The Journal of Neuroscience, April 1995, 75(4): 3053-3064

The Activation of Protein Kinase A Pathway Selectively Inhibits Anterograde Axonal Transport of Vesicles but Not Mitochondria Transport or Retrograde Transport ~II viva

Yasushi Okada, Reiko Sato-Yoshitake, and Nobutaka Hirokawa Department of Anatomy and Cell Biology, Faculty of Medicine, University of Tokyo, Hongo, Tokyo, 113, Japan

To shed light on how axonal transport is regulated, we ex- is an especially well developed intracellular organelle transport amined the possible roles of protein kinase A (PKA) in viva system. Many studieshave been focused on the mechanismof suggested by our previous work (Sato-Yoshitake et al., this transport, but little is known about its regulation. As a pu- 1992). Pharmacological probes or the purified catalytic tative mechanismfor the regulation, we have proposeda model subunit of PKA were applied to the permeabilized-reacti- from our recent studies(Hirokawa et al., 1990, 1991) that the vated model of crayfish walking leg giant axon, and the anterogrademotor kinesin is decommissionedat the axon ter- effect was monitored by the quantitative video-enhanced minal, and that the retrogrademotor cytoplasmic dynein is trans- light microscopy and the quantitative electron microscopy. ported, presumably in an inactive form, by the anterogradeor- Dibutyryl cyclic AMP caused concentration-dependent ganelle transport to the axon terminal, where it should be transient reduction in the number of anterogradely trans- activated to support the retrograde organelletransport. ported small vesicles, while the retrogradely transported Our present study was undertakento examine this regulation organelles and anterogradely transported mitochondria mechanism.One plausiblemechanism is through protein kinases showed no decrease. This transient selective inhibition of and phosphatases,as is true with fish scale chromophores.In anterograde vesicle transport was reversed by the appli- these cells, pigment granule dispersion and aggregation along cation of a specific inhibitor of PKA (KT5720) in a concen- microtubules are regulated through the protein kinase A (PKA) tration-dependent manner, and was reproduced by the ap system (Lynch et al., 1986a,b; Rodzial and Haimo, 1986a,b; plication of the purified catalytic subunit of PKA and Sammaket al., 1992). It is not unreasonableto supposethat it augmented by the application of adenosine 5’-0-(34hiotrL might regulate the direction of the fast axonal transport as well, phosphate) (ATPyS). Corresponding biochemical study becausePKA is reported to be enriched in brain, especially at showed that several axoplasmic proteins including kinesin the presynaptic terminal (Walter et al., 197X; Dunkley et al., were specifically phosphorylated by the activation of the 1988). This speculationwas supportedby our previous investi- PKA pathway. These findings suggest the possible roles of gation (Sato-Yoshitake et al., 1992) in which we demonstrated PKA in the regulation of the direction of the axonal trans- that kinesin was phosphorylated by PKA, and that the phos- port in viva. The finding that only vesicle transport but not phorylation reduced the affinity of kinesin to purified synaptic mitochondria transport was inhibited also suggests that vesicles, while its motor activities were not affected. the transport of vesicles and that of mitochondria are dif- In the present study, we examined the effect of the activation ferently regulated and might be supported by different mo- of the PKA system in vivo using a permeabilized-reactivated tors. model of crayfish walking leg giant axon (Hirokawa and Yori- [Key words: axonal transport, phosphorylation, kinesin, fuji, 1986; Forman et al., 1987). It provides a good model system protein kinase A, phosphatase, crayfish walking leg giant becauseit lacks neurofilament protein (Burton and Hinkley, axon] 1974; Hirokawa, 1986; Hirokawa and Yorifuji, 1986; Viancour et al., 1987). Neurofilament is a good substratefor PKA (Let- Numeroustypes of membrane-boundorganelles are transported errier et al., 1981); its phosphorylation can induce a substantial and distributed through the cytoplasm. Fast axonal transport, a change in the structure of the neuronal cytoskeleton, and the bidirectional transport of membranousorganelles through axon, organelle transport was greatly perturbed (mostly stimulated both in number and velocity) as reported in Aplysia growth Received Sept. 12, 1994; revised Nov. 2, 1994; accepted Nov. 8, 1994. cones (Forscher et al., 1987) (R. Sato-Yoshitake, unpublished We thank Dr. Masaki Inagaki (Tokyo Metropolitan Institute of Gerontology) for the generous gift of the catalytic subunit of PKA, Dr. G. S. Bloom (Uni- results).In the crayfish giant axon, however, no apparentchange versity of Texas) for providing anti-kinesin antibody (H2), and Dr. N. Lomax in the axonal cytoskeleton was observed even at an electron (the National Cancer Institute) for supplying us with taxol. We also thank Dr. microscopiclevel. We can thus assessthe effects of the activa- Y. Ihara (Department of Neuropathology, University of Tokyo) for technical comments on phosphorylation experiments, and the members of our laboratory, tion of the PKA system on the axonal transport system more especially Drs. H. Airawa, T. Nakata, S. Okabe, and H. Yorifuji, for their directly. invaluable advice and discussions throughout. This work was supported by a Special Grant-in-Aid for Scientific Research from the Japan Ministry of Edu- Quantitative video-enhancedlight microscopic and electron cation, Science and Culture, and by a grant from the Institute of Physical and microscopic observation of this model revealed that the activa- Chemical Research (RIKEN) to N.H. Y.O. was supported by a JSPS fellowship tion of the PKA system selectively inhibited the anterograde for Japanese Junior Scientists. Correspondence should be addressed to N. Hirokawa at the above address. vesicle transport, but the retrograde transport and the mitochon- Copyright 0 1995 Society for Neuroscience 0270.6474/95/153053-12$05.00/O dria transport were not affected. These effects by PKA activa- 3054 Okada et al. - Selective Inhibition of Axonal Transport by PKA tion, coupled with the previous in vitro results (Sato-Yoshitake min to remove contaminating axolemma debris. This axoplasm con- et al., 1992) and the corresponding biochemical results reported tained many microtubules and membranous organelles such as mito- chondria and small and large vesicles. ATP (final 2 mM) and Y-“~P-ATP here, support the idea that decommission of kinesin at the axon (final 0.3 mCi/ml) were added and incubated for 45 min in the presence terminal is performed by the release of kinesin through its phos- or absence of the catalytic subunit of PKA (final 0.05 mglml). The phorylation. samples were analyzed on a 7.5% SDS-polyacrylamide gel (SDS- PAGE) by Coomassie stain and autoradiography. The autoradiogram Materials and Methods was quantitatively analyzed with an image analyzer BAS2000 (Fuji, Tokyo, Japan). Video-enhanced light microscopy of the isolated crayjsh walking leg Purijcation of CF-kinesin from crayhsh nervous tissue. CF-kinesin giant axon. As described in our previous paper (Hirokawa and Yorifuji, was purified from crayfish nerves and ganglia by scaling down the 1986), a giant motor axon dissected from crayfish (Procambarus method already reported (Vale et al., 1985a,b; Wagner et al., 1989; Sato- clurkii) walking leg was mounted in a perfusion chamber filled with Yoshitake et al., 1992). Briefly, walking leg nerves and abdominal gan- crayfish internal medium (CFIM; 280 mM K-aspartate, 15 mM NaCl, glia were isolated from 300 crayfishes and homogenized in 10 ml PEM 15 mM HEPES, 5 mM MgCl,, 3 mM EGTA, pH 7.4) (Lieberman et al., (PIPES 100 mM, pH 6.9, EGTA 1 mM, MgCl, 1 mM, dithiothreitol 1 1981). It was observed with an Axiophot microscope (Zeiss, Ober- mM, phenylmethylsulfonylfluoride 10 FM. and leupeptin 10 pg/ml). Mi- kochen, Germany) equipped with a 63 X NA 1.40 plan-apochromat ob- crotubules, reconstituted from the clarified homogenate in the presence jective (Zeiss), an NA 1.4 condenser (Zeiss), and the G. W. Ellis fiber- of IO FM tax01 and 1 mM ATP, were removed by centrifugation, and optic light scrambler (Technical Video, Woods Hole, MA). The DIC the supernatant was incubated with 10 U/ml hexokinase and 50 mM image was projected to a video camera, Hamamatsu C-2400-07, by 6X glucose for 5 min at 25°C. The sample was further incubated with 2 relay lenses (Zeiss). With an image processor Argus-10 (Hamamatsu mM adenylyl-5’-imidodiphosphate, 10 PM taxol, and 0.3 mg/ml tubulin, Photonics, Hamamatsu, Japan), the image of moving organelles was and reconstituted microtubules were pelleted by centrifugation through contrast enhanced by differentially subtracting rolling-averaged video a 50% sucrose cushion. This pellet was extracted with 1 ml PEM con- frames. For quantification of the motility of the organelles, the objective taining 10 mM MgATP on ice, and microtubules were removed by cen- focal plane was adjusted to the center of the axon, a line perpendicular to the long axis of the axon was drawn on the video monitor, and the trifugation with 20 PM taxol. The supernatant was dialyzed against TM number of moving organelles crossing the line was counted for 10 set (50 mtvt Tris, pH 7.2, and 1 mM MgCl,), and this fraction was used in (vesicles) or 5 min (mitochondria). the following assays. Application of the pharmacological probes to the permeabilized-re- Metabolic labeling of CF-kinesin and peptide mapping. Carefully dis- activated model and the quantification of its effect by video-enhanced sected ventral nerve cord was incubated in CFIM supplemented with 1 light microscopy. Before the application of the pharmacological probes; mCi/ml ‘2P-orthophosphate for 30 min. CF-kinesin was partially puri- the isolated axon was observed as described above, and the number of fied as described above, and the 120 kDa band was excised from SDS- moving organelles was counted. Only the axons more than 20 pm in PAGE gel. At the same time, purified CF-kinesin was phosphorylated diameter and with more than 100 anterogradely moving vesicles were in vitro by 60 min incubation with 0.1 mM ATP containing 0.3 mCi/ml used for the following assays. In some preparations, the organelles in Y-‘~P-ATP and 0.05 mg/ml catalytic subunit of PKA, and excised from axons showed less smooth movement than other preparations. Such ax- SDS-PAGE gel. These in vivo and in vitro phosphorylated CF-kinesin ons were discarded, because this type of movement was often observed were partially digested by V8 protease and analyzed by a second 15% in injured axons. SDS-PAGE gel as described by Cleveland et al. (1977). Membrane-permeable probes such as dbcAMP KT5720, okadaic Assay Qf phosphatase activity in axoplusm. Purified CF-kinesin (0.1 acid, and calyculin A were directly perfused into the chamber, and the mglml) was phosphorylated by 60 min incubation with 0.1 mM ATP number of moving organelles was counted. containing 0.3 mCi/ml Y-‘~P-ATP and 0.05 ma/ml catalvtic subunit of For the application of the membrane-nonpermeable probes such as PKA. Cgkinesin and two contamination bands (70 kDa protein and the catalytic subunit of PKA and ATPyS, the permeabilized-reactivated tubulin) were strongly phosphorylated. Excess cold ATP (10 mM), phos- model was used. The axolemma was permeabilized by 30 min incu- phatase inhibitors (okdaic acid and calyculin A), and protease inhibi- bation in CFIM containing 0.015% saponin and 2 mM ATP When nec- tors were added to this sample, and it was incubated in the presence or essary, an ATP regeneration system (3 mM creatine phosphate, 1 unit/ml absence of the extruded axoplasm (1:4 v:v), and analyzed on a 7.5% creatine phosphokinase) was added to this medium to maintain an op- SDS-PAGE by Coomassie stain and autoradiography. The autoradio- timum concentration of ATP Then the permeabilizing medium contain- gram was quantitatively analyzed with a BAS2000 image analyzer ing PKA or other probes was perfused into the chamber, and the number (Fuji, Tokyo, Japan). of moving organelles was counted as above. Association of axonal organelles and kinesin. Extruded axoplasm Quantification of the effect of PKA application by electron micros- from six nerve fibers was diluted 1:lOOO with ice-cold CFIM and cen- copy. The isolated giant axon was permeabilized and reactivated as trifuged at 16,000 rpm for 30 min by desk-top centrifuge after 30 min described. After 30 min permeabilization, the catalytic subunits of PKA incubation at -1°C. The supernatant was observed by video-enhanced and ATPyS were added at final concentrations of 0.05 mg/ml and 0.02 light microscopy and negative-staining electron microscopy, and it was mM, respectively. The sample was incubated for 35 min at 25”C, and confirmed that its major component was membranous organelles other the midpoint of the axon was ligated with surgical thread. The axon than mitochondria or the debris of axolemma, and that it contained little was incubated for 5 min and fixed with 4% paraformaldehyde and 2% cytoskeletal filamentous structure. Otherwise this centrifugation step glutaraldehyde, followed by a conventional electron microscopic meth- was repeated. The organelles were collected by centrifugation (Beck- od. Thin cross sections were cut from the portion 20-60 pm proximal man TL 100.2 rotor, 75,000 rpm, 30 min) and the pellet wa< resuspended or distal to the region damaged by the ligation. Electron micrographs with 100 ul TM containing 500 mM KCl. as oreviouslv described (Sato- of 200 axons from nine independent preparations for each condition Yoshitake et al., 1992), to-remove kinesm already bound to the drgan- were taken. Two different authors independently examined the resulting elles. The sample was again centrifuged and the pellet was resuspended 1200 micrographs in a blinded fashion. Each author scored the degree with 50 ul TM. This sample was checked by video-enhanced light of accumulation of vesicles and mitochondria according to a predeter- microscopy and negative-staining electron microscopy, and many small mined scale (0 = no accumulation, 1 = slight accumulation, 2 = sig- and large vesicular organelles and occasionally mitochondria were ob- nificant accumulation, 3 = most accumulated), and the scores by the served, but microtubules or the debris of axolemma were rarely found. two authors were summed for each micrograph. Thus, the degree of Phosphorylated CF-kinesin was obtained by 60 min incubation of the organelle accumulation was semi-quantitatively scored by this “accu- purified CF-kinesin (0.1 mg/ml) with the catalytic subunit of PKA (0.05 mulation score,” and histograms were plotted to illustrate changes in mg/ml) and 2 mM ATP at 25°C. For unphosphorylated CF-kinesin, the the degree of accumulation by shifts in their peaks. For the statistical purified CF-kinesin was incubated under the same conditions but with- evaluation of the shifts, the accumulated x2 test was performed. out PKA. The CF-kinesin aliquots were centrifuged (Beckman TL100.2 Detection of phosphotylated proteins in the moplasm of the cray$sh rotor, 100,000 rpm, 30 min) to remove the aggregates. giant axon. Nerve fibers isolated from crayfish walking legs were The organelles were added to the kinesin ali’quots and incubated for squeezed with a handmade small roller onto a parafilm-coated petri dish. 30 min at 25°C. For the control, samples without organelles or kinesin About 2-3 pl of axoplasm was obtained from one nerve fiber, which were incubated under the same conditions. The samples were then cen- was diluted to 30 p,l with CFIM and centrifuged at 2000 X g for 10 trifuged (Beckman TL100.2 rotor, 100,000 rpm, 30 min) to separate the The Journal of Neuroscience, April 1995, l5(4) 3055 bound/free kinesin. In this step, cosedimentation of free kinesin with anterogradevesicle transport was transient, reaching its peak at contaminating microtubules and its nonspecific binding to the tube sur- around 30 min after dbcAMP application, and then recovering face can obscure the result. Therefore, we performed the centrifugation in the presence of ATP to reduce the binding of kinesin to microtubules, to the initial level at 40-60 min after dbcAMP application, even and siliconized tubes were used to prevent nonspecific binding. The when new medium containing the same concentration of pellets and the supernatants were analyzed by immunoblotting using dbcAMP wasperfused into the chamber.This indicated that the monoclonal anti-kinesin antibody H2 and iZsI-labeled protein A (ICN, recovery was not due to the consumptionof dbcAMF! Tokyo, Japan). The results were quantified by analyzing the autoradi- oarams of the blot with BAS2000 (Fuii, Tokvo, Japan). The inhibition by dbcAMP was concentrationdependent (Fig. ~-Other materials and reagents. The catalytic subunit of PKA, mono- 2a). At 1 mM, the anterogradevesicle transport was most inhib- clonal anti-kinesin heavy chain antibody H2, and taxol were generous ited. The higher the concentration, the more rapidly the inhibi- gifts from Dr. M. Inagaki (Tokyo Metropolitan Institute of Gerontolo- tion reached its peak and recovered (Fig. le). It appearsthat gy), Dr. G. S. Bloom (University of Texas), and Dr. N. Lomax (National increasingthe concentration of dbcAMP stimulatedboth the in- Cancer Institute), respectively. Crayfish was purchased from a local dis- tributor. Nucleotides, nucleotide analogs, and detergent were purchased hibition and its recovery. from Sigma (St. Louis, MO). Buffers were purchased from Dojindo To demonstratethat PKA is responsiblefor this inhibition by Laboratories (Kumamoto, Japan). Other reagents were purchased from dbcAMP we used a specific PKA inhibitor: KT5720. When Wako Chemical (Tokyo, Japan) unless otherwise stated. KT5720 was applied to the medium, the inhibition of the an- terogradevesicle transport by 1 mM dbcAMP was reversedin a Results dose-dependentmanner, while KT5720 alone showedno effects Observation of isolated giant axon by video-enhancedlight on organelletransport (Fig. le). IC,, for this inhibition was -50 microscopy nM. It correspondswell with the IC,,, for the inhibition of PKA, As in our previous study (Hirokawa and Yorifuji, 1986), two and was much lower than the IC,,s for the inhibition of other distinct types of organelleswere observed to move both anter- kinasessuch as protein kinase C, Ca*+-calmodulinkinase, and ogradely and retrogradely in the isolated crayfish walking leg myosin light chain kinase(Kase et al., 1987).This demonstrates giant axon observed by video-enhancedlight microscopy. One that PKA plays a key role in the effect of dbcAMP on organelle is a long (- 1 p,m) large organellemoving slowly (-0.2 bm/sec) transport. in a saccadic manner. The other is a small (smaller than the diffraction limit), spherical organelle moving rapidly (l-2 The selective inhibition by dbcAMP is causedby the activation km/set) in a smooth manner. From their shapeand size, the of PKA former is consideredto be a mitochondrion, and the latter is To study further the mechanismof the inhibition by dbcAMP, assumedto be a vesicle. This interpretation was confirmed by we useda permeabilized-reactivatedmodel. In this model, most the resultsfrom the electron microscopicobservations described cytosolic solubleproteins are washedout, and only cytoskeletal in the following section. Hence, to avoid unnecessarycomplex- and membrane-associatedcomponents remain. ity, we refer to the smaller,spherical organelles as vesicles,and Using this model, we first evaluated what effect the decrease the.larger, longer organellesas mitochondria in the following or increaseof ATP concentration might have with the organelle sectionsdescribing the resultsof light microscopicobservations. motility, becauseorganelle motility dependson ATP and its de- A quantitative measureis necessaryfor the evaluation of the pletion ceasesthe motility. In fact, in preliminary experiments drug effect. A naive one is the number of moving organelles. without ATP regenerationsystem, activation of PKA pathway Indeed, it was maintainedaround the samelevel (SD - 5% of depletedATP and organelle movementstopped. This is why we the mean) for more than 2 hr in the control conditions, but it included ATP regenerationsystem in the reactivation medium, varied very much (SD -5O-100% of the mean) from prepara- but still there remainsa possibility that a slight decreaseof ATP tion to preparation (Fig. la,c; see Fig. 4a-d, CTRL traces), concentration by pharmacologicalperturbation can influence or- which made it difficult to compare different preparations.Pre- ganelle movement. liminary studies,however, suggestedthe responseto the drug To rule out this possibility, the ATP concentration in the re- was proportional to the initial level. Therefore we usedthe rel- activation medium was altered, and organelle motility was ob- ative changesin the number of moving organellesas the mea- served. As shown in Figure 3, both anterogradeand retrograde surefor the drug effect. For this purpose, “relative motility in- vesicle transportsdecreased concentration dependently when the dex” was calculated by dividing the number of moving ATP concentration was lower than 1 ITIM. The retrograde trans- organellesby the average before drug application. In any con- port was more sensitive to the decreaseof ATP concentration ditions in this study, the SD of this index was within 10% of than the anterogradeone. Therefore, anterograde-specificinhi- the mean(mostly 3-5%), much smaller than that of raw number bition cannot be reproducedby decreasingATP concentration; (50-100%) and small enough to detect the drug effects in this that is, the observed specific inhibition of anterogradevesicle study. transport is not causedby the possibledecrease of ATP concen- tration by pharmacologicalperturbation, but rather by its phar- Application of dbcAMP selectively inhibits anterograde small macological actions. vesicle transport Second, we applied the catalytic subunit of PKA to the per- For the activation of the PKA system,we first applied dbcAMP meabilized-reactivatedmodel. To maintain the concentration of to the nonpermeabilizedisolated giant axon. DbcAMP is a mem- ATP an ATP regenerationsystem was supplementarilyadded to brane-permeableanalog of CAMP, and activates PKA. After the the reactivation medium,and the concentration of ATP was kept addition of dbcAMP, the number of anterogradely transported at l-2 ITIM, the optimum concentration for organelle transport vesiclesdecreased (Fig. lb,e), while the number of retrogradely (Fig. 3). The effect of the application of PKA was similar to transportedvesicles and anterogradelyor retrogradely transport- that by dbcAMP The anterogradevesicle transport showed a ed mitochondria showedno decrease(Fig. ld,f-h). The velocity selective and transientdecrease in number (Fig. 4a,e) but not in of transportedorganelles did not change.The inhibition of the velocity, while no significant changeswere observed in the ret- 3056 Okada et al. * Selective inhibition of Axonal Transport by PKA

a Antero-ctrl b Antero-dbcAMP C Retro-ctrl d Retro-dbcAMP

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0 0 20 40 60 0 20 40 60 0 20 40 60 Time/min Time/min Timelmin Figure I. Effects of dbcAMP on organelle transport in the crayfish giant axon. The numbers of anterogradely (a. b) and retrogradely (c, d) moving organelles were counted in the absence (a, c) and presence (b, d) of dbcAMP (1 mM). Some typical raw data are plotted against time after the drug application in a-d. Open symbols with dotted lines indicate the number of moving mitochondria, and closed symbols with solid lines show that of small vesicles. These raw data were transformed into “relative motility indices” by dividing the average values before the drug application, and the mean and SEM of eight independent preparations are plotted in e-h (e, anterograde small vesicles;j retrograde small vesicles; g, anterograde mitochondria; h, retrograde mitochondria). The values in the absence of dbcAMP ( 0) and in the presence of 1 mM dbcAMP (W) are indicated in e-h, and those in the presence of 2 mM dbcAMP (A) and in the presence of 1 mM dbcAMP and 2 PM KT5720 (*) are also shown in e andjI The application of dbcAMP caused a selective and transient reduction in the number of anterogradely transported small vesicles, and this reduction was reversed by the application of the specific PKA inhibitor KT5720. The Journal of Neuroscience, April 1995, 75(4) 3057 a 1.2 1.2 1

1 T -r

+ antero

0.2 --- 0 --- retro

0 I I I I I I I 0 0.25 0.5 1 2 0.125 0.25 0.5 1 2 3

dbcAMP /mM ATP /mM Figure 3. The effects of the alteration of ATP concentration on the anterograde (W) and retrograde ( 0 ) vesicle transport. Mean and SEM from three independent experiments are shown. Reduction of ATP con- centration has an inhibitory effect on both the anterograde and retro- b grade transport, but the latter was more sensitive. This difference in sensitivity was significant at P < 0.01 by the paired t test. Note that at 1.2 - any concentration of ATP, the relative motility index for the anterograde transport was equal to or greater than that for the retrograde one.

l-

g ATPyS augmentsthe inhibition by PKA a 0.8 - 5 z.:.:.:.:. The application of dbcAMP or PKA showed a biphasic effect . .. ” . ” . .‘. :.:.:.:.:. . :.:.:.:.:. . . :::::y:: ...‘...‘.. ,x .::::.. . . . :.:.:s:.:.* :.:.:.:.:.’...... , ..:...... ::..: on vesicle transport: inhibition and its recovery. One possible 3 .” . . ”. . :.:.:.:.:. :::::...... ,:.:.:.:.:. :.:.:.:.:. 0.6 - ;;;;;;;;;i; ....‘.‘... . . :y::::::. ,.,.,.. .~ .,., . ..,.,.,.,.,...... :..:. .:. .:. .:, . explanation for the recovery is that protein phosphatasedephos- 5 . . .‘::::. .~::::, .:::::...... ;:.*.~:...... ,:::::::::: :.:.:.:.:.. :.:.:.:.:.. . . :.:.:.:.:. .::::: .:.:.:.I.:. ),..... phorylates a protein or proteins once phosphorylatedby PKA. ,:.:.:.:.:...... ::::::.::: ..‘.‘.‘.‘...::::: :::::::::: :.:.:.:.:. ‘32 .:.:.:.:.:.. :.:.:.:.:. :...... :. ..‘.‘.‘.‘. :.:.:.:.:.. . To inspect this possibility, okadaic acid and calyculin A, broad ci ...... ‘... . ::::::::::. ‘.‘.‘.‘.‘.‘...‘.‘...... :::::::::: . @ 0.4 - :::::::::::. . . .::::. :::::, .::..... :.:.:.:.:. range phosphataseinhibitors, were used. Neither of them 02 .:.:.:.:.:. ‘.‘.‘.‘...... :::y::::’ :.:.:.:.:...‘.‘.‘.‘. .::::::::::. . I.:.:.:.:...... ‘.‘. :.:.:.:.:...... ‘.‘...... ‘...... showed a significant effect; these drugs did not interfere with .:::..:...... ’ ‘.‘.‘.‘.‘. .;:..... ” .. :.:.:.:.:...... ‘.‘..... :.‘:.‘: ...... ’ ~::::. :.:.:.:.:...... ‘.‘.‘.‘...... ::::: the inhibition by PKA. o,2 _ :$

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0.4 PKA+ys

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+ PKA+yS 0.6 , I I I a I I I 0 I I I ,_ 0 20 40 60 0 20 40 60 0 20 40 bU Time/mm Timelmin Time/min Figure 4. The effects of the application of the exogenous PKA catalytic subunit to the permeabilized-reactivated model of crayfish giant axon. a-d show typical raw data, and the mean and SEM of relative motility indices from eight independent preparations are plotted against time after PKA application (e-h). CTRL,PKA, PKA+ $9, and KT indicate the conditions: CTRL = ATPyS only; PKA = PKA only; PKA+ yS = PKA and ATP$; and KT = PKA, ATPyS, and KT5720. The application of the exogenous PKA catalytic subunit transiently and selectively inhibited the anterograde vesicle transport (I’ < 0.01 by t test at 15-30 min after PKA application). This effect was augmented by the addition of ATPyS, and its recovery was inhibited (P < 0.01 at 30-60 min). The Journal of Neuroscience, April 1995, 7~74) 3059 inhibited in the presence of ATPyS (Fig. 4a,e). This suggests alyzed by SDS-PAGE and detected by autoradiography. At least the involvement of phosphatase activity in the recovery phase. 13 bands were identified on the autoradiogram (Fig. 6, lane 1’). Their radioactivities were significantly increased in the sample EM observation con$rrned that PKA-sensitive organelles are incubated in the presence of PKA (Fig. 6, lane 2’). The addition small vesicles, but not mitochondria of KT5720 reversed this increase (Fig. 6, lane 3’). Thus these Contrast-enhanced light microscopy revealed that the antero- bands are good candidates for the target of PKA. grade organelle transport can be divided into two pharmacolog- Kinesin is reported to be a substrate of PKA in vitro (Murphy ically different systems: one is sensitive to PKA activation and et al., 1989; Sato-Yoshitake et al., 1992), and several studies the other is insensitive or less sensitive. The limited resolution have revealed that kinesin is a phosphoprotein in vivo (Hollen- of light microscopy enabled us to discriminate only two mor- beck, 1993) and that its phosphorylation level is raised by PKA phologically different populations. One consisted of small, activation (Matthies et al., 1993). Furthermore, our previous pa- spherical, smoothly moving organelles whose movement was per (Sato-Yoshitake et al., 1992) suggested the possible role of specifically inhibited by the activation of the PKA system, and kinesin phosphorylation in the regulation of the fast axonal the other was made up of larger, longer, saltatory moving or- transport. Thus kinesin is a good candidate for the substrate, and ganelles that were not inhibited by PKA. we performed the following experiments for its demonstration. For observation at a higher resolution, electron microscopy To clarify whether kinesin is really phosphorylated in crayfish was used. Ligation of the axon is an effective method for dis- giant axon, we purified crayfish kinesin (CF-kinesin) by nucle- criminating the anterogradely or retrogradely transported organ- otide-dependent binding to and release from microtubules. The elles from nontransported ones (Smith, 1980; Hirokawa et al., resulting fraction supported microtubule motility (data not 1990, 199 1). The anterogradely transported organelles accumu- shown). A 120 kDa protein was concentrated in this fraction late at the proximal portion to the ligated site, while the retro- (Fig. 6, lane 7), and this band was recognized by anti-kinesin grade ones accumulate at the distal portion. Nontransported or- heavy chain monoclonal antibody H2 (Pfister et al., 1989) (see ganelles do not accumulate. Fig. 9). We therefore consider this 120 kDa protein to be the The permeabilized-reactivated giant axon was ligated after 35 kinesin heavy chain homolog, and we refer to it as CF-kinesin. min incubation in the presence of ATPyS and the catalytic sub- This fraction was purer than the corresponding fraction from unit of PKA, and was fixed 5 min after its ligation. This prep- mammalian brain. Only tubulin and CF-kinesin were the major aration allowed us to observe only the organelles moving during components, and other contaminating bands were very subtle the period 35-40 min after PKA application, when the inhibition (Fig. 6, lane 7). Thus we used this fraction in the following of the transport reached its peak (Fig. 4a,e). Typical electron assays. We could not identify kinesin light chain (KLC) in this micrographs are demonstrated in Figure 5. First of all, unlike fraction, mainly due to the lack of anti-KLC antibody that cross- mammalian axons, no apparent changes of the cytoskeletal or- reacts with crayfish. ganization were observed with these treatments (Fig. 5a-f). Un- This CF-kinesin fraction was added to the extruded axoplasm der control conditions (Fig. 5a,d), many vesicles and mitochon- (Fig. 6, lanes 4-6). CF-kinesin was phosphorylated in vitro dria accumulated at both proximal and distal regions. Some among axoplasmic proteins and the band around 116 kDa was accumulated vesicles fused to form large membranous cisternae. demonstrated to correspond to that of CF-kinesin (Fig. 6, lanes The addition of PKA caused a decrease in the accumulation of 4’-6’). vesicular and membranous cisternae at the proximal region (Fig. 5b), while the accumulation of mitochondria showed no appar- CF-kinesin is phosphorylated by PKA in vivo ent change, and no distinct change was observed at the distal Thus CF-kinesin is a good candidate for the target of PKA in region (Fig. 5e). vivo, but is it really phosphorylated in nonperturbed crayfish The effects of the application of the pharmacological probes neuron, and is it directly phosphorylated by PKA? To answer on the accumulation of organelles at both the proximal and distal these questions, we metabolically labeled the ventral nerve gan- regions were semi-quantitatively analyzed. As shown in Figure glia with X2P-orthophosphate, purified CF-kinesin from these 5g-j, the degree of accumulation was scored and the histogram ganglia, and performed phosphopeptide mapping to compare the was calculated. No significant difference was detected in the in vivo phosphorylation site with the PKA phosphorylation site distal portion (Fig. 5hJ), as expected from the results by light in vitro. microscope. At the proximal region, the accumulation of vesicles CF-kinesin was actually phosphorylated in vivo (Fig. 7a, lane and membranous cisternae was significantly inhibited, and this 2’; 6, lanes 5-Q and the phosphopeptide mapping was identical inhibition was reversed by the application of the PKA inhibitor with CF-kinesin phosphorylated in vitro by PKA (Fig. 7b). This KT5720 (Fig. 5g), while the accumulation of mitochondria was demonstrates that CF-kinesin is a phosphoprotein in crayfish not affected (Fig. 5i). neuron, and that the 116 kDa PKA substrate in the crayfish Thus, the observation at the electron microscopic level con- axoplasm is CF-kinesin. firmed the results obtained by light microscopy that the activa- tion of the PKA system selectively inhibited the anterograde Cray$sh axoplasm has a phosphatase activity speci$c to CF- transport of vesicles. kinesin The results obtained with the contrast-enhanced light microscope Kinesin is a possible target of PKA suggested that the axoplasm has a phosphatase activity which The activation of PKA system in crayfish giant axon specifically dephosphorylates the target protein of PKA, and thus the inhi- inhibits the anterograde vesicle transport. Then what is the target bition of the anterograde vesicle transport was recovered. of PKA? To answer this question, we searched a protein band(s) This means that the target protein of PKA must be dephos- that was specifically phosphorylated by the activation of PKA phorylated by an axoplasmic factor. We therefore incubated pathway. The phosphorylated proteins in the axoplasm were an- phosphorylated CF-kinesin with extruded axoplasm. As shown h i -60, j60 . 50 50* 40 4oa 30 3oa 20 204 10 104 0 04 0123456 01 23456 0123456 0123456 Accumulation Accumulation Accumulation Accumulation The Journal of Neuroscience, April 1995, 75(4) 3061

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116-

Figure 6. Phosphorylation of crayfish axoplasmic proteins by the application of PKA under the condition corresponding to that used for the microscopic observation. Extruded axoplasm (lanes 1-3), extruded axoplasm supplemented with purified CF-kinesin (lanes 4-6), and purified CF- kinesin (lanes 7-9) were incubated in the absence of PKA (I, 4, 7), in the presence of PKA (2, 5, 8), and in the presence of PKA and KT5720 (3, 6, 9). The samples were analyzed on SDS-polyacrylamide gel by Coomassie stain (l-9), and by autoradiography (I’-9’). The positions of the molecular weight marker proteins are indicated on the left of lane I. At least 13 phosphoprotein bands were recognized in lanes I’ and 2’, and their positions are indicated on the left of lane 1’. Application of PKA increased the level of phosphorylation of these bands (lane 2’), and this increase was reversed by KT.5720 (lane 3’). Purified CF-kinesin was phosphorylated by PKA (lane 8’) even in the presence of other axoplasmic proteins (lane 5’). The position of the phosphorylated CF-kinesin band was around 116 kDa, corresponding to the phosphoprotein band around 116 kDa in lane 2’. in Figure 8, the amount of phosphatemoiety incorporated into possibility. To confirm that this is also the casewith CF-kinesin, CF-kinesin decreasedin a time-dependentmanner even in the we performed a binding experiment with crayfish axoplasmic presence of okadaic acid or calyculin A, but no significant organellesand CF-kinesin. change was found in the two other contaminating phosphopro- Crayfish axoplasmic organelleswere collected from the ex- teins p70 and tubulin. Thus the axoplasmwas demonstratedto truded axoplasmto avoid the contaminationof axolemma.Mem- have phosphataseactivity which dephosphorylatesphosphory- brane organelles were collected by sequential centrifugation. lated kinesin, one of the possibletargets of PKA. This phospha- Mitochondria were mostly removed by the first centrifugation; tase activity was not inhibited by okadaic acid or calyculin A, most microtubules, the only major filamentous cytoskeleton, correspondingwell with the observation by light microscopy. were depolymerized by the dilution and chilling just above freezing point; and most nondepolymerized microtubuleswere PKA phosphorylation of CF-kinesin reducesits affinity to also removed by the first centrifugation. Thus the pellet of the axonal organelles second centrifugation contained little cytoskeletal filamentous The above results collectively show that the phosphorylation of structure. CF-kinesin by PKA parallel the selective inhibition of antero- After washing with salt to remove already binding kinesin, grade axonal vesicle transport. Then can the phosphorylation of this organellefraction was mixed with purified CF-kinesin, and kinesin causethe inhibition? Several studiesdeny the possibility the amount of free/bound kinesin was measuredby quantitative that phosphorylation of kinesin directly inhibits its motor activ- immunoblotting with anti-kinesin heavy chain H2 antibody ity (Murphy et al., 1989; Matthies et al., 1993). In fact, the (Pfister et al., 1989). As expected, the binding of CF-kinesin to microtubule motility by kinesin was not affected by its phos- the axoplasmic organelles decreasedto 75 + 5% (mean ? phorylation (data not shown). SEM, N = 5) after the phosphorylation of CF-kinesin by PKA The secondpossibility is that the phosphorylation uncouples (Fig. 9). This supportsthe idea suggestedin our previous in vitro kinesin from its cargo. Our previous in vitro study with rodent study that PKA phosphorylation of kinesin inhibits organelle kinesin and organelle(Sato-Yoshitake et al., 1992) supportsthis motility by releasingthe motor from its cargo, though the ob- t Figure 5. Accumulation of transported organelles proximal (u-c) and distal (d-f) to the ligation. In the control group (ATPyS only), at both proximal(a) anddistal (d) portions,many mitochondria (arrow) andvesicles (small arrowhead) accumulated, and accumulated vesicles fused to form large membranous cisternae (large arrowhead). After application of PKA and ATP$S, the accumulation of vesicles decreased at the proximal portion (b), while that at the distal portion (e) and the accumulation of mitochondria had no apparent changes. The decrease of vesicle accumulation at the proximal portion was inhibited by the addition of KT5720 (c, proximal portion; f; distal portion). g-j show the histograms of the degree of accumulation scored as described in Materials and Methods (g, proximal-vesicle; /z, distal-vesicle; i, proximal-mitochondria; j, distal-mitochondria). The application of PKA (X) selectively reduced the accumulation of vesicles at the proximal portion, which caused a significant shift of the peak in g (P < 0.01 by accumulated x2 test). This reduction was reversed to the control level (0) by the addition of KT.5720 (0). No significant change in the profile of these histograms was detected in distal portions (h, j) or in the accumulation of mitochondria (i, j). 3062 Okada et al. * Selective Inhibition of Axonal Transport by PKA a b

Figure 7. Kinesin phosphorylation in viva. a, Metabolically labeled CF-kinesin was partially purified by cosedimentation with microtubules. Lanes I and 2 are Coomassie stains of crude extract and microtubule pellet, respectively. Lanes I ’ and 2’ are the corresponding autoradiogram. Arrowheads show the position of CF-kinesin band. b, Phosphopeptide mapping of PKA phosphorylated CF-kinesin (lanes I&4) and in vivo phosphorylated CF- kinesin (lanes 5-8). Phosphorylated CF-kinesin band was excised from SDS gels and digested with 0 (lanes I and 5), 3 (lanes 2 and 6), 10 (lanes 3 and 7), or 30 ng (lanes 4 and 8) of V8 protease. Peptides were separated on a second 15% SDS gel (Cleveland et al., 1977), and exposed for autoradiography. Nine phosphopeptide bands were identified (arrowheads) in both PKA phosphorylated (lanes I-4) and in vivo phosphorylated (lanes 5-8) preparations, suggesting that the in vivo phosphorylation site(s) is the same as the in vitro PKA phosphorylation site(s). Open arrowhead shows the position of the border between the stacking and the separation gels. The positions of molecular weight markers are indicated on the left of lane I: iO0, 116, 97, 66, 45, 31, 21, and 14 kDa &om the top. served change in the binding affinity was a little bit smaller, First, the phosphataseinhibitors that Bloom et al. (1993) and probably due to the impurity of the organellefraction. we usedare okadaic acid or calyculin A. They inhibit only pro- tein phosphatases1, 2A, and 2B, and are not effective to other Discussion phosphatases.This is why we used ATPyS to inhibit all the Many types of regulatory machinery are postulated in the fast putative phosphatases,and its application indeed augmentedthe axonal transport, but only a few studieshave shedany light on PKA action. However, we could not detect any significant spe- it. Phosphorylation/dephosphorylationis supposedto play a role cific effects by ATPyS alone.This would be due to the permea- in the regulation of fast axonal transportfrom the analogy of the bilization and the doseof ATP+. By permeabilization, soluble regulation of the pigment granule movement in fish scale chro- proteinsincluding kinase were washedout. The doseof ATPyS matophores(Lynch et al., 1986a,b;Rodzial and Haimo, 1986a,b; was arrangedto be as low as possibleto avoid its direct inhib- Sammaket al., 1992), but no positive evidence was reported yet. itory effect on motor protein; it was arrangedto have no appar- Bloom and his colleagues(1993) have reportedthe possiblereg- ent effect in the control experiments. ulatory roles of small GTP-binding proteins in the squid giant Second, Bloom et al. (1993) and we observed that the appli- axon, but they could not detect any effect by the inhibition of cation of PKA inhibitor alone causedno increasein the antero- the kinase/phosphatasesystem. In this paper, we first report that grade vesicle transport. This might be partly due to the very low the activation of PKA pathway selectively inhibits the antero- basalPKA activity in the axon; PKA might be already inhibited grade vesicle transport by using crayfish giant axon as the ex- by the regulatory mechanism.However, we consider that the perimental model. chief reasonlies in the intrinsic limit of video light microscopy. The resolutionof light microscopy is limited. Therefore, when Activation of PKA pathway selectively inhibits anterograde too many organellesare moving in the samedirection, they will vesicle transport create a group of moving overlapped Airy disks. Becauseit is The application of dbcAMP or exogenousPKA causeda tran- difficult to correctly estimatethe number of organellesforming sient but selective’inhibition of the anterogradevesicle transport. each Airy disks, only the number of Airy disks was counted This inhibition was completely reversed by the application of insteadof that of organelles.Thus, the number counted in this KT5720, and was augmentedby ATPyS. Thus it is natural to assay might be an underestimatedvalue, especially when too assumethat this inhibition is causedby the activation of PKA, many organellesare moving. In fact, judging from the apparent but then why did phosphataseinhibitors and PKA inhibitors optical density of the Airy disks in our system, we considerthat alone have no significant effect on organelletransport? We con- this overlapping effect was obvious when the number of moving sider that it is due to the following reasons. vesiclesexceeds 200. This intrinsic limitation of video micros- The Journal of Neuroscience, April 1995, 15(4) 3063

1 2 3 4 5 6 1' 2' 3' 4' 6' 6' 1 2 3 4

Figure 9. The binding of CF-kinesin to axonal organelles was weak- ened by the phosphorylation of CF-kinesin. Purified CF-kinesin and purified axonal organelles were incubated, and free kinesin and bound kinesin were separated by centrifugation. The amount of free (3, 4) and bound (I, 2) kinesin was quantitated by immunoblotting with anti-ki- nesin heavy chain monoclonal antibody H2 (Pfister et al., 1989). Lanes I and 3 show the result using unphosphorylated kinesin, and lanes 2 and 4 show that with phosphorylated kinesin. The amount of kinesin bound to the organelles was reduced to 75 + 10% (mean ? SEM, N = 5, P < 0.01 by paired t test) by phosphorylation.

architecture even at an electron microscopic level. Considering that only anterogradevesicle transport was selectively inhibited, it is natural to assumethat the target of PKA phosphorylation is some proteins specifically involved in anterogradevesicle transport. Thus it is unlikely that cytoskeletal proteins are the PKA target responsiblefor the inhibition. Therefore, the PKA target would be a protein whose phos- phorylation selectively inhibits anterograde vesicle transport. 20 That is, the anterograde vesicle transport can be regulated i through the phosphorylation state of this PKA target protein. 00 This is the first positive result supporting the idea that axonal 0 10 20 30 40 50 transport is regulated through phosphorylation as in other cel- Time /min lular processesincluding pigment granule movement. For further analysis,it is necessaryto identify the PKA target. Figure 8. Phosphatase activity in the crayfish axoplasm. Purified CF- It will be among the proteinsidentified in Figure 6, and we have kinesin was phosphorylated as in lanes 8 and 8’ in Figure 6. After the demonstratedthat one of them is kinesin, which is supposedto addition of excess cold ATP, okadaic acid, calyculin A, and protease be regulated by PKA phosphorylation (Sato-Yoshitake et al., inhibitors, the sample was then incubated in the absence (I, 3, 5) or in the presence (2, 4, 6) of the extruded axoplasm for 10 min (I, Z), 30 1992). Kinesin is a phosphoproteinin vivo whose phosphory- min (3, 4), and 50 min (5, 6). They were analyzed on SDS-polyacryl- lation was augmentedby PKA activation. The site of phosphor- amide gel by Coomassie stain (1-6) and by autoradiogram (I’-6’). The ylation in vivo is the sameor similar to the PKA phosphorylation position of CF-kinesin and two other contamination bands (~70 and site in vitro, and the phosphorylation reducedthe affinity to its tubulin) are indicated. The autoradiogram was quantitatively analyzed and the relative level of phosphorylation was plotted. Circle, square, cargo. These results are consistent with our previous in vitro and triangle symbols indicate kinesin, ~70, and tubulin, respectively. results with rodent kinesin (Sato-Yoshitake et al., 1992), and The closed symbols with dotted line and the open symbols with solid they strongly support our hypothesis on the regulation of the line are in the presence and the absence of axoplasm, respectively. Mean direction of axonal transport that phosphorylation regulatesthe and SEM of three independent experiments are shown. CF-kinesin was selectively and significantly dephosphorylated in the presence of axo- decommissionof kinesin at the axon terminal by releasing it plasm (p < 0.01 at 30 and 50 min by paired t test). from its cargo (Sato-Yoshitakeet al., 1992). Therefore, kinesin is a good candidatefor the PKA target responsiblefor the reg- ulation of axonal transport, although we could not demonstrate copy can limit the detection of the increase or the subtle decrease the direct relation between kinesin phosphorylation and the in- in the anterograde vesicle transport, for the number of moving hibition of anterogradevesicle transport. Furthermore, this does vesicles in unperturbed axons was around this limit. not mean that other putative PKA target proteins are not in- volved in the regulation. Further study will be necessaryfor their Phosphorylation regulatesanterograde vesicle transport characterization and the analysis on the relation between their What causesthe selective inhibition of anterogrademotility by phosphorylation and the inhibition of vesicle transport. PKA? It seemsunlikely that this inhibition is causedby nonphy- Different responseof vesiclesand mitochondria to PKA siological hyperphosphorylation of axonal proteins. PKA acti- The movementof mitochondria is apparently different from that vation can causemetabolic effects, but the selective inhibition of vesicles.Vesicles move very smoothly and rarely changedi- could not be reproduced by altering ATP concentration. PKA rection (Breuer et al., 1973, while the movementof mitochon- can also phosphorylatecytoskeletal proteins, which perturbs ax- dria is very saccadic. They move for relatively short distance onal transport in Aplysia (Forscher et al., 1987) and in mam- and then stop for a while. Their reversal of direction is not a malian neuron (Sato-Yoshitake,unpublished results). In our sys- rare event (Hirokawa and Yorifuji, 1986; Forman et al., 1987). tem, no apparent change was observed in the cytoskeletal It is not unnatural to supposethat the direction of movement is 3064 Okada et al. * Selective Inhibition of Axonal Transport by PKA differently regulated. Only a few studies have focused on this Leterrier J-E Liem RKH, Shelanski ML (1981) Preferential phosphor- ylation of the 150,000 molecular weight component of neurofilaments difference, but our results clearly show that the movement of by a cyclic AMP-dependent, microtubule-associated protein kinase. vesicles and mitochondria is differently regulated. J Cell Biol 90:755-760. One possible and attractive explanation is that this is because Lieberman EM, Villegas J, Villegas GM (1981) The nature of the the motors for anterograde vesicle transport and mitochondria membrane potential of glial cells associated with the medial giant transport are different. Recent molecular and cell biological axon of the crayfish. Neuroscience 6:261-271. Lynch TJ, Taylor JD, Tchen TT (1986a) Regulation of pigment organ- studies have revealed that in neural cells and many other types elle translocation. I. Phosphorylation of the organelle-associated pro- of cells there exist new motor molecules besides kinesin and tein ~57. J Biol Chem 261:4204-4211. dynein, and it is supposed that these motors carry many different Lynch TJ, Taylor JD, Wu B-Y, Tchen TT (1986b) Regulation of pig- cargoes (Stewart et al., 1991; Aizawa et al., 1992; Kondo et al., ment organelle translocation. II. Participation of a CAMP-dependent Reticulomyxu, protein kinase. J Biol Chem 261:42124216. 1994; Sekine et al., 1994). In for example, it is Matthies HJG, Miller RJ, Palfrey HC (1993) Calmodulin binding to reported that the bidirectional movement of mitochondria, even and CAMP-dependent phosphorylation of kinesin light chains mod- its plus-end-directedmovement, is supported by a variant of ulate kinesin ATPase activity. J Biol Chem 268: 11176-l 1187. cytoplasmic dynein (Euteneuer et al., 1988). Furthermore, we Murphy DB, McNiven MA, Wallis KT, Kuznetsov SA, Gelfand VI have recently identified a new motor protein, KIFlB, which is (1989) The phosphorylation of kinesin does not affect its ATPase and translocatinn activities. J Cell Biol 109:80a. a good candidate for mitochondrial transport (Nangaku et al., Nairn AC, Hemmmgs HC Jr, Greengard P (1985) Protein kinases in 1994). The differential regulation of vesicle and mitochondria the brain. Annu Rev Biochem 54:931-976. transportswill be an important questionfor the future challenge. Nangaku M, Sato-Yoshitake R, Okada Y, Noda Y, Takemura R, Ya- mazaki H, Hirokawa N (1994) KIFlB: a new microtubule plus-end References directed monomeric motor for transport of mitochondria. Cell, in press. Aizawa H, Sekine Y, Takemura R, Zhang Z, Nangaku M, Hirokawa N Nestler EJ, Greengard P (1989) Protein phosphorylation and the reg- (1992) Kinesin family in murine central nervous system. J Cell Biol ulation of neuronal function. 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