Direct observations of the mechanical behaviors of the cytoskeleton in living fibroblasts.

Cytoskeletal proteins tagged with green fluorescent protein were used to directly visualize the mechanical role of the cytoskeleton in determining cell shape. Rat embryo (REF 52) fibroblasts were deformed using glass needles either uncoated for purely physical manipulations, or coated with laminin to induce attachment to the cell surface. Cells responded to uncoated probes in accordance with a three-layer model in which a highly elastic nucleus is surrounded by cytoplasmic microtubules that behave as a jelly-like viscoelastic fluid. The third, outermost cortical layer is an elastic shell under sustained tension. Adhesive, laminin-coated needles caused focal recruitment of actin filaments to the contacted surface region and increased the cortical layer stiffness. This direct visualization of actin recruitment confirms a widely postulated model for mechanical connections between extracellular matrix proteins and the actin cytoskeleton. Cells tethered to laminin-treated needles strongly resisted elongation by actively contracting. Whether using uncoated probes to apply simple deformations or laminin-coated probes to induce surface-to-cytoskeleton interaction we observed that experimentally applied forces produced exclusively local responses by both the actin and microtubule cytoskeleton. This local accomodation and dissipation of force is inconsistent with the proposal that cellular tensegrity determines cell shape.

Axonal outgrowth of cultured neurons is not limited by growth cone competition.

We have examined the question of scarcity-driven competition for outgrowth among growth cones of a single neuron. We measured spontaneous neurite elongation rates from 85 hours of videotape of the arbors of 31 chick sensory neurons in culture. These rate measurements were analyzed in ten minute periods that allowed cell bodies to be classified as to the number of their growth cones and the elongation to be analyzed as a series of discrete events. Comparing periods in which neurons maintained simple bipolar morphology we find no temporal competition between the two growth cones. That is, periods of above-average growth by one growth cone are not compensated by below-average growth during the same period by its sibling growth cone. Analyzing all outgrowth from a neuron based on its number of growth cones shows that net elongation rate from a single cell body is a linear function of the number of growth cones from 1 to 11. These observations suggest that growth cones behave independently and are not limited by availability of structural precursors. A surplus pool of structural precursors available for normal growth is also indicated by the high capacity for growth from single neurites when experimentally stimulated by mechanical tension. In addition, towing one or more neurites at above average rates does not cause any decline in simultaneous growth cone-mediated outgrowth from a single neuron compared to the 2-3 hour period prior to experimentally induced elongation. This high capacity for growth combined with the often observed, intermittant growth behavior of individual growth cones suggests that neurite outgrowth is intrinsically limited primarily by poor growth cone 'performance,' not scarcity-driven competition. We postulate that growth cones are poor 'tractors,' exerting too little tension to exploit the available capacity for axonal elongation.

Cytomechanics of neurite outgrowth from chick brain neurons.

Mechanical tension is a direct and immediate stimulus for neurite initiation and elongation from peripheral neurons. We report here that the relationship between tension and neurite outgrowth is equally initimate for embryonic chick forebrain neurons. Culture of forebrain neurons was unusually simple and reliable, and some of these cells undergo early events of axonal-dendritic polarity. Neurite outgrowth can be initiated de novo by experimental application of tension to the cell margin of forebrain neurons placed into culture 8-12 hours earlier, prior to spontaneous neurite outgrowth. Experimentally induced neurite elongation from these neurons shows the same robust linear relationship between elongation rate and magnitude of applied tension as peripheral neurons, i.e. both show a fluid-like growth response to tension. Although forebrain and sensory neurons manifest a similar distribution of growth sensitivity to tension (growth rate/unit tension), chick forebrain neurons initiated and elongated neurites at substantially lower net tensions than peripheral neurons. This is because, unlike peripheral neurons, there is no minimum threshold tension required for elongation in forebrain neurons; all positive tensions stimulate neurite outgrowth. Consistent with this observation, chick forebrain neurons showed weak retractile behavior in response to slackening compared to sensory neurons. Neurites that were slackened showed only transient elastic behavior and never actively produced tension, as do chick sensory neurons after slackening. We conclude that tension is an important regulator of both peripheral and central neuronal growth, but that elastic behavior is much weaker for forebrain neurons than peripheral neurons from the same developing organism. These data have significance for the understanding of the morphogenetic events of brain development.

Osmotic dilution stimulates axonal outgrowth by making axons more sensitive to tension.

Mechanical tension is a potent stimulator of axonal growth rate, which is also stimulated by osmotic dilution. We wished to determine the relationship, if any, between osmotic stimulation and tensile regulation of axonal growth. We used calibrated glass needles to apply constant force to elongate axons of cultured chick sensory neurons. We find that a neurite being pulled at a constant force will grow 50-300% faster following a 50% dilution of inorganic ions in the culture medium. That is, osmotic dilution appears to cause axons to increase their sensitivity to applied tensions. Experimental interventions suggest that this effect is not mediated by dilution of extracellular calcium, or to osmotic stimulation of adenylate cyclase, or to osmotic stimulation of mechanosensitive ion channels. Rather, experiments measuring the static tension normally borne by neurites suggest a direct mechanical effect on the cytoskeletal proteins of the neurite shaft. Our results are consistent with a formal thermodynamic model for axonal growth in which removing a compressive load on axonal microtubules promotes their assembly, thus promoting axonal elongation.

Cytomechanics of axonal development.

Mechanical tension is a robust regulator of axonal development of cultured neurons. We review work from our laboratory, using calibrated glass needles to measure or apply tension to chick sensory neurons, chick forebrain neurons, and rat PC12 cells. We survey direct evidence for two different regimes of tension effects on neurons, a fluid-like growth regime, and a nongrowth, elastic regime. Above a minimum tension threshold, we observe growth effects of tension regulating four phases of axonal development: 1. Initiation of process outgrowth from the cell body; 2. Growth cone-mediated elongation of the axon; 3. Elongation of the axon after synaptogenesis, which normally accommodates the skeletal growth of vertebrates; and 4. Axonal elimination by retraction. Significantly, the quantitative relationship between the force and the growth response is surprisingly similar to the simple relationship characteristic of Newtonian fluid mechanical elements: elongation rate is directly proportional to tension (above the threshold), and this robust linear relationship extends from physiological growth rates to far-above-physiological rates. Thus, tension apparently integrates the complex biochemistry of axonal elongation, including cytoskeletal and membrane dynamics, to produce a simple "force input/growth output" relationship. In addition to this fluid-like growth response, peripheral neurons show elastic behaviors at low tensions (below the threshold tension for growth), as do most cell types. Thus, neurites could exert small static forces without diminution for long periods. In addition, axons of peripheral neurons can actively generate modest tensions, presumably similar to muscle contraction, at tensions near zero. The elastic and force-generating capability of neural axons has recently been proposed to play a major role in the morphogenesis of the brain.

Measurements of growth cone adhesion to culture surfaces by micromanipulation.

Neurons were grown on plastic surfaces that were untreated, or treated with polylysine, laminin, or L1 and their growth cones were detached from their culture surface by applying known forces with calibrated glass needles. This detachment force was taken as a measure of the force of adhesion of the growth cone. We find that on all surfaces, lamellipodial growth cones require significantly greater detachment force than filopodial growth cones, but this differences is, in general, due to the greater area of lamellipodial growth cones compared to filopodial growth cones. That is, the stress (force/unit area) required for detachment was similar for growth cones of lamellipodial and filopodial morphology on all surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces, which had a significantly lower stress of detachment than on other surfaces. Surprisingly, the forces required for detachment (760-3,340 mudynes) were three to 15 times greater than the typical resting axonal tension, the force exerted by advancing growth cones, or the forces of retraction previously measured by essentially the same method. Nor did we observe significant differences in detachment force among growth cones of similar morphology on different culture surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces. These data argue against the differential adhesion mechanism for growth cone guidance preferences in culture. Our micromanipulations revealed that the most mechanically resistant regions of growth cone attachment were confined to quite small regions typically located at the ends of filopodia and lamellipodia. Detached growth cones remained connected to the substratum at these regions by highly elastic retraction fibers. The closeness of contact of growth cones to the substratum as revealed by interference reflection microscopy (IRM) did not correlate with our mechanical measurements of adhesion, suggesting that IRM cannot be used as a reliable estimator of growth cone adhesion.

Mechanical tension as a regulator of axonal development.

We review studies from our laboratory over the last 6 years that indicate the mechanical tension on the axons of cultured neurons is a regulator and stimulator of axonal elongation and retraction. Using calibrated glass needles to measure or apply tension, we have accumulated direct evidence for tension as a regulator of four different phases of axonal development: 1) axonal initiation; 2) growth cone-mediated elongation; 3) growth after the growth cone reaches its target; and 4) axonal retraction. Our results can be summarized by a model in which tension levels behave as a three position controller, like a double-pole, double-throw electric switch. The three settings of this switch are separated by tension thresholds: 1) Above the upper threshold, tension acts as a stimulator for axonal elongation and initiation. The growth rate of the neurite is directly proportional to the magnitude of tension on the neurite. Similar levels of tension can initiate neurites de novo from chick sensory neurons. These tension-induced axons are normal in their axial array of microtubules and in the development of a motile growth cone. Under normal conditions of growth, our evidence supports the notion that the growth cone stimulates axonal elongation by acting as a tractor, pulling on the neurite; 2) The "switch" also has a setting for axonal retraction, which occurs at tension magnitudes below some different, lower tension threshold. Our evidence indicates that such axonal retraction involves active force generation by the neurite shaft; and 3) Between the two thresholds, the switch is in a neutral position and the neurite behaves passively as a viscoelastic solid. That is, the neurite stretches in response to tension but there is no true growth, i.e. no microtubule assembly or membrane addition etc. Thus, it seems tension can be regarded as a kind of "second messenger" whose level regulates axonal development. The mechanism of action of developmental neurotoxicants may be to alter the production of, or the sensitivity to, tension. At the least, this evidence that mechanical force regulates axonal growth provides a new avenue of investigation into neurotoxic mechanisms.

Investigation of microtubule assembly and organization accompanying tension-induced neurite initiation.

Pulling on the margin of embryonic chick sensory neurons induces neurite formation de novo. We find that these neurites contain microtubules within minutes after the application of tension and apparently normal microtubule arrays within 10-20 min. We wished to determine whether these microtubules reflected existing microtubules that were reorganized, e.g. pulled into the neurite by the applied forces, or whether they reflected primarily new assembly of tubulin. We investigated tension-induced neurite initiation in the presence of 4 nM vinblastine, a concentration that poisons net microtubule assembly but does not depolymerize extant polymers, thus separating new assembly from movements of existing microtubules. We find that vinblastine seriously compromises the ability of chick sensory neurons to initiate neurites in response to tension. The few poisoned neurites that did form were abnormal in several respects. In contrast to unpoisoned cells, poisoned neurites were prone to stretching and breaking while pulling, as though they lacked normal structural support. Indeed, poisoned neurites possessed only short microtubule fragments. We conclude that the microtubule array seen in tension-induced neurites reflects primarily new microtubule assembly, rather than existing microtubules that were reorganized to invade the neurite. This implies that tension applied to unpoisoned chick sensory neurons rapidly stimulates new microtubule assembly concomitant with neurite initiation. Examination of the tension-induced microtubules shows that both their spatial pattern and their acetylation are similar to that reported for normal growth cone-mediated neurites.

A cytomechanical investigation of neurite growth on different culture surfaces.

We have examined the relationship between tension, an intrinsic stimulator of axonal elongation, and the culture substrate, an extrinsic regulator of axonal elongation. Chick sensory neurons were cultured on three substrata: (a) plain tissue culture plastic; (b) plastic treated with collagen type IV; and (c) plastic treated with laminin. Calibrated glass needles were used to increase the tension loads on growing neurites. We found that growth cones on all substrata failed to detach when subjected to two to threefold and in some cases 5-10-fold greater tensions than their self-imposed rest tension. We conclude that adhesion to the substrate does not limit the tension exerted by growth cones. These data argue against a "tug-of-war" model for substrate-mediated guidance of growth cones. Neurite elongation was experimentally induced by towing neurites with a force-calibrated glass needle. On all substrata, towed elongation rate was proportional to applied tension above a threshold tension. The proportionality between elongation rate and tension can be regarded as the growth sensitivity of the neurite to tension, i.e., its growth rate per unit tension. On this basis, towed growth on all substrata can be described by the simple linear equation: elongation rate = sensitivity x (applied tension - tension threshold) The numerical values of tension thresholds and neurite sensitivities varied widely among different neurites. On all substrata, thresholds varied from near zero to greater than 200 mudynes, with some tendency for thresholds to cluster between 100 and 150 mudynes. Similarly, the tension sensitivity of neurites varied between 0.5 and 5.0 microns/h/mudyne. The lack of significant differences among sensitivity or threshold values on the various substrata suggest to use that the substratum does not affect the internal "set points" of the neurite for its response to tension. The growth cone of chick sensory neurons is known to pull on its neurite. The simplest cytomechanical model would assume that both growth cone-mediated elongation and towed growth are identical as far as tension input and elongation rate are concerned. We used the equation above and mean values for thresholds and sensitivity from towing experiments to predict the mean growth cone-mediated elongation rate based on mean rest tensions. These predictions are consistent with the observed mean values.

An absolute rate theory model for tension control of axonal elongation.

This paper extends our previous thermodynamic model for the effect of mechanical force on the microtubule assembly that accompanies axonal (neurite) elongation of neurons. Based on the previous treatment, experimental data, and the formalism of absolute rate theory, we derive an exact expression for how tension on the neurite affects mechanical force in the microtubule, and in turn, how these affect the rate of microtubule assembly and neurite outgrowth in cultured neurons. This prediction approximates the experimentally observed linear relationship between growth rate and experimentally applied tension, and predicts the previously postulated three-position integral control.

Growth cone motility.

The exact nature of growth cone motility is far from understood but progress has been made in several areas. It now appears that growth cones pull and not push; we will review the biophysical basis of growth cone movement. Current ideas on the regulation of growth cone motility and the relationship between motility and axon pathfinding are also discussed.

Tensile regulation of axonal elongation and initiation.

Neurites of chick sensory neurons in culture were attached by their growth cones to glass needles of known compliance and were subjected to increasing tensions as steps of constant force; each step lasted 30-60 min and was 25-50 mu dyn greater than the previous step. After correcting for elastic stretching, neurite elongation rate increased in proportion to tension magnitude greater than a tension threshold. The value of the tension threshold required for growth varied between 25 and 560 mu dyn, with most between 50 and 150 mu dyn. The growth sensitivity of neurites to tension was surprisingly high: an increase in tension of 1 mu dyn increased the elongation rate an average of about 1.5 microns/hr. The linear relationship between growth rate and tension provides a simple control mechanism for axons to accommodate tissue expansion in growing animals that consistently maintains a moderate rest tension on axons. Styrene microspheres treated with polyethyleneimine were used to label the surface of neurites in order to determine the site and pattern of surface addition during the experimental "towed growth" regime. New membrane is added interstitially throughout the neurite, but different regions of neurite vary widely in the amount of new membrane added. This contrasts with membrane addition specifically at the distal end in growth-cone-mediated growth. The different sites for membrane addition in growth mediated by towing and by the growth cone indicate that the membrane addition process is sensitive to the mode of growth. We confirmed the finding of Bray (1984) that neurites can be initiated de novo by application of tension to the cell margin of chick sensory neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

On the cytomechanics and fluid dynamics of growth cone motility.

Following a brief review of the controversy concerning the physical mechanism of growth cone advance, we present cytomechanical data to support a version of the classic model of growth cone motility. In this model, the growth cone is pulled forward by filopodial tension. Observations of growth cone behavior and axonal guidance suggest that this model should include fluid flow mechanisms as well as the original solid, elastic mechanism. Recent data are reviewed on the similarity of the fluid behavior of cytoplasm and of suspensions of cytoskeletal filaments. The thixotropic behavior of cytoplasm is used to develop a model for lamellipodial protrusion caused by filopodial tension.

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.

Liquid crystal domains and thixotropy of filamentous actin suspensions.

The thixotropic properties of filamentous actin suspensions were examined by a step-function shearing protocol. Samples of purified filamentous actin were sheared at 0.2 sec-1 in a cone and plate rheometer. We noted a sharp stress overshoot upon the initiation of shear, indicative of a gel state, and a nearly instantaneous drop to zero stress upon cessation of shear. Stress-overshoot recovery was almost complete after 5 min of "rest" before samples were again sheared at 0.2 sec-1. Overshoot recovery increased linearly with the square root of rest time, suggesting that gel-state recovery is diffusion limited. Actin suspensions subjected to oscillatory shearing at frequencies from 0.003 to 30 radians/sec confirmed the existence of a 5-min time scale in the gel, similar to that for stress-overshoot recovery. Flow of filamentous actin was visualized by polarized light observations. Actin from 6 mg/ml to 20 mg/ml showed the "polycrystalline" texture of birefringence typical for liquid crystal structure. At shear rates less than 1 sec-1, flow occurred by the relative movement of irregular, roughly ellipsoidal actin domains 40-140 microns long; the appearance was similar to moving ice floes. At shear rates greater than 1 sec-1, domains decreased in size, possibly by frictional interactions among domains. Eventually domains flow in a "river" of actin aligned by the flow. Our observations confirm our previous domain-friction model for actin rheology. The similarities between the unusual flow properties of actin and cytoplasm argue that cytoplasm also may flow as domains.

Extracellular matrix allows PC12 neurite elongation in the absence of microtubules.

Several groups have shown that PC12 will extend microtubule-containing neurites on extracellular matrix (ECM) with no lag period in the absence of nerve growth factor. This is in contrast to nerve growth factor (NGF)-induced neurite outgrowth that occurs with a lag period of several days. During this lag period, increased synthesis or activation of assembly-promoting microtubule-associated proteins (MAPs) occurs and is apparently required for neurite extension. We investigated the growth and microtubule (MT) content of PC12 neurites grown on ECM in the presence or absence of inhibitors of neurite outgrowth. On ECM, neurites of cells with or without prior exposure to NGF contain a normal density of MTs, but frequently contain unusual loops of MTs in their termini that may indicate increased MT assembly. On ECM, neurites extend from PC12 cells in the presence of 10 microM LiCl at significantly higher frequency than on polylysine. On other substrates, LiCl inhibits neurite outgrowth, apparently by inhibiting phosphorylation of particular MAPs (Burstein, D. E., P. J. Seeley, and L. A. Greene. 1985. J. Cell Biol. 101:862-870). Although 35-45% of 60 Li(+)-neurites examined were found to contain a normal array of MTs, 25-30% were found to have a MT density approximately 15% of normal. The remaining 30% of these neurites were found to be nearly devoid of MTs, containing only occasional, ambiguous, short tubular elements. We also found that neurites would extend on ECM in the presence of the microtubule depolymerizing drug, nocodazole. At 0.1 micrograms/ml nocodazole, cells on ECM produce neurites that contain a normal density of MTs. This is in contrast to the lack of neurite outgrowth and retraction of extant neurites that this dose produces in cells grown on polylysine. At 0.2 microgram/ml nocodazole, neurites again grew out in substantial number and four of five neurites examined ultrastructurally were found to be completely devoid of microtubules. We interpret these results by postulating that growth on ECM relieves the need for MTs to serve as compressive supports for neurite tension (Dennerll, T. J., H. C. Joshi, U. L. Steel, R. E. Buxbaum, and S. R. Heidemann. 1988. J. Cell Biol. 107:665). Because compression destabilizes MTs and favors disassembly, this would tend to increase MT assembly relative to other conditions, as we found. Additionally, if MTs are not needed as compressive supports, neurites could grow out in their absence, as we also observed.