Lytic Granules from Cytotoxic T Cells Exhibit Kinesin-Dependent Motility on Microtubules in Vitro

Journal of Cell Science 104, 151-162 (1993) 151 Printed in Great Britain © The Company of Biologists Limited 1993

Lytic granules from cytotoxic T cells exhibit kinesin-dependent motility on microtubules in vitro

J. K. Burkhardt1,*, J. M. McIlvain, Jr2, M. P. Sheetz2 and Y. Argon1,2,† Departments of 1Immunology and 2Cell Biology, Duke University Medical Center, Durham, NC 27710, USA

*Present address: Department of Cell Biology, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-6900 Heidelberg, FRG †Author for correspondence at Department of Immunology, Box 3010, Duke University Medical Center, Durham, NC 27710, USA

SUMMARY

One major mechanism of cell-mediated cytolysis is the strongly biased toward microtubule plus-ends. Inacti- polarized secretion of lytic granules, a process which is vation of cytoplasmic dynein had little effect on granule highly dependent on microtubules. We isolated lytic binding and movement, but immuno-depletion of granules from murine cytotoxic T cells and tested their kinesin from the motor preparation inhibited granule ability to bind to and move along microtubules in vitro. binding by 50%. These results indicate that most gran- In the presence of a motor-containing supernatant, the ule movement in this assay is mediated by kinesin. The granules bound to the microtubules and moved along speed and direction of granule movement in vitro are them at an average maximal rate of 1 m/second. Vir- sufficient to account for the release of lytic granules in tually every granule could bind to microtubules, and the intact T cell. This model system should be valuable about half translocated within a few seconds of binding. for studying the interactions of secretory granules with Motility required exogenous cytosolic motors, hydrolyz- microtubules, and for identifying the regulatory factors able nucleotides, and an intact granule membrane. involved. Although the motor preparation used to support granule movement contains both plus- and minus-end- Key words: organelle motility, cytolysis, microtubules, secretory directed motor proteins, granule movement was granules, kinesin

INTRODUCTION 1982; Kupfer et al., 1983). If the microtubules are allowed to repolymerize, cytolytic activity returns (Kupfer et al., A major mechanism by which natural killer (NK) cells and 1983). Thus, the exocytosis of lytic granules by CTL rep- cytotoxic T lymphocytes (CTL) kill their targets is by reg- resents one of the clearest cases of microtubule involve- ulated exocytosis of specialized granules, termed lytic gran- ment in regulated secretion. ules (reviewed by Henkart, 1985). When a cytolytic lym- In lymphocytes, as in most cell types, microtubules are phocyte recognizes a virus-infected cell or a tumor cell as arrayed with their fast-growing (plus) ends at the periphery a suitable target, cross-linking of surface receptors triggers and their slow-growing (minus) ends at the cell center a rapid reorganization of the killer cell cytoskeleton and (Bergen et al., 1980). This provides a radially polarized secretory apparatus. Within five minutes, the microtubule cytoskeletal scaffold along which membranous organelles organizing center (MTOC), the Golgi complex, and the lytic can bind and move. Several proteins which can mediate granules face the bound target (Carpen et al., 1982; Geiger organelle-microtubule interactions have been identified, pri- et al., 1982; Kupfer et al., 1985; Yannelli et al., 1986). In marily as a result of studies that have reconstituted these this way the lymphocyte assumes polarity with respect to interactions in vitro. These proteins include several its target. As soon as this polarity has been established, organelle-microtubule binding proteins (Mithieux and some of the granules move away from the MTOC and fuse Rousset, 1989; Kreis, 1990) and two major motor proteins, with the plasma membrane, releasing their contents in the kinesin and cytoplasmic dynein (Brady, 1985; Vale et al., space between the killer cell and the target (Frey et al., 1985a; Lye et al., 1987; Paschal and Vallee, 1987). Kinesin 1982; Zagury, 1982). The proteins released from the gran- has been shown to support the motility of organelles toward ules then induce perforations in the target cell membrane the microtubule plus-end (Vale et al., 1985b; Porter et al., and degrade the target cell’s DNA (Millard et al., 1984; 1987), while cytoplasmic dynein supports organelle motil- Podack and Konigsberg, 1984; Hayes et al., 1989), thereby ity in the opposite direction (Paschal and Vallee, 1987; killing the target. Agents that depolymerize microtubules, Schroer et al., 1989). including colchicine, nocodazole, vinblastine and vin- Two basic roles have been attributed to the microtubule cristine, inhibit cell-mediated cytotoxicity (Katz et al., cytoskeleton in membrane traffic: positioning stationary 152 J. K. Burkhardt and others organelles in the cytosol and mediating the directional recombinant human interleukin 2 (Dupont, gift from Dr. A. movement of motile organelles (reviewed by Vale, 1987; Hollingsworth, U. Nebraska) and 10% Con A supernatant, pro- Kelly, 1990). In many cell types the Golgi complex, lyso- duced by culturing rat splenocytes for 2 days at 2 ´ 106/ml in the somes and late endosomes cluster near the MTOC, and presence of 5 mg/ml Con A (gift from Dr. D. Howell, Duke Uni- depolymerization of microtubules disrupts their distribution versity). These culture conditions have been shown to support the (Matteoni and Kreis, 1987; Swanson et al., 1987; Ho et al., preferential differentiation of CTL from a mixed population of cells (Erard et al., 1985; Hardt et al., 1985). After 2 days in cul- 1989). It seems likely that minus-end-directed motors are ture, cells were adjusted daily to 3 ´ 105/ml with medium and responsible for establishing the clustering of organelles at growth factors, without additional lectin stimulation. Cells were the MTOC (Matteoni and Kreis, 1987; Ho et al., 1989; cultured for a total of 5-6 days, during which time their numbers Bomsel et al., 1990). Plus-end-directed motors are likely to increased by more than 25-fold and the cells developed granules play a role in organelle positioning as well, since antibod- detectable by fluorescence and electron microscopy. FACS analy- ies to kinesin can disrupt the distribution of tubular lyso- sis revealed that the expanded cultures contained greater than 95% somes along microtubules in the cell periphery (Hollenbeck CD8 positive cells. In parallel with the development of granules, and Swanson, 1990). Microtubules also direct the outward the CTL developed cytolytic activity. Cytolysis was completely movement of organelles. The best studied example of this dependent on calcium, indicating that granule exocytosis was the is fast axonal transport, where secretory vesicles and mito- main lytic mechanism used (Henkart, 1985). Though these cultures survived for 10 days, cells after 5-6 days were judged to be chondria are transported along microtubules to the nerve in optimal condition, and were therefore used for granule prepa- terminal (Sheetz et al., 1989). In non-neuronal cells, micro- rations. tubule-based movement is not required for secretion in gen- Primary cultures of chicken embryo fibroblasts (CEF) were pre- eral, but it is critical for directed transport of secretory vesi- pared from 11-day embryos as described by Kelley and Sch- cles to specific membrane domains (Rindler et al., 1987; lessinger (1978) and maintained in Earle’s minimal essential Achler et al., 1989; Eilers et al., 1989; Kreis et al., 1989). medium, supplemented with 5% fetal calf serum, penicillin and To understand how cells regulate the timing and direc- streptomycin (all from Gibco). After 2 days, primary cultures were tionality of particular organelle movements, we set out to frozen in 30% FCS, 10% DMSO, 60% Iscove’s modified Dul- reconstitute the binding and movement of a purified popu- becco’s modified medium (Gibco) and maintained under liquid lation of organelles on microtubules. The lytic granules of nitrogen. Secondary cultures were prepared from these frozen cells, and used within 2-3 days of plating. CTL are ideally suited to this purpose, for two reasons. First, the granules have a distinct size and structure, which Preparation of lytic granules enable their purification with relative ease (Millard et al., Lytic granules were prepared by a modification of the procedure 1984; Podack and Konigsberg, 1984). Second, CTL are 8 9 likely to have a well developed mechanism for regulating of Millard et al. (1984). A total 2 ´ 10 to 1 ´ 10 CTL were washed with balanced salts solution, adjusted to 2 ´ 108/ml in dis- microtubule-based granule movements, since the position- ruption buffer (0.25 M sucrose, 4 mM EGTA, 10 mM Hepes, pH ing and secretion of the granules is vital for CTL function. 7.4) containing protease inhibitors, and disrupted by 10 passes Therefore, we isolated lytic granules from CTL and exam- through a ball-bearing homogenizer at 4˚C. Nuclei and unbroken ined in vitro their interaction with microtubules. We show cells were removed by centrifugation at 1000 g in a Sorvall that in the presence of exogenous motors, lytic granules RT6000B centrifuge and the resulting pellet was washed with dis- bind to microtubules and translocate along them in vitro. ruption buffer and centrifuged again. Percoll (Pharmacia) was Consistent with their secretory destination, the movement made isotonic by the addition of 1/10 vol. of 2.5.M sucrose and of lytic granules in vitro is toward the plus-end of the micro- 1/100 vol. of 1 M Hepes, pH 7.4, and 20 ml gradients of 48% tubule, and is mediated preferentially by kinesin. (v/v) isotonic Percoll in disruption buffer were prepared. Each gradient was overlaid with lysate from 1 ´ 108 to 3 ´ 108 cells, and gradients were centrifuged at 60,000 g for 30 minutes at 4˚C using a 70Ti rotor (Beckman L3-50 ultracentrifuge). Fractions (0.8 ml) MATERIALS AND METHODS were collected from the bottom of the tubes, and analyzed as described below. For dense granule preparations, fractions 3-7 of Cell culture the gradient (d =1.12 to 1.09 g/ml) were pooled, diluted with one volume of disruption buffer, and concentrated by centrifugation To generate quantities of granular CTL suitable for fractionation, for 45 minutes at 85,000 g at 4˚C using a Beckman Ti70.1 rotor. primary cultures of splenic T cells were polyclonally stimulated The visible granule band was collected, and granules were con- with lectin and lymphokines by an adaptation of the procedure of centrated again by centrifugation for 30 minutes at 100,000 g at Hardt et al. (1985). C57Bl/6 mice were obtained from the National 4˚C using a TLA-45 rotor (Beckman TL-100 ultracentrifuge). Cancer Institute Animal Program. Spleens were removed asepti- cally, and splenocytes teased into RPMI-1640 (Sigma) containing 10% fetal calf serum, non-essential amino acids, penicillin, strep- Analysis of membrane fractions tomycin (all from JRH Biosciences) and b-mercaptoethanol Density of gradient fractions was calculated on the basis of refrac- (Kodak). Precursors of cytotoxic cells were positively selected by tive index. The protein profile of the gradients was determined as panning for CD8 + cells as described by Sprent and Shafer (1985) TCA-precipitable counts from cells metabolically labelled with using monoclonal anti-CD8 antibody 31M (gift from Dr. R. Kur- [35S]methionine. CTL (5 ´ 106) were labelled for 10 hours (greater lander, Duke Univ.). Typically, 5-8% of the starting splenocytes than one generation time) in medium supplemented as usual, were recovered after panning. Cells were seeded for culture at 1 except that it contained 5% dialyzed newborn calf serum, 1/10 the ´ 106/ml in medium supplemented as described above. Con- normal amount of unlabelled methionine, and 75 mCi/ml canavalin A (1 mg/ml; Con A, Aldrich) was added as a polyclonal [35S]methionine (Translabel, ICN). Radiolabelled cells were mitogen. To further stimulate growth and differentiation of cyto- mixed with an excess of unlabelled cells and fractionated as usual. toxic T cells, medium was also supplemented with 20 units/ml Samples were adjusted to 0.5% NP-40, 0.1 M Tris, pH 8.0, pre- Kinesin-based motility of lytic granules 153 cipitated with 10% TCA, and relative protein levels were deter- motile organelles pulled themselves out of the trap. The behavior mined by scintillation counting. The granule marker enzymes BLT of granules within 30 seconds of binding was observed and esterase and perforin/cytolysin, and the mitochondrial marker suc- recorded. cinate dehydrogenase were assayed using the microassays To determine the orientation of granule movement, granules described by Young et al. (1987). Plasma membrane was detected were mixed with kinesin-coated beads and the direction of their by fractionating cells, which had first been surface biotinylated movement on the same microtubule was compared. Kinesin- (Lisanti et al., 1989), and blotting with 125I-streptavidin. Endo- coated beads were prepared by mixing carboxylated beads (Poly- plasmic reticulum was monitored by blotting with rabbit sciences, 0.143 mm diameter) with purified squid kinesin in the anti-ribophorin I (gift from Dr. D. Meyer, UCLA). presence of NaPMEE (PMEE containing 80 mM NaCl) and incubating 5 minutes at 25˚C. The binding was blocked by adding Electron microscopy FCS to 10% and incubating an additional 5 minutes. Unbound Granule fractions prepared as described above were mixed with protein was removed by pelleting beads through a cushion of 5% an equal volume of ice-cold fixative (2% glutaraldehyde, 2% sucrose in NaPMEE. Coated beads were resuspended in PMEE osmium tetroxide, 0.25 M sodium cacodylate, pH 7.4), and fixed containing 150 mg/ml casein as a stabilizing agent. The usefulness in suspension for 30 minutes at 4˚C, as described by Millard et of these beads as standards for directionality relies on knowing al. (1984). Membranes were then pelleted in an Eppendorf micro- that their movements are due to kinesin, even in the presence of centrifuge, washed with cacodylate buffer, and embedded in agar. the cytosolic motor preparation. We therefore prepared control Membranes were stained en bloc with uranyl acetate, dehydrated, beads, coated in parallel with heat-inactivated kinesin. These con- and embedded in EMBED-812. Silver sections were obtained trol beads exhibited very low binding and movement along micro- using a Reichert Ultracut E ultramicrotome and observed with a tubules, even in the presence of the mixed motor extract. Philips EM-300 electron microscope operating at 80 kV. As an alternative assay for directed granule movement, sea urchin axonemes were prepared according to Bell et al. (1982), Preparation of cytosol from chicken embyro and used as seeds for microtubule polymerization. A mixture of fibroblasts NEM-conjugated tubulin and unconjugated tubulin was used to favor growth from the plus-end of the axoneme (Vale and The cytosolic motor fraction was prepared as previously described Toyoshima, 1988). After the addition of taxol to stabilize micro- 8 (Schroer et al., 1989). Briefly, approximately 4 ´ 10 secondary tubules, these axonemes were used in the standard motility assay passage CEF were collected by trypsinization, and washed with in place of randomly oriented microtubules. 35 mM PIPES, pH 7.4, 5 mM MgSO4, 5 mM EGTA, 0.5 mM EDTA, 1 mM DTT, and protease inhibitors (PMEE). The pelleted Protease, detergent and antibody treatment of cells were resuspended in an equal volume of ice-cold PMEE, and granule surfaces passed 6 times through a ball-bearing homogenizer. Nuclei and unbroken debris were pelleted at 1000 g, and the supernatant (S1), For protease treatment, purified granules were incubated for 1 hour was centrifuged for 30 minutes at 100,000 g using a TL55 rotor at 4˚C with 20 mg/ml trypsin (type XIII, Sigma). Prior to assay- in a Beckman TL100 ultracentrifuge (all at 4˚C). 1 mM GTP ing motility, trypsin was inactivated for 10 minutes with an excess (Sigma) and 20 mM taxol (gift from Dr. Nancita Lomax, National (300 mg/ml) Trasylol (FBA Pharmaceuticals). This quantity of Cancer Institute) were added to the resulting supernatant (S2), and Trasylol was sufficient to inhibit 20 mg/ml trypsin completely in the mixture was incubated for 15 minutes at 37˚C to polymerize a colorimetric enzyme assay (Young et al., 1987). For control endogenous microtubules. Assembled microtubules were removed digests, trypsin and Trasylol were mixed for 10 minutes, and the by centrifugation at 100,000 g for 5 minutes in a Beckman airfuge. mixture was then incubated with granules for 1 h at 4˚C. Deter- The resulting supernatant (S3) was maintained at 4˚C and used as gent treatment was performed by mixing granules with 0.02% the source of cytosolic motors. (v/v) Triton X-100, and incubating on ice for 10 minutes prior to assaying motility. For antibody blocking studies, purified granules Organelle motility assays were incubated for 1 hour at 4˚C with a polyspecific rabbit anti- granule antiserum (Reynolds et al., 1987; gift from Dr. P. Henkart, Bovine brain tubulin was purified by phosphocellulose chro- National Institutes of Health, Bethesda, MD) diluted either 1:100 matography (Williams and Lee, 1982), and stored at - 70˚C until or 1:30 in PMEE. Goat anti-rabbit Ig antiserum was used in par- use. Microtubules were polymerized by incubating tubulin at 2-4 allel as a negative control. mg/ml in PMEE containing 20 mM taxol and 1 mM GTP at 37˚C for 15 minutes. Assays were prepared by spotting 1 ml of diluted UV photocleavage microtubules, 3 ml of cytosolic motors, 1.2 ml 10 mM ATP, and Samples of organelles and S3 cytosol were incubated for 15 min- 1 ml of organelles directly onto a glass coverslip. Movement was utes in the presence of 2 mM ATP and 100 mM sodium vanadate, visualized using video-enhanced DIC microscopy (Allen et al., and exposed to long wave ultraviolet light for 50 minutes as 1981; Kuo et al., 1991). Since the activity of different cytosolic described by Schroer et al. (1989). motor preparations was somewhat variable, all comparisons were made from samples analysed in parallel. When indicated, substi- Immunodepletion of kinesin tutions were made for the cytosolic motor preparation. These included purified kinesin isolated from either chick brain (Schroer SUK4 (anti-kinesin) Sepharose (Ingold et al., 1988) or Protein et al., 1988) or squid optic lobe (Vale et al., 1985a), cytoplasmic A/Sepharose (Sigma) was pre-equilibrated with PMEE, and 300 dynein isolated from chick brain (Schroer et al., 1989), and 0.5 ml of S3 was added to 50 ml of each packed resin. Slurries were mg/ml casein (Sigma). Motility rates were determined by identi- incubated for 1 hour at 4˚C, and the supernatant was removed and fying short time intervals during which organelles travelled in a subjected to a second round of depletion with fresh affinity resin. straight line, and dividing the distance travelled by the time inter- Further depletion removed no more kinesin. Resin was washed val. three times with PMEE containing 0.5 mg/ml casein and bound For some experiments, organelles were trapped and presented material was eluted with 0.2 M glycine-HCl, pH 2.5. to microtubules using the laser trap described by Kuo et al. (1991). Laser power was adjusted to minimal levels, such that tightly Gel electrophoresis and Western blotting bound organelles could not be removed from microtubules, and 7.5% SDS-polyacrylamide gels were run using the Biorad mini- 154 J. K. Burkhardt and others gel apparatus and the buffers of Laemmli (1970). Western blot- around d = 1.105 ± 0.02 g/ml, and a light peak around d = ting was performed as described previously (Dabora and Sheetz, 1.075 ± 0.02 g/ ml (Fig. 1A). In addition to granzyme A, 1988), using alkaline phosphatase-conjugated secondary reagents. the dense peak was enriched for perforin/cytolysin, as measured by hemolytic activity. A pool of fractions 3-7 of RESULTS this peak, containing 5 ´ 106 cell equivalents, was sufficient to completely lyse 1 ´ 107 red blood cells in 1 hour Purification of lytic granules at 37˚C. The light peak of BLT-esterase activity co- As a source of lytic granules, we used murine CTL grown migrated with a major peak of total proteins (Fig. 1B). In in large-scale cultures. CTL homogenates were fractionated addition to granule proteins, this peak contained the mito- by Percoll density centrifugation, as described by Millard chondrial protein succinate dehydrogenase (Fig. 1A), as et al. (1984). A representative fractionation is shown in Fig. well as ER and plasma membrane markers (detected 1. The gradient fractions were monitored for BLT esterase, immunologically, data not shown). None of these markers which is the enzymatic activity of the major lytic granule was detectable in the dense granule fractions. protease, granzyme A (Pasternack and Eisen, 1985). As The purity of granules in the dense peak is illustrated in described previously (Millard et al., 1984), this procedure the electron micrograph shown in Fig. 2. The pooled mate- yielded two peaks of lytic granules, a dense peak centered rial contained almost exclusively granules with electron- dense cores. The size of the granules was relatively uni- form (mean diameter: 0.45 ± 0.1 mm). Occasional granules exhibited multivesicular regions surrounding the dense core (Fig. 2, asterisk), although these are difficult to distinguish with the fixation procedure used. The granules were surrounded by a membrane bilayer, or by several membrane lamellae (Fig. 2, arrow), which in most granules appeared to be intact. The size and morphology of the dense core granules were very similar to the type I granules as observed in CTL and NK cells (e.g. see Fig. 1 of Burkhardt et al., 1990). No mitochondria, Golgi elements, endoplas- mic reticulum or other contaminating organelles were observed in these fractions. Thus, the dense core granules isolated by this procedure are highly purified and morpho- logically intact. On the basis of the specific activity of BLT esterase, we estimate that the dense granules in fractions 3- 7 were enriched by more than 200-fold. These pooled fractions were therefore used as the source of lytic granules for the following in vitro motility studies.

Fig. 1. Fractionation of CTL by Percoll density centrifugation. Post-nuclear supernatants from 3 ´ 108 CTL were layered onto gradients of Percoll and centrifuged as described in Materials and methods. 0.8 ml fractions were collected from the bottom of the gradient. (A) BLT esterase (᭹) was measured as a marker for lytic granules, and succinate dehydrogenase activity (᭿) was Fig. 2. Electron micrograph of the organelles present in the pooled determined as a mitochondrial marker. (B) Density of gradient dense granule fractions. The granules contain an electron-dense fractions (᭿) was determined from their refractive indices, and core, often surrounded by several membrane lamellae (arrow). protein content (᭹) was determined by metabolically labeling Occasional granules contain a multivesicular cortical region (*). cells with [35S]methionine to steady state and measuring the TCA Note the absence of contaminating organelles. Arrowheads, precipitable radioactivity in each fraction. Percoll particles. Kinesin-based motility of lytic granules 155

Fig. 3. Video-enhanced contrast DIC microscopy of a motility assay containing microtubules polymerized from bovine brain, CEF cytosol, ATP and lytic granules. Photos were taken from the monitor every 4 seconds. The granule marked with an arrow moves several mm along a microtubule during the sequence. Note that the microtubule to which this granule is bound is also gliding along the coverslip, so that actual granule motility must be measured relative to the microtubule end (*). The second granule is relatively stationary in an area where several microtubules intersect; it begins to translocate along a microtubule at the end of the series (compare E and F).

Lytic granules move on microtubules in vitro mm/second (at 25˚C). Much of the variation in this average To determine whether lytic granules can bind to and move rate is due to retardation of granule movement by obstacles along microtubules in vitro, granules were added to a motil- in the web of microtubules. Because of this, a more useful ity assay containing microtubules polymerized from bovine parameter is the maximal rate of granule movement, which brain tubulin, ATP, and a cytosolic extract from CEF cells. was 1.0 ± 0.2 mm/second. Using either value, the rate of This extract contains both kinesin and cytoplasmic dynein, lytic granule translocation is in good agreement with rates and has been shown to support the motility of mixed mem- reported for kinesin- and cytoplasmic dynein-driven motil- branous organelles (Dabora and Sheetz, 1988; Schroer et ity of organelles and motor-coated beads (Vale et al., 1985a; al., 1989). In the presence of the cytosolic extract, the dense Schroer et al., 1989). core granules bound to the microtubules and translocated along them. Fig. 3 shows a series of video images from one Motility requires cytosol, hypotonic buffer and such assay. During this sequence, one granule (arrow) hydrolyzable nucleotides moves several micrometers along a microtubule. The The binding of granules to microtubules was absolutely second granule in the ﬁeld has encountered an area where dependent on the addition of exogenous cytosolic extract; several microtubules intersect; it begins to move down a if BSA was substituted for cytosol, the granules remained microtubule between Fig. 3E and F. Granules frequently free or became stuck to the glass coverslip. Substitution of switched from one microtubule to another, but they did not casein for cytosol prevented the granules from binding non- reverse direction. From sequences like the one shown in speciﬁcally to the coverslip, as previously reported for squid Fig. 3, the average rate of granule translocation was calcu- axoplasmic organelles (Schnapp et al., 1991). However, lated to be 0.7 ± 0.3 mm/second, with a range of 0.3 to 1.2 binding of granules to microtubules was not observed in 156 J. K. Burkhardt and others

Table 1. Laser trap quantitation of granule binding Condition N % Bound % Moving Untreated 50 90 36 Trypsin 25 28 0 Trypsin + inhibitor 25 72 24

A standard motility assay containing granules, cytosol, microtubules and ATP was prepared as described in Materials and methods. Granules were randomly selected from above the plane of the coverslip using a single-beam gradient laser trap, and brought into proximity with the web of microtubules bound to the coverslip. The behavior of granules as they touched the microtubules was scored. Bound means granules that became ﬁrmly bound to microtubules within 3-5 attempts. Moving means granules that translocated along microtubules within 30 seconds of binding. N, number of granules.

and McIntosh, 1989). The slowed rate of granule movement is expected, since kinesin hydrolyses GTP more slowly that ATP (Kuznetsov and Gelfand, 1986). Fig. 4. Nucleotide dependence of motility. Granule motility was measured using the standard assay, except that GTP or AMP-PNP Virtually every granule can interact with was substituted for the standard ATP. Each nucleotide was used at microtubules a final concentration of 2 mM in the assay. AMP-PNP + ATP, Our standard motility assay necessarily samples only a por- assay set up initially with AMP-PNP and perfused with an excess tion of the lytic granules, i.e. that population near enough of 2 mM ATP. Motility was measured a few seconds after perfusion. For each condition, the number of granules bound to to the coverslip to bind to the attached microtubules. Since microtubules and the number moving along microtubules per 300 at any given time the majority of granules in the chamber mm2 field were counted. In each case 10-20 fields were observed, are not bound to microtubules, we wished to determine what in two or more independent samples. proportion of granules was capable of interacting with microtubules in vitro. Using a sample prepared as for the standard assay, unbound granules diffusing above the plane the presence of casein unless cytosol was also added (data of the coverslip were optically trapped with a single-beam not shown). gradient laser trap (Ashkin, 1992), and brought into appo- As in other organelle motility systems (Vale et al., 1985a; sition with microtubules. As shown in Table 1, 90% of the Dabora and Sheetz, 1988), granule motility was favored by randomly sampled granules bound to microtubules when hypotonic buffer conditions, and was inhibited by the given an opportunity. Most of the organelles bound tightly, addition of KCl. A concentration of 5-10 mM KCl in the as determined by our inability to pull them away from the assay did not inhibit granule binding, but inhibited granule microtubule using the laser trap. Of those granules that movement by over 70%. Addition of 50 mM KCl to the bound, 40% translocated along the microtubule within a assay diminished binding to 20% of control levels, and few seconds. This level of motility is very comparable to completely abolished movement. Since the motor-depen- that observed in our standard assay (e.g. see Fig. 4, ATP). dent binding of microtubules to the coverslip was also These results demonstrate conclusively that the vast major- inhibited by KCl, it is likely that KCl acts directly on the ity of lytic granules isolated from CTL can interact directly motor proteins, as opposed to the granule membranes. with microtubules. ATP was not required for granule binding, but was required for motility. This effect was more pronounced if Integrity of the granule membrane is required for a non-hydrolyzable ATP analog, AMP-PNP, was substi- microtubule binding tuted for ATP in the assay (Fig. 4). In the presence of AMP- To determine whether an intact granule membrane is PNP, granule binding was permitted or even enhanced, but important for motility, granules were incubated with Triton motility was abolished. Thus, ATP hydrolysis, and not X-100 for 10 minutes at 4˚C, and their movement on micro- simply ATP binding, is necessary for granule movement. tubules was assessed using the laser trap assay. Incubation The effects of AMP-PNP were reversible; perfusion of ATP of the granules with as little as 0.02% Triton X-100 was into an AMP-PNP-arrested assay restored motility after a sufficient to completely abolish granule motility (data not lag of several seconds. This result is consistent with motor- shown). Limited proteolysis of the granule preparation was mediated movement in other systems (Lasek and Brady, used to test whether the interaction with microtubules 1985; Vale et al., 1985c). GTP could substitute for ATP in requires the participation of granule membrane proteins. supporting granule motility. The numbers of granules that The granules were incubated with trypsin as described in bound to microtubules and moved in the presence of GTP Materials and methods, and the enzyme was inactivated were very similar to those with ATP (Fig. 4), but the rate prior to setting up the assay. Trypsin treatment greatly of granule movement was consistently slower with GTP diminished granule binding, and the few granules that did (data not shown). This finding is consistent with kinesin- bind, did not move (Table 1). This inhibition was due to mediated granule motility, since the ability to utilize GTP proteolysis of the granule membranes, since control gran- for force production is a characteristic of kinesin (Warner ules that were incubated with previously inactivated trypsin Kinesin-based motility of lytic granules 157

Fig. 5. Directional motility of granules on microtubules polymerized from sea urchin sperm flagellar axonemes. Microtubules were grown from sea urchin flagellar axonemes (Axo) under conditions that favor growth from the plus-end (Vale and Toyoshima, 1988). In A, a granule (arrow) was selected from above the plane of focus using a laser trap, and placed onto a microtubule extending from the axoneme. Immediately after binding to the microtubule, the granule broke free of the trap and proceeded toward the microtubule plus-end (B-D). The time in hours:minutes:seconds is indicated in each frame. bound and moved at near-normal frequencies. Similar assigned, on the basis of the movement of the beads, and results were obtained if granules were treated with chy- the direction of granule movements on the microtubules motrypsin or proteinase K (data not shown). was then scored. To ensure that bead movement was As an independent method of disrupting interactions of kinesin-mediated, the beads were coated under conditions granule membrane proteins with microtubule motors, gran- that minimize further binding of cytosolic motors, as ules were preincubated with a polyspecific rabbit anti-gran- described in Materials and methods. We estimate that no ule antiserum (Reynolds et al., 1987). At a dilution of 1:100, more than 5% of beads moved toward the microtubule this antiserum reduced granule binding in the laser trap minus-ends. Wherever possible, the polarity of a given assay to 25% of control levels. Preincubation of granules microtubule was assigned, based upon the movement of with a 1:30 dilution of the antiserum completely abolished multiple beads. Of the 29 granules scored, 25 (86%) moved granule binding. In contrast, a control antiserum at the same in the same direction as the kinesin-coated beads (toward dilution showed minimal inhibition. Taken together, these the microtubule plus-end). Of the four granules that results show that the interaction of granules with micro- appeared to move in the opposite direction, two were scored tubules and microtubule motors requires an intact and on the basis of a single bead movement. Thus, 86% is a accessible granule membrane. lower estimate for plus-end-directed granule movement in this assay. This result using randomly oriented microtubules Most granule movements are plus-end-directed agrees well with the data obtained using the axoneme assay. In order to determine the direction of granule movement, It is important to note that the cytosolic extract used in these we analyzed the movement of granules under conditions assays contains both plus-end- and minus-end-directed where the polarity of the microtubules was known. Micro- motors (see Figs 6A and 7A) and that it supports bidirec- tubules were grown from axonemal seeds under conditions tional movement of mixed organelles (Schroer et al., 1989, where growth at the plus-end is favored (Vale and and data not shown). Therefore, the granules must prefer- Toyoshima, 1988), and used in an assay containing stan- entially utilize a plus-end-directed motor selected from the dard proportions of granules, ATP and cytosolic motors. mixture. Since the axonemes were sparse, granules were applied to the microtubules using the laser trap. In each of 4 cases Granule motility requires kinesin, but not where granules bound to the axonemal microtubules, the cytoplasmic dynein granule moved toward the free (plus) end of the micro- On the basis of the directionality data, it seems likely that tubule (Fig. 5). cytoplasmic dynein, the major minus-end-directed motor in For reasons which we do not yet understand, granule the CEF cytosol, contributes little to granule motility in binding to the axonemal microtubules was always poor, vitro. To assess the contribution of cytoplasmic dynein, the making this assay unsuitable for quantitative analysis. We cytosolic extract and the granules were incubated with 100 therefore adapted our standard motility assay to allow quan- mM sodium vanadate and irradiated with ultraviolet light, titation of granule direction on randomly oriented micro- a procedure which covalently cleaves dyneins at their tubules. The standard motility assay was performed, except nucleotide binding sites (Schroer et al., 1989). To ensure that carboxylated beads coated with purified kinesin were that the UV-vanadate treatment successfully cleaved the included as an internal standard for plus-end-directed cytoplasmic dynein, the cytosol used in these experiments movement. The polarity of individual microtubules was was immunoblotted with an antibody that recognizes dynein 158 J. K. Burkhardt and others

Fig. 6. Effects of UV-vanadate inactivation of cytoplasmic dynein on motility. Granules or S3 cytosolic extract were incubated with 100 mM sodium vanadate in the presence of ATP and ultraviolet light. (A) Portions of the untreated cytosol (S3) and the treated Fig. 7. Effects of immunodepletion of kinesin on motility. (A) cytosol (S3 + UV-VO4) were analyzed by gel electrophoresis and Immunodepletion of kinesin was performed by two successive immuno-blotting with an antibody to the heavy chain of rounds of incubation with SUK4 anti-kinesin coupled to cytoplasmic dynein. The covalent cleavage of dynein is indicated Sepharose. As a control, S3 was incubated with Protein 3 by the shift in mobility from > 400 ´ 10 Mr (Dyn) to the 230 ´ A/Sepharose in parallel. Each sample was analyzed by gel 3 10 Mr heavy UV fragment (HUV). (B) Cytosol and/or granules electrophoresis and immunoblotting with SUK4 anti-kinesin were treated with UV-VO4 and used in a standard motility assay antibody. An independent antibody was also used to evaluate the containing microtubules and ATP. Untreated granules and blots, with identical results (not shown). S3, cytosol prior to untreated cytosol (untreated); untreated granules and UV-VO4- depletion; post PA, cytosol mock-depleted with Protein treated cytosol (S3 + UV-VO4); or UV-VO4-treated granules and A/Sepharose; post SUK4, cytosol depleted with SUK4 Sepharose; untreated cytosol (Granules + UV-VO4). SUK4 eluate, material that bound to the SUK4 Sepharose; Kin, 3 110 ´ 10 Mr kinesin heavy chain. (B) Results of assay using each of the treated cytosol preparations to support motility. Post SUK4 + kinesin, cytosol depleted with SUK4 Sepharose, supplemented heavy chain. As expected, the uncleaved dynein heavy with purified chick brain kinesin. 3 chain migrated as a polypeptide of Mr > 400 ´ 10 (Fig. 6A, S3). After UV-treatment, it shifted to a mobility of 230 3 ´ 10 Mr (Fig. 6A, S3 + UV-VO4), the so-called heavy UV- 1989). To ask whether granule motility requires kinesin, we fragment (the antibody employed does not detect the 200 used an anti-kinesin antibody coupled to Sepharose to 3 ´ 10 Mr light UV-fragment). The ability of treated gran- immunodeplete kinesin from the cytosol. As a control for ules to bind and move in the presence of treated motors the effects of the immunodepletion procedure itself, Protein was then assessed using the random microtubule assay. As A/Sepharose was used in parallel. As shown in Fig. 7A, shown in Fig. 6B, granule binding and motility proceeded kinesin was specifically removed by the SUK4 anti-kinesin normally using the cleaved motor preparation. UV-vana- Sepharose, but not by the Protein A/Sepharose. Fig. 7B date treatment of granules also had no effect. Since dynein shows the effects of kinesin depletion on granule motility. cleaved in this way is inactive as a motor, we conclude that In the presence of the kinesin-depleted motor preparation, lytic granule motility in vitro is largely independent of func- granule binding was reduced by 50%, and the number of tional cytoplasmic dynein. motile organelles was proportionately decreased. The effect The major plus-end-directed motor in the CEF cytosolic was specific to the anti-kinesin affinity resin, since the extract is kinesin (Dabora and Sheetz, 1988; Schroer et al., cytosol incubated with Protein A/Sepharose supported Kinesin-based motility of lytic granules 159 normal levels of granule binding. A small but significant (Smith, 1980; Tsukita and Ishikawa, 1980). However, to number of granules continued to bind and move along our knowledge this is the first study to show that a homo- microtubules in the presence of kinesin-depleted cytosol. geneous population of organelles exhibits directional speci- These granules could represent a subset of organelles that ficity in vitro. We now show that purified lytic granules utilize a second motor protein. More likely, this result is move preferentially toward the microtubule plus-ends, and due to small amounts of residual kinesin, either on the gran- that they do so by selectively utilizing kinesin. ule membranes or in the motor preparation. Traces of Several pieces of evidence indicate that granule motility kinesin are still detectable in the cytosol (Fig. 7A), even in this assay is driven almost exclusively by kinesin. First, after three rounds of immunodepletion. the vast majority of granule movements are toward the To determine whether purified motor proteins are suffi- microtubule plus-end, as expected for kinesin-mediated cient to support granule movement, kinesin and cytoplas- motility (Vale et al., 1985b; Porter et al., 1987). Second, mic dynein isolated from embryonic chick brain and kinesin GTP can substitute for ATP in supporting granule motility, isolated from squid optic lobe were used in place of cytosol and kinesin, unlike the dynein family of motors, can utilize in the standard motility assay. In some cases, casein was GTP for force production (Warner and McIntosh, 1989). added as a stabilizing carrier protein. Although each of the Third, motility is not affected by specific inactivation of motor preparations was active as judged by its ability to dynein-like motors (Gibbons et al., 1987; Lye et al., 1987). support microtubule gliding, none could support granule- Finally, immuno-depletion of kinesin from the cytosolic microtubule interactions (data not shown). This result was motor preparation substantially inhibits granule motility. expected for cytoplasmic dynein, based on the results of Urrutia et al. (1991) recently showed that in the presence UV-inactivation. In the case of kinesin, this result indicates of purified kinesin, disrupted chromaffin granule mem- that other factors are required, in addition to kinesin, to sup- branes can move on microtubules in vitro. Our work port granule movement. As shown in Fig. 7B, purified chick extends these findings by showing that lytic granules also brain kinesin was also unable to support motility if it was utilize kinesin, and that the granules selectively utilize added back to the kinesin-depleted cytosol. This result kinesin when presented with a mixture of microtubule suggests that the missing co-factor is a kinesin binding pro- motors. The fibroblast cytosolic extract used in the motil- tein that is removed from the cytosol along with kinesin. ity assay contains a mixture of two oppositely directed Taken together with the directionality data, our results motor proteins, kinesin and cytoplasmic dynein. Both with motor inactivation and depletion indicate that granule motors are active, as indicated by the ability of the extract motility in vitro depends on kinesin and another, as yet to support bidirectional movement of mixed organelle unidentified, activating factor. preparations. Therefore, the predominantly plus-end- directed movement we observed cannot be explained by the properties of the motor preparation alone. Indeed, if the DISCUSSION extract has a tendency to predispose directional movement, it is toward the minus-end, since Schroer et al. (1989). We have reconstituted in vitro the microtubule-mediated observed 90% minus-end-directed movement of mixed movement of lytic granules purified from CTL. The behav- organelles using the same preparation. Thus, some property ior of the granules in vitro is remarkable homogeneous, and of the lytic granules themselves dictates their preferential is consistent with their behavior in vivo. In the in vitro use of kinesin in this assay. The granule membrane assay, nearly every granule could interact with microtubules undoubtedly contributes to this specificity, since binding and virtually all movement was toward the plus-ends of and motility are abolished by treatment of the granules with microtubules. This directional preference is in keeping with protease, detergent, or a polyspecific anti-granule anti- the secretory nature of lytic granules, since in intact T cells, serum. microtubules are oriented with their minus-ends at the MTOC and their plus-ends at the cell surface (Bergen et Association of kinesin with granule membranes al., 1980). Granule movement in vitro required ATP, as As motility systems using well-characterized organelles does the lytic cycle in intact CTL (Roder et al., 1980). become available, it will be important to understand how Moreover, the speed of granule movement in vitro (about motor proteins are partitioned between membrane-bound 1 mm/second) is sufficient to account for the release of gran- and soluble pools, and between active and inactive forms. ules within a few seconds, well within the observed range Our findings indicate that the lytic granules prepared by for delivery of the “lethal hit” in cytolysis. Thus, the prop- Percoll density centrifugation do not bear sufficient levels erties of granule movement in vitro agree well with the of active motor proteins to mediate their motility in the function of lytic granules in vivo. absence of exogenous cytosol. During the isolation procedure, the granules were not exposed to high salt or to other Directional specificity by selective motor use conditions that would release peripherally associated pro- It has long been assumed that organelles of different types teins such as kinesin. We therefore think it likely that the move with directional specificity on microtubules; i.e. granules are not decorated with active motors in the rest- secretory granules move towards microtubule plus-ends ing T cell. In support of this conclusion, immunofluores- while lysosomes move toward microtubule minus-ends. cence microscopy of T cells stained with anti-kinesin anti- Directional preference has been demonstrated on a popula- bodies does not reveal a granular distribution (J. Burkhardt tion level in axons, where secretory vesicles and endocytic and S. Hester, unpublished results). As with chromaffin organelles accumulate on opposite sides of a ligation granules (Urrutia et al., 1991) and mixed organelle prepa- 160 J. K. Burkhardt and others rations from CEF cells (Dabora and Sheetz, 1988; Schroer tion (Kupfer et al., 1985; Yannelli et al., 1986; Lye et al., et al., 1989), it appears that the major pool of active motor 1987). Presumably, granule-microtubule interactions must proteins is soluble, with a relatively minor fraction bound change from a state where quiescent granules cluster at the to organelle membranes. Perhaps kinesin becomes bound minus-ends of microtubules to one where they rapidly move to the granule membrane only after the cell is stimulated toward the microtubule plus-ends. This dramatic shift is to secrete. This hypothesis can be readily addressed using likely to be regulated by signal transduction events that the CTL system, since these cells can be stimulated to occur after engagement of the T cell receptor, including an degranulate in a variety of ways. activation of protein kinases and a transient rise in cytosolic free calcium (Weiss et al., 1986; Hsi et al., 1989). As Evidence for kinesin-regulatory factors discussed above, phosphorylation of kinesin or its acces- Our results indicate that kinesin is necessary, but not suf- sory factors may be important for activating movement of ficient, for lytic granule motility. When added in place of granules toward the cell surface. Proteins responsible for the cytosolic extract, purified kinesin did not support gran- clustering of granules at the MTOC may also be targets for ule movement. Purified kinesin also failed to reconstitute regulation, since at least one microtubule binding protein granule motility when added back to the kinesin-depleted is released from microtubules by phosphorylation (Rickard cytosol. Similar results were obtained by Schroer et al. and Kreis, 1991). The increase in cytosolic calcium levels (1988) using a mixed organelle preparation. There are two could also stimulate granule motility. Haverstick et al. possible interpretations of these findings. First, granule (1991) have shown that treatment of CTL with calcium motility may require an additional activating factor which ionophore induces cytoplasmic redistribution of granules, is removed from the cytosol during immunodepletion with and our own preliminary data indicate that granule motil- anti-kinesin, and which is not supplied with highly purified ity is stimulated by small increases in calcium concentra- kinesin. This would be analogous to the requirement of tion. Thus, taken together, the effects of increased kinase purified cytoplasmic dynein for the activating factor dyn- activity and elevated free calcium are excellent candidates actin (Gill et al., 1991; Schroer and Sheetz, 1991). Indeed, for regulators of granule motility in vivo. one study indicates that a single activating factor may stim- It is important to point out that the behavior of granules ulate both kinesin and cytoplasmic dynein, and that this in the presence of CEF cytosol resembles that expected for factor partially co-purifies with kinesin (Schroer and Sheetz, granules in CTL that have been stimulated to secrete. Since 1991). An alternate possibility is that post-translational the CTL were not stimulated prior to lysis, we assume that modification of kinesin regulates its interaction with this effect is due to the use of CEF cytosol, which must organelles, and the purification procedure that we employ somehow mimic the cytosol of stimulated CTL. For does not preserve the active kinesin species. Phosphoryla- unknown reasons, CEF cells are an exceptionally rich tion is a likely candidate, since it has been recently shown source of active motors, superior to other cell types, includ- that kinesin heavy and light chains are phosphorylated in ing other fibroblasts and lymphocytes. Nonetheless, we vivo (Farshori and Goode, 1991; Hollenbeck, 1991). These have recently found that cytosol from CTL and T cell two possibilities are not mutually exclusive, and studies are hybridomas can also support granule motility. As with the underway to test their relative importance for CEF cytosol, granule movement is constitutive, suggesting granule-microtubule interactions. that regulatory factors that inhibit granule motility (or that direct granules towards the centriolar region) are missing Regulation of granule-microtubule interactions in from the reconstituted system. vivo While the CEF extract supports high levels of motility In the intact T cell, lytic granules probably interact with of all organelle preparations tested, not all organelles microtubules in multiple ways depending on the physio- behave like the dense granules in this assay. For example, logical state of the cell. Prior to 1982; Geiger et al., 1982; when the fractions of the Percoll gradient which contain Kupfer et al., 1985; McKinnon et al., 1988). Similar clus- light granules and other organelles are assayed in the pres- tering of late endocytic organelles has been attributed to the ence of the CEF cytosol, the organelles move in a bidirec- action of microtubule binding proteins (Mithieux and Rous- tional fashion, and ER-like networks form with time. It set, 1989; Kreis, 1990) and minus-end-directed microtubule therefore seems that the motile behavior of the dense gran- motors (Matteoni and Kreis, 1987; Bomsel et al., 1990). ules is produced by specific interaction of cytosolic factors The granules closely resemble these endocytic organelles (determining the secretory state of activity) and granule in function and composition (Burkhardt et al., 1990), and membrane components (determining the direction of move- may share common protein machinery for maintaining their ment). resting distribution. Since all these organelles are acidic The regulation of granule-microtubule interactions is (Mellman et al., 1986; Burkhardt et al., 1990), one attrac- likely to be a complex process involving a variety of cel- tive possibility is that interaction with minus-end-directed lular factors. The relative ease with which lytic granules motors is linked to acidification. Consistent with this idea, can be isolated and the wealth of information about signal treatment of intact CTLs with the protonophore CCCP neu- transduction in lymphocytes makes this system well suited tralizes granule pH and disrupts granule reorientation for determining how granule motility is regulated in vivo. during cytolysis (McKinnon et al., 1988). When a T cell is stimulated by binding to a target cell, We thank P. Henkart and D. Meyer for gifts of antibodies, R. the MTOC and the clustered granules reorient to face the Kurlander and D. Howell for help with culturing CTL, and D. target, and the granules move to the cell surface for secre- McClay for providing sea urchin sperm. Purified kinesin and cyto- Kinesin-based motility of lytic granules 161 plasmic dynein were generously provided by S. Kuo, S. Hamm- intracellular signals for cytotoxic T lymphocyte-mediated Alvarez and C. Martenson. S. Hester and R. Phang provided valu- killing:Independent roles for protein kinase C, Ca2+ influx, and Ca2+ able assistance with electron microscopy and video analysis. release from internal stores. J. Immunol. 146, 3306-3313. Finally, we thank D.B. Amos and members of the Sheetz and Hayes, M. P., Berrebi, G. A. and Henkart, P. A. (1989). Induction of Argon laboratories for helpful advice and criticism. This work was target cell DNA release by the cytotoxic T lymphocyte granule protease supported in part by grants from the American Cancer Society to granzyme A. J. Exp. Med.170, 933-946. Henkart, P. A. (1985). 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