Deformation and flow of membrane into tethers extracted from neuronal growth cones.

Membrane tethers are extracted at constant velocity from neuronal growth cones using a force generated by a laser tweezers trap. A thermodynamic analysis shows that as the tether is extended, energy is stored in the tether as bending and adhesion energies and in the cell body as "nonlocal" bending. It is postulated that energy is dissipated by three viscous mechanisms including membrane flow, slip between the two monolayers that form the bilayer, and slip between membrane and cytoskeleton. The analysis predicts and the experiments show a linear relation between tether force and tether velocity. Calculations based on the analytical results and the experimental measurements of a tether radius of approximately 0.2 micron and a tether force at zero velocity of approximately 8 pN give a bending modulus for the tether of 2.7 x 10(-19) N.m and an extraordinarily small "apparent surface tension" in the growth cone of 0.003 mN/m, where the apparent surface tension is the sum of the far-field, in-plane tension and the energy of adhesion. Treatments with cytochalasin B and D, ethanol, and nocodazole affect the apparent surface tension but not bending. ATP depletion affects neither, whereas large concentrations of DMSO affect both. Under conditions of flow, data are presented to show that the dominant viscous mechanism comes from the slip that occurs when the membrane flows over the cytoskeleton. ATP depletion and the treatment with DMSO cause a dramatic drop in the effective viscosity. If it is postulated that the slip between membrane and cytoskeleton occurs in a film of water, then this water film has a mean thickness of only approximately 10 A.     

Characteristics of a membrane reservoir buffering membrane tension.

When membrane-attached beads are pulled vertically by a laser tweezers, a membrane tube of constant diameter (tether) is formed. We found that the force on the bead (tether force) did not depend on tether length over a wide range of tether lengths, which indicates that a previously unidentified reservoir of membrane and not stretch of the plasma membrane provides the tether membrane. Plots of tether force vs. tether length have an initial phase, an elongation phase, and an exponential phase. During the major elongation phase, tether force is constant, buffered by the "membrane reservoir." Finally, there is an abrupt exponential rise in force that brings the tether out of the trap, indicating depletion of the membrane reservoir. In chick embryo fibroblasts and 3T3 fibroblasts, the maximum tether lengths that can be pulled at a velocity of 4 microm/s are 5.1 +/- 0. 3 and 5.0 +/- 0.2 microm, respectively. To examine the importance of the actin cytoskeleton, we treated cells with cytochalasin B or D and found that the tether lengths increased dramatically to 13.8 +/- 0.8 and 12.0 +/- 0.7 microm, respectively. Similarly, treatment of the cells with colchicine and nocodazole results in more than a twofold increase in tether length. We found that elevation of membrane tension (through osmotic pressure, a long-term elevation of tether force, or a number of transitory increases) increased reservoir size over the whole cell. Using a tracking system to hold tether force on the bead constant near its maximal length in the exponential phase, the rate of elongation of the tethers was measured as a function of tether force (membrane tension). The rate of elongation of tethers was linearly dependent on the tether force and reflected an increase in size of the reservoir. Increases in the reservoir caused by tension increases on one side of the cell caused increases in reservoir size on the other side of the cell. Thus, we suggest that cells maintain a plasma membrane reservoir to buffer against changes in membrane tension and that the reservoir is increased with membrane tension or disruption of the cytoskeleton.     

Extension of filopodia by motor-dependent actin assembly.

A variety of mechanisms have been proposed to explain the forward extension of cytoplasm in advancing cells and axonal growth cones, including actin polymerization and osmotic swelling. Based on our observations of the filopodia of cultured neuronal growth cones, we propose a mechanism involving motor-induced extension and retraction. We observed that filopodia (actin-based protrusions 0.2-0.5 mu in diameter) extend and retract from growth cone lamellae at the same rate. Further, force is generated at the tips of filopodia which is sufficient to produce compressive buckling of the proximal portion of the filopodium. From our analysis of these movements we suggest that a motor protein powers both the extension and retraction of filopodia.     

Cytoskeletal remodeling during growth cone-target interactions

Reorganization of the cytoskeleton of neuronal growth cones in response to environmental cues underlies the process of axonal guidance. Most previous studies addressing cytoskeletal changes during growth cone pathfinding have focused on the dynamics of a single cytoskeletal component. We report here an investigation of homophilic growth cone- target interactions between Aplysia bag cell neurons using digitally enhanced video microscopy, which addresses dynamic interactions between actin filaments and microtubules. After physical contact of a growth cone with a physiological target, mechanical coupling occurred after a delay; and then the growth cone exerted forces on and displaced the target object. Subsequent to coupling, F-actin accumulation was observed at the target contact zone, followed by preferential microtubule extension to the same site. After successful target interactions, growth cones typically moved off highly adhesive poly-L- lysine substrates into native target cell surfaces. These events were associated with modulation of both the direction and rate of neurite outgrowth: growth cone migration was typically reoriented to a trajectory along the target interaction axis and rates of advance increased by about one order of magnitude. Directed microtubule movements toward the contact site appeared to be F-actin dependent as target site-specific microtubule extension and bundling could be reversibly randomized by micromolar levels of cytochalasin B in a characteristic manner. Our results suggest that target contacts can induce focal F-actin assembly and reorganization which, in turn, guides target site-directed microtubule redistribution.     

Eukaryotic membrane tethers revisited using magnetic tweezers

Membrane nanotubes, under physiological conditions, typically form en masse. We employed magnetic tweezers (MTW) to extract tethers from human brain tumor cells and compared their biophysical properties with tethers extracted after disruption of the cytoskeleton and from a strongly differing cell type, Chinese hamster ovary cells. In this method, the constant force produced with the MTW is transduced to cells through super-paramagnetic beads attached to the cell membrane. Multiple sudden jumps in bead velocity were manifest in the recorded bead displacement-time profiles. These discrete events were interpreted as successive ruptures of individual tethers. Observation with scanning electron microscopy supported the simultaneous existence of multiple tethers. The physical characteristics, in particular, the number and viscoelastic properties of the extracted tethers were determined from the analytic fit to bead trajectories, provided by a standard model of viscoelasticity. Comparison of tethers formed with MTW and atomic force microscopy (AFM), a technique where the cantilever-force transducer is moved at constant velocity, revealed significant differences in the two methods of tether formation. Our findings imply that extreme care must be used to interpret the outcome of tether pulling experiments performed with single molecular techniques (MTW, AFM, optical tweezers, etc). First, the different methods may be testing distinct membrane structures with distinct properties. Second, as soon as a true cell membrane (as opposed to that of a vesicle) can attach to a substrate, upon pulling on it, multiple nonspecific membrane tethers may be generated. Therefore, under physiological conditions, distinguishing between tethers formed through specific and nonspecific interactions is highly nontrivial if at all possible.     

Strength in the periphery: growth cone biomechanics and substrate rigidity response in peripheral and central nervous system neurons

There is now considerable evidence of the importance of mechanical cues in neuronal development and regeneration. Motivated by the difference in the mechanical properties of the tissue environment between the peripheral (PNS) and central (CNS) nervous systems, we compare substrate-stiffness-dependent outgrowth and traction forces from PNS (dorsal root ganglion (DRG)) and CNS (hippocampal) neurons. We show that neurites from DRG neurons display maximal outgrowth on substrates with a Young's modulus of ～1000 Pa, whereas hippocampal neurite outgrowth is independent of substrate stiffness. Using traction force microscopy, we also find a substantial difference in growth cone traction force generation, with DRG growth cones exerting severalfold larger forces compared with hippocampal growth cones. The traction forces generated by DRG and hippocampal growth cones both increase with increasing stiffness, and DRG growth cones growing on substrates with a Young's modulus of 1000 Pa strengthen considerably after 18–30 h. Finally, we find that retrograde actin flow is almost three times faster in hippocampal growth cones than in DRG. Moreover, the density of paxillin puncta is significantly lower in hippocampal growth cones, suggesting that stronger substrate coupling of the DRG cytoskeleton is responsible for the remarkable difference in traction force generation. These findings reveal a differential adaptation of cytoskeletal dynamics to substrate stiffness in growth cones of different neuronal types, and highlight the potential importance of the mechanical properties of the cellular environment for neuronal navigation during embryonic development and nerve regeneration.     

Label-free DNA sequencing using Millikan detection

A label-free method for DNA sequencing based on the principle of the Millikan oil drop experiment was developed. This sequencing-by-synthesis approach sensed increases in bead charge as nucleotides were added by a polymerase to DNA templates attached to beads. The balance between an electrical force, which was dependent on the number of nucleotide charges on a bead, and opposing hydrodynamic drag and restoring tether forces resulted in a bead velocity that was a function of the number of nucleotides attached to the bead. The velocity of beads tethered via a polymer to a microfluidic channel and subjected to an oscillating electric field was measured using dark-field microscopy and used to determine how many nucleotides were incorporated during each sequencing-by-synthesis cycle. Increases in bead velocity of approximately 1% were reliably detected during DNA polymerization, allowing for sequencing of short DNA templates. The method could lead to a low-cost, high-throughput sequencing platform that could enable routine sequencing in medical applications.     

The Local Forces Acting on the Mechanotransduction Channel in Hair Cell Stereocilia

In hair cells, mechanotransduction channels are located in the membrane of stereocilia tips, where the base of the tip link is attached. The tip-link force determines the system of other forces in the immediate channel environment, which change the channel open probability. This system of forces includes components that are out of plane and in plane relative to the membrane; the magnitude and direction of these components depend on the channel environment and arrangement. Using a computational model, we obtained the major forces involved as functions of the force applied via the tip link at the center of the membrane. We simulated factors related to channels and the membrane, including finite-sized channels located centrally or acentrally, stiffness of the hypothesized channel-cytoskeleton tether, and bending modulus of the membrane. Membrane forces are perpendicular to the directions of the principal curvatures of the deformed membrane. Our approach allows for a fine vectorial picture of the local forces gating the channel; membrane forces change with the membrane curvature and are themselves sufficient to affect the open probability of the channel.     

The Local Forces Acting on the Mechanotransduction Channel in Hair Cell Stereocilia

In hair cells, mechanotransduction channels are located in the membrane of stereocilia tips, where the base of the tip link is attached. The tip-link force determines the system of other forces in the immediate channel environment, which change the channel open probability. This system of forces includes components that are out of plane and in plane relative to the membrane; the magnitude and direction of these components depend on the channel environment and arrangement. Using a computational model, we obtained the major forces involved as functions of the force applied via the tip link at the center of the membrane. We simulated factors related to channels and the membrane, including finite-sized channels located centrally or acentrally, stiffness of the hypothesized channel-cytoskeleton tether, and bending modulus of the membrane. Membrane forces are perpendicular to the directions of the principal curvatures of the deformed membrane. Our approach allows for a fine vectorial picture of the local forces gating the channel; membrane forces change with the membrane curvature and are themselves sufficient to affect the open probability of the channel.     

Neutrophil-bead collision assay: pharmacologically induced changes in membrane mechanics regulate the PSGL-1/P-selectin adhesion lifetime

Visualization of flowing neutrophils colliding with adherent 1-mum-diameter beads presenting P-selectin allowed the simultaneous measurement of collision efficiency (epsilon), membrane tethering fraction (f), membrane tether growth dynamics, and PSGL-1/P-selectin binding lifetime. For 1391 collisions analyzed over venous wall shear rates from 25 to 200 s(-1), epsilon decreased from 0.17 to 0.004, whereas f increased from 0.15 to 0.70, and the average projected membrane tether length, L(tether)(m), increased from 0.35 mum to approximately 2.0 mum over this shear range. At all shear rates tested, adhesive collisions lacking membrane tethers had average bond lifetimes less than those observed for collisions with tethers. For adhesive collisions that failed to form membrane tethers, the regressed Bell parameters (consistent with single bond Monte Carlo simulation) were zero-stress off-rate, k(off)(0) = 0.56 s(-1) and reactive compliance, r = 0.10 nm, similar to published atomic force microscopy (AFM) measurements. For all adhesion events (+/- tethers), the bond lifetime distributions were more similar to those obtained by rolling assay and best simulated by Monte Carlo with the above Bell parameters and an average of 1.48 bonds (n = 1 bond (67%), n = 2 (22%), and n = 3-5 (11%)). For collisions at 100 s(-1), pretreatment of neutrophils with actin depolymerizing agents, latrunculin or cytochalasin D, had no effect on epsilon, but increased L(tether)(m) by 1.74- or 2.65-fold and prolonged the average tether lifetime by 1.41- or 1.65-fold, respectively. Jasplakinolide, an actin polymerizing agent known to cause blebbing, yielded results similar to the depolymerizing agents. Conversely, cholesterol-depletion with methyl-beta-cyclodextrin or formaldehyde fixation had no effect on epsilon, but reduced L(tether)(m) by 66% or 97% and reduced the average tether lifetime by 30% or 42%, respectively. The neutrophil-bead collision assay combines advantages of atomic force microscopy (small contact zone), aggregometry (discrete interactions), micropipette manipulation (tether visualization), and rolling assays (physiologic flow loading). Membrane tether growth can be enhanced or reduced pharmacologically with consequent effects on PSGL-1/P-selectin lifetimes.     

The restricted spatial and temporal expression of a nervous-system-specific antigen involved in axon outgrowth during development of the grasshopper

To identify molecules important for pathfinding by growing axons, monoclonal antibodies (mAb) have been generated against embryonic grasshopper tissue. One mAb, 2B2, shows labeling exclusively in the nervous system. It recognizes a surface epitope on neuronal growth cones, filopodia and axons in the central nervous system (CNS). Initially, the antigen is expressed on all processes of the CNS; after 70% of embryonic development, localization of the 2B2 mAb is restricted to a small subset of axon tracts within the ganglia. Immunoprecipitation from embryonic membrane extracts with the 2B2 mAb reveals a unique band of 160 x 10(3) Mr. Functional studies with the 2B2 mAb demonstrate that the antigen is important in growth cone-axon interactions during process outgrowth. Growth cones that extend along axonal substrata are either blocked in growth or grow along an aberrant pathway when embryos are cultured in the presence of the 2B2 mAb. However, pioneer neurons that extend processes on non-neuronal substrata grow normally.     

Growth of neurites without filopodial or lamellipodial activity in the presence of cytochalasin B

To examine the role in neurite growth of actin-mediated tensions within growth cones, we cultured chick embryo dorsal root ganglion cells on various substrata in the presence of cytochalasin B. Time-lapse video recording was used to monitor behaviors of living cells, and cytoskeletal arrangements in neurites were assessed via immunofluorescence and electron microscopic observations of thin sections and whole, detergent-extracted cells decorated with the S1 fragment of myosin. On highly adhesive substrata, nerve cells were observed to extend numerous (though peculiarly oriented) neurites in the presence of cytochalasin, despite their lack of both filopodia and lamellipodia or the orderly actin networks characteristic of typical growth cones. We concluded that growth cone activity is not necessary for neurite elongation, although actin arrays seem important in mediating characteristics of substratum selectivity and neurite shape.     

Membrane tether extraction from human umbilical vein endothelial cells and its implication in leukocyte rolling

During the rolling of human neutrophils on the endothelium, tethers (cylindrical membrane tubes) are likely extracted from the neutrophil. Tether extraction reduces the force imposed on the adhesive bond between the neutrophil and endothelium, thereby facilitating the rolling. However, whether tethers can be extracted from the endothelium is still unknown. Here, with the micropipette-aspiration technique, we show that tethers can be extracted from either suspended or attached human umbilical vein endothelial cells. We also show that a linear relationship between the pulling force and tether growth velocity exists and this relationship does not depend on the receptor type (used to impose point forces), tumor necrosis factor-alpha stimulation, or cell attachment state. With linear regression, we determined that the threshold force was 50 pN and the effective viscosity was 0.50 pN.s/microm. Therefore, tethers might be simultaneously extracted from the neutrophil and endothelial cell during the rolling and, more importantly, the endothelial cell might contribute much more to the total composite tether length than the neutrophil. Compared with tether extraction from the neutrophil alone, simultaneous tether extraction results in a larger increase in the lifetime of the adhesive bond, and thus further stabilizes the rolling of neutrophils under high physiological shear stresses.     

Membrane tether formation from blebbing cells

Membrane tension has been proposed to be important in regulating cell functions such as endocytosis and cell motility. The apparent membrane tension has been calculated from tether forces measured with laser tweezers. Both membrane-cytoskeleton adhesion and membrane tension contribute to the tether force. Separation of the plasma membrane from the cytoskeleton occurs in membrane blebs, which could remove the membrane-cytoskeleton adhesion term. In renal epithelial cells, tether forces are significantly lower on blebs than on membranes that are supported by cytoskeleton. Furthermore, the tether forces are equal on apical and basolateral blebs. In contrast, tether forces from membranes supported by the cytoskeleton are greater in apical than in basolateral regions, which is consistent with the greater apparent cytoskeletal density in the apical region. We suggest that the tether force on blebs primarily contains only the membrane tension term and that the membrane tension may be uniform over the cell surface. Additional support for this hypothesis comes from observations of melanoma cells that spontaneously bleb. In melanoma cells, tether forces on blebs are proportional to the radius of the bleb, and as large blebs form, there are spikes in the tether force in other cell regions. We suggest that an internal osmotic pressure inflates the blebs, and the pressure calculated from the Law of Laplace is similar to independent measurements of intracellular pressures. When the membrane tension term is subtracted from the apparent membrane tension over the cytoskeleton, the membrane-cytoskeleton adhesion term can be estimated. In both cell systems, membrane-cytoskeleton adhesion was the major factor in generating the tether force.     

Microtubule-associated Protein 2c Reorganizes Both Microtubules and Microfilaments into Distinct Cytological Structures in an Actin-binding Protein-280–deficient Melanoma Cell Line

The emergence of processes from cells often involves interactions between microtubules and microfilaments. Interactions between these two cytoskeletal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal microtubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates interactions between microfilaments and microtubules. To approach this problem in vivo, we used the human melanoma cell, M2, which lacks actin-binding protein-280 (ABP-280) and forms membrane blebs, which are not seen in wild-type or ABP-transfected cells. The microinjection of tau or mature MAP2 rescued the blebbing phenotype; MAP2c not only caused cessation of blebbing but also induced the formation of two distinct cellular structures. These were actin-rich lamellae, which often included membrane ruffles, and microtubule-bearing processes. The lamellae collapsed after treatment with cytochalasin D, and the processes retracted after treatment with colchicine. MAP2c was immunocytochemically visualized in zones of the cell that were devoid of tubulin, such as regions within the lamellae and in association with membrane ruffles. In vitro rheometry confirmed that MAP2c is an efficient actin gelation protein capable of organizing actin filaments into an isotropic array at very low concentrations; tau and mature MAP2 do not share this rheologic property. These results suggest that MAP2c engages in functionally specific interactions not only with microtubules but also with microfilaments.     

Axon extension in the fast and slow lanes: substratum-dependent engagement of myosin II functions

Axon extension involves the coordinated regulation of the neuronal cytoskeleton. Actin filaments drive protrusion of filopodia and lamellipodia while microtubules invade the growth cone, thereby providing structural support for the nascent axon. Furthermore, in order for axons to extend the growth cone must attach to the substratum. Previous work indicates that myosin II activity inhibits the advance of microtubules into the periphery of growth cones, and myosin II has also been implicated in mediating integrin-dependent cell attachment. However, it is not clear how the functions of myosin II in regulating substratum attachment and microtubule advance are integrated during axon extension. We report that inhibition of myosin II function decreases the rate of axon extension on laminin, but surprisingly promotes extension rate on polylysine. The differential effects of myosin II inhibition on axon extension rate are attributable to myosin II having the primary function of mediating substratum attachment on laminin, but not on polylysine. Conversely, on polylysine the primary function of myosin II is to inhibit microtubule advance into growth cones. Thus, the substratum determines the role of myosin II in axon extension by controlling the functions of myosin II that contribute to extension.     

Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. I. Membrane separation from the cytoskeleton

We have used a biomembrane force probe decorated with P-selectin to form point attachments with PSGL-1 receptors on a human neutrophil (PMN) in a calcium-containing medium and then to quantify the forces experienced by the attachment during retraction of the PMN at fixed speed. From first touch to final detachment, the typical force history exhibited the following sequence of events: i), an initial linear-elastic displacement of the PMN surface, ii), an abrupt crossover to viscoplastic flow that signaled membrane separation from the interior cytoskeleton and the beginning of a membrane tether, and iii), the final detachment from the probe tip by usually one precipitous step of P-selectin:PSGL-1 dissociation. In this first article I, we focus on the initial elastic response and its termination by membrane separation from the cytoskeleton, initiating tether formation. Quantifying membrane unbinding forces for rates of loading (force/time) in the elastic regime from 240 pN/s to 38,000 pN/s, we discovered that the force distributions agreed well with the theory for kinetically limited failure of a weak bond. The kinetic rate for membrane unbinding was found to increase as an exponential function of the pulling force, characterized by an e-fold scale in force of approximately 17 pN and a preexponential factor, or apparent unstressed off rate, of approximately 1/s. The rheological properties of tether growth subsequent to the membrane unbinding events are presented in a companion article II.     

Transmission of growth cone traction force through apCAM-cytoskeletal linkages is regulated by Src family tyrosine kinase activity.

Tyrosine kinase activity is known to be important in neuronal growth cone guidance. However, underlying cellular mechanisms are largely unclear. Here, we report how Src family tyrosine kinase activity controls apCAM-mediated growth cone steering by regulating the transmission of traction forces through receptor-cytoskeletal linkages. Increased levels of tyrosine phosphorylation were detected at sites where beads coated with apCAM ligands were physically restrained to induce growth cone steering, but not at unrestrained bead binding sites. Interestingly, the rate and level of phosphotyrosine buildup near restrained beads were decreased by the myosin inhibitor 2,3-butanedione-2-monoxime, suggesting that tension promotes tyrosine kinase activation. While not affecting retrograde F-actin flow rates, genistein and the Src family selective tyrosine kinase inhibitors PP1 and PP2 strongly reduced the growth cone's ability to apply traction forces through apCAM-cytoskeletal linkages, assessed using the restrained bead interaction assay. Furthermore, increased levels of an activated Src family kinase were detected at restrained bead sites during growth cone steering events. Our results suggest a mechanism by which growth cones select pathways by sampling both the molecular nature of the substrate and its ability to withstand the application of traction forces.     

Tether extrusion from red blood cells: integral proteins unbinding from cytoskeleton

We investigate the mechanical strength of adhesion and the dynamics of detachment of the membrane from the cytoskeleton of red blood cells (RBCs). Using hydrodynamical flows, we extract membrane tethers from RBCs locally attached to the tip of a microneedle. We monitor their extrusion and retraction dynamics versus flow velocity (i.e., extrusion force) over successive extrusion-retraction cycles. Membrane tether extrusion is carried out on healthy RBCs and ATP-depleted or -inhibited RBCs. For healthy RBCs, extrusion is slow, constant in velocity, and reproducible through several extrusion-retraction cycles. For ATP-depleted or -inhibited cells, extrusion dynamics exhibit an aging phenomenon through extrusion-retraction cycles: because the extruded membrane is not able to retract properly onto the cell body, each subsequent extrusion exhibits a loss of resistance to tether growth over the tether length extruded at the previous cycle. In contrast, the additionally extruded tether length follows healthy dynamics. The extrusion velocity L depends on the extrusion force f according to a nonlinear fashion. We interpret this result with a model that includes the dynamical feature of membrane-cytoskeleton association. Tether extrusion leads to a radial membrane flow from the cell body toward the tether. In a distal permeation regime, the flow passes through the integral proteins bound to the cytoskeleton without affecting their binding dynamics. In a proximal sliding regime, where membrane radial velocity is higher, integral proteins can be torn out, leading to the sliding of the membrane over the cytoskeleton. Extrusion dynamics are governed by the more dissipative permeation regime: this leads to an increase of the membrane tension and a narrowing of the tether, which explains the power law behavior of L(f). Our main result is that ATP is necessary for the extruded membrane to retract onto the cell body. Under ATP depletion or inhibition conditions, the aging of the RBC after extrusion is interpreted as a perturbation of membrane-cytoskeleton linkage dynamics.     

Concentration of membrane antigens by forward transport and trapping in neuronal growth cones

Formation of the nervous system requires that neuronal growth cones follow specific paths and then stop at recognition signals, sensed at the growth cone's leading edge. We used antibody-coated gold particles viewed by video-enhanced differential interference contrast microscopy to observe the distribution and movement of two cell surface molecules, N-CAM and the 2A1 antigen, on growth cones of cultured cortical neurons. Gold particles are occasionally transported forward at 1-2 microns/s to the leading edge where they are trapped but continue to move. Concentration at the edge persists after cytochalasin D treatment or ATP depletion, but active movements to and along edges cease. We also observed a novel outward movement of small cytoplasmic aggregates at 1.8 microns/s in filopodia. We suggest that active forward transport and trapping involve reversible attachment of antigens to and transport along cytoskeletal elements localized to edges of growth cones.