behaviors of cells in metazoans are context dependent, thus large-scale multi-cellular modeling is often necessary, for which cellular automata are natural candidates. Two related issues are involved in cellular automata based multi-cellular modeling: how to introduce differential equation based quantitative computing to precisely describe cellular activity, and upon it, how to solve the heavy time consumption issue in simulation.     

Filopodial focal complexes direct adhesion and force generation towards filopodia outgrowth

Filopodia are key structures within many cells that serve as sensors constantly probing the local environment. Although filopodia are involved in a number of different cellular processes, their function in migration is often analyzed with special focus on early processes of filopodia formation and the elucidation of filopodia molecular architecture. An increasing number of publications now describe the entire life cycle of filopodia, with analyses from the initial establishment of stable filopodium-substrate adhesion to their final integration into the approaching lamellipodium. We and others can now show the structural and functional dependence of lamellipodial focal adhesions as well as of force generation and transmission on filopodial focal complexes and filopodial actin bundles. These results were made possible by new high resolution imaging techniques as well as by recently developed elastomeric substrates and theoretical models. The data additionally provide strong evidence that formation of new filopodia depends on previously existing filopodia through a repetitive filopodial elongation of the stably adhered filopodial tips. In this commentary we therefore hypothesize a highly coordinated mechanism that regulates filopodia formation, adhesion, protein composition and force generation in a filopodia dependent step by step process.     

Surfing along Filopodia: A Particle Transport Revealed by Molecular-Scale Fluctuation Analyses

Filopodia perform cellular functions such as environmental sensing or cell motility, but they also grab for particles and withdraw them leading to an increased efficiency of phagocytic uptake. Remarkably, withdrawal of micron-sized particles is also possible without noticeable movements of the filopodia. Here, we demonstrate that polystyrene beads connected by optical tweezers to the ends of adherent filopodia of J774 macrophages, are transported discontinuously toward the cell body. After a typical resting time of 1–2 min, the cargo is moved with alternating velocities, force constants, and friction constants along the surface of the filopodia. This surfing-like behavior along the filopodium is recorded by feedback-controlled interferometric three-dimensional tracking of the bead motions at 10–100 kHz. We measured transport velocities of up to 120 nm/s and transport forces of ∼70 pN. Small changes in position, fluctuation width, and temporal correlation, which are invisible in conventional microscopy, indicate molecular reorganization of transport-relevant proteins in different phases of the entire transport process. A detailed analysis implicates a controlled particle transport with fingerprints of a nanoscale unbinding/binding behavior. The manipulation and analysis methods presented in our study may also be helpful in other fields of cellular biophysics.     

Hybrid metabolic flux analysis: combining stoichiometric and statistical constraints to model the formation of complex recombinant products

Background  

Stoichiometric models constitute the basic framework for fluxome quantification in the realm of metabolic engineering. A recurrent bottleneck, however, is the establishment of consistent stoichiometric models for the synthesis of recombinant proteins or viruses. Although optimization algorithms for in silico metabolic redesign have been developed in the context of genome-scale stoichiometric models for small molecule production, still rudimentary knowledge of how different cellular levels are regulated and phenotypically expressed prevents their full applicability for complex product optimization.     

Myosin-X is an unconventional myosin that undergoes intrafilopodial motility

Filopodia are thin cellular protrusions that are important in cell motility and neuronal growth cone guidance. The actin filaments that make up the core of a filopodium undergo continuous retrograde flow towards the cell body. Surface receptors or particles can couple to this retrograde flow and can also move forward to the tips of filopodia, although the molecular basis of forward transport is unknown. We report here that myosin-X (Myo10 or M10), the founding member of a novel class of myosins, localizes to the tips of filopodia and undergoes striking forward and rearward movements within filopodia, which we term intrafilopodial motility. The movements of the GFP-M10 puncta correspond to forward and rearward movements of phase-dense granules along the filopodia. Finally, overexpressing full-length M10 (but not truncated forms of M10) causes an increase in the number and length of filopodia, indicating that M10 or its cargo may function in filopodial dynamics. The localization and movements of M10 strongly suggest that it functions as a motor for intrafilopodial motility.     

Target recognition by the archenteron during sea urchin gastrulation

During sea urchin gastrulation filopodia are sent out by secondary mesenchyme cells (SMCs) at the tip of the archenteron in continual cycles of extension, attachment, and retraction. Eventually the archenteron ceases its elongation and its tip localizes to the animal pole region of the embryo (Gustafson and Kinnander, 1956, Exp. Cell Res. 11, 36-57; Dan and Okazaki, 1956, Biol. Bull. 110, 29-42). We have investigated the mechanisms and specificity of this localization by analyzing filopodial behavior and by experimental manipulation of the interaction of the archenteron with the animal pole region. When the tip of the archenteron nears the animal pole, some filopodia make contact with a well-defined locus within this region. Filopodia that make contact with the locus remain attached 20-50 times longer than attachments observed at any other site along the blastocoel wall. The SMCs bearing the long-lived filopodia eventually change their phenotype by flattening and spreading onto this region. Several lines of experimental evidence indicate that contact with the animal pole locus, or "target" region, is crucial for the change in phenotype of the SMCs: (1) the phenotypic change can be induced precociously by bringing the animal pole region within reach of the tip of the archenteron early in gastrulation. Precocious contact with other regions of the blastocoel wall does not induce a similar change. (2) The phenotypic change can be delayed by placing the animal pole out of reach late in gastrulation, resulting in artificial prolongation of exploratory behavior by filopodia. (3) Ectopic combinations of animal pole ectoderm and archenterons in fused multiple embryos and chimaeras result in attachment of archenterons to the nearest available target, and (4) freely migrating SMCs are observed to migrate randomly within the blastocoel, then stop at the animal pole and undergo the change in phenotype. Filopodia rapidly attach to the animal pole when the shape of early gastrulae is altered such that the animal pole is less than 35 microns from the tip of the archenteron, even though such attachments only occur in normal embryos at the 2/3-3/4 gastrula stage. Since it has previously been shown that the archenteron elongates autonomously to 2/3 of its final length (Hardin, 1988, Development 103, 317-324), it appears that autonomous extension of the archenteron is required to place filopodia close enough to the animal pole to allow them to interact with it.(ABSTRACT TRUNCATED AT 400 WORDS)     

Filopodia and adhesion in cancer cell motility

Slender bundled actin containing plasma membrane protrusions, called filopodia, are important for many essential cellular processes like cell adhesion, migration, angiogenesis and the formation of cell-cell contacts. In migrating cells, filopodia are the pioneers at the leading edge which probe the environment for cues. Integrins are cell surface adhesion receptors critically implicated in cell migration and they are transported actively to filopodia tips by an unconventional myosin, myosin-X. Integrin mediated adhesion stabilizes filopodia and promotes cell migration even though integrins are not essential for filopodia initiation. Myosin-X binds also PIP3 and this regulates its activation and localization to filopodia. Filopodia stimulate cell migration in many cell types and increased filopodia density has been described in cancer. Furthermore, several proteins implicated in filopodia formation, like fascin, are also relevant for cancer progression. To investigate this further, we performed a meta-analysis of the expression profiles of 10 filopodia-linked genes in human breast cancer. These data implicated that several different filopodia inducing genes may contribute in a collective manner to cancer progression and the high metastasis rates associated with basal-type breast carcinomas.     

Statistical model comparison applied to common network motifs

Background  

Network motifs are small modules that show interesting functional and dynamic properties, and are believed to be the building blocks of complex cellular processes. However, the mechanistic details of such modules are often unknown: there is uncertainty about the motif architecture as well as the functional form and parameter values when converted to ordinary differential equations (ODEs). This translates into a number of candidate models being compatible with the system under study. A variety of statistical methods exist for ranking models including maximum likelihood-based and Bayesian methods. Our objective is to show how such methods can be applied in a typical systems biology setting.     

Toward the Structure of Dynamic Membrane-Anchored Actin Networks: An Approach Using Cryo-Electron Tomography

In the cortex of a motile cell, membrane-anchored actin filaments assemble into structures of varying shape and function. Filopodia are distinguished by a core of bundled actin filaments within finger-like extensions of the membrane. In a recent paper by Medalia et al¹ cryo-electron tomography has been used to reconstruct, from filopodia of Dictyostelium cells, the 3-dimensional organization of actin filaments in connection with the plasma membrane. A special arrangement of short filaments converging toward the filopod''s tip has been called a “terminal cone”. In this region force is applied for protrusion of the membrane. Here we discuss actin organization in the filopodia of Dictyostelium in the light of current views on forces that are generated by polymerizing actin filaments, and on the resistance of membranes against deformation that counteracts these forces.Key Words: actin network, cytoskeleton, Dictyostelium, electron tomography, filopodia, membrane bending     

SH2B1 increases the numbers of IRSp53-induced filopodia

BackgroundFilopodia are actin-rich membrane protrusions that play instrumental roles in development, cell migration, pathogen detection, and wound healing. During neurogenesis, filopodium formation precedes the formation of dendrites and spines. The insulin receptor substrate protein of 53 kDa (IRSp53) has been implicated in regulating the formation of filopodia. Our previous results suggest that a signaling adaptor protein SH2B1β is required for neurite outgrowth of hippocampal neurons and neurite initiation of PC12 cells. Thus, we hypothesize that IRSp53 and SH2B1β may act together to regulate filopodium formation.MethodsTo determine the contribution of IRSp53 and SH2B1β in the formation of filopodia, we transiently transfect IRSp53 and/or SH2B1β to 293T cells. Cell morphology and protein distribution are assessed via confocal microscopy and subcellular fractionation. Total numbers of filopodia and filopodium numbers per perimeter are calculated to show the relative contribution of IRSp53 and SH2B1β.ResultsIn this study, we show that SH2B1β interacts with IRSp53 and increases the number of IRSp53-induced filopodia. One mechanism for this enhancement is that IRSp53 recruits SH2B1β to the plasma membrane to actively promote membrane protrusion. The increased numbers of filopodia likely result from SH2B1-mediated cytoplasmic extension and thus increased cell perimeter as well as IRSp53-mediated filopodium formation.ConclusionsTaken together, this study provides a novel finding that SH2B1β interacts with IRSp53-containing complexes to increase the number of filopodia.General significanceA better understanding of how SH2B1β and IRSp53 promote filopodium formation may have clinical implication in neurogenesis and regeneration.     

Human Lsg1 defines a family of essential GTPases that correlates with the evolution of compartmentalization

Background  

Compartmentalization is a key feature of eukaryotic cells, but its evolution remains poorly understood. GTPases are the oldest enzymes that use nucleotides as substrates and they participate in a wide range of cellular processes. Therefore, they are ideal tools for comparative genomic studies aimed at understanding how aspects of biological complexity such as cellular compartmentalization evolved.