The cytoskeletons of isolated, neuronal growth cones

Abstract

We have examined by electron microscopy the cytoskeletons of growth cones isolated from neonatal rat forebrain by the method of Gordon-Weeks and Lockerbie [Gordon-Weeks and Lockerbie (1984) Neuroscience 13, 119-136]. When fixed in suspension with conventional fixatives, isolated growth cones contain a central region filled with a branching system of smooth endoplasmic reticulum and a cortical region immediately beneath the plasma membrane that is relatively free of organelles and is composed of an amorphous granular cytoplasm. The filopodia of isolated growth cones are also devoid of organelles and contain a cytoplasm that is similar in appearance to that in the cortical region. No microtubules or neurofilaments have been found in these growth cones. When isolated growth cones were prepared for electron microscopy by a method which preserves actin filaments [Boyles, Anderson and Hutcherson (1985) J. Histochem. Cytochem. 33, 1116-1128], microfilaments were found throughout the cortical cytoplasm. In the filopodia, the microfilaments were bundled together and oriented longitudinally. Filopodial microfilament bundles often extended into the body of the growth cone and could traverse it completely. Inclusion of Triton X-100 (1% v/v) in the fixative solubilized the membranes and soluble cytoplasmic proteins of growth cones, allowing an unobscured view of the microfilament cytoskeleton including the core bundle of microfilaments in filopodia. Suspended within the cytoskeleton were the coats of coated vesicles. These were particularly numerous at the broad bases of filopodia. Microfilaments bound heavy meromyosin and were cytochalasin B (2.0 X 10(-7) M) sensitive. Individual microfilaments branched and within filopodia they were extensively cross-linked by thin (7 nm) filaments. Microtubules and neurofilaments were not seen in these cytoskeletons despite the fact that the fixative contained a Ca2+ chelator. When growth cones were preincubated in taxol (14 microM) their cytoskeletons were found to contain microtubules. These were located mainly in the centre of the growth cone, were absent from the filopodia and were contiguous with microfilaments. We conclude that the cytoskeletons of isolated neuronal growth cones from neurones of the central nervous system are mainly composed of actin microfilaments. Although microtubules are not normally present, there is a pool of soluble tubulin which will form microtubules in the presence of taxol. This may imply that those microtubule-associated proteins that promote tubulin polymerization are absent in the growth cone or are below the concentration threshold for polymerization.