Tension and compression in the cytoskeleton of PC 12 neurites.

Abstract

We report in this article that the retraction of PC 12 neurites, unlike that of other cultured neurons, is due to tension within the neurite. Retraction is rapid and independent of metabolic energy. Transection of one arm of a branched neurite immediately causes the remaining arm to take up a new equilibrium position between attachment points. Similarly, detachment of one growth cone of a cell causes the cell body to move to a new equilibrium position between the remaining neurites. These observations provide direct evidence for the suspension of the cell soma among a network of tensioned neurites. We used retraction as an assay for neurite tension to examine the role of actin filaments and microtubules in neurite support and elongation. Our data suggest that microtubules (MTs) within PC 12 neurites are under compression, supporting tension within the actin network. Treatment of cells with drugs that disrupt actin networks, cytochalasin D or erythro-9-[3-(2-hydroxynonyl)]adenosine eliminates retraction regardless of the absence of MTs, lack of adhesion to the substratum, or integrity of the neurite. Conversely, stimulation of actin polymerization by injection of phalloidin causes retraction of neurites. Treatments that depolymerize MTs, nocodazole or cold, cause retraction of neurites, which suggests that microtubules support this tension, i.e., are under compression. Stabilization of MTs with taxol stabilizes neurites to retraction and under appropriate circumstances can drive neurite extension. Taxol-stimulated neurite extension is augmented by combined treatment with anti-actin drugs. This is consistent with the actin network's normally exerting a force opposite that of MT assembly. Cytochalasin and erythro-9-[3-(2-hydroxynonyl)] adenosine were found to increase slightly the dose of nocodazole required for MT depolymerization. This is consistent with the postulated balance of forces and also suggests that alteration of the compression borne by the microtubules could serve as a local regulator for MT polymerization during neurite outgrowth.There is general agreement that axonal growth is intimately dependent upon the assembly and spatial organization of the neuronal cytoskeleton (3,16). Despite this agreement, the role of the cytoskeletal elements in axonal growth is poorly understood. The mechanism for integrating the polymerization of actin and microtubules (MTs) ~ is completely unknown. However, several different lines of evidence point to a local, i.e. controlled within the axon, mechanism for the integration of cytoskeletal assembly with axonal growth: axons and neurites severed from the cell body can regrow (18,24,28); new growth cones appear on the normally quiescent sides of neurites in response to MT depolymerization (4, 28); neurite outgrowth can be guided by local changes in substratum adhesiveness (17); and isolated neurites or growth cones can respond to a gradient of nerve growth factor (NGF) (6, 23).We recently reported that rate and extent of neurite outgrowth of PC 12 rat pheochromocy...

Dendritic Fibroblasts in Three-dimensional Collagen Matrices

“…If the network already was formed, however, then disrupting the actin cytoskeleton did not cause withdrawal of extensions but did cause actin clustering. Disrupting the actin cytoskeleton of bipolar chick embryo fibroblasts in collagen matrices (Tomasek and Hay, 1984) or neurites on planar surfaces was shown to have a similar effect on actin organization (Joshi et al, 1985). The appearance of these actin clusters suggests that the extensions are subject to some kind of as yet unexplained topographic organization.…”

Microtubule-based filopodium-like protrusions form after axotomy

“…It has been suggested that tension exerted by the actin network of the growth cone inhibits the elongation of microtubules (Joshi et al, 1985), and it has been shown that weakening of this network with cytochalasin promotes microtubule advance (Forscher and Smith, 1988;Burmeister et al, 199 1). The emergence of blebs after axotomy suggests local cortical weakening (Albrect-Buehler, 1987).…”

Mechanical control of tissue morphogenesis during embryological development

“…This ability to orient cell movement through cell distortion likely plays a central role in control of tissue patterning, as evidenced by the finding that tension application similarly promotes capillary outgrowth and elongation along the tension field lines in 3D matrices (Korff and Augustin, 1999). Cell-generated tensional forces also direct nerve outgrowth, axon fate and 3D organization of neuron arbors in vivo as well as in vitro (Bray, 1979, Joshi et al, 1985, Condron and Zinn, 1997, Lamoureux et al, 2002. In contrast, complete basement membrane dissolution that leads to cell retraction and rounding produces tissue involution during angiogenesis inhibition , as well as during regression of Mullerian duct (Trelstad et al, 1982) and mammary gland (Wicha et al, 1980).…”