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Fig. 7 GFP-«5 integrin receptors in a live growth cone growing on fibronectin colocalize with ¡1 integrin receptors after fixation. (A) GFP-«5 integrin distribution in a growth cone just prior to fixation. Note streaks of «5 integrin receptor clusters resembling focal contacts (arrow). (B) Immunofluorescent staining for ¡1 integrin receptors after fixation reveals that GFP-«5 integrin clusters in the live growth cone colocalize ¡1 integrin clusters (arrow). Neighboring nonneuronal cells that were not expressing the GFP-«5 integrin also express clustered ¡1 integrin receptors. (C) A merged live GFP image with a fixed immunofluorescence image reveals colocalizations (arrow). Scale: 10 ^m. (See Color Insert.)

Fig. 7 GFP-«5 integrin receptors in a live growth cone growing on fibronectin colocalize with ¡1 integrin receptors after fixation. (A) GFP-«5 integrin distribution in a growth cone just prior to fixation. Note streaks of «5 integrin receptor clusters resembling focal contacts (arrow). (B) Immunofluorescent staining for ¡1 integrin receptors after fixation reveals that GFP-«5 integrin clusters in the live growth cone colocalize ¡1 integrin clusters (arrow). Neighboring nonneuronal cells that were not expressing the GFP-«5 integrin also express clustered ¡1 integrin receptors. (C) A merged live GFP image with a fixed immunofluorescence image reveals colocalizations (arrow). Scale: 10 ^m. (See Color Insert.)

time-lapse sequence. Xenopus neurons and nonneuronal cells are fixed by the rapid perfusion of room temperature 4% paraformaldehyde/4% sucrose, followed by standard immunocytochemical procedures. Rapid fixation results in only moderate changes in cell morphology during this process but should be performed immediately following live imaging. We have used this technique to localize integrin receptor clusters to sites of Ca2+ transient activity in growth cone filopo-dia (Gomez et al., 2001) and because Fluo-4 fluorescence is lost upon fixation, this wavelength remains available, along with others, to localize multiple cellular elements with immunofluorescence. This approach also serves as an excellent test for the proper localization of fluorescent fusion proteins, as proteins tracked in live cells can be stained subsequently with specific antibodies after fixation (Fig. 7).

D. Ultraviolet Photolysis

Manipulation of cell physiology by UV photolysis of caged compounds is a powerful method used to study the acute effects of well-defined stimuli on cell motility. Many photosensitive reagents are available commercially, such as caged Ca2+ (MP-EGTA) and cAMP (Molecular Probes), and still others are being synthesized by individual investigators using a variety of techniques (Marriott, 1998). Caged Ca2+ has been a particularly useful tool in studying the effects of user-defined Ca2+ transients on growth cone motility. This approach allows one to examine the downstream effectors of spatially and temporally distinct Ca2+ signals. Imposed Ca2+ transients are detected by coloading Fluo-4, whereas the effects on growth cone behaviors can be studied with DIC optics or with various expressed fluorescent proteins. However, caution must be taken when using caged compounds, as growth cones are sensitive to UV light. Therefore, UV-only controls are important to determine the Ca2+-dependent component of behavioral changes in response to photorelease.

UV photolysis of caged compounds is simplified on a confocal microscope, as these systems typically have separate light paths for laser-scanning imaging and wide-field fluorescence excitation. We use an Olympus AX-70 upright microscope equipped with a fiber-launched 100-W mercury arc lamp on our Fluoview 500 laser-scanning confocal system. Excitation and neutral density filters to select and attenuate photolysis wavelengths, respectively, are positioned in the optical path. We use narrow bandpass filters (either 360 ± 25 or 380 ± 6.5) to release Ca2+ efficiently while minimizing UV damage. The region exposed to UV light is adjusted using the field diaphragm, an aperture iris located at the conjugate focal plane between the illuminator and the specimen. UV exposure can typically be restricted to areas as small as 10-15ym in diameter when using a 100x objective and can be determined by imaging a solution of caged-FITC dextran (Molecular Probes) sandwiched between coverslips. A dichroic mirror (Chroma) reflects light shorter than 400 nm toward the sample while transmitting longer wavelengths. A programmable shutter (Uniblitz) controls both the duration of single UV pulses and the delay between a series of pulses. It is worth noting that the total power of light delivered to the UV spot appears slightly higher toward the center, which may be due to the mechanism of iris opening and increased light defraction near the edge of the field diaphragm.

Photoactivation of caged Ca2+ provides higher spatial and temporal control of stimuli compared to alternative techniques (i.e., local or bath perfusion of Ca2+ channel activators). We use caged Ca2+ to locally generate Ca2+ transients at the tips of filopodia to determine the effects of these signals on growth cone guidance. Growth cones typically have many filopodia distributed in a fan shape at the leading edge of the advancing neurite. Thus, a growth cone can be positioned such that only filopodia on one side are exposed to the uncaging stimulus (Gomez et al., 2001). We use local release of caged Ca2+ to determine how imposed Ca2+ transients at the tips of filopodia on one side of the growth cone affect the direction of neurite outgrowth. We position NP-EGTA-loaded growth cones so their direction of growth is toward a ^12-ym spot of pulsed UV light. To generate brief Ca2+ transients, we program the shutter to open for 100-200 ms at a frequency of 3-6/min. Fluorescent images of GFP expressing growth cones or DIC images of untransfected growth cones are collected every 15-60 s for 30 min of growth. Using this paradigm, we find that growth cones loaded with NP-EGTA

consistently turn to avoid the region of pulsed UV light and typically do so after only filopodia enter the spot. In contrast, unloaded growth cones do not exhibit significant turning, although some UV-dependent responses are observed.

V. Summary

This chapter described techniques that distinguish X. laevis as a unique and highly effective model system, which we use to study the development of spinal neurons in culture. The principal advantages of Xenopus as a model system are the speed and ease of ectopic gene expression, as well as the versatility and amenability of its neuronal culture. Work with fluorescent fusion proteins seems particularly well suited for studying the mechanism of growth cone motility using Xenopus neurons, as labeled neurons can be examined within 36 h of fertilization. The molecular tools available to label or modify cells are expanding rapidly with no end in sight. Color variants of fluorescent fusion proteins engineered to utilize FRET to report physiological activity or protein function continue to be developed. These unique reporter constructs, as well as tailor-made DN and CA receptors, signaling components, and effector molecules, can be expressed easily in Xenopus to study the molecular mechanisms governing growth cone motility in culture. Although this chapter focused principally on in vitro studies, the Xenopus spinal cord is also an excellent model for studying in vivo axon guidance. Many of the tools developed and used in vitro can ultimately be tested in vivo. With such versatility and so many technical advances, it seems that this developmental model system will continue to be a productive organism of study over the next century.

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