Axon Growth: Roles of Microfilaments and Microtubules

Abstract

Abstract. The motile tips of elongating axons consist of growth cones from which microspikes protrude. Cytochalasin B causes retraction of microspikes, rounding-up of growth cones, and cessation of axon elongation. Drug withdrawal is followed by resumption of growth cone and microspike activity and of axon elongation. In contrast, colchicine causes shortening and retraction of axons, but it does not initially affect the tips. Growth cones and microspikes of elongating axons contain a network of 50 A microfilaments, the pattern of which is altered by cytochalasin treatment. These experiments indicate that both structural integrity of the axon and continuing function of its motile tip are essential elements in axonal elongation.The tips of elongating axons consist of expanded regions called "growth cones" from which project long slender microspikes (filopodia). These microspikes continually wave about, extend, and retract as the growth cone moves over a substratum.1 Although it is assumed that growth cones and microspikes play a significant role in axon extension, it has not been previously possible to analyze their functions.We have investigated the effects of cytochalasin B on axon elongation since this drug halts cell movement,2 a process similar in many respects to growth cone movement. Cytochalasin B is also known to inhibit cytokinesis3 and morphogenetic movements,4 apparently by disrupting contractile 50 A microfilaments. The drug's effects upon nerve cells are compared with those of colchicine and colcemid, which disrupt microtubules' and cause retraction of elongating axons.6Methods. Nerve ganglion culture: Lumbosacral dorsal root ganglia from 8-day-old white leghorn chick embryos were cultured in Grobstein tissue culture dishes7 (Microchemical Specialties). The semisolid culture medium consisted of 0.05 ml of 1% agar in Hanks' balanced salt solution plus 0.1 ml of medium 199 containing 10% embryo extract,8 20% fetal calf serum, and antibiotics (100 units/ml penicillin, 100 Ag/ml streptomycin, and 2.5 ug/ml amphotericin B). Nerve growth factor was present as a 1: 300,000 homogenate of submandibular glands from highly inbred BALB/c adult male mice;9 this concentration of homogenate was found to be optimal for producing rapid outgrowth of axons.Cell cultures: Dorsal root ganglia and the ventral halves of spinal cords were dissociated by a modification of Scott's procedure.10 Lumbosacral dorsal root ganglia (80-120) from 8-day-old embryos were incubated at 370C in 0.25% trypsin solution (Grand Island Biological). After 1 hr, the trypsin was aspirated, culture medium containing serum was added, and the ganglia were repeatedly flushed through a narrow bore pipet. The resulting cell suspension was pelleted at 500 g for 2 min and resuspended.After two such washes, the final dilution was made to approximately 105 cells/ml in nerve 1206

Disorganized Microtubules Underlie the Formation of Retraction Bulbs and the Failure of Axonal Regeneration

“…Microtubules play a key role in axonal growth and guidance (Yamada et al, 1970;Forscher and Smith, 1988;Suter et al, 1998;Dent et al, 1999;Dent and Gertler, 2003). They form the backbone of the axonal shafts and core domain of growth cones, giving stability to those structures and enabling organelle transport (Hirokawa and Takemura, 2005).…”

The Effects of Cytochalasin B on the Microfilaments of Baby Hamster Kidney (Bhk-21) Cells

“…Cytochalasin B (CB)I (1) causes the disruption of microfilaments in the cleavage furrow of marine eggs end HeLa cells (2)(3)(4), and the reversible disorganization of microfilaments in several types of embryonic cultures (5)(6)(7)(8) . It has been suggested that these microfilaments are involved in primitive contractile processes and that they are similar to actin (8)(9)(10) .…”

Cytochalasin B inhibits actin-related gelation of HeLa cell extracts.

“…These movements seem to be related to a membraneassociated microfilament system (14,27,28,36,38) that has frequently been shown to contain actin (2,22,24,29,30). Many morphological studies showing disruption of thin filament arrays (3,6,7,18,31,32,39,40) and a few studies with purified actin (16,21,33,34) or myosin (footnote 2 and reference 26) suggest that cytochalasins can interact directly with proteins of the microfilament system. However, because of the multiple and sometimes contradictory effects of cytochalasins (4,5,8,12), no in vivo effect has yet been related unambiguously to direct interaction with the microfilament system.…”