Growth cone behavior and production of traction force

The growth cone must push its substrate rearward via some traction force in order to propel itself forward. To determine which growth cone behaviors produce traction force, we observed chick sensory growth cones under conditions in which force production was accommodated by movement of obstacles in the environment, namely, neurites of other sensory neurons or glass fibers. The movements of these obstacles occurred via three, different, stereotyped growth cone behaviors: (a) filopodial contractions, (b) smooth rearward movement on the dorsal surface of the growth cone, and (c) interactions with ruffling lamellipodia. More than 70% of the obstacle movements were caused by filopodial contractions in which the obstacle attached at the extreme distal end of a filopodium and moved only as the filopodium changed its extension. Filopodial contractions were characterized by frequent changes of obstacle velocity and direction. Contraction of a single filopodium is estimated to exert 50-90 microdyn of force, which can account for the pull exerted by chick sensory growth cones. Importantly, all five cases of growth cones growing over the top of obstacle neurites (i.e., geometry that mimics the usual growth cone/substrate interaction), were of the filopodial contraction type. Some 25% of obstacle movements occurred by a smooth backward movement along the top surface of growth cones. Both the appearance and rate of movements were similar to that reported for retrograde flow of cortical actin near the dorsal growth cone surface. Although these retrograde flow movements also exerted enough force to account for growth cone pulling, we did not observe such movements on ventral growth cone surfaces. Occasionally obstacles were moved by interaction with ruffling lamellipodia. However, we obtained no evidence for attachment of the obstacles to ruffling lamellipodia or for directed obstacle movements by this mechanism. These data suggest that chick sensory growth cones move forward by contractile activity of filopodia, i.e., isometric contraction on a rigid substrate. Our data argue against retrograde flow of actin producing traction force.     

A Combination of Diffusion and Active Translocation Localizes Myosin 10 to the Filopodial Tip

Myosin 10 is an actin-based molecular motor that localizes to the tips of filopodia in mammalian cells. To understand how it is targeted to this distinct region of the cell, we have used total internal reflection fluorescence microscopy to study the movement of individual full-length and truncated GFP-tagged molecules. Truncation mutants lacking the motor region failed to localize to filopodial tips but still bound transiently at the plasma membrane. Deletion of the single α-helical and anti-parallel coiled-coil forming regions, which lie between the motor and pleckstrin homology domains, reduced the instantaneous velocity of intrafilopodial movement but did not affect the number of substrate adherent filopodia. Deletion of the anti-parallel coiled-coil forming region, but not the EKR-rich region of the single α-helical domain, restored intrafilopodial trafficking, suggesting this region is important in determining myosin 10 motility. We propose a model by which myosin 10 rapidly targets to the filopodial tip via a sequential reduction in dimensionality. Molecules first undergo rapid diffusion within the three-dimensional volume of the cell body. They then exhibit periods of slower two-dimensional diffusion in the plane of the plasma membrane. Finally, they move in a unidimensional, highly directed manner along the polarized actin filament bundle within the filopodium becoming confined to a single point at the tip. Here we have observed directly each phase of the trafficking process using single molecule fluorescence imaging of live cells and have quantified our observations using single particle tracking, autocorrelation analysis, and kymographs.     

Filopodial actin bundles are not necessary for microtubule advance into the peripheral domain of Aplysia neuronal growth cones

Filopodial actin bundles guide microtubule assembly in the growth cone peripheral (P) domain and retrograde actin-network flow simultaneously transports microtubules rearward. Therefore, microtubule-end position is determined by the sum of microtubule assembly and retrograde transport rates. However, how filopodia actually affect microtubule assembly dynamics is unknown. To address this issue we quantitatively assessed microtubule and actin dynamics before and after selective removal of filopodia. Filopodium removal had surprisingly little effect on retrograde actin-flow rates or underlying network structures, but resulted in an approximate doubling of peripheral microtubule density and deeper penetration of microtubules into the P domain. The latter stemmed from less efficient coupling of microtubules to remaining actin networks and not from a change in microtubule polymer dynamics. Loss of filopodia also resulted in increased lateral microtubule movements and a more randomized microtubule distribution in the P domain. In summary, filopodia do not seem to be formally required for microtubule advance; however, their presence ensures radial distribution of microtubules in the P domain and facilitates microtubule transport by retrograde flow. The resulting dynamic steady state has interesting implications for rapid microtubule-positioning responses in the P domain.     

Region-specific microtubule transport in motile cells

Photoactivation and photobleaching of fluorescence were used to determine the mechanism by which microtubules (MTs) are remodeled in PtK2 cells during fibroblast-like motility in response to hepatocyte growth factor (HGF). The data show that MTs are transported during cell motility in an actomyosin-dependent manner, and that the direction of transport depends on the dominant force in the region examined. MTs in the leading lamella move rearward relative to the substrate, as has been reported in newt cells (Waterman-Storer, C.M., and E.D. Salmon. 1997. J. Cell Biol. 139:417-434), whereas MTs in the cell body and in the retraction tail move forward, in the direction of cell locomotion. In the transition zone between the peripheral lamella and the cell body, a subset of MTs remains stationary with respect to the substrate, whereas neighboring MTs are transported either forward, with the cell body, or rearward, with actomyosin retrograde flow. In addition to transport, the photoactivated region frequently broadens, indicating that individual marked MTs are moved either at different rates or in different directions. Mark broadening is also observed in nonmotile cells, indicating that this aspect of transport is independent of cell locomotion. Quantitative measurements of the dissipation of photoactivated fluorescence show that, compared with MTs in control nonmotile cells, MT turnover is increased twofold in the lamella of HGF-treated cells but unchanged in the retraction tail, demonstrating that microtubule turnover is regionally regulated.     

Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

Endothelial cell migration is an important step during angiogenesis, and its dysregulation contributes to aberrant neovascularization. The bone morphogenetic proteins (BMPs) are potent stimulators of cell migration and angiogenesis. Using microarray analyses, we find that myosin-X (Myo10) is a BMP target gene. In endothelial cells, BMP6-induced Myo10 localizes in filopodia, and BMP-dependent filopodial assembly decreases when Myo10 expression is reduced. Likewise, cellular alignment and directional migration induced by BMP6 are Myo10 dependent. Surprisingly, we find that Myo10 and BMP6 receptor ALK6 colocalize in a BMP6-dependent fashion. ALK6 translocates into filopodia after BMP6 stimulation, and both ALK6 and Myo10 possess intrafilopodial motility. Additionally, Myo10 is required for BMP6-dependent Smad activation, indicating that in addition to its function in filopodial assembly, Myo10 also participates in a requisite amplification loop for BMP signaling. Our data indicate that Myo10 is required to guide endothelial migration toward BMP6 gradients via the regulation of filopodial function and amplification of BMP signals.     

Myosin IIIB uses an actin-binding motif in its espin-1 cargo to reach the tips of actin protrusions

Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity [1]. We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia [2], MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip localization is lost when we remove the ESPN1 C terminus actin-binding site. We also demonstrate that, like MYO3A [2], MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip localization reveals a novel mechanism for the cell to regulate myosin tip localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations.     

Relationship between plasma membrane mobility and substrate attachment in the crawling movement of spermatozoa from Caenorhabditis elegans

Caenorhabditis elegans sperm are nonflagellated cells that lack actin and myosin yet can form pseudopods to propel themselves over solid substrates. Surface-attached probes such as latex beads, lectins, and antimembrane protein monoclonal antibodies move rearward over the dorsal pseudopod surface of sessile cells. Using monoclonal antibodies against membrane proteins of C. elegans sperm to examine the role of localized membrane assembly and rearward flow in crawling movement, we determined that substrates prepared by coating glass with antimembrane protein antibodies, but not naked glass or other nonmembrane-binding proteins, promote sperm motility. Sperm locomotion is inhibited in a concentration-dependent fashion when cells are bathed with soluble antimembrane protein monoclonal antibodies but not with antimouse Ig antibodies or a monoclonal antibody against a sperm cytoplasmic protein. Our results suggest that C. elegans sperm crawl by gaining traction with substrate-attached ligands via their surface proteins and by using the motor that moves those proteins rearward on unattached cells to pull the entire cell forward. Continuous insertion of new proteins at the front of the cell and their subsequent adhesion to the substrate allows this process to continue.     

Investigation of the mechanism of retraction of the cell margin and rearward flow of nodules during mitotic cell rounding.

We have studied two types of cell motility directed toward the cell center: retraction of the cell margin and rearward flow of small cytoplasmic nodules during mitotic cell rounding in Potoroo tridactylis kidney (PtK2) cells by time-lapse video microscopy, drug treatments, and photoactivation of fluorescence. Nodules flow rearward on thin, actin-rich fibers (retraction fibers) exposed as the cell margin retracts. Retraction of the cell margin and rearward flow of nodules require intact actin filaments, but are insensitive to an inhibitor of myosin function (butanedione monoxime). Using photoactivation of fluorescence marking, we have determined that actin filaments in the majority of retraction fibers remain stationary while the cell margin retracts and nodules flow rearward. The pointed ends of retraction fiber actin filaments face the cell center. We argue that nodule motility is driven by a novel actin-based force that perhaps also partially contributes to retraction of the cell margin during cell rounding at mitosis.     

Keratocytes pull with similar forces on their dorsal and ventral surfaces

As cells move forward, they pull rearward against extracellular matrices (ECMs), exerting traction forces. However, no rearward forces have been seen in the fish keratocyte. To address this discrepancy, we have measured the propulsive forces generated by the keratocyte lamella on both the ventral and the dorsal surfaces. On the ventral surface, a micromachined device revealed that traction forces were small and rearward directed under the lamella, changed direction in front of the nucleus, and became larger under the cell body. On the dorsal surface of the lamella, an optical gradient trap measured rearward forces generated against fibronectin-coated beads. The retrograde force exerted by the cell on the bead increased in the thickened region of the lamella where myosin condensation has been observed (Svitkina, T.M., A.B. Verkhovsky, K.M. McQuade, and G. G. Borisy. 1997. J. Cell Biol. 139:397-415). Similar forces were generated on both the ventral (0.2 nN/microm(2)) and the dorsal (0.4 nN/microm(2)) surfaces of the lamella, suggesting that dorsal matrix contacts are as effectively linked to the force-generating cytoskeleton as ventral contacts. The correlation between the level of traction force and the density of myosin suggests a model for keratocyte movement in which myosin condensation in the perinuclear region generates rearward forces in the lamella and forward forces in the cell rear.     

Microtubule-mediated Src tyrosine kinase trafficking in neuronal growth cones

Src family tyrosine kinases are important signaling enzymes in the neuronal growth cone, and they have been implicated in axon guidance; however, the detailed localization, trafficking, and cellular functions of Src kinases in live growth cones are unclear. Here, we cloned two novel Aplysia Src kinases, termed Src1 and Src2, and we show their association with both the plasma membrane and the microtubule cytoskeleton in the growth cone by live cell imaging, immunocytochemistry, and cell fractionation. Activated Src2 is enriched in filopodia tips. Interestingly, Src2-enhanced green fluorescent protein–positive endocytic vesicles and tubulovesicular structures undergo microtubule-mediated movements that are bidirectional in the central domain and mainly retrograde in the peripheral domain. To further test the role of microtubules in Src trafficking in the growth cone, microtubules were depleted with either nocodazole or vinblastine treatment, resulting in an increase in Src2 plasma membrane levels in all growth cone domains. Our data suggest that microtubules regulate the steady-state level of active Src at the plasma membrane by mediating retrograde recycling of endocytosed Src. Expression of constitutively active Src2 results in longer filopodia that protrude from smaller growth cones, implicating Src2 in controlling the size of filopodia and lamellipodia.     

Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines

The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin–coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain.     

Src and cortactin promote lamellipodia protrusion and filopodia formation and stability in growth cones

Src tyrosine kinases have been implicated in axonal growth and guidance; however, the underlying cellular mechanisms are not well understood. Specifically, it is unclear which aspects of actin organization and dynamics are regulated by Src in neuronal growth cones. Here, we investigated the function of Src2 and one of its substrates, cortactin, in lamellipodia and filopodia of Aplysia growth cones. We found that up-regulation of Src2 activation state or cortactin increased lamellipodial length, protrusion time, and actin network density, whereas down-regulation had opposite effects. Furthermore, Src2 or cortactin up-regulation increased filopodial density, length, and protrusion time, whereas down-regulation promoted lateral movements of filopodia. Fluorescent speckle microscopy revealed that rates of actin assembly and retrograde flow were not affected in either case. In summary, our results support a model in which Src and cortactin regulate growth cone motility by increasing actin network density and protrusion persistence of lamellipodia by controlling the state of actin-driven protrusion versus retraction. In addition, both proteins promote the formation and stability of actin bundles in filopodia.     

GEF at work: Vav in protruding filopodia.

The Dbl family proto-oncogene vav is a nucleotide exchange factor for Rho family GTPases and is involved in triggering cytoskeletal changes contributing to the alterations of cell shape and motility, as well as in the induction of gene expression. In vitro and in vivo Vav is regulated by multiple tyrosine phosphorylation and binding to phosphatidylinositol phosphates. Although recruitment of Vav to the plasma membrane appears important for the activation of Vav function, there is little information on the precise subcellular localization of Vav in living cells. Employing live video fluorescence and immunoelectron microscopy, we show that GFP-tagged full-length Vav, and several mutants in which the N-terminal regulatory calponin homology (CH) domain has been deleted, specifically localize to the tips of filopodia. This localization was congruent with a high content of tyrosine phosphorylation in these regions. Consistent with earlier observations, mutants lacking the C-terminal SH domain region were unable to translocate to the filopodia tips. The enrichment in filopodial tips persisted despite their lateral movement but was dependent on forward growth. Upon retraction, the signal was rapidly lost, indicating that Vav undergoes a specific and transient translocation in response to actin-based, protrusive events in filopodia.     

Roles of type II myosin and a tropomyosin isoform in retrograde actin flow in budding yeast

Retrograde flow of cortical actin networks and bundles is essential for cell motility and retrograde intracellular movement, and for the formation and maintenance of microvilli, stereocilia, and filopodia. Actin cables, which are F-actin bundles that serve as tracks for anterograde and retrograde cargo movement in budding yeast, undergo retrograde flow that is driven, in part, by actin polymerization and assembly. We find that the actin cable retrograde flow rate is reduced by deletion or delocalization of the type II myosin Myo1p, and by deletion or conditional mutation of the Myo1p motor domain. Deletion of the tropomyosin isoform Tpm2p, but not the Tpm1p isoform, increases the rate of actin cable retrograde flow. Pretreatment of F-actin with Tpm2p, but not Tpm1p, inhibits Myo1p binding to F-actin and Myo1p-dependent F-actin gliding. These data support novel, opposing roles of Myo1p and Tpm2 in regulating retrograde actin flow in budding yeast and an isoform-specific function of Tpm1p in promoting actin cable function in myosin-driven anterograde cargo transport.     

In vitro induction of crawling in the amoeboid sperm of the nematode parasite, Ascaris suum

In a highly synchronous process, the immotile spermatids of Ascaris suum extend pseudopods and become rapidly crawling sperm when treated with an extract from the glandular vas deferens of the male under strict anaerobic conditions. Within 9-12 min, a pseudopod develops, elongates rapidly, and exhibits a continuous flow of membrane specializations, the villipodia, from tip toward base. When attached to acid-washed glass, the pseudopod pulls the cell body along at speeds exceeding 70 microns/min. The pseudopod length remains constant while retrograde flow of villipodia proceeds at the same rate as the sperm's forward movement. Cohorts of about 15 villipodia form at the leading edge, move rearward together, and disappear at the junction of pseudopod and cell body. These are the terminations of branched, refringent fibers, which extend the length of the pseudopod. The latter are the fiber complexes that form its cytoskeleton (Sepsenwol et al.: Journal of Cell Biology 108:55-66, 1989). Locomoting cells sometimes change direction when another crawls by and follow each other. When cells are exposed to air, forward movement ceases in a predictable pattern: the forward extension of the leading edge ceases, the pseudopod shortens from the base, and the cell body continues to be pulled forward. These data contribute to a model for Ascaris sperm amoeboid motility in which independent processes of continuous extension at the leading edge and continuous shortening at the base of the pseudopod act to propel the cell forward.     

Cell migration does not produce membrane flow

We have previously reported that rearward migration of surface particles on slowly moving cells is not driven by membrane flow (Sheetz, M. P., S. Turney, H. Qian, and E. L. Elson. 1989. Nature (Lond.). 340:284-288) and recent photobleaching measurements have ruled out any rapid rearward lipid flow (Lee, J., M. Gustafsson, D. E. Magnussen, and K. Jacobson. 1990. Science (Wash. DC.) 247:1229-1233). It was not possible, however, to conclude from those studies that a slower or tank-tread membrane lipid flow does not occur. Therefore, we have used the technology of single particle tracking to examine the movements of diffusing particles on rapidly locomoting fish keratocytes where the membrane current is likely to be greatest. The keratocytes had a smooth lamellipodial surface on which bound Con A-coated gold particles were observed either to track toward the nuclear region (velocity of 0.35 +/- 0.15 micron/s) or to diffuse randomly (apparent diffusion coefficient of [3.5 +/- 2.0] x 10(-10) cm2/s). We detected no systematic drift relative to the cell edge of particles undergoing random diffusion even after the cell had moved many micrometers. The average net particle displacement was 0.01 +/- 2.7% of the cell displacement. These results strongly suggest that neither the motions of membrane proteins driven by the cytoskeleton nor other possible factors produce a bulk flow of membrane lipid.     

Regulated actin cytoskeleton assembly at filopodium tips controls their extension and retraction.

The extension and retraction of filopodia in response to extracellular cues is thought to be an important initial step that determines the direction of growth cone advance. We sought to understand how the dynamic behavior of the actin cytoskeleton is regulated to produce extension or retraction. By observing the movement of fiduciary marks on actin filaments in growth cones of a neuroblastoma cell line, we found that filopodium extension and retraction are governed by a balance between the rate of actin cytoskeleton assembly at the tip and retrograde flow. Both assembly and flow rate can vary with time in a single filopodium and between filopodia in a single growth cone. Regulation of assembly rate is the dominant factor in controlling filopodia behavior in our system.     

Comparative behavior of membrane protein-antibody complexes on motile fibroblasts: implications for a mechanism of capping

A characteristic feature of fibroblast locomotory activity is the rearward transport across the leading lamella of various materials used to mark the cell surface. The two processes most frequently invoked as explanations for this transport phenomenon, called capping, are (a) retrograde membrane flow arising from directed membrane insertion and (b) rearward cortical cytoskeletal flow arising from cytoskeletal assembly and contraction. The retrograde lipid flow hypothesis, the most current form of the membrane flow scheme, makes explicit predictions about the movement of membrane proteins subjected to the postulated rearward lipid flow. Several of these predictions were tested by comparing the behavior of four membrane proteins, Pgp-1, Thy- 1, H-2, and influenza HA0, identified by fluorescent antibodies. With the exception of Pgp-1, these proteins were uniformly distributed under nonaggregated conditions but were capped when aggregated into patches. In contrast, Pgp-1 was capped in similar time frames in both nonaggregated and aggregated states where the lateral diffusion coefficients were very different. Furthermore, the capping behavior of two tagged membrane proteins was markedly different yet both had similar diffusion coefficients. The results from these tests disprove the bulk membrane flow hypothesis and are at odds with explicit predictions of the retrograde lipid flow hypothesis for the mechanism of capping. This work, therefore, supports the alternative cytoskeletal- based mechanism for driving capping. Requirements for coupling cytoskeletal movement to membrane components are discussed.     

Slipping or gripping? Fluorescent speckle microscopy in fish keratocytes reveals two different mechanisms for generating a retrograde flow of actin

Fish keratocytes can generate rearward directed traction forces within front portions of the lamellipodium, suggesting that a retrograde flow of actin may also occur here but this was not detected by previous photoactivation experiments. To investigate the relationship between retrograde flow and traction force generation, we have transfected keratocytes with GFP-actin and used fluorescent speckle microscopy, to observe speckle flow. We detected a retrograde flow of actin within the leading lamellipodium that is inversely proportional to both protrusion rate and cell speed. To observe the effect of reducing contractility, we treated transfected cells with ML7, a potent inhibitor of myosin II. Surprisingly, ML7 treatment led to an increase in retrograde flow rate, together with a decrease in protrusion and cell speed, but only in rapidly moving cells. In slower moving cells, retrograde flow decreased, whereas protrusion rate and cell speed increased. These results suggest that there are two mechanisms for producing retrograde flow. One involves slippage between the cytoskeleton and adhesions, that decreases traction force production. The other involves slippage between adhesions and the substratum, which increases traction force production. We conclude that a biphasic relationship exists between retrograde actin flow and adhesiveness in moving keratocytes.     

Mechanism of lateral movement of filopodia and radial actin bundles across neuronal growth cones

We investigated the motion of filopodia and actin bundles in lamellipodia of motile cells, using time-lapse sequences of polarized light images. We measured the velocity of retrograde flow of the actin network and the lateral motion of filopodia and actin bundles of the lamellipodium. Upon noting that laterally moving filopodia and actin bundles are always tilted with respect to the direction of retrograde flow, we propose a simple geometric model for the mechanism of lateral motion. The model establishes a relationship between the speed of lateral motion of actin bundles, their tilt angle with respect to the direction of retrograde flow, and the speed of retrograde flow in the lamellipodium. Our experimental results verify the quantitative predictions of the model. Furthermore, our observations support the hypothesis that lateral movement of filopodia is caused by retrograde flow of tilted actin bundles and by their growth through actin polymerization at the tip of the bundles inside the filopodia. Therefore we conclude that the lateral motion of tilted filopodia and actin bundles does not require a separate motile mechanism but is the result of retrograde flow and the assembly of actin filaments and bundles near the leading edge of the lamellipodium.