Evidence for fascin cross-links between the actin filaments in coelomocyte filopodia

Coelomocyte filopodia are composed primarily of actin and a 57000 D protein, which is antigenically related to the actin cross-linking protein found in needles produced in vitro from extracts of sea urchin oocytes. In this report we present electron micrographs of the filopodia which show longitudinal striations corresponding to bundled, F-actin filaments and transverse bands corresponding to a cross-linking protein. The periodicities of the spacings are 90 and 130 Å respectively. Interestingly, the filaments in the filopodia are less well ordered than those in needles, probably as a result of the speed with which the filopodia are assembled in the transforming coelomocytes. These results indicate that the well ordered needles are a good in vitro model for important in vivo structures.     

Role of the actin bundling protein fascin in growth cone morphogenesis: localization in filopodia and lamellipodia

Growth cones at the distal tips of growing nerve axons contain bundles of actin filaments distributed throughout the lamellipodium and that project into filopodia. The regulation of actin bundling by specific actin binding proteins is likely to play an important role in many growth cone behaviors. Although the actin binding protein, fascin, has been localized in growth cones, little information is available on its functional significance. We used the large growth cones of the snail Helisoma to determine whether fascin was involved in temporal changes in actin filaments during growth cone morphogenesis. Fascin localized to radially oriented actin bundles in lamellipodia (ribs) and filopodia. Using a fascin antibody and a GFP fascin construct, we found that fascin incorporated into actin bundles from the beginning of growth cone formation at the cut end of axons. Fascin associated with most of the actin bundle except the proximal 6--12% adjacent to the central domain, which is the region associated with actin disassembly. Later, during growth cone morphogenesis when actin ribs shortened, the proximal fascin-free zone of bundles increased, but fascin was retained in the distal, filopodial portion of bundles. Treatment with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which phosphorylates fascin and decreases its affinity for actin, resulted in loss of all actin bundles from growth cones. Our findings suggest that fascin may be particularly important for the linear structure and dynamics of filopodia and for lamellipodial rib dynamics by regulating filament organization in bundles.     

Formation of filopodia in coelomocytes: localization of fascin, a 58,000 dalton actin cross-linking protein.

Echinoderm coelomocytes or phagocytes purified in the petaloid stage will attach to a glass substrate and form large circumferential lamellIpodia. Hypotonic shock will induce quantitative transformation to a filopodial form. Differentiation of the peripheral cytoplasm begins at the cell edge, when phase dense rods composed of actin filaments start to form. These structures, which eventually form the cores of filopodia, continue to grow, lengthen and extend deeper into the cytoplasm. In the final stage, the plasma membrane retracts down around a core to form a filopodium. Antibody against a 58,000 dalton protein isolated from sea urchin egg actin gels has been used to study a rather striking redistribution of this protein in the peripheral cytoplasm of the coelomocyte during the transformation sequence. This protein is known to organize actin filaments in vitro into linear paracrystalline arrays with a distinct 11 nm banding pattern by forming cross-links between adjacent actin filaments. In the early stage of the transformation, indirect immunofluorescence indicates a random distribution of this protein in the circumferential lamellipodia. Organization is first seen at the cell edge, where fluorescent rods coincident with the phase-dense structures start to form. These rods lengthen, extend deeper into the cytoplasm and become more intensely fluorescent. After membrane retraction, cells with individual, intensely stained filopodia are visible. The known chemistry of the actin cross-linking protein (M_r = 58,000) and its redistribution during the transformation sequence are consistent with the idea that this protein functions to organize F actin into filopodial cores by cross-linking adjacent actin filaments. We have named this protein “fascin,” because it organizes actin filaments, both in vivo and in vitro, into linear arrays or fascicles. Antibody staining shows a second population of these actin cross-linking molecules localized in the perinuclear cytoplasm. In this region, fascin appears to function to maintain a stable circumnuclear cage structure which is part of the coelomocyte cytoskeleton.     

Intrinsic pump-conduit behavior of lymphangions

Lymphangions, segments of lymphatic vessels bounded by valves, have characteristics of both ventricles and arteries. They can act primarily like pumps when actively transporting lymph against a pressure gradient. They also can act as conduit vessels when passively transporting lymph down a pressure gradient. This duality has implications for clinical treatment of several types of edema, since the strategy to optimize lymph flow may depend on whether it is most beneficial for lymphangions to act as pumps or conduits. To address this duality, we employed a simple computational model of a contracting lymphangion, predicted the flows at both positive and negative axial pressure gradients, and validated the results with in vitro experiments on bovine mesenteric vessels. This model illustrates that contraction increases flow for normal axial pressure gradients. With edema, limb elevation, or external compression, however, the pressure gradient might reverse, and lymph may flow passively down a pressure gradient. In such cases, the valves may be forced open during the entire contraction cycle. The vessel thus acts as a conduit, and contraction has the effect of increasing resistance to passive flow, thus inhibiting flow rather than promoting it. This analysis may explain a possible physiological benefit of the observed flow-mediated inhibition of the lymphatic pump at high flow rates.     

S100A4 downregulates filopodia formation through increased dynamic instability

Cell migration requires the initial formation of cell protrusions, lamellipodia and/or filopodia, the attachment of the leading lamella to extracellular cues and the formation and efficient recycling of focal contacts at the leading edge. The small calcium binding EF-hand protein S100A4 has been shown to promote cell motility but the direct molecular mechanisms responsible remain to be elucidated. In this work, we provide new evidences indicating that elevated levels of S100A4 affect the stability of filopodia and prevent the maturation of focal complexes. Increasing the levels of S100A4 in a rat mammary benign tumor derived cell line results in acquired cellular migration on the wound healing scratch assay. At the cellular levels, we found that high levels of S100A4 induce the formation of many nascent filopodia, but that only a very small and limited number of those can stably adhere and mature, as opposed to control cells, which generate fewer protrusions but are able to maintain these into more mature projections. This observation was paralleled by the fact that S100A4 overexpressing cells were unable to establish stable focal adhesions. Using different truncated forms of the S100A4 proteins that are unable to bind to myosin IIA, our data suggests that this newly identified functions of S100A4 is myosin-dependent, providing new understanding on the regulatory functions of S100A4 on cellular migration.     

S100A4 downregulates filopodia formation through increased dynamic instability

Cell migration requires the initial formation of cell protrusions, lamellipodia and/or filopodia, the attachment of the leading lamella to extracellular cues and the formation and efficient recycling of focal contacts at the leading edge. The small calcium binding EF-hand protein S100A4 has been shown to promote cell motility but the direct molecular mechanisms responsible remain to be elucidated. In this work, we provide new evidences indicating that elevated levels of S100A4 affect the stability of filopodia and prevent the maturation of focal complexes. Increasing the levels of S100A4 in a rat mammary benign tumor derived cell line results in acquired cellular migration on the wound healing scratch assay. At the cellular levels, we found that high levels of S100A4 induce the formation of many nascent filopodia, but that only a very small and limited number of those can stably adhere and mature, as opposed to control cells, which generate fewer protrusions but are able to maintain these into more mature projections. This observation was paralleled by the fact that S100A4 overexpressing cells were unable to establish stable focal adhesions. Using different truncated forms of the S100A4 proteins that are unable to bind to myosin II A, our data suggests that this newly identified functions of S100A4 is myosin-dependent, providing new understanding on the regulatory functions of S100A4 on cellular migration.Key words: filopodia, S100A4, cell migration, focal adhesion, cancer progression, myosin IIA     

Role of fascin in filopodial protrusion

In this study, the mechanisms of actin-bundling in filopodia were examined. Analysis of cellular localization of known actin cross-linking proteins in mouse melanoma B16F1 cells revealed that fascin was specifically localized along the entire length of all filopodia, whereas other actin cross-linkers were not. RNA interference of fascin reduced the number of filopodia, and remaining filopodia had abnormal morphology with wavy and loosely bundled actin organization. Dephosphorylation of serine 39 likely determined cellular filopodia frequency. The constitutively active fascin mutant S39A increased the number and length of filopodia, whereas the inactive fascin mutant S39E reduced filopodia frequency. Fluorescence recovery after photobleaching of GFP-tagged wild-type and S39A fascin showed that dephosphorylated fascin underwent rapid cycles of association to and dissociation from actin filaments in filopodia, with t(1/2) < 10 s. We propose that fascin is a key specific actin cross-linker, providing stiffness for filopodial bundles, and that its dynamic behavior allows for efficient coordination between elongation and bundling of filopodial actin filaments.     

The making of filopodia

Filopodia are rod-like cell surface projections filled with bundles of parallel actin filaments. They are found on a variety of cell types and have been ascribed sensory or exploratory functions. Filopodium formation is frequently associated with protrusion of sheet-like actin filament arrays called lamellipodia and membrane ruffles, but, in comparison to these structures, the molecular details underpinning the initiation and maintenance of filopodia are only just beginning to emerge. Recent advances have improved our understanding of the molecular requirements for filopodium protrusion and have yielded insights into the inter-relationships between lamellipodia and filopodia, the two 'sub-compartments' of the protrusive actin cytoskeleton.     

Cripto-1 localizes to dynamic and shed filopodia associated with cellular migration in glioblastoma cells

Cripto-1 is a protein participating in tissue orientation during embryogenesis but has also been implicated in a wide variety of cancers, such as colon, lung and breast cancer. Cripto-1 plays a role in the regulation of different pathways, including TGF-β/Smad and Wnt/β-catenin, which are highly associated with cell migration both during embryonal development and cancer progression. Little is known about the detailed subcellular localization of cripto-1 and how it participates in the directional movement of cells. In this study, the subcellular localization of cripto-1 in glioblastoma cells was investigated in vitro with high-resolution microscopy techniques. Cripto-1 was found to be localized to dynamic and shed filopodia and transported between cells through tunneling nanotubes. Our results connect the refined subcellular localization of cripto-1 to its functions in cellular orientation and migration.     

Modeling the formation of in vitro filopodia

Filopodia are bundles of actin filaments that extend out ahead of the leading edge of a crawling cell to probe its upcoming environment. In vitro experiments (Vignjevic et al. in J Cell Biol 160:951–962, 2003) have determined the minimal ingredients required for the formation of filopodia from the dendritic-like morphology of the leading edge. We model these experiments using kinetic aggregation equations for the density of growing bundle tips. In mean field, we determine the bundle size distribution to be broad for bundle sizes smaller than a characteristic bundle size above which the distribution decays exponentially. Two-dimensional simulations incorporating both bundling and cross-linking measure a bundle size distribution that agrees qualitatively with mean field. The simulations also demonstrate a nonmonotonicity in the radial extent of the dendritic region as a function of capping protein concentration, as was observed in experiments, due to the interplay between percolation and the ratcheting of growing filaments off a spherical obstacle.     

Organization of actin networks in intact filopodia

Filopodia are finger-like extensions of the cell surface that are involved in sensing the environment, in attachment of particles for phagocytosis, in anchorage of cells on a substratum, and in the response to chemoattractants or other guidance cues. Filopodia present an excellent model for actin-driven membrane protrusion. They grow at their tips by the assembly of actin and are stabilized along their length by a core of bundled actin filaments. To visualize actin networks in their native membrane-anchored state, filopodia of Dictyostelium cells were subjected to cryo-electron tomography. At the site of actin polymerization, a peculiar structure, the "terminal cone," is built of short filaments fixed with their distal end to the filopod's tip and with their proximal end to the flank of the filopod. The backbone of the filopodia consists of actin filaments that are shorter than the entire filopod and aligned in parallel or obliquely to the filopod's axis. We hypothesize that growth of the highly dynamic filopodia of Dictyostelium is accompanied by repetitive nucleation of actin polymerization at the filopod tip, followed by the rearrangement of filaments within the shaft.     

Roles of fascin in cell adhesion and motility

Many cell interactions depend on the assembly of cell protrusions; these include cell attachment and migration in the extracellular matrix, cell-cell communication, and the ability of cells to sense their local environment. Cell protrusions are extensions of the plasma membrane that are supported internally by actin-based structures that impart mechanical stiffness. Fascin is a small, globular actin-bundling protein that has emerging roles in diverse forms of cell protrusions and in cytoplasmic actin bundles. The fascin-actin interaction is under complex regulation from the extracellular matrix, peptide factors and other actin-binding proteins. Recent developments advance our understanding of the multifaceted regulation of fascin and the roles of fascin-containing structures in cell adhesion, motility and invasion in the life of vertebrate organisms.     

Lamellipodia and filopodia in metastasis and invasion

Advances in our understanding of the mechanisms of lamellipodia and filopodia assembly have led to a better concept of how cells move, including how the actin cytoskeleton might be important for the motility of metastatic cancer cells. The cytoskeleton is a potentially interesting target for prevention of invasion and metastasis. As key proteins are uncovered which regulate the assembly of actin-based structures, these need to be considered in light of whether they represent potential invasion and metastasis proteins.     

Robust patterns in the stochastic organization of filopodia

Background  

Filopodia are actin-based cellular projections that have a critical role in initiating and sustaining directional migration in vertebrate cells. Filopodia are highly dynamic structures that show a rich diversity in appearance and behavior. While there are several mathematical models of filopodia initiation and growth, testing the capacity of these theoretical models in predicting empirical behavior has been hampered by a surprising shortage of quantitative data related to filopodia. Neither is it clear how quantitatively robust the cellular filopodial network is and how perturbations alter it.     

Modulators of endothelial cell filopodia

Filopodia are an important feature of actively motile cells, probing the pericellular environment for chemotactic factors and other molecular cues that enable and direct the movement of the cell. They also act as points of attachment to the extracellular matrix for the cell, generating tension that may act to pull the cell forward and/or stabilize the cell as it moves. Endothelial cell motility is a critical aspect of angiogenesis, but only a limited number of molecules have been identified as specific regulators of endothelial cell filopodia. Recent reports, however, provide evidence for the involvement of PECAM-1, an endothelial cell adhesion and signaling molecule, in the formation of endothelial cell filopodia. This commentary will focus on these studies and their suggestion that at least two PECAM-1-regulated pathways are involved in the processes that enable filopodial protrusions by endothelial cells. Developing a more complete understanding of the role of PECAM-1 in mediating various endothelial cell activities, such as the extension of filopodia, will be essential for exploiting the therapeutic potential of targeting PECAM-1.     

On the mechanical stabilization of filopodia

We studied force-induced elongation of filopodia by coupling magnetic tweezers to the tip through the bacterial coat protein invasin, which couples the force generator to the actin bundles (through myosin X), thus impeding the growth of the actin plus end. Single force pulses (15–30 s) with amplitudes between 20 and 600 pN and staircase-like force scenarios (amplitudes, ∼50 pN; step widths, 30 s) were applied. In both cases, the responses consist of a fast viscoelastic deflection followed by a linear flow regime. The deflections are reversible after switching off the forces, suggesting a mechanical memory. The elongation velocity exhibits an exponential distribution (half-width <v_1/2>, ∼0.02 μm s⁻¹) and did not increase systematically with the force amplitudes. We estimate the bending modulus (0.4 × 10⁻²³ J m) and the number of actin filaments (∼10) by analyzing filopodium bending fluctuations. Sequestering of intracellular Ca²⁺ by BAPTA caused a strong reduction in the amplitude of elongation, whereas latrunculin A resulted in loss of the elastic response. We attribute the force-independent velocity to the elongation of actin bundles enabled by the force-induced actin membrane uncoupling and the reversibility by the treadmilling mechanism and an elastic response.     

Mechanism of actin filament bundling by fascin

Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four β-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in β-trefoil domains 1 and 3. The site in β-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in β-trefoil-3 is related by pseudo-2-fold symmetry to that in β-trefoil-1. The two sites are ～5 nm apart, resulting in a distance between actin filaments in the bundle of ～8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.     

Intrinsic fluctuations within cortical systems account for intertrial variability in human behavior

The resting brain is not silent, but exhibits organized fluctuations in neuronal activity even in the absence of tasks or stimuli. This intrinsic brain activity persists during task performance and contributes to variability in evoked brain responses. What is unknown is if this intrinsic activity also contributes to variability in behavior. In the current fMRI study, we identify a relationship between human brain activity in the left somatomotor cortex and spontaneous trial-to-trial variability in button press force. We then demonstrate that 74% of this brain-behavior relationship is attributable to ongoing fluctuations in intrinsic activity similar to those observed during resting fixation. In addition to establishing a functional and behavioral significance of intrinsic brain activity, these results lend new insight into the origins of variability in human behavior.     

Testis fascin (FSCN3): a novel paralog of the actin-bundling protein fascin expressed specifically in the elongate spermatid head

During spermiogenesis, significant morphological changes occur as round spermatids are remodeled into the fusiform shape of mature spermatozoa. These changes are correlated with a reorganization of microfilaments and microtubules in the head and tail regions of elongating spermatids. There is also altered expression of specialized actin- and tubulin-associated proteins. We report the characterization of a novel, spermatid-specific murine paralog of the actin-bundling protein fascin (FSCN1); this paralog is designated testis fascin or FSCN3. Testis fascin is distantly related to fascins but retains its primary sequence organization. cDNA clones of mouse testis fascin predict a 498 amino acid protein of molecular mass 56 kD that shares 29% identity with mouse fascin. Mapping of murine and human FSCN3 genes shows localization to the 7q31.3 chromosome. Northern analysis indicates that FSCN3 expression is highly specific to testis and that in situ hybridization further restricts expression to elongating spermatids. Antibodies raised against recombinant FSCN3 protein identify a band at 56 kD in testis, epididymis, and epididymal spermatozoa, suggesting that testis fascin persists in mature spermatozoa. In accord with the in situ hybridization results, immunofluorescent microscopy localizes testis fascin protein to areas of the anterior spermatid head that match known distributions of F-actin in the dorsal and ventral subacrosomal spaces. It is possible that testis fascin may function in the terminal elongation of the spermatid head and in microfilament rearrangements that accompany fertilization.