Neurites of PC12 and chick dorsal root ganglion neurons behave as viscoelastic solids in response to applied forces. This passive behavior can be modeled with three mechanical elements; a relatively stiff, undamped spring in series with a Voight element composed of a less stiff spring in parallel with a dashpot. In response to applied tensions greater than 100 microdynes, PC12 cells show lengthening behavior distinct from and in addition to the passive viscoelastic response. We interpret this as "towed growth" (Bray, D. 1984. Dev. Biol. 102:379-389) because the neurites can become twice as long without obvious thinning of the neurite and because in two cases neurite tensions fell below original rest tensions, a result that cannot be obtained with passive viscoelastic elements. The rate of towed growth showed a linear dependence of growth rate with applied tensions in 8 of 12 PC12 neurites exposed to applied tension greater than 100 microdynes. Both PC12 and chick sensory neurons showed evidence of retraction when neurite tensions were suddenly diminished. This response was measured as tension recovery after slackening in chick sensory neurites. In 62% of the cases, tension recovery exceeded and sometimes doubled the preexperimental steady-state tension. Our data indicate that this response is active tension generation by the neurite shaft. We conclude that neurite length is regulated by axial tension in both elongation and retraction. Our data suggest a three-way controller: above some tension set point, the neurite is stimulated to elongate. Below some different, lower tension threshold the neurite is stimulated to retract. Between these two tension thresholds, the neurite responds passively as a viscoelastic solid.

There is controversy over whether axonal elongation is the result of a pulling growth cone and the role of tension in axonal elongation. Earlier in this decade, the consensus was that axons or neurites elongated from tension generated by forward motility of the growth cone. It was presumed that contractile filopodia were the source of the tension moving the growth cone. But this view was challenged by experiments showing that neurites elongate, albeit abnormally, in the presence of cytochalasin, which inhibits growth-cone and filopodial movements. Additionally, high resolution, video-enhanced observations of growth-cone activity argued against filopodial shortening as a source of tension, suggesting instead that an extrusion of cytoplasm rather than a pulling process, is the key event in neurite elongation. Studies of slow axonal transport, however, indicate that much slower cytoskeletal pushing underlies axonal elongation. We report here direct measurements of neurite force as a function of growth-cone advance which show that they are linearly related and accompanied by apparent neurite growth. No increase in force occurs in neurites whose growth cone fails to advance.

The polarity orientation of cellular microtubules is widely regarded to be important in understanding the control of microtubule assembly and microtubule-based motility in vivo . We have used a modification of the method of Heidemann and McIntosh (Nature (Lond .) . 286:517-519) to determine the polarity orientation of axonal microtubules in postganglionic sympathetic fibers of the cat. In fibers from three cats we were able to visualize the polarity of 68% of the axonal microtubules ; of these, 96% showed the same polarity orientation . Our interpretation is that the rapidly growing end of all axonal microtubules is distal to the cell body . We support Kirschner's hypothesis on microtubule organizing centers (J . Cell Biol. 86 : 330-334), although this interpretation raises questions about the continuity of axonal microtubules. Our results are inconsistent with a number of models for axonal transport based on force production on the surface of microtubules in which the direction of force is determined by the polarity of microtubules .

Abstract. We assessed the mechanical properties of PC-12 neurites by applying a force with calibrated glass needles and measured resulting changes in neurite length and deflection of the needle. We observed a linear relationship between force and length change that was not affected by multiple distensions and were thus able to determine neurite spring constants and initial, nondistended, rest tensions. 81 out of 82 neurites showed positive rest tensions ranging over three orders of magnitude with most values clustering around 30-40 lxdynes. Treatment with cytochalasin D significantly reduced neurite rest tensions to an average compression equal to 14% of the former tension and spring constants to an average of 17 % of resting values. Treatment with nocodazole increased neurite rest tensions to an average of 282 % of resting values but produced no change in spring constant. These observations suggest a particular type of complementary force interaction underlying axonal shape; the neurite actin network under tension and neurite microtubules under compression. Thermodynamics suggests that microtubule (MT) assembly may be regulated by changes in compressive load. We tested this effect by releasing neurite attachment to a polylysine-coated surface with polyaspartate, thus shifting external compressive support onto internal elements, and measuring the relative change in MT polymerization using quantitative Western blotting. Neurons grown on polylysine or collagen without further treatment had a 1:2 ratio of soluble to polymerized tubulin. When neurites grown on polylysine were treated with 1% polyaspartate for 15-30 min, 80% of neurites retracted, shifting the soluble: polymerized tubulin ratio to 1:1. Polyaspartate treatment of cells grown on collagen, or grown on polylysine but treated with cytochalasin to reduce tension, caused neither retraction nor a change in the soluble:polymerized tubulin ratio. We suggest that the release of adhesion to the dish shifted the compressive load formerly borne by the dish onto MTs causing their partial depolymerization. Our observations are consistent with the possibility that alterations in MT compression during growth cone advance integrates MT assembly with the advance.INCE the pioneering studies of Yamada et al. (59), many investigators have found a clear cut "division of labor" in the role of the cytoskeleton in growth cone motility and axonal elongation. Anti-microtubule (MT) I drugs cause neurites to collapse but have no effect on growth cone motility functions, e.g. ruffling, microspike activity, etc. (8,17,24,30,31,49). Conversely, anti-actin drugs inhibit growth cone motility functions but neurites remain extended (8,24,31,49). Further, drugs that disrupt the actin network stabilize the neurite to retraction, while drugs that disrupt MTs cause retraction. Also, drugs that augment actin assembly cause retraction while drugs that augment MT assembly stabilize neurites to retraction and sometimes cause extension (15,31,35,49). Similarly, the networks move at different rate...

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)