[19] Application of caged fluorescein-labeled tubulin to studies of microtubule dynamics and transport of tubulin molecules in axons

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motor proteins in vitro. Hence many protocols for laser pulse photolysis with these proteins have been described. Combination of photolysis with other biophysical signals that probe molecular events is a fertile area for investigation. Many of the considerations in photolysis experiments on skeletal muscle and motor proteins are applicable to other types of muscle and other enzymes. Acknowledgments This work was supported by NIH Grant HL15835 to the Pennsylvania Muscle Institute and the Yanagida Biomotron Project E R A T O JST. We thank Drs. J. W. Walker and G. C. R. Ellis-Davies for access to unpublished data, Drs. J. E. T. Corrie and D. R. Trentham for helpful comments on the manuscript, and Ms. Kimberly L. Dopke for help with the manuscript.

[19] A p p l i c a t i o n o f C a g e d F l u o r e s c e i n - L a b e l e d T u b u l i n to Studies of Microtubule Dynamics and Transport of Tubulin Molecules in Axons B y TAKESHI FUNAKOSHI a n d N O B U T A K A H I R O K A W A

Introduction Microinjection of caged fluorescein-labeled cytoskeleton associated proteins into cultured ceils and subsequent photoactivation of the fluorescence in these conjugates has proved to be a very powerful tool in studying the dynamics of the microtubule and actin cytoskeleton in living cells, t-4 Caged fluorescein-labeled tubulin has been successfully applied to study microtubule dynamics in mitotic spindles and neuronal axons. This approach has allowed us to investigate molecular mechanisms of mitosis and the origin of the slow transport of tubulin molecules in nerve axons. This article describes methods and techniques using caged fluorescein-labeled tubulin for investigations of the dynamics and transport of axonal tubulin molecules in neurons. In these studies, caged fluorescein-labeled tubulin is introduced into cultured neurons by microinjection and, after a recovery period that also allows for diffusion of the caged fluorescein tubulin throughout the cell, l T. J. Mitchison, J. Cell Biol. 109, 637 (1989). 2 j. A. Theriot and T, J. Mitchison, Nature 352, 126 (1991). 3 S. Okabe and N. Hirokawa, Z Cell Biol. 117, 105 (1992). 4 j. Sabry, T. P. O'Connor, and M. W. Kirschner, Neuron 14, 1247 (1995).

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small segments of axons are exposed to a brief pulse of near-ultraviolet light. The caged protection groups on the fluorescein absorb these photons, and in a dark reaction the ether bond that links the caged groups to the phenolic groups of fluorescein is cleaved, liberating highly fluorescent tubulin conjugates only in the irradiated segment. The fate of the photoactivated tubulin molecules was observed with low-light level fuorescence microscopy or with electron microscopy by staining with antibody directed against fluorescein. Preparation of Caged Fluorescein-Labeled Tubulin Caged ftuorescein-labeled tubulin is prepared according to the method of Mitchison 1 with slight modifications. Briefly, phosphocellulose-purified hog brain tubulin is labeled with Bis caged-fluorescein Sulfo-OSu (Dojindo Laboratories, Japan) in solution at high pH (pH 8.5). This fluorescein derivative is not in itself fluorescent, but generates highly fluorescent carboxyfluorescein after irradiation with near-ultraviolet light (photoactivation). After the labeling reaction, the tubulin preparation is subjected to two cycles of polymerization and depolymerization to select for assembly competent conjugates. Labeled tubulin preparations are analyzed after photoactivation by sodium dodecyl sulfate gel electrophoresis to confirm that the protein is labeled with the caged fluorescein reagent and is free of unlabeled caged fluorescein. Free caged carboxyfluorescein runs at the front of the gel and can be easily detected by irradiating the gel under an ultraviolet light transilluminator. Cell Culture Cultured mouse dorsal root ganglion (DRG) neurons are used in these experiments. These neurons have large cell bodies (25-60/xm), and the growth rate of their axons plated on laminin-coated coverslips is very fast, with a protrusion rate of 10-80/zm/hr. 3 1. About 20 dorsal root ganglions are isolated from adult mice 5'6 and kept in Hanks' balanced salt solution (HBSS, GIBCO, Grand Island, NY) at 4°. To minimize injury to these neurons, this step is completed within 45 min. 2. Dorsal root ganglions are washed twice with Ca 2÷, Mg2+-free HBSS (Gibco). s S. S. S. Goldenberg and U. De Boni, J. Neurobiol. 14, 195 (1983). 6S. Okabe and N. Hirokawa,J. Neurosci. 11, 1918 (1991).

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3. Ganglions are treated with 0.25% (w/v) collagenase in Ca 2÷, Mg 2+free HBSS for 1 hr at 37°. 4. Ganglions are further incubated with 0.25% (w/v) trypsin in Ca 2÷, Mg2+-free HBSS for 15 min. 5. After washing with culture medium (MEM, Nissui, Tokyo, Japan) supplemented with glutamine and 15 mM HEPES, 5% horse serum, and 5% new calf serum, the ganglions are homogenized in the culture medium by pipetting several times through a Pasteur pipette. The pipetting should be performed gently to avoid damaging cells. If the ganglions cannot be resuspended easily, harsher treatment with protease (with shaking) is recommended. 6. Suspended cells are plated on laminin-coated glass coverslips. Healthy cells should be smooth and round, whereas heavily damaged cells have a wrinkled appearance. The addition of Nerve Growth Factor (100 ng/ml) to the culture medium is optional. Glass coverslips are secured with vacuum grease on the bottom of plastic dishes that have been drilled with a hole smaller than the diameter of the coverslip (Dow Coming, high vacuum silicone grease). Care should be taken as some kinds of adhesive agents are toxic to the cells. For electron microscopic analysis, we use CELLocate micro-grid coverslips (Eppendorf) in order to identify the axons of cells irradiated with near-ultraviolet light. The square meshes etched onto these coverslips are transferred to the resin block and allow us to easily and precisely identify the photoactivated axons after embedding. Microinjection of Caged Fluorescein-Labeled Tubulin Into Neurons 3,7 Neurons that do not show any axonal processes or those with minor sprouts are microinjected with bis-caged carboxyfluorescein-labeled tubulin 3-8 hr after plating and are incubated for a further 10-20 hr. This ensures that microinjected-caged fluorescein tubulin is equally distributed throughout the neuron. To identify microinjected neurons, cells are coinjected with caged fluorescein-labeled tubulin (50-100/xM) and rhodamine-bovine serum albumin (BSA) (0.5 mg/ml) in injection buffer (50 mM potassiumglutamate, i00 mM KCI, and 1 mM MgCl2). Photoactivation and Low Light Level Video Microscopy Photoactivation is performed as described by Mitchison and colleagues (see also the chapter in this volume) with slight modifications. I'3,s Essen7T. Funakoshi, S. Takeda, and N. Hirokawa,J. Cell BioL 133, 1347 (1996). 8T. Umeyama,S. Okabe, Y. Kanai, and N. Hirokawa,J. Cell Biol. 120, 451 (1993).

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PHOTOACTIVATION OF TUBULIN MOLECULES IN NEURONS

tially, the 365-nm line of a mercury lamp selected with a bandpass filter (390-nm long pass) is introduced into the epifluorescent light path of an inverted microscope (Axiovert; Carl Zeiss, Inc.) via a dichroic mirror positioned between the original mirror box and the field diaphragm (Fig. 1). A handmade slit is placed at the point where the slit makes an image at the field diaphragm. The beam of the mercury lamp is focused onto the

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slit using a lens. We can easily illuminate a small region of an axon (3-5 /zm wide) using this microscope with a 100× objective lens (Plan-Neofluar, Zeiss). To observe the fluorescence of cells we use a 100-W halogen lamp, the excitation-emission filter set for fluorescein and/or rhodamine, and a cooled CCD camera (C3640; Hamamatsu Photonics) with an exposure time of 1 sec. The halogen lamp is turned on only to record the fluorescence region in order to limit photobleaching of the dye and any accompanying damage to the cells. Mercury lamp epi-illumination of the sample is used in combination with a low power objective lens to identify injected cells. Photomicroscopic Observation Following Photoactivation3.8 Although fluorescence microscopy does not allow us to resolve a single microtubule in an axon, as microtubules are so densely packed, we can observe a single cell many times after photoactivation.

Approach 1. Illuminate a small segment of the axon of the microinjected cell with near-ultraviolet light. Fluorescein-labeled tubulin molecules are generated in the illuminated segment. 2. Observe the fluorescence images immediately after and at appropriate time intervals following photoactivation. Narrow bars of fluorescent tubulin marked in the axon by photoactivation do not move from the irradiation site even if in extending axons, although the fluorescence intensity of the marked tubulin does decay gradually (Fig. 2). These observations strongly imply that most of the axonal microtubules are stationary, and we suggest that the intensity decay reflects the polymerization-depolymerization turnover of microtubules in the photoactivated segments. The limited spatial resolution and sensitivity of fluorescence light microscope make it difficult to detect any population of tubulin molecules that move out of the photoactivated segments. Electron Microscopic Observation Following Photoactivation7 This method allows us to observe the fate of photoactivated molecules at very high resolution with high sensitivity. To detect the population of mobile tubulin molecules that might have been overlooked in observations with the fluorescence microscope, we determined the distribution of fluoresceinated tubulin molecules in the same photoactivated axons using elec-

[ 191

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FI~. 2. Photoactivation of an extending axon of a mouse DRG neuron. (a-e) Fluorescence images showingthe marks (arrows) made on the microtubules by the photoactivation technique remain stationary as the axon moves forward. (f and g) DIC images of the same axon. Arrows indicate the position of the photoactivated region. During this experimental run, there was a rapid extension of the axon. Elapsed time (minutes) after photoactivation is shown in the lower left-hand corner. Reproduced from S. Okabe and N. Hirokawa, Z Cell Biol. 117, 105 (1992) by copyright permission of Rockefeller University Press. Bars: 10/zm.

tron microscope-based detection of an antiflorescein antibody. Although this m e t h o d does not allow us to observe a single axon at m a n y time points after the photoactivation, it provides both the resolution and the sensitivity to detect transported tubulin molecules in a single axon. Antiserum against fluorescein was produced using fluorescein-labeled keyhole limpet hemocyanin for the antigen. The antibody directed against the fluorescein hapten was affinity purified using a CNBr-Sepharose affinity column (Pharmacia, Piscataway, NJ) coupled with fluorescein-conjugated BSA. We employ two different procedures to address the issue of whether tubulin molecules are transported in the axon. To unambiguously observe single microtubules, we permeabilize cells using taxol before fixation, as previously described 9 with some slight modifications. This procedure extracts soluble cytosolic protein from axons (Fig. 3). To preserve the soluble population of tubulin molecules (tubulin oligomers or heterodimers), cells are fixed without a permeabilizing agent. U n d e r this condition, using highresolution electron microscopy, a transported population of tubulin molecules can be seen (Fig. 4). 9 p. W. Baas and M. M. Black, J. Cell Biol. 111, 495 (1990).

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FIG. 3. Behavior of photoactivated microtubules in extending axons. One hour after the photoactivation of small regions of the axon, the cells were permeabilized, fixed, and stained by antifluorescein antibody. (a and b) Photoactivated region. Microtubules decorated with gold particles (arrowheads) were observed. (c and d) Examples of the areas outside the photoactivated region: (c) an area approximately 4/zm distal to the photoactivated region and (d) an area approximately 4/zm proximal to the photoactivated region. No microtubules with gold labels were observed outside of the photoactivated regions. Reproduced from T. Funakoshi, S. Takeda, and N. Hirokawa, J. Cell Biol. 133,1347 (1996) by copyright permission of Rockefeller University Press.

FIG. 4. Transported tubulin molecules in the axon. (a-c) One minute after the photoactivation of a small section of the axon; cells were fixed without the preceding permeabilization and visualized using an antifluorescein antibody. Many gold particles were observed outside of the photoactivated region. (a) Photoactivated region. Many gold particles can be seen. (b) An area about 20 tzm distal and (c) an area about 20 t~m proximal to the photoactivated region. (a-c) Arrowheads indicate some of the gold particles. (d) An axon photoactivated after fixation. The photoactivated region is underlined. (a-c) Many gold labels appear outside of the photoactivated regions; the density of gold label is higher in the distal region than in the proximal region. (a-c) Large spots are used to exaggerate the positions of the gold particles, as they are too small to be resolved at this magnification. Reproduced from T. Funakoshi, S. Takeda, and N. Hirokawa, J. Cell Biol. 133~ 1347 (1996) by copyright permission of Rockefeller University Press. Bars: 1 ~m.

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The procedure to observe single microtubules is as follows: 1. Illuminate a small segment of the axon with a beam of near-ultraviolet light. 2. After appropriate time intervals, permeabilize and fix the cells according to the method described previously. 9 Permeabilize the cells for 5 min with 1% Triton X-100 in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCI2, pH 6.9) containing 10 txM taxol and 0.2 M NaC1. 3. Fix the cells for 30 min by adding an equal volume of 1% glutaraldehyde in PHEM. 4. Wash with phosphate-buffered saline (pH 7.2) (PBS) and treat the cells with 50 mM glycine in PBS for 30 min. 5. Incubate the cells with blocking solution containing 5% skimmed milk in PBS. 6. Incubate the cells overnight at 4 ° with the antifluorescein antibody. Antiserum is used at a dilution of 1 : 500 in PBS with 0.5% skimmed milk. 7. Wash the cells for 1 hr with PBS. 8. Incubate the cells with a 5-nm gold-conjugated anti-rabbit antibody (Amersham Life Science) for 5 hr at a dilution of 1 : 10 in PBS at 37°. 9. Wash the cells completely with PBS and fix again with 1% glutaraldehyde in PBS and process for silver enhancement (Amersham). 10. Osmify (for 1 min), block stain, dehydrate, and embed the cells. 11. Serially section the cells and examine the block. The procedure to preserve the soluble population of tubulin molecules is as follows: 1. Illuminate a small segment of the axon with a UV beam. 2. Fix the cells 1-20 min after photoactivation with 0.1% glutaraldehyde and 2% paraformaldehyde in PEM. 3. Treat the neurons with 1% Triton X-100 in PBS for 30 min. 4. Wash with PBS. 5. Incubate with 50 mM glycine in PBS for 30 rain. 6. Incubate the cells with blocking solution containing 5% skimmed milk in PBS for 30 rain. 7. Incubate the samples overnight with affinity-purified antifluorescein antibody in BSA-PBS (0.5% (w/v) BSA and 0.1% (w/v) gelatin in PBS) containing 0.5% (w/v) skimmed milk at 4 °. Completely wash the cells with BSA-PBS containing 0.5% skimmed milk. 8. Incubate the cells overnight at 4° with a 1.4-nm gold-conjugated antirabbit Fab' fragment (Nanoprobes, Inc., Nanogold anti-rabbit) diluted 500 times in BSA-PBS containing 1% skimmed milk, 500 mM NaC1, and 0.05%

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Tween 20 (the 5 nM gold-conjugated antibody usually employed for immunoelectron microscopy does not penetrate D R G neurons that have not been permeabilized before fixation). 9. Completely wash (about 1 hr) the cells with BSA-PBS containing skimmed milk, NaC1, and Tween 20 and then with PBS. 10. Fix the cells again with 1% glutaraldehyde in PBS. 11. Silver enhancement in developer solution (30% gum arabic, 0.85% hydroquinone, 0.11% silver lactate, citric acid monohydrate, 25.5 g/liter, sodium citrate dihydrate, 23.5 g/liter) 1° for about 1 hr. 12. Block stain, dehydrate, and embed cells without osmification for serial sectioning and observation. To evaluate this procedure and to check whether labels found outside of the photoactivated regions really represent moving tubulin molecules in the axons, we illuminated small axonal regions of previously fixed cells with near-ultraviolet light, stained and observed in the electron microscope as described earlier. In these axons, no gold labels were found out of the photoactivated regions (Fig. 4d). No translocated microtubules were detected in cells that had been fixed after permeabilization. In photoactivated axons of cells fixed without permeabilization, where fluorescently labeled oligomers, heterodimers, and polymers are preserved, a significantly higher amount of gold label was found in regions distal to the photoactivated regions than in the proximal region. These data indicate that tubulin molecules are not transported as polymers, but as heterodimers or oligomers by an active mechanism rather than by simple diffusion.

10 M. A. Hayat, "Principles and Techniques of Electron Microscopy." CRC Press, Boca Raton, FL, 1989.

[20] T W o - P h o t o n A c t i v a t i o n o f C a g e d C a l c i u m w i t h Submicron, Submillisecond Resolution

By E D W A R D

B. BROWN and WATT W. WEBB

In~oduc~on Photolabile calcium chelators, or "cages," are a popular tool to explore the calcium dynamics of a variety of biological systems. On absorption of a single UV or short-wavelength visible photon a calcium cage can undergo a conformational shift and release a calcium ion. This release can take

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