No abstract

In the present report we used the calcium indicator fura-2 to compare intracellular levels of free calcium in growth cones of isolated Helisoma neurons under a variety of experimental conditions. We tested whether 2 different signals that inhibit growth cone motility--action potentials and serotonin--changed calcium levels in growth cones. Electrical stimulation of the cell body caused a rise in calcium levels at the growth cone. After brief stimulation, calcium levels quickly recovered to normal values, whereas longer stimulation periods required longer recovery times. The application of serotonin to growth cones caused an increase in calcium levels that was selective for growth cones of neurons whose outgrowth was inhibited by serotonin, but not for neurons whose outgrowth was not affected. We also found that motile growth cones had higher free calcium levels than growth cones that had spontaneously stopped growing. Furthermore, the distribution of calcium in neurons that contained motile growth cones was heterogeneous; calcium levels were always higher in the growth cone than in the neurite or soma. These data indicate that calcium levels in growth cones vary in different states of outgrowth and that calcium levels can be modulated by both electrical and chemical signals.

Ît is commonly believed that growth cone turning during pathfinding is initiated by reorganization of actin filaments in response to guidance cues, which then affects microtubule structure to complete the turning process. However, a major unanswered question is how changes in actin cytoskeleton are induced by guidance cues and how these changes are then translated into microtubule rearrangement. Here, we report that local and specific disruption of actin bundles from the growth cone peripheral domain induced repulsive growth cone turning. Meanwhile, dynamic microtubules within the peripheral domain were oriented into areas where actin bundles remained and were lost from areas where actin bundles disappeared. This resulted in directional microtubule extension leading to axon bending and growth cone turning. In addition, this local actin bundle loss coincided with localized growth cone collapse, as well as asymmetrical lamellipodial protrusion. Our results provide direct evidence, for the first time, that regional actin bundle reorganization can steer the growth cone by coordinating actin reorganization with microtubule dynamics. This suggests that actin bundles can be potential targets of signaling pathways downstream of guidance cues, providing a mechanism for coupling changes in leading edge actin with microtubules at the central domain during turning.

The E. coli single-stranded DNA-binding protein (SSB) binds to the fork DNA helicases RecG and PriA in vitro. Typically for binding to occur, 1.3 M ammonium sulfate must be present, bringing into question the validity of these data as these are non-physiological conditions. To determine whether SSB can bind to these helicases, we examined binding in vivo. First, using fluorescence microscopy, we show that SSB localizes PriA and RecG to the vicinity of the inner membrane in the absence of DNA damage. Localization requires that SSB be in excess over the DNA helicases and the SSB C-terminus and both PriA and RecG be present. Second, using purification of tagged complexes, our results demonstrate that SSB binds to PriA and RecG in vivo, in the absence of DNA. We propose that this may be the “storage form” of RecG and PriA. We further propose that when forks stall, RecG and PriA are targeted to the fork by SSB which, by virtue of its high affinity for single stranded DNA, allows these helicases to out compete other proteins. This ensures their actions in the early stages of the rescue of stalled replication forks.

Electrical activity may regulate a number of neuronal functions in addition to its role in transmitting signals along nerve cells. The hypothesis that electrical activity affects neurite elongation in sprouting neurons was tested by stimulating individual snail neurons isolated in cell culture. The findings demonstrated that growth cone advance, and thus neurite elongation, is reversibly stopped during periods when action potentials are experimentally evoked. A decrease in filopodial number and growth cone area was also observed. Thus, action potentials can mediate the cessation of neurite outgrowth and thereby may influence structure and connectivity within the nervous system.