This subproject does not utilize Center technology but is supported by staff expertise. In the long term we aim to combine the high quality imaging available - Oldenbourg's lab with the role of biocurrents - neural pathfinding and cellular differentiation. The neuronal growth cone appears at the tip of dendrites and axons where it plays an important role in the navigation of dendritic and axonal growth. Growth cones are rich in actin fibers which are presumably involved in growth cone movement. Careful observations of fluorescent labeled actin filaments with a fluorescent microscope or of native actin bundles with a video-enhanced differential interference contrast (DIC) microscope demonstrated important aspects of actin -related dynamics, such as retrograde flow (1), treadmilling of actin (2), and involvement of myosin V in filopodial elongation (3). However, the molecular mechanisms of growth cone movement are still in debate, because it is difficult to visualize actin bundles in living cells. A new methodology is needed for understanding the mechanism of growth cone movement. Therefore, we observed actin bundles in living growth cones with the Oldenbourg's New Pol-Scope, which has been shown to visualize single microtubules and actin bundles and can estimate the number of filaments in a bundle (4, 5, 6). In contrast to the traditional polarized light micro-scope, the New Pol-Scope visualizes birefringent components independent of their orientation, and measures their retardance with high sensitivity and resolution over the whole field of view (7). We used primary cell cultures of Aplysia bag cell neurons which form relatively large growth cones. We prepared cultures according to the method of Kaczmarek and Strumwasser (8). The cultured cells formed growth cones for 1-3 days. The Aplysia growth cone in culture is shaped like a thin lamelipodia containing radially aligned actin bundles and a space filling network of actin filaments (9). The New Pol-Scope image shows a growth cone in which several different birefringent components can be distinguished. Very prominent are radially aligned birefringent fibers that have the same location as the previously reported actin bundles. These actin bundles show lateral association as well as growth and retraction patterns. We can also recognize faint, cloud-like birefringent structures located in the space between bundles. These biregringent clouds are possibly areas of partially aligned actin filaments which are part of the space filling actin network. In addition to the actin induced birefringence, we saw the high birefringence of the central region of the nerve process, which is filled with aligned microtubules and vesicles. At the leading edge of the growth cone, the cell membrane is imaged as a birefringent double layer. This has to be interpreted with caution since the detailed structure of the double layer contains an optical artifact (edge birefringence) caused by the refractive index mismatch between the cytosol and extracellular medium (10). Filamentous actin in a neuronal growth cone is known to show a constant retrograde flow (1). In time lapse movies of the New Pol-Scope images we recognized the retrograde flow in growth cones through the movement of, e. g., morphological kinks in radially aligned fibers and of the birefringent cloud-like areas. To confirm whether or not these structures are actin based, we examined the effect of externally applied cytochalasin B. Cytochalasin B is well known to prevent polymerization of actin filaments. When cytochalasin B was added to the external medium, all radially aligned fibers and cloud-like birefringent structures disappeared within 1-2 minutes. Both, the radially aligned fibers and birefringent clouds reappeared several minutes after removal of the cytochalasin B from the extrecellular medium. Moreover, in fixed non-cytochalasin treated cells, the radially aligned fibers were fluorescently labeled with rhodamine phalloidin, which selectively binds to actin filaments. These results suggest that the radial bundles and cloud-like structures are indeed actin based. In this report, we have demonstrated that the New Pol-Scope can visualize actin based structures in the growth cone. we are currently analyzing the quantitative results provided by the Pol-Scope with regard to the actin dynamics in living growth cones. References 1. Forscher, P., and S. J. Smith. 1988. J. Cell Biol. 107: 1505-1516. 2. Theriot, J. A., and T. J. Mitchison. 1991. Nature 352: 126-131. 3. Wang,F-S., J.S.Wolenski, R.E. Cheney, M.S. Mooseker, and D.G. Jay. 1996. Science 273:660-663. 4. Tran, P., E. D. Salmon, and R. Oldenbourg. 1995. Biol. Bull. 189: 206. 5. Oldenbourg, R., E. D. Salmon, and P. Tran. 1997. Biophys. J. In press. 6. Katoh, K., K. Yamada, F. Oosawa, and R. Oldenbourg. 1996. Biol. Bull. 191: 270-271. 7. Oldenbourg, R. 1996. Nature 381: 811-812. 8. Kaczmarek, L.K., M. Finbow, J.P. Revel, and F. Strumwasser. 1979. J. Neurobiol.10:535-550. 9. Lewis, A. K., and P. C. Bridgeman. 1992. J. Cell Biol. 119: 1219-1243. 10. Oldenbourg, R. 1991. Biophys. J. 60 629-641.
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