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Mol Biol Cell. Oct 1997; 8(10): 2077–2088.
PMCID: PMC25673

The Involvement of the Intermediate Chain of Cytoplasmic Dynein in Binding the Motor Complex to Membranous Organelles of Xenopus Oocytes

J. Richard McIntosh, Monitoring Editor

Abstract

Cytoplasmic dynein is one of the major motor proteins involved in intracellular transport. It is a protein complex consisting of four subunit classes: heavy chains, intermediate chains (ICs), light intermediate chains, and light chains. In a previous study, we had generated new monoclonal antibodies to the ICs and mapped the ICs to the base of the motor. Because the ICs have been implicated in targeting the motor to cargo, we tested whether these new antibodies to the intermediate chain could block the function of cytoplasmic dynein. When cytoplasmic extracts of Xenopus oocytes were incubated with either one of the monoclonal antibodies (m74–1, m74–2), neither organelle movement nor network formation was observed. Network formation and membrane transport was blocked at an antibody concentration as low as 15 μg/ml. In contrast to these observations, no effect was observed on organelle movement and tubular network formation in the presence of a control antibody at concentrations as high as 0.5 mg/ml. After incubating cytoplasmic extracts or isolated membranes with the monoclonal antibodies m74–1 and m74–2, the dynein IC polypeptide was no longer detectable in the membrane fraction by SDS-PAGE immunoblot, indicating a loss of cytoplasmic dynein from the membrane. We used a panel of dynein IC truncation mutants and mapped the epitopes of both antibodies to the N-terminal coiled-coil domain, in close proximity to the p150Glued binding domain. In an IC affinity column binding assay, both antibodies inhibited the IC–p150Glued interaction. Thus these findings demonstrate that direct IC–p150Glued interaction is required for the proper attachment of cytoplasmic dynein to membranes.

INTRODUCTION

Microtubule-based motor proteins provide the machinery for most membrane trafficking within the cytoplasm of higher eukaryotes, and the microtubule cytoskeleton provides the polarized framework on which an oriented transport can take place. Oriented transport is achieved by two classes of motor proteins with opposite directionality, kinesins and cytoplasmic dyneins. Cytoplasmic dynein is a minus-end–directed motor complex that is responsible for centripetal transport. Cytoplasmic dynein has been colocalized with components of the Golgi apparatus (Corthesy-Theulaz et al., 1992 blue right-pointing triangle; Fath et al., 1994 blue right-pointing triangle; Vaisberg et al., 1996 blue right-pointing triangle), endocytic vesicles (Lin and Collins, 1992 blue right-pointing triangle), lysosomes (Lin and Collins, 1992 blue right-pointing triangle), and mitochondria (Hirokawa et al., 1990 blue right-pointing triangle). Furthermore, Aniento et al. (1993) blue right-pointing triangle have demonstrated that cytoplasmic dynein is essential for the fusion of endocytic vesicles.

The extension of tubular networks of the endoplasmic reticulum (ER) is another process that most likely will depend on microtubular motors (Lee and Chen, 1988 blue right-pointing triangle; Lee et al., 1989 blue right-pointing triangle; Terasaki and Reese, 1994 blue right-pointing triangle). The ER, which is linked to microtubules via cross-bridges (Franke, 1971 blue right-pointing triangle), represents the largest membrane system within a cell. Experiments examining recovery from nocodazole treatment in cultured cells have demonstrated that the extension of the ER tubules is preceded by the polymerization of microtubules (Lee et al., 1989 blue right-pointing triangle). The extension of the ER networks is oriented toward the cell periphery along microtubular tracks (Terasaki and Reese, 1994 blue right-pointing triangle) and it is believed to be caused by the plus-end–directed motor kinesin (Dabora and Sheetz, 1988 blue right-pointing triangle; Vale and Hotani, 1988 blue right-pointing triangle; Toyoshima et al., 1992 blue right-pointing triangle). ER network formation has also been reconstituted in vitro in cytoplasmic extracts of cultured cells (Dabora and Sheetz, 1988 blue right-pointing triangle) and of Xenopus oocytes (Allan and Vale, 1991 blue right-pointing triangle). Interestingly, in the case of Xenopus oocytes, Allan and Vale (1991 blue right-pointing triangle, 1994) blue right-pointing triangle have demonstrated that cytoplasmic dynein but not kinesin is involved in the formation and maintenance of ER networks (Allan and Vale, 1994 blue right-pointing triangle; Allan, 1995 blue right-pointing triangle) in vitro. It is currently not understood why Xenopus ER should move exclusively by cytoplasmic dynein. The large size of the Xenopus oocyte may, however, require an egg-specific function (Allan, 1995 blue right-pointing triangle). Cytoplasmic extracts of Xenopus oocytes provide, therefore, an excellent in vitro system to study the function of cytoplasmic dynein.

Cytoplasmic dynein is a large complex consisting of four subunit classes: heavy chains, intermediate chains (ICs), light intermediate chains, and light chains (Paschal et al., 1987 blue right-pointing triangle). Although cytoplasmic dynein interacts with microtubules dynamically via the catalytic heavy chains, it had been postulated that ICs link the motor complex to membranous organelles (King et al., 1991 blue right-pointing triangle; Paschal et al. 1992 blue right-pointing triangle). In a previous study, we have demonstrated that the ICs are localized at the base of the motor complex (Steffen et al., 1996 blue right-pointing triangle). The position of the ICs is, therefore, in complete agreement with them being responsible for docking the motor protein to cargo.

Dynein-dependent vesicle transport in vitro depends on the presence of dynactin (Gill et al., 1991 blue right-pointing triangle). More recent work suggests that dynactin might function as the cytoplasmic dynein “receptor” (Vaughan and Vallee, 1995 blue right-pointing triangle; Echeverri et al., 1996 blue right-pointing triangle). By using blot overlay and immunoprecipitation assays and immuno-affinity column chromatography, it was found that dynein IC interacted with dynactin and the interaction was mediated via the p150Glued subunit (Karki and Holzbaur, 1995 blue right-pointing triangle; Vaughan and Vallee, 1995 blue right-pointing triangle). To determine whether the ICs play an important part in the function of cytoplasmic dynein, we investigated the formation of membrane networks in cytoplasmic extracts of Xenopus eggs by using our monoclonal antibodies (m74–1 and m74–2) specific for the 74-kDa dynein IC. We demonstrate that IC-specific antibodies prevented the interaction between dynactin and dynein IC in vitro and disrupted the dynein–membrane interaction. Our data, therefore, indicate that the IC–p150Glued interaction plays a critical role in binding the motor complex to membranous organelles.

MATERIALS AND METHODS

Antibodies

Monoclonal antibodies m74–1 and m74–2 specific for dynein IC that had been characterized previously (Steffen et al., 1996 blue right-pointing triangle) were obtained from cell culture supernatant. Monoclonal cell lines were cultured in Ultrodoma medium (BioWittaker, Bender, Vienna, Austria) containing 2% fetal calf serum depleted of IgGs at 5% CO2. Antibodies were isolated from cell culture supernatant by protein G column chromatography. Purified antibodies were dialyzed into phosphate-buffered saline (PBS: 10 mM sodium phosphate, 150 mM NaCl, 2.7 mM KCl, pH 7.4), concentrated to about 10 mg/ml with Centricon 10 filters (Amicon, Witten, Germany), and stored in aliquots at −80°C. Fab fragments were generated according to Mage (1980) blue right-pointing triangle by incubating protein G-purified antibody with 50 mM cysteine, 1 mM EDTA, and 3 U/ml papain attached to agarose beads for 90 min at 37°C. The completion of the digest was monitored by SDS-PAGE.

Monoclonal antibody LEP100 developed by Dr. D.M. Fambrough was obtained from the Developmental Studies Hybridoma Bank (maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, and the Department of Biological Science, University of Iowa, Iowa City, IA, under contract N01-HD-6–2915 from the National Institute of Child Health and Human Development). Monoclonal antibody m150–1 specific for p150Glued was raised against ATP-extracted microtubule-associated proteins from bovine brain as described earlier (Steffen et al., 1996 blue right-pointing triangle). An affinity-purified polyclonal antibody raised against bacterially expressed heavy chain from Dictyostelium was obtained from E. Vaisberg (Vaisberg et al., 1993 blue right-pointing triangle).

Isolation of Xenopus Egg Cytosol

Interphase cytoplasmic extract was isolated from Xenopus oocytes according to Allan (1993) blue right-pointing triangle and Murray (1991) blue right-pointing triangle. The eggs were washed in MMR/4 buffer [MMR buffer: 100 mM NaCl, 2 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 5 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), 0.1 mM EDTA, pH 7.8]. The jelly coats were removed with freshly prepared 2% cysteine (pH 7.8). The eggs were washed again three times with MMR/4 buffer. Interphase eggs were obtained by activation in the presence of 0.4 μg/ml ionophore A23187 for 5 min, washing in MMR/4 buffer containing 100 μg/ml cycloheximide, and incubation at room temperature for 20 min. The eggs were washed twice in ice-cold acetate buffer [100 mM potassium acetate, 2.5 mM magnesium acetate, 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 mM HEPES, 50 mM sucrose, 1 mM dithiothreitol (DTT), pH 7.2]. Cytochalasin B (50 μg/ml) and protease inhibitors (10 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml Nα-p-tosyl-l-arginine methyl ester) were added to the last washing step. Eggs were settled by centrifugation at 150 × g for 1 min followed by a centrifugation at 600 × g for 30 s. Excess buffer was removed and eggs were crushed by centrifugation at 10,000 rpm in a Beckman SW60 rotor for 20 min. Cytosol was removed, 0.05 volume of the energy mixture (150 mM creatine phosphate, 20 mM Mg-ATP, 2 mM EGTA), protease inhibitors, and cytochalasin B (50 μg/ml) were added. The cytosol was frozen as 50- to 100-μl aliquots in liquid N2 and stored at −80°C.

Fractionation of Cytoplasm and SDS-PAGE

Cytoplasmic extract was fractionated by flotation through a sucrose density gradient. Twenty microliters of cytosol was mixed with 0.5 ml of 60% sucrose in acetate buffer, placed at the bottom of a step gradient (0.5 ml of 58% sucrose, 1 ml of 50% sucrose, and 0.5 ml of 15% sucrose in acetate buffer) in a Beckman TLS-55 centrifuge tube, and centrifuged at 55,000 rpm for 30 min at 4°C. The membrane fraction at the 15–50% sucrose interface was removed from the top with a hypodermic needle and a peristaltic pump. The membrane fractions were precipitated with ethanol or pelleted at 100,000 × g and then analyzed by SDS-PAGE and immunoblot. Electrotransfer to nitrocellulose was carried out according to Towbin et al. (1979) blue right-pointing triangle at 70 V for 3 h by using a Tris(hydroxymethyl)aminomethane (Tris)/glycine buffer containing 25 mM Tris, 192 mM glycine, and 10% methanol. The antigens were detected with a goat anti-mouse and a goat anti-rabbit peroxidase-conjugated antibody (Bio-Rad, Richmond, CA) and visualized using a luminescence assay (Pierce, Rockford, IL). In some experiments cytoplasmic extracts were incubated for 3 h on ice with 0.5 mg/ml antibody prior to flotation in the presence or absence of protein kinase inhibitors (150 μM H7, 10 μM staurosporin) and/or phosphatase inhibitors (1 μM calyculin, 10 μM microcystein, 1 μM okadaic acid).

Quantification of SDS-PAGE and SDS-PAGE Immunoblot

Membrane fractions were isolated from equal volumes of cytoplasmic extracts. The relative proportion of the membrane fraction was determined by gel densitometry using a flat bed scanner HP Scanjet IIp (Hewlett-Packard, Vienna, Austria) and the NIH Image software (National Institutes of Health, Bethesda, MD). The SDS-PAGE immunoblots were normalized to the major membrane proteins on corresponding SDS-PAGE gels to compensate for slight variations in loading between lanes. Also, where equal loading was necessary, the protein loading was adjusted prior to SDS-PAGE immunoblot.

Isolation and Purification of Cytoplasmic Dynein and Tubulin

Cytoplasmic dynein was isolated via its affinity to taxol-stabilized microtubules and purified by sucrose density gradient (5–25%) centrifugation (Paschal et al. 1987 blue right-pointing triangle). Taxol was kindly provided to us by Jill Johnson (National Cancer Institute, Bethesda, MD). Brain stem (100–150 g) from bovine brain was homogenized in an equal volume of PEM buffer [100 mM piperazine-N,N′-bis(2-ethanesulfonic acid), pH 7.3, 1 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 10 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM PMSF, 10 μg/ml Nα-p-tosyl-l-arginine methyl ester] and then centrifuged at 10,000 × g for 15 min and 80,000 × g for 30 min at 4°C. Microtubules were polymerized at 37°C in the presence of 1 U/ml hexokinase and 10 mg/ml glucose by adding 10 μM taxol and then centrifuged through a 10% sucrose cushion in PEM at 80,000 × g for 40 min at 25°C. Dynein was released by resuspending taxol-stabilized microtubules in 0.05 tissue volume of extraction buffer (PEM supplemented with 20 mM Mg-ATP and 5 μM taxol) followed by an incubation at room temperature for 30 min. Released microtubule-associated proteins were separated from microtubules by centrifugation at 100,000 × g for 30 min at 25°C. The supernatant was then centrifuged through a 5–25% continuous sucrose density gradient in a Beckman SW-28 rotor at 100,000 × g (27,000 rpm) for 18 to 19 h. Up to 0.1 mM Mg-ATP was included in the sucrose gradient to enhance the stability of cytoplasmic dynein. Fractions of about 0.8 ml were collected from the bottom of the gradient.

Taxol-stabilized microtubules obtained as leftover from the dynein preparation were used as a source of tubulin. Microtubules were depolymerized by resuspending and incubating them in PEM buffer containing a final concentration of 300 mM NaCl and 3 mM CaCl2 for 90 min on ice according to Collins (1991) blue right-pointing triangle. The depolymerized microtubules were centrifuged at 100,000 × g for 60 min. The supernatant was adjusted to 250 mM NaCl and 0.2 mM GTP and then further purified by DEAE-Sephadex column chromatography. DEAE-Sephadex A-50 was equilibrated in PEM buffer containing 250 mM NaCl. Tubulin was bound to the column, washed with 250 mM NaCl in PEM buffer, eluted with 500 mM NaCl in PEM buffer, and then dialyzed against PEM containing 0.1 mM GTP and 0.5 mM DTT. Aliquots were frozen in liquid N2 and stored at −80°C. The purity of cytoplasmic dynein and tubulin was examined by SDS-PAGE.

Video Microscopy

Microscope flow chambers were built as described by Blocker et al. (1996) blue right-pointing triangle. An 11-mm circular cover glass (Menzel, Braunschweig, Germany) was placed onto a glass microscope slide with two pieces of double-sided tape (Scotch, 3 M, St. Paul, MN) forming a 2- to 3-μl chamber. Chambers were perfused with Xenopus cytoplasmic extracts diluted 1:4 (vol/vol) with acetate buffer containing 2 mM ATP. Preparations were observed with video-enhanced differential interference contrast (VEC-DIC) microscopy (Allen et al., 1981 blue right-pointing triangle, 1985 blue right-pointing triangle; Weiss et al., 1989 blue right-pointing triangle). A Nikon Diaphot 300 inverted microscope (Nikon, Düsseldorf, Germany) equipped with an oil-immersion condenser (numerical aperature 1.4), 100× DIC PlanApo oil objective (numerical aperature 1.4), and xenon lamp (XBO 100). A Hamamatsu C2400–07 Newvicon camera was used for acquiring DIC images (Hamamatsu Photonics Deutschland, Herrsching, Germany). The video signals were first subjected to analogue contrast enhancement (Allen et al., 1981 blue right-pointing triangle, 1985 blue right-pointing triangle; Weiss et al., 1989 blue right-pointing triangle), followed by real-time digital image processing in the following steps: subtraction of an out-of-focus background (mottle pattern), accumulation or averaging of images to increase signal to noise ratio, and finally selection of the desired range of gray levels (histogram stretching or digital contrast enhancement; Allen and Allen, 1983 blue right-pointing triangle; Weiss and Maile, 1993 blue right-pointing triangle). The analogue and digital processing of VEC-DIC signals was performed by using the ARGUS 20 real-time image processor (Hamamatsu Photonics Deutschland). All observations were recorded in real time with a Panasonic AG-6730 SVHS video recorder. Single-frame images were captured from video tape or directly from ARGUS 20 image processor onto a Power MacIntosh 8500 (Apple Computer, Cupertino CA) equipped with a LG-3 frame grabber (Scion, Frederick, MD) using IPLab Spectrum software (Scanalytics, Vienna, VA).

Affinity Column Binding Assay

The affinity column binding assay was performed essentially as described by Karki and Holzbaur (1995) blue right-pointing triangle. Three identical columns were constructed by packing 200 μl of CH-Sepharose 4B (Pharmacia, Uppsala, Sweden) beads cross-linked with purified recombinant dynein IC at a concentration of 2 mg/ml. After thorough equilibration of the columns with PHEM buffer [50 mM sodium piperazine-N,N′-bis(2-ethanesulfonic acid), 50 mM sodium HEPES, 1 mM EDTA, 2 mM MgCl2, pH 6.9] 2 mg bovine serum albumin (BSA) was added to the first column, 700 μg of antibody m74–1 was added to the second column, and 700 μg of antibody m74–2 was added to the third column. After washing with 10 column volumes of PHEM, 500 μl of rat brain cytosol was loaded onto each column. After thorough washing (100 column volumes) with HEM buffer (50 mM sodium HEPES, 1 mM EDTA, 2 mM MgCl2, pH 6.9), the columns were eluted with 500 μl of 1 M NaCl, and the resulting fractions were precipitated with trichloroacetic acid. The precipitates were each resuspended in 30 μl of 1× Laemmli sample buffer and neutralized with saturated Tris. A 15-μl aliquot of the precipitate was loaded and resolved by SDS-PAGE. The proteins were transferred onto Immobilon-P (Millipore, Bedford, MA), Coomassie-stained, and subsequently probed with affinity-purified rabbit polyclonal antibodies to 150Glued and centractin (Holleran et al., 1996 blue right-pointing triangle; Tokito et al., 1996 blue right-pointing triangle).

Expression of Dynein IC cDNA

The coding region of IC-1a (Paschal et al., 1992 blue right-pointing triangle) was subcloned into the pET-14b expression vector (Novagene, Madison, WI) by using standard cloning techniques (Vaughan and Vallee, 1995 blue right-pointing triangle). Truncation mutants of IC-1a were prepared in pET-14b by using a combination of internal restriction sites and site-directed mutagenesis (Vaughan and Vallee, 1995 blue right-pointing triangle). After transformation into the expression strain BL21(DE3) and induction with 0.4 mM isopropyl β-d-thiogalactoside, bacteria were lysed in a French press and expressed proteins were purified by using nickel-affinity chromatography. Purified recombinant polypeptides were immobilized on Immobilon-P (Millipore) membranes by using a slot-blot apparatus (BRL, Gaithersburg, MD). Immunoblots were performed by standard techniques and results were visualized by using ECL (Kirkegaard & Perry, Gaithersburg, MD). A pan-IC antiserum was generated against full-length IC-1a and shown to react with all truncation mutant polypeptides (Vaughan and Vallee, 1995 blue right-pointing triangle).

RESULTS

ER Network Formation and Maintenance

Incubation of cytoplasmic extract from Xenopus oocytes leads to the formation of tubular membrane networks (Allan and Vale, 1991 blue right-pointing triangle). The formation of these networks is depended on the attachment of microtubules to the glass surface, the extension of membrane tubules along these microtubules, and the fusion of these membrane tubules (Allan and Vale, 1994 blue right-pointing triangle). Figure Figure11 demonstrates the formation of a typical network of cytoplasmic extracts isolated from interphase Xenopus oocytes. Polymerized microtubules became visible at the surface of the cover glass within minutes of placing the diluted cytoplasmic extract in a microscope flow chamber. Shortly thereafter, membrane plaques became attached to the surface and membrane tubules were pulled out by moving along microtubule tracks. Evenly spread membrane networks were established within 20 min and the density of the networks continued to increase over time. The membrane networks underwent constant rearrangements and stayed motile for at least 3 h (period of observation). During the whole period, movement of membrane tubules along microtubules and vesicle motility could be observed.

Figure 1
Formation of a tubular network in crude cytoplasmic extracts of Xenopus oocytes. Cytoplasmic extract was diluted 1:4 with acetate buffer containing 2 mM ATP and placed in a microscope flow chamber. The same area was monitored over a period of 60 min, ...

Importance of Cytoplasmic Dynein for ER Network Formation

In a previous study, the 74-kDa intermediate chain of bovine cytoplasmic dynein was localized to the base of the motor complex by direct immunogold labeling (Steffen et al., 1996 blue right-pointing triangle). To investigate the role of the dynein IC in docking the motor complex onto the membrane and thereby influencing the formation of membranous networks, monoclonal antibodies specific for dynein IC (m74–1 and m74–2) were added to cytoplasmic extract prior to network formation. The cytoplasmic extract was placed in a microscope flow chamber and monitored by VEC-DIC microscopy for up to 1 h after the addition of the antibodies. A normal membrane network formed in control samples containing 0.5 mg/ml mouse antibody LEP100 (Figure (Figure2).2). The motile activity of the membrane tubules was indistinguishable from samples lacking control antibodies. In contrast, no membrane networks were observed in the presence of 0.5 mg/ml of either monoclonal antibody m74–1 or m74–2 (Figure (Figure2).2). Although the antibody m74–1 and m74–2 interfered with the formation of membrane networks, the polymerization of microtubules and their attachment to the glass surface remained unaffected (Figure (Figure2).2). Because both monoclonal antibodies m74–1 and m74–2 appeared to have identical effects in blocking the formation of membrane networks, only m74–2 was used in the follow up experiments.

Figure 2
Effect of anti-dynein antibodies on the formation of membrane networks in cytoplasmic extracts of Xenopus oocytes. VEC-DIC images of cytoplasmic extracts supplemented 0.5 mg/ml antibody after an incubation for 60 min in a microscope flow chamber. (A and ...

As described above, the ER network formation was inhibited with an antibody concentration of 0.5 mg/ml. To determine the efficiency of the antibodies in interfering with the network formation, the titer of the antibodies was gradually reduced from 125 μg/ml to 7.5 μg/ml (Figure (Figure3).3). Complete inhibition of membrane network formation was observed at an antibody concentration of ~15 μg/ml. At an antibody concentrations of ~7 μg/ml, short membrane tubules could be observed occasionally (Figure (Figure3E).3E).

Figure 3
Determination of minimal concentration of antibody m74–2 for blocking the formation of tubular networks. VEC-DIC images of cytoplasmic extract supplemented with 125 μg/ml (A), 62.5 μg/ml (B), 31.5 μg/ml (C), 15.5 μg/ml ...

Allan (1995) blue right-pointing triangle has reported that a large portion of the cytoplasmic dynein is found in a nonmembrane bound pool. It is therefore conceivable that the divalent IgG might deplete the pool of soluble dynein by immunoprecipitation, thereby removing the motor protein from the membrane fraction. To determine whether the effect seen by the antibody was due to an interference with a specific function of dynein IC rather than due to a mere immunoprecipitation of the soluble cytoplasmic dynein, protein G-purified antibody m74–2 was fragmented into Fab fragments by Sephadex-immobilized papain. When Fab fragments of m74–2 were added to the cytoplasmic extract, identical results were obtained as had been obtained for whole IgGs; the formation of membrane networks was blocked (our unpublished results).

The lack of network formation caused by the IC-specific antibodies could be interpreted either by an interference with the motor activity (ATPase activity) or by an interference with the dynein–membrane interaction. Dynein-dependent microtubule gliding was investigated in the presence of IC-specific antibodies to determine whether the antibodies might interfere with the motor activity. As demonstrated in Figure Figure2,2, the antibodies m74–1 and m74–2 did not affect the polymerization of microtubules or their attachment onto the glass surface. Furthermore, microtubule gliding was observed as well. In cytoplasmic extracts of Xenopus oocytes, the directionality of microtubule movement could not be determined due to the high density of microtubules and most importantly due the lack of an appropriate marker of microtubule polarity. To determine whether m74–2 interfered with dynein-dependent microtubule motility, microtubule gliding was analyzed by using taxol-stabilized microtubules and cytoplasmic dynein from bovine brain. In this assay, the antibody m74–2 did not cause an inhibition of microtubule gliding. Instead, the velocity of gliding microtubules was slightly elevated in the presence of the antibody. In control samples microtubules moved with a velocity of 0.62 ± 0.14 μm/sec (mean ± SD; n = 51), and they moved with a velocity of 0.82 ± 0.2 μm/sec (mean ± SD; n = 46) in samples treated with m74–2, indicating that the antibody did not block the ATPase activity.

Blocking of Dynein–Membrane Interaction

The lack of network formation in the presence of IC-specific antibodies appeared to be due to interference with the dynein–membrane interaction. To gain a better understanding of how the antibody m74–2 might interfere with the formation of membrane networks, the effect of the antibodies on the binding of cytoplasmic dynein to membranous organelles was investigated. The monoclonal antibodies m74–2 (see above) and 70.1 (Steuer et al., 1990 blue right-pointing triangle) were used to identify cytoplasmic dynein within the membrane fractions. On the basis of immunoblot data obtained from bovine brain microsomes, these two antibodies recognized different epitopes. The monoclonal antibody m74–2 raised against the 74-kDa intermediate chain of sucrose-density-gradient–purified cytoplasmic dynein (Steffen et al., 1996 blue right-pointing triangle) detected a doublet band of 72/74-kDa in bovine brain microsomes. In contrast, monoclonal antibody 70.1 detected just the 74-kDa polypeptide band in bovine brain microsomes. In membrane fractions from Xenopus oocytes, both antibodies (m74–2 and 70.1) detected a single polypeptide band of about 83-kDa (Figure (Figure4C).4C). As pointed out by Allan (1995) blue right-pointing triangle, the 83-kDa polypeptide band represents dynein IC in Xenopus oocytes.

Figure 4
Dissociation of cytoplasmic dynein from membranes of cytoplasmic extracts of Xenopus oocytes analyzed by SDS-PAGE immunoblot. (A) Undiluted cytoplasmic extracts of interphase oocytes were supplemented with 0.5 mg/ml monoclonal antibody and incubated on ...

Undiluted cytoplasmic extracts were incubated with 0.5 mg/ml monoclonal antibody for 3 h on ice in the presence of protease inhibitors. To obtain a membrane fraction free of soluble proteins or cosedimenting protein complexes, the membrane fractions were isolated by flotation through a sucrose step gradient (Niclas et al., 1996 blue right-pointing triangle) and then analyzed by SDS-PAGE immunoblot. Although the 83-kDa IC was observed in membrane fractions of untreated samples and samples treated with a control antibody (Figure (Figure4A,4A, lanes 2 and 5), it was no longer detectable in membrane fractions of samples treated with antibodies m74–1 and m74–2 (Figure (Figure4B,4B, lanes 3 and 4). Identical results were obtained, if the immunoblot was carried out with the monoclonal antibodies m74–1 and 70.1 (Figure (Figure4C).4C). The absence of the 83-kDa antigen in m74–2-treated membrane fractions indicated a dissociation of cytoplasmic dynein from the membranes. To determine, therefore, whether the loss of the m74–2 antigen from the membrane fraction had been caused by blocking the rebinding of cytoplasmic dynein due to the presence of the monoclonal antibody m74–2, membranes were isolated by flotation prior to incubation with m74–2. The treatment of isolated membrane fractions with the monoclonal antibody m74–2 resulted again in a complete loss of the 83-kDa IC (Figure (Figure4B).4B). Immunoblot replicas of antibody-treated membrane samples were also probed with a polyclonal antibody against the heavy chain and two different monoclonal antibodies against the intermediate chain, to determine whether the whole motor complex was removed from the membrane (Figure (Figure4C).4C). By using the luminescence immunoblot assay, neither of these antibodies detected subcomponents of cytoplasmic dynein in m74–2-treated membrane fractions.

As demonstrated by video microscopy, 15 μg/ml m74–2 was sufficient to block ER network formation (Figure (Figure3).3). For the network formation assay, the cytoplasmic extract was diluted 1:5, and undiluted extract was used for the membrane binding study. To compare the data from motility assay and membrane binding study, we determined the molar ratio of the antibody per 1 ml of undiluted cytoplasmic extract and found that 0.39 nmol of m74–2 was sufficient to block ER network formation. Because the biochemical experiments were carried out on ice to prevent microtubule polymerization, an ~10-fold excess of antibody was used to disrupt the dynein–membrane interaction (Figure (Figure4).4). To be able to compare both sets of experiment, cytoplasmic extract was incubated with 0.66 nmol/ml of m74-2 and analyzed after 5, 10, and 30 min of incubation. A loss of ~90% of dynein IC was already observed after an incubation of 10 min (our unpublished observation), indicating a similar activity of the antibody in the membrane binding studies.

Interaction of Cytoplasmic Dynein with Dynactin

It has been demonstrated that dynactin, a regulator for the dynein-dependent membrane transport (Gill et al., 1991 blue right-pointing triangle), can bind to dynein IC via the p150Glued subcomponent (Karki and Holzbaur, 1995 blue right-pointing triangle; Vaughan and Vallee, 1995 blue right-pointing triangle). Because the IC-specific antibody m74–2 was able to influence the dynein–membrane interaction, the association of the regulatory component dynactin with the membrane fractions was also investigated. The monoclonal antibody m150–1 raised against p150Glued from bovine brain recognized two polypeptide bands of ~150 kDa and ~160 kDa in microsomes of bovine brain (Figure (Figure4C,4C, lane 1). In membrane fractions from Xenopus oocytes, the antibody m150–1 detected only a single polypeptide band of about 165-kDa. A significant reduction in the association of dynactin with membrane was observed in m74–2 treated cytoplasmic extracts by using the monoclonal antibody m150–1 to detect dynactin (Figure (Figure4B,4B, lanes 1′ and 2′). When compared with untreated membranes, only about 20% of the dynactin p150Glued remained with the membranes after treatment with the antibody m74–2. The use of phosphatase inhibitors and protein kinase inhibitors did not influence the dissociation of dynactin caused by m74–2 (our unpublished results).

The effect of the antibody m74–2 on the dynein–dynactin interaction was further investigated by an affinity column binding assay. Immobilized bacterially expressed dynein IC was employed as affinity matrix. The column matrix was equilibrated with BSA, m74–1, or m74–2 before loading whole brain extract. The wash fractions and the eluted fractions were analyzed by SDS-PAGE immunoblot using antibodies to dynactin p150Glued and centractin. As shown in Figure Figure5,5, both antibodies m74–1 and m74–2 prevented the binding of dynactin to the column matrix, whereas in the presence of BSA, the dynactin p150Glued and centractin were retained by the dynein IC, demonstrating that both antibodies m74–1 and m74–2 interfered with the dynein–dynactin interaction.

Figure 5
Monoclonal antibodies 74-1 and 74-2 block dynein IC–dynactin interaction in vitro. Rat brain cytosol (lane 1) was loaded onto a dynein IC affinity column (see MATERIALS AND METHODS) that was pretreated with BSA (lanes 2–4), m74–1 ...

Determination of Epitopes for m74–1 and m74–2

Binding studies using the IC affinity column indicate that both antibodies interfere with the interaction of dynein IC with dynactin p150Glued. This interaction has been mapped to the N-terminal domain of the ICs containing a coiled-coil structure (Vaughan and Vallee, 1995 blue right-pointing triangle). To test this possibility, we mapped the epitopes of the two monoclonal antibodies by using a panel of IC truncation mutants. A pan-IC antibody generated against full-length IC-1A detected all peptide fragments (Figure (Figure6),6), as reported previously (Vaughan and Vallee, 1995 blue right-pointing triangle). Both monoclonal antibodies detected all fragments down to amino acids 1–66 yet failed to detect a fragment containing amino acids 61–643 (Figure (Figure6).6). These findings suggest that the epitopes for the monoclonal antibodies m74–1 and m74–2 lie within the N-terminal coiled-coil domain overlapping with the region dictating the IC–p150Glued interaction.

Figure 6
Identification of the binding site of monoclonal antibodies m74–1 and m74–2 on dynein IC. (A) Immunoblot of truncation mutants of expressed dynein IC labeled with monoclonal antibody m74–1 and m74–2 or polyclonal antibody ...

DISCUSSION

We have demonstrated here for the first time that monoclonal antibodies specific for dynein IC are able to block the function of cytoplasmic dynein to form ER networks in cytoplasmic extracts of Xenopus oocytes in vitro. The dynein IC-specific antibodies caused a dissociation of cytoplasmic dynein from the membrane surface and thereby resulted in a disruption of the function of cytoplasmic dynein to generate membrane networks in vitro. These observations, therefore, support the hypothesis that dynein IC plays an essential part in docking the motor complex on to its target; a model which had been supported by the immuno-gold localization of the intermediate chain to the base of the motor complex (Steffen et al., 1996 blue right-pointing triangle).

To address the question of how the monoclonal antibodies m74–1 and m74–2 might be able to disrupt the association of cytoplasmic dynein with membranes, we discuss first the possible interaction of cytoplasmic dynein with membranous organelles. Two models are still under discussion, a binding of the motor complex onto a dynein-specific receptor or a charge interaction of cytoplasmic dynein with the membrane surface (Lacey and Haimo, 1992 blue right-pointing triangle). The model of an electrostatic interaction of cytoplasmic dynein with membranes has been supported by a binding study using purified cytoplasmic dynein and phospholipid vesicles (Lacey and Haimo, 1994 blue right-pointing triangle) and the model was, furthermore, substantiated by the presence of a lysine-rich region at the N-terminal end of dynein IC (Paschal et al., 1992 blue right-pointing triangle; Trivinos-Lagos et al., 1993 blue right-pointing triangle). Electrostatic interaction with the surface of membranes has also been proposed for myosin (Adams and Pollard, 1989 blue right-pointing triangle; Hayden et al., 1990 blue right-pointing triangle; Doberstein and Pollard, 1992 blue right-pointing triangle; Li et al., 1994 blue right-pointing triangle). On the other hand, conflicting data concerning a possible dynein receptor were obtained from binding studies of cytoplasmic dynein to protease-treated vesicles. Muresan et al. (1996) blue right-pointing triangle demonstrated that cytoplasmic dynein can bind to trypsin-treated KI-extracted vesicles from squid axoplasm. In contrast, Yu et al. (1992) blue right-pointing triangle and Blocker et al., (1997) blue right-pointing triangle found the binding of cytoplasmic dynein to membrane was α-chymotrypsin-sensitive.

Little is known about the exchange between soluble- and membrane-bound cytoplasmic dynein. In our studies of membranes isolated from Xenopus oocytes, the antibody m74–2 caused a dissociation of cytoplasmic dynein from the membrane. If we assume that the soluble and membrane-bound dyneins are constantly exchanged within the cytoplasm, then the loss of membrane-bound cytoplasmic dynein due to the presence of m74–2 could very well be explained by blocking the binding sites needed for an electrostatic interaction with phospholipids. The antibody m74–2 also caused a loss of cytoplasmic dynein from isolated membranes, suggesting a more active role of the antibody in membrane–dynein interaction. However, we cannot completely rule out the possibility that the antibody blocked the rebinding of dynein to the membrane.

Recently it had been proposed that dynactin might represent the “receptor” for the cytoplasmic dynein (Karki and Holzbaur, 1995 blue right-pointing triangle; Vaughan and Vallee, 1995 blue right-pointing triangle). The dynactin complex was initially described as an activator for dynein-mediated vesicle transport (Gill et al., 1991 blue right-pointing triangle; Schroer and Sheetz, 1991 blue right-pointing triangle). The dynactin p150Glued contains a binding site for microtubules (Waterman-Storer et al., 1995 blue right-pointing triangle) and for dynein IC (Karki and Holzbaur, 1995 blue right-pointing triangle; Vaughan and Vallee, 1995 blue right-pointing triangle). As demonstrated herein, dynein IC-specific antibodies displaced the cytoplasmic dynein from the membrane and also blocked the in vitro binding of dynactin onto immobilized dynein IC. By epitope mapping, the binding sites for the monoclonal antibodies m74–1 and m74–2 have been localized to the N terminus of the dynein IC, in close proximity to the binding site for dynactin p150Glued. Because of the close proximity of binding sites for antibodies and dynactin p150Glued, it is not surprising that m74–1 and m74–2 disrupt the dynein–dynactin interaction. A dissociation of cytoplasmic dynein from squid membrane vesicles has also been achieved with monoclonal dynein IC antibody m74-1 and a polyclonal antibody against p150Glued (Waterman-Storer et al., 1997 blue right-pointing triangle). Binding of cytoplasmic dynein to dynactin as a primary target does not, however, exclude an additional electrostatic interaction of dynein with negatively charged membrane regions as has been suggested by binding studies with phospholipids (Lacey and Haimo, 1994 blue right-pointing triangle). A treatment of Xenopus membranes with the antibody m74–2 revealed that the membrane association of dynactin was affected as well, indicating that both molecules, cytoplasmic dynein and dynactin, are needed for a proper membrane interaction. Although dynactin might provide the primary target for cytoplasmic dynein (Vaughan and Vallee, 1995 blue right-pointing triangle), an additional stabilization of the binding could be achieved by an electrostatic interaction of cytoplasmic dynein with the membrane.

In summary, our data indicate that the ICs play an important role in docking cytoplasmic dynein onto membranous organelles. The use of dynein IC-specific antibodies that interfered with the dynein–dynactin interaction resulted in a dissociation/loss of cytoplasmic dynein from the membrane, indicating that the dynactin complex represents the primary target for cytoplasmic dynein. Because the anti-dynein monoclonal antibody m74–2 also affected the membrane binding of the dynactin complex, we propose that both complexes, dynactin and cytoplasmic dynein, are needed for a proper attachment to the membrane surface.

ACKNOWLEDGMENTS

We thank Josef Gotzmann, Berhard Schierer, and Bärbel Redlich for providing technical support; Drs. Julie L. Hodgkinson and George M. Langford for critical reading of the manuscript; Drs. Mike Koonce and Eugene Vaisberg for antibodies specific for dynein heavy chain; and the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute for the generous gift of taxol. This study was supported in part by an Established Investigator Award from the American Heart Association and National Institutes of Health grant GM-48661 to E.L.F.H., a Predoctoral Fellowship from the American Heart Association to S.K., a Human Frontier Science Program grant to S.A.K., a grant from the University Jubilaumsstiftung to W.S., and grant We790 from the Deutsche Forschungs-Gemeinschaft to D.G.W.

REFERENCES

  • Adams RJ, Pollard TD. Binding of myosin I to membrane lipids. Nature. 1989;340:565–568. [PubMed]
  • Allan V. Assay of membrane motility in interphase and metaphase Xenopus extracts. Methods Cell Biol. 1993;39:203–226. [PubMed]
  • Allan V. Protein phosphatase 1 regulates the cytoplasmic dynein-driven formation of endoplasmic reticulum networks in vitro. J Cell Biol. 1995;128:879–891. [PMC free article] [PubMed]
  • Allan V, Vale R. Cell cycle control of microtubule-based membrane transport and tubule formation in vitro. J Cell Biol. 1991;113:347–359. [PMC free article] [PubMed]
  • Allan V, Vale R. Movement of membrane tubules along microtubules in vitro: evidence for specialized sites of motor attachment. J Cell Sci. 1994;107:1885–1897. [PubMed]
  • Allen RD, Allen NS. Video-enhanced microscopy with a computer frame memory. J Microscopy. 1983;129:3–17. [PubMed]
  • Allen RD, Allen NS, Travis JL. Video-enhanced contrast, differential interference contrast (AVEC-DIC) microscopy: a new method capable of analyzing microtubule-related motility in the reticulopodial network of Allogromia laticollaris. Cell Motil Cytoskeleton. 1981;1:291–302. [PubMed]
  • Allen RD, Weiss DG, Hayden JH, Brown DT, Fujiwake H, Simpson M. Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasmic transport. J Cell Biol. 1985;100:1736–1752. [PMC free article] [PubMed]
  • Aniento F, Emans N, Griffiths G, Gruenberg J. Cytoplasmic dynein-dependent vesicular transport from early to late endosomes. J Cell Biol. 1993;123:1373–1387. [PMC free article] [PubMed]
  • Blocker A, Severin FF, Habermann A, Hyman AA, Griffiths GG, Burkhardt JK. Microtubule-associated protein-dependent binding of phagosomes to microtubules. J Biol Chem. 1996;271:3803–3811. [PubMed]
  • Blocker A, Severin FF, Burkhardt JK, Bingham JB, Yu H, Olivo JC, Schroer TA, Hyman AA, Griffiths G. Molecular requirements for bi-directional movement of phagosomes along microtubules. J Cell Biol. 1997;137:113–129. [PMC free article] [PubMed]
  • Collins CA. Reversible assembly purification of taxol-treated microtubules. Methods Enzymol. 1991;196:246–253. [PubMed]
  • Corthesy-Theulaz I, Pauloin A, Pfeffer SR. Cytoplasmic dynein participates in the centrosomal localization of the Golgi complex. J Cell Biol. 1992;118:1333–1345. [PMC free article] [PubMed]
  • Dabora S, Sheetz MP. The microtubule-dependent formation of a tubulovesicular network with characteristics of the ER from cultured cell extracts. Cell. 1988;54:27–35. [PubMed]
  • Doberstein SK, Pollard TD. Localization and specificity of the phospholipid and actin binding sites on the tail of Acanthamoeba myosin IC. J Cell Biol. 1992;117:1241–1249. [PMC free article] [PubMed]
  • Echeverri CJ, Paschal BM, Vaughan KT, Vallee RB. Molecular characterization of the 50-kDa subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J Cell Biol. 1996;132:617–633. [PMC free article] [PubMed]
  • Fath KR, Trimbur GM, Burgess DR. Molecular motors are differentially distributed on Golgi membranes from polarized epithelial cells. J Cell Biol. 1994;126:661–675. [PMC free article] [PubMed]
  • Franke WW. Cytoplasmic microtubules linked to endoplasmic reticulum with cross-bridges. Exp Cell Res. 1971;66:486–489. [PubMed]
  • Gill SR, Schroer TA, Szilak I, Steuer ER, Sheetz MP, Cleveland DW. Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein. J Cell Biol. 1991;115:1639–1650. [PMC free article] [PubMed]
  • Hayden SM, Wolenski JS, Mooseker MS. Binding of brush border myosin I to phospholipid vesicles. J Cell Biol. 1990;111:443–451. [PMC free article] [PubMed]
  • Hirokawa N, Sato-Yoshitake R, Yoshida T, Kawashima T. Brain dynein (MAP-1C) localizes on both anterogradely and retrogradely transported membranous organelles in vivo. J Cell Biol. 1990;111:1027–1037. [PMC free article] [PubMed]
  • Holleran EA, Tokito MK, Karki S, Holzbaur ELF. Centractin (ARP1) associates with spectrin revealing a potential mechanism to link dynactin to intracellular organelles. J Cell Biol. 1996;135:1815–1829. [PMC free article] [PubMed]
  • Karki S, Holzbaur LF. Affinity chromatography demonstrates a direct binding between cytoplasmic dynein and the dynactin complex. J Biol Chem. 1995;270:28806–28811. [PubMed]
  • King SM, Wilkerson CG, Witman GB. The Mr 78,000 intermediate chain of Chlamydomonas outer arm dynein interacts with α-tubulin in situ. J Biol Chem. 1991;266:8401–8407. [PubMed]
  • Lacey ML, Haimo LT. Cytoplasmic dynein is a vesicle protein. J Biol Chem. 1992;267:4793–4798. [PubMed]
  • Lacey ML, Haimo LT. Cytoplamic dynein binds to phospholipid vesicles. Cell Motil Cytoskeleton. 1994;28:205–212. [PubMed]
  • Lee C, Chen LB. Dynamic behavior of endoplasmic reticulum in living cells. Cell. 1988;54:37–46. [PubMed]
  • Lee C, Ferguson M, Chen LB. Construction of the endoplasmic reticulum. J Cell Biol. 1989;109:2045–2055. [PMC free article] [PubMed]
  • Li DQ, Miller M, Chantler PD. Association of a cellular myosin-II with anionic phospholipids and the neuronal plasma membrane. Proc Natl Acad Sci USA. 1994;91:853–857. [PMC free article] [PubMed]
  • Lin SX, Collins CA. Immunolocalization of cytoplasmic dynein in lysosomes in cultured cells. J Cell Sci. 1992;110:125–137. [PubMed]
  • Mage MG. Preparation of Fab fragments from IgGs of different animal species. Methods Enzymol. 1980;70:142–150. [PubMed]
  • Muresan V, Godek CP, Reese TS, Schnapp BJ. Plus-end motors override minus-end motors during transport of squid axon vesicles on microtubules. J Cell Biol. 1996;135:383–397. [PMC free article] [PubMed]
  • Murray A. Cell cycle extracts. Methods Cell Biol. 1991;36:581–605. [PubMed]
  • Niclas J, Allan VJ, Vale RD. Cell cycle regulation of dynein association with membrane modulates microtubule-based organelle transport. J Cell Biol. 1996;133:585–593. [PMC free article] [PubMed]
  • Paschal BM, Mikami A, Pfister KK, Vallee RB. Homology of the 74-kDa cytoplasmic dynein subunit with a flagellar dynein polypeptide suggests an intracellular targeting function. J Cell Biol. 1992;118:1133–1143. [PMC free article] [PubMed]
  • Paschal BM, Shpetner HS, Vallee RB. MAP1C is a microtubule-associated ATPase which translocates microtubules in vitro and has dynein-like properties. J Cell Biol. 1987;105:1273–1282. [PMC free article] [PubMed]
  • Schroer TA, Sheetz MP. Two activators of microtubule-based vesicle transport. J Cell Biol. 1991;115:1309–1318. [PMC free article] [PubMed]
  • Steffen W, Hodgkinson JL, Wiche G. Immunogold localization of the intermediate chain within the protein complex of cytoplasmic dynein. J Struct Biol. 1996;117:227–235. [PubMed]
  • Steffen, W., Langford, G.M., Weiss, D.G., and Kuznetsov, S.A. (1997). Inhibition of microtubule-dependent minus-end directed transport of axoplasmic organelles by a dynein intermediate chain specific antibody. Biol. Bull. (in press).
  • Steuer ER, Wordeman L, Schroer TA, Sheetz MP. Localization of cytoplasmic dynein in mitotic spindles and kinetochores. Nature. 1990;345:266–269. [PubMed]
  • Terasaki M, Reese TS. Interactions among endoplasmic reticulum, microtubules, and retrograde movements of the cell surface. Cell Motil Cytoskeleton. 1994;29:291–300. [PubMed]
  • Tokito MK, Howland DS, Lee VM, Holzbaur EL. Functionally distinct isoforms of dynactin are expressed in human neurons. Mol Biol Cell. 1996;7:1167–1180. [PMC free article] [PubMed]
  • Toyoshima I, Yu H, Steuer ER, Sheetz MP. Kinectin, a major kinesin-binding protein on ER. J Cell Biol. 1992;118:1121–1131. [PMC free article] [PubMed]
  • Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA. 1979;76:4350–4354. [PMC free article] [PubMed]
  • Trivinos-Lagos L, Collins CA, Chrisholms RL. Cloning of dynein intermediate chain: multiple isoforms are expressed during Dictyostelium development. Mol Biol Cell. 1993;4:47a. (Abstract).
  • Vaisberg EA, Grissom PM, McIntosh JR. Mammalian cells express three distinct dynein heavy chains that are localized to different cytoplasmic organelles. J Cell Biol. 1996;133:831–842. [PMC free article] [PubMed]
  • Vaisberg EA, Koonce MP, McIntosh JR. Cytoplasmic dynein plays a role in mammalian mitotic spindle formation. J Cell Biol. 1993;123:849–858. [PMC free article] [PubMed]
  • Vale RD, Hotani H. Formation of membrane networks in vitro by kinesin-driven microtubule movement. J Cell Biol. 1988;107:2233–2242. [PMC free article] [PubMed]
  • Vaughan KT, Vallee RB. Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued. J Cell Biol. 1995;131:1507–1517. [PMC free article] [PubMed]
  • Waterman-Storer CM, Karki S, Holzbaur ELF. The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1) Proc Natl Acad Sci USA. 1995;92:1634–1638. [PMC free article] [PubMed]
  • Waterman-Storer, C.M., Kuznetsov, S.A., Karki, S., Tabb, J.S., Weiss, D.G., Langford, G.M., and Holzbaur, E.L.F. (1997). The interaction between cytoplasmic dynein and dynactin is required for fast axonal transport. Proc. Natl. Acad. Sci. (in press). [PMC free article] [PubMed]
  • Weiss DG, Maile W. Electronic Light Microscopy. D.M. Shotton, New York: Wiley-Liss; 1993. Principle, practice, and application of video-enhanced contrast microscopy; pp. 105–140.
  • Weiss DG, Maile W, Wick RA. Video microscopy. In: Lacey AJ, editor. Light Microscopy in Biology. A Practical Approach. Oxford, United Kingdom: IRL Press at Oxford University Press; 1989. pp. 221–278.
  • Yu H, Toyoshima I, Steuer ER, Sheetz MP. Kinesin and cytoplasmic dynein binding to brain microsomes. J Biol Chem. 1992;267:20457–20464. [PubMed]

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