Tension and compression in the cytoskeleton of PC-12 neurites. II: Quantitative measurements.

Abstract

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...

Cyclic stretch-induced reorganization of the cytoskeleton and its role in enhanced gene transfer

“…10,15 We found that when microtubules undergo a dynamic deformation in the form of biaxial cyclic stretch, significant remodeling of the microtubule network occurs. Analysis via Western blot indicated that tubulin in the TX-100 insoluble fraction (i.e.…”

“…10 Likewise, depolymerization is increased in cells that undergo compressive stresses, with a twofold decrease in polymerized tubulin seen in cells undergoing active compression. 15 Unlike the microfilaments, which showed structural reorganization by fluorescence microscopy but did not decrease in number as measured biochemically, microtubules decreased in abundance after as little as 30 min of stretch. By 24 h, polymerized tubulin in stretched cells was o20% than in their unstretched counterparts, suggesting that tubulin depolymerization also plays a role in enhanced gene delivery.…”

Leading Tip Drives Soma Translocation via Forward F-Actin Flow during Neuronal Migration

“…In non-neuronal cells, proper cell migration requires microtubule severing at the minus end and the release of microtubules from the centrosome (Abal et al, 2002;Toyo-Oka et al, 2005;Sudo and Maru, 2008), processes that may help to remove the restriction of microtubule arrays on nucleus advance. Moreover, pharmacological disruption of microtubules increases F-actin-mediated contractility in many different cell types, including PC12 cells (Dennerll et al, 1988), fibroblasts (Danowski, 1989), retinal photoreceptor cells (Madreperla and Adler, 1989), and cardiac muscle cells (Tsutsui et al, 1993). In Xenopus oocytes, cortical F-actin flow could be suppressed by microtubule polymerization and enhanced by microtubule depolymerization (Canman and Bement, 1997;Benink et al, 2000).…”

Tensegrity II. How structural networks influence cellular information processing networks

“…3 bottom). In fact, this precise response has been demonstrated in various types of cultured cell, including nerve cells and smooth muscle cells (Joshi et al, 1985;Buxbaum and Heidemann, 1988;Dennerll et al, 1988;Dennerll et al, 1989;Putnam et al, 1998;Putnam et al, 2001;Kaverina et al, 2002). Although altering stresses across integrins does not alter microtubule polymerization in liver cells, the steady-state level of soluble tubulin monomer changes in a manner consistent with a similar stressinduced change in the critical concentration of tubulin (Mooney et al, 1994).…”