FRAP analysis of the stability of the microtubule population along the neurites of chick sensory neurons.

In order to study microtubule turnover in elongating neurites, chick embryo sensory neurons were microinjected with x-rhodamine tubulin, and after 6-12 hours, short segments along chosen neurites were photobleached at multiple sites. Previous studies [Lim et al., 1989; 1990] indicated that recovery of fluorescence (FRAP) in neurites occurs by the dynamic turnover of stationary microtubules. In all cases, distal bleached zones recovered fluorescence faster than bleached zones more proximally located along the same neurites. Bleached zones at growth cones completely recovered in 30-40 minutes, while bleached zones located more proximally usually recovered in 50-120 minutes. In the most proximal regions of long neurites, recovery of fluorescence was often incomplete, indicating that a significant fraction of the microtubules in these regions were very stable. These studies indicate that there are differences in microtubule stability along the length of growing neurites. These differences may arise from the combined effects of 1) modifications that stabilize and lengthen microtubules in maturing neurites and 2) the dynamic instability of the distally oriented microtubule plus ends.

Insulin-like growth factor-I and its N-terminal modified analogues induce marked gut growth in dexamethasone-treated rats.

The effects of insulin-like growth factor-I (IGF-I) on the gut of 150 g dexamethasone-treated rats were compared with those of two analogues with reduced affinity for IGF-binding proteins, des(1-3)IGF-I and LR3IGF-I, an N-terminal-extended variant. Administration of IGF-I for 7 days to rats made catabolic by co-treatment with dexamethasone induced a dose-dependent increase in total gut weight, with the highest dose of IGF-I (695 micrograms/day) increasing gut weight by up to 60%, and gut weight as a fraction of body weight by up to 32%. Effects were apparent in all regions of the gut examined, including the stomach, small intestine and colon. Histological and biochemical analyses of the intestine showed that cross-sectional mass, rather than gut length, was increased, and proportional increases in wet weight, protein and DNA content per unit length were measured in both the mucosa and muscularis layers. The rate of duodenal protein synthesis measured on day 7 of treatment was not increased by IGF-I treatment. The IGF-I analogues had qualitatively similar effects to IGF-I, but were consistently severalfold more potent, providing evidence that IGF-binding proteins reduce the biological activity of exogenous IGF-I in the gut. The results indicate that the gut is one of the most sensitive IGF-I target tissues, and that potency in vivo correlates with a reduced interaction with IGF-binding proteins.

Identification of a tektin-like protein associated with neurofilaments in the developing chick nervous system.

A 160-kD polypeptide, which is recognized by an affinity-purified polyclonal antibody to the 55-kD tektin-A polypeptide from sea urchin sperm flagellar microtubules, is associated with neurofilaments in embryonic chick nerve cells. Antibodies to tektin-A and monoclonal antibodies to the neurofilament triplet proteins colocalize to filaments in cultured nerve cells and to filaments in extracts of chick spinal cord, using indirect immunofluorescence microscopy and immunogold electron microscopy. The antigen reacting with anti-tektin-A in chick brain and spinal cord extracts has been identified as a 160-kD polypeptide by SDS-PAGE and has been shown to be distinct from the known neurofilament-triplet proteins by two-dimensional immunoblot analysis. These data suggest that a unique protein with limited sequence homology to tektin-A is a component of the neuronal cytoskeleton and is incorporated into or associated with neurofilaments.

A test of microtubule translocation during neurite elongation.

In a previous study using PC-12 cells (Lim, S. S., P. J. Sammak, and G. G. Borisy, 1989. J. Cell Biol. 109:253-263), we presented evidence that the microtubule component of the neuronal cytoskeleton is differentially dynamic but stationary. However, neurites of PC-12 cells grow slowly, hindering a stringent test of slow axonal transport mechanisms under conditions where growth was substantial. We therefore extended our studies to primary cultures of dorsal root ganglion cells where the rate of neurite outgrowth is rapid. Cells were microinjected with X-rhodamine-labeled tubulin 7-16 h after plating. After a further incubation for 6-18 h, the cells were photobleached with an argon ion laser. Using a cooled charged couple device and video microscopy, the cells were monitored for growth of the neurite and movement and recovery of fluorescence in the bleached zone. As for PC-12 cells, all bleached zones in the neurite recovered their fluorescence, indicating that incorporation of tubulin occurred along the neurite. Despite increases in neurite length of up to 70 microns, and periods of observation of up to 5 h, no movement of bleached zones was observed. We conclude that neurite elongation cannot be accounted for by the transport of a microtubule network assembled only at the cell body. Rather, microtubules turn over all along the length of the neurite and neurite elongation occurs by net assembly at the tip.

Distribution of laminin in the developing peripheral nervous system of the chick.

During axonal elongation in the developing peripheral nervous system, the temporal and spatial distribution of adhesive molecules in extracellular matrices and on neighboring cell surfaces may provide "choices" of pathways for growth cone migration. The extracellular matrix glycoprotein laminin appears in early embryos and mediates neuronal adhesion and neurite extension in vitro. In this study, we have examined the distribution of laminin at early periods of peripheral nervous system development. The distribution of laminin, demonstrated by immunostaining frozen sections of chick embryos, was compared to the distribution of fibronectin and of early peripheral neurites as revealed with an antibody to a neurofilament-associated protein. Laminin is present in the neural tube basement membrane, in early ganglia, and in developing dorsal and ventral roots, where the laminin staining pattern parallels that of neurofilaments. In early ganglia and nerve roots, laminin immunostaining defines loose "meshworks" rather than basement membranes, which seem to form slightly later in these structures. In contrast, fibronectin is absent in neural tube basement membrane, ganglia, and nerve roots, although it is present along neural crest migratory pathways and in intersomitic spaces. Our observations of laminin distribution are consistent with the possibility that laminin provides an adhesive surface for neurite extension at some stages of early peripheral nervous system development.