Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells

High levels of the neuron-specific protein kinase C substrate, B-50 (= GAP43), are present in neurites and growth cones during neuronal development and regeneration. This suggests a hitherto nonelucidated role of this protein in neurite outgrowth. Comparable high levels of B-50 arise in the pheochromocytoma PC12 cell line during neurite formation. To get insight in the putative growth-associated function of B-50, we compared its ultrastructural localization in naive PC12 cells with its distribution in nerve growth factor (NGF)- or dibutyryl cyclic AMP (dbcAMP)-treated PC12 cells. B-50 immunogold labeling of cryosections of untreated PC12 cells is mainly associated with lysosomal structures, including multivesicular bodies, secondary lysosomes, and Golgi apparatus. The plasma membrane is virtually devoid of label. However, after 48-h NGF treatment of the cells, B-50 immunoreactivity is most pronounced on the plasma membrane. Highest B-50 immunoreactivity is observed on plasma membranes surrounding sprouting microvilli, lamellipodia, and filopodia. Outgrowing neurites are scattered with B-50 labeling, which is partially associated with chromaffin granules. In NGF-differentiated PC12 cells, B-50 immunoreactivity is, as in untreated cells, also associated with organelles of the lysosomal family and Golgi stacks. B-50 distribution in dbcAMP-differentiated cells closely resembles that in NGF-treated cells. The altered distribution of B-50 immunoreactivity induced by differentiating agents indicates a shift of the B-50 protein towards the plasma membrane. This translocation accompanies the acquisition of neuronal features of PC12 cells and points to a neurite growth-associated role for B-50, performed at the plasma membrane at the site of protrusion.     

Life at the leading edge

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

Changes in the distribution of adenylate cyclase activity in PC 12 cells under the influence of nerve growth factor

Changes in distribution of adenylate cyclase in PC 12 cells under the influence of nerve growth factor (NGF) have been studied using cytochemical methods. The adenylate cyclase activity was predominantly associated with the plasma membrane. In cell cultures without NGF the activity was revealed on the contacting surfaces of cell aggregates; single grains of reaction product were revealed on exposed cell surface only in cultures with a high cell density. One day after administration of NGF, the adenylate cyclase activity on exposed cell surface increased, and three days later the whole cell surface was covered with lead sediment. The enzyme activity was also revealed in growth cones, filopodia and microcytospheres. The role of adenydlate cyclase system in neuron-like differentiation of PC 12 cells is discussed.     

Alpha-actinins, calspectin (brain spectrin or fodrin), and actin participate in adhesion and movement of growth cones

We have used biochemical and immunocytochemical techniques to investigate the possible involvement of membrane cytoskeletal elements such as alpha-actinin, calspectin (brain spectrin or fodrin), and actin in growth cone activities. During NGF-induced differentiation of PC12 cells, alpha-actinin increased in association with neurite outgrowth and was predominantly distributed throughout the entire growth cone and the distal portion of neurites. Filopodial movements were sensitive to Ca2+ flux. Two types of alpha-actinin, with Ca2(+)-sensitive and -insensitive actin binding abilities, were identified in the differentiated cells. Ca2(+)-sensitive alpha-actinin and actin filaments were concentrated in filopodia. The Ca2(+)-insensitive protein was distributed from the body of the growth cone to the distal portion of neurites, corresponding to the substratum-adhesive sites. The location of calspectin in growth cones was similar to that of the Ca2(+)-insensitive alpha-actinin. These results are consistent with the hypothesis that Ca2(+)-sensitive alpha-actinin and actin filaments are involved in Ca2(+)-dependent filopodial movement and Ca2(+)-insensitive alpha-actinin and calspectin are associated with adhesion of growth cones.     

Peripheral hyaline blebs (podosomes) of macrophages

The plasmalemma and hyaline ectoplasm together constitute the sensory and motor organ of macrophages. The purpose of this study was to isolate this cell fraction in order to analyze it biochemically and functionally. Brief sonification of warmed rabbit lung macrophages caused release of heterodisperse hyaline blebs and filopodia, which were easily collected by differential centrifugation. Viewed in the electron microscope, these structures consisted of membrane-bounded sacs principally containing actin filaments. Some contained secondary lysosomes. They were enriched threefold over whole cell homogenates in specific adenylate cyclase activity and in trichloroacetic-acid-precipitable (125)I when derived from cells labeled with 125(I) by means of a lactoperoxidase-catalyzed reaction. These markers were found to have identical isopycnic densitites when macrophage homogenates were subjected to sedimentation in a focusing sucrose density gradient system, and these markers had densities distinct from those of other cytoplasmic organelles. These markers were therefore assumed to be associated with macrophage plasma membranes. The specific β- glucuronidase activity of the bleb fraction was similar to that of homogenates, but the blebs had considerably lower specific succinic dehydrogenase activity and RNA content, and DNA was undetectable. Electrophoresis of blebs solubilized in sodium dodecyl sulfate on polyacrylamide gels revealed polypeptides co-migrating with macrophage actin-binding protein, myosin, and actin; blebs also had EDTA-activated adenosine triphosphatase activity characteristic of myosin. The concentrations of actin-binding protein and myosin were higher in blebs than in cells or cytoplasmic extracts, whereas actin concentrations were similar (relative to extracts) or only slightly greater (than in cells). Blebs and intact cells had high lactate dehydrogenase activities in the presence but not the absence of Triton X-100. Blebs and cells oxidased 1-[(14)C]glucose, and the rate of glucose oxidation was increased substantially in the presence of latex beads. We conclude that intact sacs of plasmalemma encasing contractile proteins and cytoplasmic enzymes can be isolated from macrophages. They are enriched in myosin and actin-binding protein, indicating that the contractile apparatus is regulated in the cell periphery. These structures have the capacity to respond to environmental signals. We suggest the name "podosomes" for them because of their resemblance to macrophage pseudopodia. We propose that podosome formation results from rapid dissolution of the cortical gel when the membrane is in an actively extended configuration.     

Role of the growth-associated protein B-50/GAP-43 in neuronal plasticity

The neuronal phosphoprotein B-50/GAP-43 has been implicated in neuritogenesis during developmental stages of the nervous system and in regenerative processes and neuronal plasticity in the adult. The protein appears to be a member of a family of acidic substrates of protein kinase C (PKC) that bind calmodulin at low calcium concentrations. Two of these substrates, B-50 and neurogranin, share the primary sequence coding for the phospho- and calmodulin-binding sites and might exert similar functions in axonal and dendritic processes, respectively. In the adult brain, B-50 is exclusively located at the presynaptic membrane. During neuritogenesis in cell culture, the protein is translocated to the growth cones, i.e., into the filopodia. In view of many positive correlations between B-50 expression and neurite outgrowth and the specific localization of B-50, a role in growth cone function has been proposed. Its phosphorylation state may regulate the local intracellular free calmodulin and calcium concentrations or vice versa. Both views link the B-50 protein to processes of signal transduction and transmitter release.     

Differences in cortical actin structure and dynamics document that different types of blebs are formed by distinct mechanisms

Bleb formation has been studied by specifically targeting major factors controlling this process, such as microtubule disassembly, local actin depolymerization, and increased pressure. At least two different types of blebs (types 1 and 2) formed by different mechanisms and possibly a third type (type 3) can be documented at the front of living polarized cells expressing green fluorescent protein-actin and/or in fixed and stained cells. Type 1 blebs (membrane/cortex dissociation blebs) formed by dissociation of the plasma membrane from cortical actin develop cytoplasmic actin layers associated with restriction rings. They can be induced by the microtubule-disassembling agent colchicine. Type 2 blebs (cortical actin disassembly blebs) form after disassembly of the cortical actin layer in the presence of latrunculin A. Restriction rings without a cytoplasmic actin layer occur in a transition zone between the intact cortical actin layer of the cell body and the compromised actin layer of the bleb. Evidence for a third type of bleb (type 3), showing an intact cortical actin layer but no cytoplasmic actin layer and no recognizable relationship between the actin cytoskeleton and the restriction ring, has been obtained by passive cell deformation in micropipettes, which increases pressure. Repolymerization of the cortical actin layer does not necessarily result in bleb retraction. Once formed, restriction rings do not narrow, suggesting that they result from isometric contraction. A simplified classification scheme has been developed to relate the type of bleb to specific signals or cell functions. Its application shows that spontaneously blebbing cells form almost exclusively type 1 blebs.     

Ezrin Ser66 phosphorylation regulates invasion and metastasis of esophageal squamous cell carcinoma cells by mediating filopodia formation

BackgroundEzrin, links the plasma membrane to the actin cytoskeleton, and plays an important role in the development and progression of human esophageal squamous cell carcinoma (ESCC). However, the roles of ezrin S66 phosphorylation in tumorigenesis of ESCC remain unclear.MethodsDistribution of ezrin in membrane and cytosol fractions was examined by analysis of detergent-soluble/-insoluble fractions and cytosol/membrane fractionation. Both immunofluorescence and live imaging were used to explore the role of ezrin S66 phosphorylation in the behavior of ezrin and actin in cell filopodia. Cell proliferation, migration and invasion of ESCC cells were investigated by proliferation and migration assays, respectively. Tumorigenesis, local invasion and metastasis were assessed in a nude mouse model of regional lymph node metastasis.ResultsEzrin S66 phosphorylation enhanced the recruitment of ezrin to the membrane in ESCC cells. Additionally, non-phosphorylatable ezrin (S66A) significantly prevented filopodia formation, as well as caused a reduction in the number, length and lifetime of filopodia. Moreover, functional experiments revealed that expression of non-phosphorylatable ezrin (S66A) markedly suppressed migration and invasion but not proliferation of ESCC cells in vitro, and attenuated local invasion and regional lymph node metastasis, but not primary tumor growth of ESCC cells in vivo.ConclusionEzrin S66 phosphorylation enhances filopodia formation, contributing to the regulation of invasion and metastasis of esophageal squamous cell carcinoma cells.     

Immunocytochemical evidence for colocalization in neurite growth cones of actin and myosin and their relationship to cell-substratum adhesions

Sensory neurons from 8- to 11-day chick embryos were cultured on polyornithine-treated coverslips, fixed with glutaraldehyde, and stained for immunofluorescent localization of actin. Actin was distributed in a fibrous form in the growth cones, extending into filopodia and lamellipodial expansions of the growth cone margin. Often, these actin fibers were located at sites of linear adhesions to the glass substratum, as viewed by interference reflection optics. Our antisera to myosin did not recognize myosin in glutaraldehyde-fixed cells, and paraformaldehyde, which preserves the antigenicity of myosin, did not fix embryonic neurons well. Thus, myosin was localized in NGF-stimulated PC12 cells, whose morphology is better preserved by paraformaldehyde. Within the growth cones of PC12 neurites, actin and myosin are distributed into fibrous arrays which resemble the actin fibers seen in the growth cones of sensory neurons. Thus, actomyosin-like contractile forces may be exerted in neurite growth cones. These forces may act in concert with cell-substratum adhesive bonds to move the growth cone across the substratum or move organelles within the growth cone.     

Requirement of a vasodilator-stimulated phosphoprotein family member for cell adhesion,the formation of filopodia,and chemotaxis in dictyostelium

We have examined the function of a member of the vasodilator-stimulated phosphoprotein family of proteins (DdVASP) in Dictyostelium. Ddvasp null cells lack filopodia, whereas targeting DdVASP to the plasma membrane with a myristoyl tag results in a significant increase in filopodia. The proline-rich domain-Ena/VASP homology 2 structure is required for both actin polymerization activity and filopodia formation. Ddvasp null cells exhibit a chemotaxis defect, which appears to be due to a defect in the ability of the cells to properly adhere to the substratum and to suppress lateral pseudopod extension. We demonstrate that during chemotaxis, the anterior approximately 50% of the cell lifts from the substratum and remains elevated for up to 1 min. These defects lead to a significant decrease in chemotaxis efficiency. DdVASP localizes to the leading edge in migrating cells and to the tips of filopodia. In addition, Ddvasp null cells have a defect in particle adhesion but internalize particles normally. Our results provide new insights into the function of DdVASP in controlling the actin cytoskeleton during chemotaxis and filopodia formation.     

Vinculin-deficient PC12 cell lines extend unstable lamellipodia and filopodia and have a reduced rate of neurite outgrowth

We have studied the role of vinculin in regulating integrin-dependent neurite outgrowth in PC12 cells, a neuronal cell line. Vinculin is a cytoskeletal protein believed to mediate interactions between integrins and the actin cytoskeleton. In differentiated PC12 cells, the cell body, the neurite, and the growth cone contain vinculin. Within the growth cone, both the proximal region of "consolidation" and the more distal region consisting of lamellipodia and filopodia contain vinculin. To study the role of vinculin in neurite outgrowth, we generated vinculin-deficient isolates of PC12 cell lines by transfection with vectors expressing antisense vinculin RNA. In some of these cell lines, vinculin levels were reduced to 18-23% of normal levels. In the vinculin-deficient cell lines, neurite outgrowth on laminin was significantly reduced. In time-lapse analysis, growth cones advanced much more slowly than normal. Further analysis indicated that this deficit could be explained in large part by changes in the behaviors of filopodia and lamellipodia. Filopodia were formed in normal numbers, extended at normal rates, and extended to approximately normal lengths, but were much less stable in the vinculin deficient compared to control PC12 cells. Similarly, lamellipodia formed and grew nearly normally, but were dramatically less stable in the vinculin- deficient cells. This can account for the reduction in rate of growth cone advance. These results indicate that interactions between integrins and the actin-based cytoskeleton are necessary for stability of both filopodia and lamellipodia.     

Actin polymerization and intracellular solvent flow in cell surface blebbing

The cortical actin gel of eukaryotic cells is postulated to control cell surface activity. One type of protrusion that may offer clues to this regulation are the spherical aneurysms of the surface membrane known as blebs. Blebs occur normally in cells during spreading and alternate with other protrusions, such as ruffles, suggesting similar protrusive machinery is involved. We recently reported that human melanoma cell lines deficient in the actin filament cross-linking protein, ABP-280, show prolonged blebbing, thus allowing close study of blebs and their dynamics. Blebs expand at different rates of volume increase that directly predict the final size achieved by each bleb. These rates decrease as the F-actin concentration of the cells increase over time after plating on a surface, but do so at lower concentrations in ABP-280 expressing cells. Fluorescently labeled actin and phalloidin injections of blebbing cells indicate that a polymerized actin structure is not present initially, but appears later and is responsible for stopping further bleb expansion. Therefore, it is postulated that blebs occur when the fluid-driven expansion of the cell membrane is sufficiently rapid to initially outpace the local rate of actin polymerization. In this model, the rate of intracellular solvent flow driving this expansion decreases as cortical gelation is achieved, whether by factors such as ABP-280, or by concentrated actin polymers alone, thereby leading to decreased size and occurrence of blebs. Since the forces driving bleb extension would always be present in a cell, this process may influence other cell protrusions as well.     

Accumulation of actin in subsets of pioneer growth cone filopodia in response to neural and epithelial guidance cues in situ

Directed outgrowth of neural processes must involve transmission of signals from the tips of filopodia to the central region of the growth cone. Here, we report on the distribution and dynamics of one possible element in this process, actin, in live growth cones which are reorienting in response to in situ guidance cues. In grasshopper embryonic limbs, pioneer growth cones respond to at least three types of guidance cues: a limb axis cue, intermediate target cells, and a circumferential band of epithelial cells. With time-lapse imaging of intracellularly injected rhodamine-phalloidin and rhodamine-actin, we monitored the distribution of actin during growth cone responses to these cues. In distal limb regions, accumulation of actin in filopodia and growth cone branches accompanies continued growth, while reduction of actin accompanies withdrawal. Where growth cones are reorienting to intermediate target cells, or along the circumferential epithelial band, actin selectively accumulates in the proximal regions of those filopodia that have contacted target cells or are extending along the band. Actin accumulations can be retrogradely transported along filopodia, and can extend into the central region of the growth cone. These results suggest that regulation and translocation of actin may be a significant element in growth cone steering.     

Fluxes of Water through Aquaporin 9 Weaken Membrane-Cytoskeleton Anchorage and Promote Formation of Membrane Protrusions

All modes of cell migration require rapid rearrangements of cell shape, allowing the cell to navigate within narrow spaces in an extracellular matrix. Thus, a highly flexible membrane and a dynamic cytoskeleton are crucial for rapid cell migration. Cytoskeleton dynamics and tension also play instrumental roles in the formation of different specialized cell membrane protrusions, viz. lamellipodia, filopodia, and membrane blebs. The flux of water through membrane-anchored water channels, known as aquaporins (AQPs) has recently been implicated in the regulation of cell motility, and here we provide novel evidence for the role of AQP9 in the development of various forms of membrane protrusion. Using multiple imaging techniques and cellular models we show that: (i) AQP9 induced and accumulated in filopodia, (ii) AQP9-associated filopodial extensions preceded actin polymerization, which was in turn crucial for their stability and dynamics, and (iii) minute, local reductions in osmolarity immediately initiated small dynamic bleb-like protrusions, the size of which correlated with the reduction in osmotic pressure. Based on this, we present a model for AQP9-induced membrane protrusion, where the interplay of water fluxes through AQP9 and actin dynamics regulate the cellular protrusive and motile activity of cells.     

Mechanics and dynamics of actin-driven thin membrane protrusions

Motile cells explore their surrounding milieu by extending thin dynamic protrusions, or filopodia. The growth of filopodia is driven by actin filament bundles that polymerize underneath the cell membrane. We compute the mechanical and dynamical features of the protrusion growth process by explicitly incorporating the flexible plasma membrane. We find that a critical number of filaments are needed to generate net filopodial growth. Without external influences, the filopodium can extend indefinitely up to the buckling length of the F-actin bundle. Dynamical calculations show that the protrusion speed is enhanced by the thermal fluctuations of the membrane; a filament bundle encased in a flexible membrane grows much faster. The protrusion speed depends directly on the number and spatial arrangement of the filaments in the bundle and whether the filaments are tethered to the membrane. Filopodia also attract each other through distortions of the membrane. Spatially close filopodia will merge to form a larger one. Force-velocity relationships mimicking micromanipulation experiments testing our predictions are computed.     

12/15-lipoxygenase translocation enhances site-specific actin polymerization in macrophages phagocytosing apoptotic cells

The enzyme 12/15-lipoxygenase (12/15-LO) introduces peroxyl groups in a position-specific manner into unsaturated fatty acids in certain cells, but the role of such enzymatic lipid peroxidation remains poorly defined. Here we report a novel function for 12/15-LO in mouse peritoneal macrophages. When macrophages were coincubated with apoptotic cells, the enzyme translocated from cytosol to the plasma membrane and was more extensively concentrated at sites where macrophages bound apoptotic cells, colocalizing with polymerized actin of emerging filopodia. Disruption of F-actin did not prevent the 12/15-LO translocation. In contrast, inhibition of the 12/15-LO activity, or utilization of genetically engineered macrophages in which the 12/15-LO gene has been disrupted, greatly reduced actin polymerization in phagocytosing macrophages. Lysates of 12/15-LO-deficient macrophages had significantly lower ability to promote in vitro actin polymerization than the lysates of wild type macrophages. These studies suggest that the 12/15-LO enzyme plays a major role in local control of actin polymerization in macrophages in response to interaction with apoptotic cells.     

Increased Neurite Outgrowth Induced by Inhibition of Protein Tyrosine Kinase Activity in PC12 Pheochromocytoma Cells

Abstract: Genistein and other inhibitors of protein tyrosine kinases were examined for effects on neurite elongation and growth cone morphology in the rat PC12 pheochromocytoma cell line. Genistein increased the rate of neurite elongation in PC12 cells grown on a collagen/polylysine substratum after priming with nerve growth factor (NGF), but had no effect on undifferentiated cells. Steady-state levels of phosphotyrosine-modified proteins (105, 59, 52, and 46 kDa) were reduced in NGF-primed cells by genistein treatment. The target of genistein action did not appear to be the NGF receptor/ trk tyrosine kinase because the presence of NGF in cultures of NGF-primed cells was not necessary for genistein-stimulated neurite outgrowth. The tyrosine kinase inhibitors tyrphostin RG508964 and herbimycin A also increased the rate of neurite elongation in NGF-primed PC12 cells. Video-enhanced differential interference contrast microscopy revealed that growth cones of genistein-treated cells had less complex morphologies and were less dynamic than untreated cells, with short filopodia restricted to the leading edge, unlike untreated cells whose growth cones exhibited longer, more numerous filopodia and lamellipodia, which remodeled continuously. These results suggest that protein tyrosine kinase activity in PC12 cells negatively regulates neurite outgrowth and directly or indirectly affects growth cone morphology.     

An Ultrastructural Study of Cell-to-Cell Membrane Interaction at Early Stages of Postnatal Ontogenesis of Cortical Brain Neurons in White Rats

In early postnatal ontogenesis of cerebral cortex (visual area) of the white rat, a wide distribution of different types of membrane contacts have been found between developing nervous cells and their processes. The following types of contacts were observed: 1. Penetration of thin filopodia into specialized invaginations having all the features of coated vesicles; 2. Contacts of filopodia with thickened surface membrane; 3. Contacts of opposit filopodia; 4. Contacts of membranes with reciprocal invaginations alternating with filopodia or surface blebs.
These types of membrane interaction were regularly distributed along the surface of cells and their processes, and were situated in close approximation to typical tight junctions and other adhesional complexes. As a rule, filopodia were components of axon branches, and almost all invaginations were situated on plasma membranes of cell bodies or on dendrites, although sometimes there were invaginations on axon profiles and filopodia on dendrites.
It is suggested that the distribution and structural specialization of these membrane contacts reflect their participation in the process of programmed cell-to-cell recognition that precedes the formation of synaptic contact. Other reports and the current data reveal the special morphogenetic role of membrane communication in the formation and stability of integrative cell systems.     

Mutagenesis of ser41 to ala inhibits the association of GAP-43 with the membrane skeleton of GAP-43-deficient PC12B cells: Effects on cell adhesion and the composition of neurite cytoskeleton and membrane

To investigate the molecular basis for GAP-43 function in axon outgrowth, we produced a mutant, GAP-43 (Ala₄₁), whose interaction with calmodulin in vitro was unaffected by increasing Ca²⁺ concentrations, and stably transfected it into GAP-43-deficient PC12B cells. Several lines that expressed wild-type or mutant protein at levels that resembled endogenous GAP-43 expression in PC12 controls were subcloned and characterized. GAP-43 (Ala₄₁) was significantly more extractable with Nonidet P-40 and less tightly associated with the membrane skeleton than the wild-type protein. Furthermore, GAP-43 (Ala₄₁) expression by PC12B cells profoundly affected their phenotype: First, observation of living cells using video-enhanced microscopy revealed irregular plasma membranes with numerous blebs and protrusions and neurites that appeared thin and varicose. Second, both the cells' ability to remain attached to laminin substrates and the amount of α1β1 integrin expressed on the cell surface was significantly decreased. Finally, peripherin transport, which is abnormal in PC12B cells, could be rescued by transfection of wild-type GAP-43 but not the GAP-43 (Ala₄₁) mutant. The phenotypic abnormalities resemble other cell types in which membrane skeleton/plasma membrane interactions have been functionally decoupled, and our results are consistent with the notion that these interactions may be abnormal in GAP-43 (Ala₄₁)-expressing PC12B cells, either as a direct consequence of the mutation or arising secondarily to the altered availability of calmodulin in the growing neurite. © 1996 John Wiley & Sons, Inc.     

Coordination of Membrane and Actin Cytoskeleton Dynamics during Filopodia Protrusion

Leading edge protrusion of migrating cells involves tightly coordinated changes in the plasma membrane and actin cytoskeleton. It remains unclear whether polymerizing actin filaments push and deform the membrane, or membrane deformation occurs independently and is subsequently stabilized by actin filaments. To address this question, we employed an ability of the membrane-binding I-BAR domain of IRSp53 to uncouple the membrane and actin dynamics and to induce filopodia in expressing cells. Using time-lapse imaging and electron microscopy of IRSp53-I-BAR-expressing B16F1 melanoma cells, we demonstrate that cells are not able to protrude or maintain durable long extensions without actin filaments in their interior, but I-BAR-dependent membrane deformation can create a small and transient space at filopodial tips that is subsequently filled with actin filaments. Moreover, the expressed I-BAR domain forms a submembranous coat that may structurally support these transient actin-free protrusions until they are further stabilized by the actin cytoskeleton. Actin filaments in the I-BAR-induced filopodia, in contrast to normal filopodia, do not have a uniform length, are less abundant, poorly bundled, and display erratic dynamics. Such unconventional structural organization and dynamics of actin in I-BAR-induced filopodia suggests that a typical bundle of parallel actin filaments is not necessary for generation and mechanical support of the highly asymmetric filopodial geometry. Together, our data suggest that actin filaments may not directly drive the protrusion, but only stabilize the space generated by the membrane deformation; yet, such stabilization is necessary for efficient protrusion.