Retrograde but not anterograde bead movement in intact axons requires dynein

Retrograde but Not Anterograde Bead Movement in Intact Axons Requires Dynein

Chang Wang,* David J. Asai, and Kenneth R. Robinsont

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1 392

SUMMARY

Dynein and kinesin have been implicated as the molecular motors that are responsible for the fast transport of axonal membranous organelles and vesicles. Experi- ments performed in vitro with partially reconstituted preparations have led to the hypothesis that kinesin moves organelles in the anterograde direction and dynein moves them in the retrograde direction. However, the molecular basis of transport directionality remains unclear. In the experiments described here, carboxylated fluorescent beads were injected into living Mauthner axons of lamprey and the beads were observed to move in both the anterograde and retrograde directions. The bead movement in both directions required intact microtubules, occurred at velocities approaching organelle fast

transport in vivo, and was inhibited by vanadate at concentrations that inhibit organelle fast transport. When living axons were injected with micromolar concentrations of vanadate and irradiated at 365 nm prior to bead injections, a treatment that results in the V l photolysis of dynein, the retrograde movement of the beads was specifically abolished. Neither the ultraviolet irradiation alone nor the vanadate alone produced the retrograde- specific inhibition. These results support the hypothesis that dynein is required for retrograde, but not anterograde, transport in vivo. Keywords: dynein, axonal transport, vanadate, movement, lamprey.

8 1995 John Wiley & Sans, Inc.

INTRODUCTION

Fast axonal transport is essential for nerve function; it is the mechanism by which organelles and membranous vesicles are transported in both directions (reviewed in Grafstein and Forman, 1980). Anterograde fast transport supplies the axon termi- nus with neurotransmitters and other secretory materials synthesized in the cell body and may be responsible for the transport of ion channels that are inserted into the plasma membrane of the axon. Retrograde fast transport forms the endo- cytic pathway, recycling old proteins and membranes and carrying internalized ligand-receptor complexes to the soma for processing.

Received June 27, 1994: accepted January 19, 1995 Journal of Neurobiology, Vol. 27, No. 2, pp. 2 16-226 (1995) Q 1995 John Wiley & Sons, Inc. CCC 0022-3034/95/0202 16-1 1

* Present address: Department of Neurobiology. Northwest- ern University, Evanston, IL 60208

To whom correspondence should be addressed.

216

It is now clear that fast axonal transport in both directions is effected by cytoskeletal motor proteins (reviewed in Vale, 1987; Schroer, 1992; Hirokawa, 1993). Although actin and myosin operate in vesicle movements over short distances (Kuznetsov et al., 1992), microtubules are the principal tracks for anterograde and retrograde transport. Microtu- bules have an intrinsic structural polarity that is re- flected in the relative kinetics of assembly at the ends of the microtubules. In axons, bundles of microtubules are aligned uniformly with their minus ends toward the cell body (Black and Baas, 1989). The organization of the microtubule tracks and the in vitro properties of kinesin and dynein lead to a hypothesis that provides a molecular explanation of directionality: the plus-end-directed microtubule motor, conventional kinesin, produces anterograde transport and the minus-end-directed motor, cytoplasmic dynein, produces movement in the retrograde direction (Vale et al., 1985a; Pas- chal and Vallee, 1987; Sheetz et al., 1989; Schnapp et al., 1992).

Dynein Requirements in Intact Axons 21 7

Although the model is attractive, the evidence for it is mostly circumstantial (Coy and Howard, 1994). Both motors are present in nerve cells, and immunocytochemistry indicates that the motors are on the surfaces of axonal vesicles (Hirokawa et al., 1990, 199 1). In vitro, each motor produces microtubule-based movements at rates similar to fast transport (Vale et al., 1992). Additionally, in reconstituted, cell-free systems, cytosolic fractions of chick embryo fibroblasts (Schroer et al., 1989) and squid giant axons (Schnapp and Reese, 1989) pro- mote microtubule-based movement that is inhibited by ultraviolet (UV) irradiation in the presence of vanadate, a treatment that selectively photolyzes dynein (Gibbons et al., 1989).

The molecular basis for the directionality of fast transport remains unclear (summarized by Brady, 199 1). When the information gained from in vitro experiments is applied to intact axonal transport, the results are disappointingly ambiguous. For example, dynein and kinesin have significantly different sensitivities to vanadate in vitro, yet vanadate does not distinguish between the two directions in vivo (Forman et al., 1983). In vitro experiments suggest that low concentrations of adenyll- imidodiphosphate (AMP-PMP) should selectively have little effect on dynein-mediated motility, but AMP-PNP does not discriminate between movements in both directions in extruded axoplasm (La- sek and Brady, 1985). N-ethylmaleimide, although capable of producing differential inhibition of kinesin and dynein in vitro, cannot distinguish between anterograde and retrograde motility in the axoplasm (Pfister et al., 1989). An antibody directed specifically against kinesin inhibited movement in both directions (Brady et al., 1990) and a mutation of Drosophila conventional kinesin does not impair the transport of material to the axon ter- minus but affects other neuronal functions (Gho et al., 1992). The existence of multiple kinesin-like proteins in neuronal cells and the fact that some members of the kinesin family produce movement to the minus ends of microtubules provide the im- petus to consider alternative models for axonal transport. As pointed out by others (Coy and How- ard, 1994), transport might be the consequence of different kinesins carrying specific cargoes in both directions in the axon, and dynein may have little to do with axonal transport.

In the present study, we have examined axonal transport of fluorescent beads injected into living axons, after the strategy of Adams and Bray (1 983). The Mauthner axons of the larval lamprey spinal cord were used in this study. These axons combine

optical transparency with a large diameter, present- ing the opportunity to introduce and visualize beads in the living cells. Quantitative video fluorescence microscopy was applied to demonstrate that the injected beads moved in both the anterograde and retrograde directions at velocities corre- sponding to fast axonal transport. Bead movement required intact microtubules. Injection of vanadate inhibited movements in both directions in a non- discriminatory fashion, which is in contrast to the significant difference in vanadate sensitivities ex- hibited by isolated kinesin and dynein. Vanadate- mediated UV treatment of living cells, under conditions that specifically photolyze dynein, resulted in the inhibition of retrograde bead movement with no effect on anterograde movement. This is the first example of inhibition in vivo of a molecular motor that produces a cessation of axonal movement in only one direction.

MATERIALS AND METHODS

Spinal Cord Preparation

Larval lampreys were anesthetized by a 0. I % solution of tricaine methanesulfonate. They were then washed and dissected in lamprey saline ( 1 1 I m M sodium chloride (NaCI), 1.8 m M magnesium chloride (MgCI2), 2.6 m M calcium chloride, . I m M potassium chloride, 2.5 m M N- 2 - hydroxyethylpiperazine- N - 2 - ethanesulfonicacid (HEPES) pH 7.4) (Strautman et al., 1990). The spinal cord from brain to first fin was removed, bathed in lamprey saline, and pinned down in a chamber. Under these conditions, the spinal cord could be kept for several days at 10°C in saline.

Solutions

Carboxylated fluorescent beads (Polysciences, YO; 0.2 pm diameter, 2.5%) werediluted 1 to 1 with 2 X injection buffer (IB; 10 mMNaC1, 140 mMpotassium glutamate, 1 m M MgC12, 1 mM ethyleneglycol-bis-(p-aminoethy1)- N,N,N:N’-tetraacetic acid {EGTA), 10 m M HEPES, pH 7 .5 ) . Bead clusters were removed from the suspension by filtering it through 1.2 pm Millipore filters.

Colchicine solution for injection was composed of 2 Mcolchic ine and 1.3 m M 6-carboxylate-fluorescein in 1B. To make lumicolchicine, colchicine (4 mM in IB) was irradiated with 265 nm UV light for I h on ice. The final lumicolchicine concentration for injection was 2 mMin IB with fluorescein.

Vanadate concentrations in the micropipette, when co-injected with beads, were 10.8 mM, 16.3 mM, 21.6 mM, or 1.5 M in injection buffer with 1.3 mM fluorescein in IB. The micropipette concentration of vanadate

218 Wang et a!.

in the UV irradiation experiments was 2 1.6 m M with 1.3 mM fluorescein. The vanadate solutions were made immediately before use. Norepinephrine concentration was 1.5 A4 with 1.3 m M fluorescein.

Microinjection

Micropipettes for bead injection were pulled from 1.5 mm thin wall capillaries with filament (World Precision Instruments, Sarasota, FL) using a vertical pipette puller (David Kopf Instruments, Tujunga, CA). The diameter of the tip of the electrode was about 1 pm, and the resistance was about 8 MQ when filled with injection buffer. To avoid tip clogging due to the evaporation ofwater, the electrode was dipped into IB for 20 sand then back-filled with beads. This buffer was then extruded before injection. If beads were co-injected with a drug, the electrode was dipped into the drug buffer instead of injection buffer and then back-filled with beads.

Beads were pressure injected into a Mauthner axon of a freshly dissected spinal cord with a single pulse at 1.90 X lo5 Pa and duration of 50 ms. The membrane poten- tial of the axon, which was usually around -60 mV when electrodes were filled with IB, was measured when impal- ing to determine if the electrode was in the cell and to assess the physiological state of the axon.

Micropipettes for vanadate injection during the UV- vanadate experiments were pulled from 1.5 mm thick wall capillaries with filament. The resistance of the electrodes was between 14 and 22 MQ when filled with injection buffer. The diameter of the tip was less than I pm.

Vanadate was pressure injected into axons before UV irradiation. The solution was injected with 20 to 40 pulses at 1.90 X lo5 Pa. The duration of each pulse was between 10 and 20 ms, depending on the resistance of the electrode and whether the pipette was stable during the injection. The injection was done relatively slowly (2 to 3 min) so that vanadate had time to diffuse. The intra- axonal concentration right after injection (about 30 s after injection) was between 250 and 450 pM. After 25 min of UV irradiation, the vanadate concentration in the injected area was more than 80 p M .

Image Processing and Data Analysis

The video image processing system consisted of a Nikon Diaphot TMD inverted fluorescence microscope (X 10 or X20 objectives), a KS-1380 microchannel-plate image intensifier, and VS-2000N Newvicon camera (Videoscope International, Herndon, VA). Software for image processing running on a Sun 3 workstation was by G. W. Hannaway and Associates (Boulder, CO).

All the images taken in the experiments had back- grounds removed and were corrected for nonuniform gain and illumination before analyzing the results. In each experiment, images of the injection area, background. and light source were recorded. The background was taken from the area on the spinal cord close to the

injection area with the same gain and magnification. The light source image was always recorded at as high a gain (usually 35) as possible without neutral density filters and with the rhodamine filter cube and X20 objective. All of these images were the averages of 100 frames to reduce the noise. Since it only took about 3 s to collect 100 frames, there was no significant bead movement during the collection of images. All the images were averaged over 100 frames to reduce the noise. During data analysis, experimental images were processed by subtracting the background and then dividing by light source image. This process eliminated the unevenness of the image on the screen due to the uneven illumination.

The distance that the beads moved was calculated using the image processing system. In most of the cases, there were some beads, presumably attached to the plasma membranes, that never moved. This region was brighter than the rest of the image; therefore, the center of this area was used as the reference point marking where the beads were injected. The image processing system also allowed tracing the contour of a given intensity. With this function, the edge ofthe bead group was found using the lowest intensity that could separate different bead groups on the screen. Since the long axis of the axon image was always parallel to the horizontal axis of the screen, the center of the bead group was calculated from the difference between the X values of the farthest and closest points of the group. The horizontal distance between the center of the bead group and the point where beads were injected was used as the distance beads moved.

The total intensity within an area was calculated by integrating the intensity of all the pixels in the area. To correlate the intensity ofthe bead group with the number of beads in the group, a known concentration of beads was injected as a measurable sphere into a drop of oil. Total intensity of this bead droplet was measured, and since the bead droplet was spherical in oil, its volume could be determined. Since the number of beads per unit volume was known, the fluorescence intensity of each bead could be calculated.

The concentration of drug was determined by the fluorescence intensity of fluorescein, which was co-injected with drug. To calibrate the drug concentration, the same drug solution was diluted 200 to 1600 times and used to fill 50 pm diameter microcapillaries. The mean fluorescence intensity of fluorescein in the capillary was then used as a measure of the drug concentration. Since both the drug and fluorescein diffused after injection, fluorescence intensity depended on the time after injection the measurement was done. To compare the drug concentrations, all the concentrations were expressed as the drug concentrations at 3 min after injection by using the equation: C = C, erf(h/2fi) (Crank, 1975), where Co is the injection-site intensity soon after injection, C is the intensity at time t, h is the half-width of the droplet injected (20 pm), and D is the diffusion coefficient, which we measured in axons as 2.2 X cm2/s. We assumed

Djnein Reqitivemmts in Intuct Axons 219

that the drug had a similar diffusion constant to fluorescein. Many small organic compounds have diffusion co- efficients within a factor of 3 of each other. Since the fluorescence images were usually taken after injection (within 3 min), any variations in diffusion would con- tribute only a small effect on the calculated concentration.

UV Irradiation of Spinal Cord

The spinal cord injected with vanadate or fluorescein was irradiated at 365 nm for about 25 min on ice. Ruores- cence images were taken immediately before and after irradiation to determine the vanadate concentration. Beads were then injected in the same location where the vanadate was injected. Vanadate concentration was calculated from the mean fluorescence intensity in a 500 pm length ofthe axon, centered on the point of vanadate injection. The mean vanadate concentration during UV irradiation was calculated using the diffusion function given in the previous paragraph.

RESULTS

Bead Movements in the Living Axon Mimic Fast Transport in Both Directions

Fluorescent carboxylated beads (0.2 pm in diameter) were pressure injected into Mauthner axons of the larval sea lamprey (Petromyzon marinus) spinal cord. These axons are each about 40 pm in diameter and are optically clear, facilitating the microinjection. Following the injection, there was a period during which the beads appeared to be sus- pended in the axon. The beads then appeared to attach to structures, presumably microtubules, in the center of the axon and began to move. These beads migrated as two distinct groups, one in each direction. Figure 1 shows, in pseudocolor, fluorescence images of beads in an axon, with the color indicating the fluorescence intensity. The bright spot at the injection site is due to beads that never moved after the injection, perhaps because they be- came attached to fixed structures such as the plasma membrane. Because the beads moved away from the injection site in two discrete groups, the movement was active and not simply due to diffusion.

Video image processing allowed the determination of the distance and velocity of bead movements. The distance between the bright spot at the injection site, which did not move, and the center of each bead group was the transport distance of the group. In about 60% of the experiments, the

Figure 1 Bead movement as a function of time. Pic- tures were taken at different times after beads were injected into an axon. The time when the image was taken is indicated on each picture. The bright spot in the center is where the beads were injected. Each division of the scale is 10 pm. The numbers connected to the color scale indicate the gray value the color represents. “An” indicates the anterograde direction and “Re” indicates the retrograde direction.

beads maintained a constant velocity over the en- tire observation period (more than 20 rnin). Figure l(a) illustrates an experiment in which the beads moved continuously for about 20 min at a constant velocity. In the remaining 40% of the cases, the velocity of the beads decreased 10 to 12 min after the injection (data not shown). These two patterns of movement velocity could be observed even in con- secutive injections into the same axon. Attempts were made to determine the reason for the reduced velocity (the number of beads injected, the temper-

220 Wangetal

Table 1 Summary of Results

Type of [Vanadate] Animal ( P A 4 No. va f SE (pm/s) Pa Vr _+ SE (pm/s) Pr

s. 1. 0 41 0.100 f 0.0086 0.10 1 _+ 0.0086 b. 1. 0 4 0.183 t 0.042 0.009 0.226 t 0.046 0.000 s. 1. 2-50 7 0.135 f 0.03 1 0.15 0.12 I t 0.025 0.40 s. 1. 60- I20 I 0.055 k 0.0097 0.04 1 0.047 t 0.0 1 1 0.0 15 s. 1. 140-200 3 0.0 15 t 0.039 0.014 0.022 t 0.030 0.020 s. 1. I150 1 0.006 -0.025

Bead movement velocities with different vanadate concentrations. s. 1. indicates sea lamprey, b. I. indicates brook lamprey.) [Van- adate] gives the vanadate concentration in the axon at 3 rnin after injection. v a and v r are the average anterograde and retrograde bead velocities between 2 and 10 rnin after injection. SE is the standard error. Pa and Pr are the significance values when the velocities are compared with the control (vanadate concentration = 0, s. I.). They are calculated with Student's t test.

ature of the bathing solution), but no conclusive result was obtained. Therefore, the average velocity between 2 and 10 rnin after injection was used to define the characteristic velocity of bead transport. In sea lamprey axons, the mean velocity was about 0.1 pm s-' for transport in both directions (Table 1). In contrast, when we injected beads into larval brook lamprey (Lumpetru paneril ) axons, which are larger than the sea lamprey axons, the measured velocity was almost twice as fast (Table l). These velocities are substantially less than fast axonal transport velocities but approaching the reported instantaneous velocities of injected beads in the crab giant axon (Adams and Bray, 1983). Spe- cies-dependent differences in transport velocities may be related to differences in axon diameters (Al- len et al., 1982).

Bead Transport Requires Intact Microtubules

To determine if the bead movements depended on microtubules, the beads were co-injected with colchicine, an agent that promotes the disassembly of microtubules (Banks et al., 197 1). The concentration of colchicine in the axon was estimated by measuring the fluorescence intensity of fluorescein, which was mixed in the colchicine solution. Since both drug and fluorescein concentrations at the injection site change with time due to diffusion, 3 rnin after injection was used as a standard time for determining concentrations. Higher doses of colchicine (4 pM and greater at 3 rnin after injection) completely abolished bead movement [see Fig. 3(b)], whereas in a lower concentration (2 pM at 3 rnin after injection) a small fraction of the beads moved (data not shown). These colchicine concentrations are comparable to the concentrations that

disassemble microtubules and inhibit axonal transport (Banks et a]., 1971; Dustin, 1978).

As a control for the colchicine experiments, lumicolchicine, the inactive isomer of colchicine (Wilson et al., 19741, at concentrations up to 18 p M , did not inhibit bead movement. One of the lumicolchicine experiments is shown in Figure 3 (c). These results indicate the bead movement in the axon depended on intact microtubules.

Bead Transport Is Inhibited by Vanadate

Vanadate was injected in an attempt to determine the adenosine triphosphate (ATP) dependence of the bead movement. Vanadate inhibits many ATPases (Simons, 1979), and it has been used to inhibit fast axonal transport (Forman et al., 1983). The enzymes that are most sensitive to vanadate include the plasma membrane Na,K-ATPase, which is inhibited by nanomolar concentrations of vanadate (Cantley et al., 1977, 1978), dynein, which is half-maximally inhibited in vitro by less than 25 pA4 vanadate (Gibbons et al., 1978), and kinesin, whose microtubule-stimulated ATPase activity is half-maximally inhibited in vitro by more than 400 pA4 vanadate (Cohn et al., 1987). Other ATPases (such as myosin) are significantly less sensitive to vanadate. Since organelles can move in the absence of an intact plasma membrane (Brady et al., 1982), it is unlikely that inhibition of bead movement by vanadate is related to an effect on the Na,K-ATPase.

Bead movement was inhibited when axons were co-injected with vanadate and beads (Table 1). Fig- ures 2 and 3 (d and e) show the results of one experiment. The concentration of vanadate that inhibited bead transport was similar to what has been reported to inhibit vesicle motility in extruded axo-

Dj'nein Reqziirements in Intact Axons 221

250 -

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-f Without Vanadate - WahVanadate

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Figure 2 Bead movement with or without vanadate. Carboxylated beads were co-injected with vanadate into one Mauthner axon, and beads with no vanadate were injected into the contralateral axon as a control. Vana- date concentration was 110 pMat 3 min after injection.

plasm (Forman et al., 1983). We note that the buffer used to dissolve the vanadate in our experiments reduced the inhibitory activity of vanadate, probably by affecting its oxidation state. In experiments in which vanadate was used to inhibit sea urchin axonemal dynein MgATPase activity, it was found that a fourfold higher concentration of vanadate was required in lamprey buffer (pH 7.4) to achieve the same degree of inhibition as that in the usual dynein assay buffer (pH 8.2). (Data not shown.)

Conditions that Produce the V1 Photolysis of Dynein Abolish the Retrograde, but Not the Anterograde, Transport of Beads in the Living Axon

In the presence of vanadate and MgATP2-, irradiation at 365 nm results in the specific scission of the dynein heavy chain (Lee-Eiford et al., 1986). This V 1 photolysis renders the dynein inactive and has been used to inhibit dynein-mediated move-

ment in reconstituted systems (Schnapp and Reese, 1989; Schroer et al., 1989). In an attempt to elucidate the mechanism or mechanisms driving the transport of the injected beads, the intact lamprey axons were treated with conditions that would produce the V1 photolysis of dynein.

Vanadate was injected into the axon and the en- tire spinal cord was irradiated at 365 nm for about 25 min; during this time, the injected vanadate continued to diffuse. After the vanadate concentration had decreased by diffusion to below the concentration required for inhibition of bead movement (Table l), beads were injected into the axon at the vanadate injection site. These experiments are summarized in Table 2 and examples are shown in Figure 3(f and 8). If the mean vanadate concentration was greater than 140 pM during the UV irradiation, retrograde movement was completely abolished and the beads only moved in the anterograde direction. If the mean vanadate concentration during the UV irradiation was between 80 and 140 pM, significantly more beads moved anterogradely than retrogradely.

An alternative to allowing vanadate sufficient time to diffuse away from the injection site was to co-inject norepinephrine with beads immediately after UV treatment. Norepinephrine reduces vanadate to an inactive form, so any nonphotolyzed motor molecules would be expected to become functional. The problem with this approach was that norepinephrine caused the beads to clump in the micropipette; however, in two experiments we successfully injected beads with norepinephrine. The vanadate concentration was about 100 pA4 and the norepinephrine concentration was about 10 mM at the time of bead injection. In one case, the beads moved only in the anterograde direction and in the other case, three times as many beads moved in the anterograde direction as in the retrograde direction. These results are consistent with those obtained by allowing vanadate to diffuse away before injecting beads (Table 2).

Two sets of control experiments were performed to rule out the possibilities that the abolishment of retrograde movement was either due to the irradiation or excess vanadate. Since fluorescein was co- injected with vanadate to determine the vanadate concentration, fluorescein alone was injected fol- lowed by UV irradiation. Even when the fluorescein concentration was increased to levels higher than those used in the vanadate-UV experiments, the beads moved unimpeded in both directions [Table 2, last row; Fig. 3(h)]. Therefore, UV irradiation alone did not inhibit retrograde movement.

222 Wung rt al.

In the absence of UV irradiation, different concentrations of vanadate, when co-injected with the beads, did not alter the proportion of beads that moved in the anterograde and retrograde directions (Table 2). Therefore, vanadate alone was not responsible for the selective inhibition of retrograde movement in the vanadate-UV-treated axons.

DISCUSSION

The results presented herein provide direct support for the idea that retrograde transport in lamprey axons is powered by dynein. When 0.2 pm fluorescent latex beads were pressure injected into the Mauthner axons, they moved in both directions at rates comparable to, but somewhat slower than, other measurements of axonal fast transport.

is indicated). “An” indicates the anterograde direction; “Re” indicates the retrograde direction. The pictures are shown in pseudocolor; the fluorescence intensity corre- sponding to each is shown in the linear color scale in the bottom left corner of (A). Each division of the scale bar is 10 pm; the scale is the same for all photographs. (A) No treatment. Group of beads moved in both anterograde and retrograde directions. (B) Beads co-injected with colchicine. Colchicine concentration at 3 rnin after injection was 6 pM. Bead movement was abolished. (C) Beads co-injected with lumicolchicine. Lumicolchicine concentration at 3 min after injection was 18 pM. Beads moved in both directions. (D) Beads co-injected with vanadate. Vanadate concentration was 110 pMat 3 rnin after injection (also see Fig. 2). Beads movement in both directions was slowed. (E) Contralateral control for (D). Beads without vanadate were injected into the contralat- era1 axon in the same spinal cord as (D). Beads moved the same distance in a shorter time. (F) Bead movement after vanadate injection and UV irradiation. The mean vanadate concentration was 140 during this period. Beads were injected after UV irradiation. Vanadate concentration at 4 min after bead injection was 75 pM. More beads moved in the anterograde than in the retrograde direction. (G) Bead movement after high level of vanadate-UV. Mean vanadate concentration was 235 ph4 during the 25 rnin UV cleavage. Vanadate concentration was 70 pMat 3 rnin after bead injection. Retrograde bead movement was abolished. (H) Bead movement after fluorescein-UV treatment. Fluorescein concentration corresponded to the fluorescein level that achieved when 350 p M of vanadate was injected. Beads still moved in both directions after UV irradiation.

Figure 3 Bead movement under different conditions. Fluorescence images of beads were taken at different times after injection (the time when the picture was taken

Tab

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224 U’ung et al

When beads were co-injected with colchicine to achieve a drug concentration of 5 p M at 3 min after injection, all bead movement was abolished, but an inactive isomer, lumicolchicine, had no effect at 18 pM. Clearly, the bead movement we observed required intact microtubules.

The co-injection of vanadate with beads reduced bead movement in both directions in a concentration-dependent manner, suggesting the involvement of ATPases with movement. To iden- tify one of the ATPases, we utilized the selective sensitivity of dynein to UV photolysis in the presence of micromolar concentrations of vanadate. By injecting vanadate at a local concentration of 140 pMand then irradiating at 365 nm, dynein should be irreversibly destroyed. The diffusion of intact dynein into the irradiated area would be expected to be much slower than the diffusion of vanadate away from its injection site. Thus, as the vanadate concentration fell, ATPases other than dynein would recover activity, whereas dynein-dependent movement would remain blocked. When fluorescent beads were injected following vanadate-UV treatment, we found that retrograde movement was selectively abolished, implicating dynein as the retrograde but not the anterograde motor.

The relationship between bead movement and actual axonal fast transport of organelles is unclear. The movement of beads that we observed is con- siderably slower than fast transport, but comparable to reported velocities of beads in vzfro. Never- theless, the analysis of bead movement has led to significant insights into microtubule-mediated transport and several features of the movement of the fluorescent beads may reveal important information about organelle transport. When beads were injected alone and in the absence of inhibi- tors, we noted a delay of varying time before the beads began to move. This is similar to what was reported for beads injected into crab axons (Adams and Bray, 1983) and may represent the time required for suflkient numbers of motor molecules to attach to the beads, or for the beads to attach to available membranous organelles. When beads were co-injected with vanadate, the beads moved a short distance before stopping. Since vanadate inhibits an ATPase by substituting for the P, after ATP hydrolysis (Shimizu and Johnson, 1983), the delay suggests that the beads are coated with multiple copies of the motor molecules and that only a small fraction of these motor molecules is required to produce movement (Howard et al., 1989). In some cases, the injected beads, after stopping in vanadate, were observed to move backward

(the negative velocity in Table 1). One explanation for this would be that the beads were coated with both anterograde and retrograde motors and that vanadate only inhibited the ATPases that were ac- tively hydrolyzing ATP. This agrees with observa- tions that dynein is found on organelles moving in either direction (Hirokawa et al., 1990) and with the observation that additional, nonmotor factors appear to regulate motor activity (Schroer et al., 1989).

Video image processing also allowed for the quantitative determination of the total fluorescence intensity in a defined area, which could be translated into the actual number of individual beads in the area (see Materials and Methods). In 27 control experiments, there was no significant di- rectional bias in the number of beads and the velocity of the beads (p > 0.2 for both cases, Student’s t test). The largest number of beads that was carried by anterograde and retrograde transport was calculated to be 3 X lo5. If the kinesin concentration in the cell is 0.5 pA4 (Brady et al., 1990), and if the anterograde transport of the beads were due to kinesin, then there would have been approximately 50 kinesin molecules bound to each bead. This as- sumes that all of the kinesin is exchangeable and capable of binding to the beads. Considering that many of the immobile beads may also have had kinesin on their surfaces, the kinesin density could have been lower.

The movement of the fluorescent beads in the lamprey axons that we report here provides an ex- cellent model system in which to study transport. Bead transport in both directions occurs at velocities similar to what is observed for membranous organelles, and the bead velocity is correlated with the diameter of the axon, a property that is observed in intact axons but not in cell-free systems (Allen et al., 1982; Vale et al., 1985b). Bead movement requires intact microtubules and is inhibited by vanadate at concentrations that inhibit organelle transport in extruded axoplasm. The video analysis of the transport of the fluorescent beads promises to lead to studies of the regulation of fast transport, since the concentrations of molecules such as calcium and cyclic AMP can be easily var- ied, measured, and quantitatively related to the motion of the beads.

This work was supported by grants from the National Science Foundation and the National Institutes of Health to K. R. R. and a grant from the National Science Foundation to D. J. A.

Djwin Requirements in Intact Axons 225

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Retrograde but not anterograde bead movement in intact axons requires dynein