Microtubule dynamics
This movie shows the dynamic behavior of microtubules in a growth cone of an Aplysia bag cell neuron cultured on poly-lysine. The cell body was injected with 1mg/ml rhodamine-labeled tubulin and after incorporation of the low level of labeled tubulin, the labeled microtubule structures were images taken with a very sensitive CCD camera in 5 second intervals for a total time of 10 min. The time lapse movie plays at 75x real time speed and shows a horizontal field of 66 microns. This fluorescence imaging technique is called “Fluorescent Speckle Microscopy” (FSM). The discontinuous labeling pattern of microtubules enables the investigator to precisely analyze all the dynamic parameters of microtubule growth and translocation. Investigating the changes in microtubule dynamics during growth cone extension and turning is critical for our understanding of the molecular and cellular mechanisms of growth cone guidance. (Movie was acquired by Aih Cheun Lee in the Suter Lab).
Growth cone steering
This movie shows a high resolution video-enhanced DIC (differential interference contrast) time-lapse sequence of a restrained bead interaction (RBI) of a cultured Aplysia bag cell neuronal growth cone. The RBI assay can be used to study growth cone steering in response to defined adhesive molecular cues. In this particular case, glass beads (5 micron in diameter) were coated with an antibody against the immunoglobulin superfamily cell adhesion molecule apCAM, which is present on the growth cone plasmamembrane. The beads are being positioned on the growth cone surface with a microneedle controlled by a micromanipulator. The bead rapidly binds to cell surface apCAM and would undergo retrograde translocation if not restrained by the microneedle as shown from the beginning of the movie. The restrained bead mimics a physiological adhesive and immobilized target site. As a response to the apCAM-specific adhesive site, the growth cone, after an initial latency period of 15 min, turns towards the restrained bead. The following changes happen exactly as in cellular growth cone target interactions: central domain extension towards the target site, leading edge growth behind the bead, slowing of F-actin flow specificically along the interaction axis, building up of tension as indicated by needle deflection as well as microtubule extension to and F-actin accumulation around the bead (not seen in the movie). This assay is provides a useful system to investigate the molecular and cellular mechanisms of growth cone turning. The horizontal field is 60 microns wide; the time compression factor is 50x. (Movie was published in Suter et al., 1998. J. Cell Biol. 141 (1): 227-240)