Purification and characterization of cytoplasmic dynein of rabbit liver

Journal of Hepatoiogy 1995; 23: 6&10 Printed in Denmark . AN rights reserved

Copyright Q Journal of Heprrtdogy 1995

Journal of Hepatology ISSN 0168-8278

Purification and characterization of cytoplasmic dynein of rabbit liver Ko Nakajima,

Masafumi Komatsu, Itaru Toyoshima, Tomoyuki Kuramitsu, Masato Funaoka, Junji Kato and Osamu Masamune

First Department

of Internal

Medicine,

Akita University

Cytoplasmic dynein is a microtubuledependent motor protein, which plays a role in intracellular transport. However, there have been few studies regarding the role of cytoplasmic dynein in the liver. Purification of cytoplasmic dynein from rabbit liver took advantage of the affinity of microtubule-dependent motor proteins for microtubules Purified dynein contained heavy chain (450 kDa), intermediate chain (75 kDa), light chains (45-58 kDa) and dynactin (150 kDa). The subunit composition was consistent with previously reported data on brain cytoplasmic dynein. Microtubules prepared from bovine brain were driven

INESIN

(1,2) and cytoplasmic dynein (3,4) are motor proteins with ATPase and play a part in microtubule-dependent intracellular transport. These motor proteins were originally detected in neural tissue but have since been found in various other cells and tissues (S-15). Hepatocytes are involved in the synthesis and secretion of various materials, and may therefore have developed an extensive intracellular transport system. However, there have been few studies of motor proteins in hepatocytes (10,ll). We have now purified cytoplasmic dynein from rabbit liver.

K

Materials and Methods Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA), unless otherwise stated. The protocols for animal experimentation described in this paper were previously approved by the Animal Research Committee, Akita University School of Medicine. All subsequent animal experiments adhered to the Received I5 September 1994; accepted II January 1995

Correspondence: Ko Nakajima, M.D., First Department of Internal Medicine, Akita University School of Medicine, l-l-l Hondo, Akita 010, Japan.

66

School of Medicine.

Tsuyoshi

Ono,

Akita, Japan

by purified cytoplasmic dynein from rabbit liver, and movements of microtubules were visualized by videoenhanced differential interference contrast microscopy. The mean velocity of the motile microtubules was 1.0920.13 &s. Our study provides evidence of rapid intracellular transport in hepatocytes controlled by cytoplasmic dynein.

Key words: Cytoplasmic dynein; Hepatocyte; Intracellular transport; Microtubules. 0 Journal of Hepatology.

‘Guidelines for Animal Experimentation’ versity.

of the Uni-

PuriJication of cytoplasmic dynein

The purification was performed as described by Schroer et al. (5). A rabbit was anesthetized with pentobarbital and its liver was removed after perfusion of 200 ml of PMEE’ buffer (35 mM PIPES, 5 mM MgS04, 1 mM EGTA, 0.5 mM EDTA, pH 7.4) (14) through the portal vein. The liver was homogenized in 0.8 volume of PMEE’ buffer containing 1 mM dithiothreitol and protease inhibitors ( 1 mM phenylmethylsulfonyl fluoride, 1 &ml pepstain A, 10 pg/ml p-tosyl-L-arginine methyl ester, 10 &ml N-tosyl-L-phenylalanine chloromethyl ketone, 1 &ml soybean trypsin inhibitor) (14). The homogenate was centrifuged at 1OOOOOXgfor 30 min and the supernatant (Sl) was recentrifuged at 165OOOxg for 1 h. Guanosine triphosphate (GTP) and taxol were added to the resulting supernatant (S2) to final concentrations of 1 mM and 20 PM, respectively, and incubated for 30 min at 37°C to polymerize microtubules. Adenylylimidodiphosphate (AMP-PNP) (final concentration 2 mM) was added and the supernatant incubated for 20 min at room temperature to bind motor proteins to the microtubules. The solution was

PuriJication qf cytoplasmic dynein

placed onto a discontinuous sucrose gradient with PMEE’ buffer containing 25%, 12.5% sucrose, and centrifuged at 1OOOOOXgfor 1 h. The precipitated microtubules were resuspended in 10 ml of PMEE’ buffer and again centrifuged at 100 OOOXgfor 30 min at 30°C. The pellet was then homogenized in 1 ml of PMEE’ buffer containing 20 mM adenosine triphosphate (ATP) and incubated for 20 min at room temperature to release motor proteins. The suspension was centrifuged at 1OOOOOxg for 30 min at 25°C. The resulting supernatant (ATP release) was put onto a 12 ml linear density gradient consisting of 515% sucrose in PMEE’ buffer and centrifuged at 1OOOOOxgfor 15 h at 4°C. Every 1 ml fraction was collected from the bottom of the centrifuge tube. Electrophoresis

Proteins were electrophoresed on SDS-PAGE with the Laemmli system (16). Gels were stained with Coomassie brilliant blue or silver (17). Pur@cation

of tubuiin

Two cycle tubulin was obtained by the method of Shelanski et al. (18). Microtubules

motility

assay

Fifty microliters of the dynein fraction was placed in a microchamber made from a slide glass and cover glass, and incubated for 10 min to bind dynein molecules to the glass surface. Microtubules were polymerized by adding 1 mM GTP and 20 PM taxol (final concentration) to the purified tubulin. Ten microliters of this microtubule solution was mixed with PMEE’ containing 20 PM taxol, 1 mM ATP and 0.1 mg/ml cytochrome C, and added to the microchamber. Microtubule motility was analyzed essentially as described by Allen et al. (19). An image of differential interference contrast microscopy (Axioplan, Carl Zeiss) was taken by video camera (Newvicon, Hamamatsu photonics), enhanced by an image processor (Argus 10, Hamamatsu photonics) and recorded to a videorecorder (sVHS, Sony). Immunoaffinity

columns

The ascites of 70.1 anti-dynein intermediate chain mAbs (IgM) (20) were treated with an equal volume of saturated ammonium sulfate for 1 h, and the pellet was dialyzed thoroughly in coupling buffer (0.1 M NaHCOs, 0.2 M NaCl, pH 8.0). About 20 mg immunoglobulins were conjugated to 1 ml of CNBr-activated Sepharose to make immunoaffinity column. The supernatant (S2) from rabbit liver and the 70.1 affinity gel were incubated for 3 h. After washing with

PMEE’, the dynein fraction was obtained by elution with 0.2 M glycine pH 4.

Results Sucrose density gradient puriJied cytoplasmic

dynein

AMP-PNP was added to the microtubule protein fraction of rabbit liver and microtubule-associated proteins and motor proteins were co-sedimented in the pellet (Fig. 1, Lane 2). The pellet was resuspended with surplus ATP and again centrifuged. Microtubules were pelleted and the supernatant (ATP release) was electrophoresed on a SDS-PAGE gel, demonstrating the presence of the 450 kDa cytoplasmic dynein heavy chain, 116 kDa kinesin heavy chain (12) and several other proteins (Fig. 1, Lane 3). Kinesin heavy chain, but not the 450 kDa cytoplasmic dynein heavy chain was released from microtubule protein by adding GTP instead of ATP (Fig. 1, Lane 5). The cytoplasmic dynein fraction purified from a sucrose density gradient of ATP release contained 450 kDa, 150 kDa, 75 kDa, 58 kDa, 52 kDa, 47 kDa, and 45 kDa polypeptides as detected on a SDS-PAGE gel (Fig. 2a, Lane 4-6. Fig. 2b). We presumed that the 450 kDa polypeptide corresponds to the heavy chain, the 75 kDa polypeptide to the intermediate chain, and the 45-58 kDa polypeptide to the light chains. The additional 150 kDa polypeptide may correspond to dyn-

Fig. 1. SDS-PAGE of fractions during the purification of cytoplasmic dynein from liver (silver stain). Lane I: Cytosolic extract (S2) from rabbit liver after high-speed centrifugation. Lane 2: AMP-PNP-treated microtubule protein fraction. Lane 3: Microtubule depleted supernatant after A TP release. Lane 4: Microtubule pellet. Lane 5: Microtubule depleted supernatant after GTP release. Lane 6. Microtubule pellet. The electrophoretic mobilities of standard proteins as well as the positions of dynein heavy chain (DHC), kinesin heavy chain (KHC), dynein intermediate chain (DIG) and tubulin (Tub) are indicated. 67

K. Nakajima et al

1

a

2 3 4 5 6

7 8 9 10 1112

kDa 20011697-

45-

Fig. 2a. SDS-PAGE of the sucrose density gradients of the ATP release fraction from microtubules b. Lane 5 of Fig. 2a is enhanced. Molecular weights of each band are indicated.

actin, which is thought to be a dynein-associated protein (21). The 75 kDa polypeptide reacted with antichicken embryo brain dynein intermediate chain mAbs (70.1) on an immunoblot (Fig. 3). The antibody reacted with the bands of lane 5 and 6 which seemed to correspond to intermediate chain binding to heavy chain, but the antibody also reacted with the bands of lane S-10, which may represent intermediate chains dissociated from heavy chain. The possibility that intermediate chains may form complexes with other proteins was also considered.

Motility

of cytoplasmic

(Coomassie

blue stain).

dynein

The gliding motility of microtubules induced by cytoplasmic dynein fractioned after sucrose velocity sedimentation was well observed by video-enhanced differential interference contrast microscopy. Each microtubule glided along its long axis (Fig. 4), and moved in a fixed direction. The mean velocity was 1.09 q-~/s (standard deviation=0.13 pm/s).

;,

_/

kDa

12

3

4

5

6

7

8

‘_

; ..*-

i

9 10 1112

(, ‘i* -;.= ,s’*iir.:*n‘+.‘i _* . .: 8. “.aij-“_;*-~j~~,~~~ Fig. 3. Reactivity of the dynein intermediate chain monoclonal antibody (70.1) with the ATP release fraction after sucrose density gradient centrtfiigation (Fig. 2a). The electrophoretic mobilities of standard proteins are noted.

68

..,

.,_

” ;,

Fig. 4. Movement of microtubules on glass in the presence of purtfied cytoplasmic dynein and I mM ATP. Translocation of a microtubule toward the upper right side can be observed. Arrow heads indicate the tip of the same microtubule. The elapsed time is displayed in the upper-left corner. Bar= 10.0 pm.

Purification of cytoplasmic dynein

Immunoafjnity

column purljied cytoplasmic dynein

SDS-PAGE analysis of immunoaffinity column purified cytoplasmic dynein revealed 450 kDa, 150 kDa and 75 kDa polypeptides as observed in sucrose density gradient purified cytoplasmic dynein (data not shown). An additional 200 kDa polypeptide was detected in the immunoaffinity column fraction but was separated from the cytoplasmic dynein fraction by sucrose density gradient centrifugation. We made mAbs to this 200 kDa protein for use in an immunoaffinity column but could not bind purified cytoplasmic dynein from rabbit liver. Therefore, it is unlikely that this 200 kDa polypeptide is a dynein-associated protein, but it may be a protein which crossreacts with the 70.1 antibodies.

Discussion Polypeptides identified as subunits of rabbit hepatic cytoplasmic dynein were purified by sucrose density gradient centrifugation and had molecular weights of 450 kDa, 150 kDa, 75 kDa, 58 kDa, 52 kDa, 47 kDa and 45 kDa. These polypeptide compositions correlate fairly well with those reported for bovine brain cytoplasmic dynein subunits (5). As shown in a previous study (14), the 150 kDa polypeptide is probably not a dynein structural protein, but rather a dynein-associated protein. Gill et al. (21) found that the 150 kDa polypeptide has dynein-activating properties; they have named it dynactin. Recently, the amino acid sequence of dynactin was determined and was shown to be homologous to the Drosophila gene Glued (22). The development of a system which incorporates a video image processor with differential interference contrast microscopy (19) enabled visualization of axonal microtubule-dependent intracellular transport in real time (23,24). The system made it possible to observe microtubules gliding in vitro on a glass surface to which an axonal soluble fraction was attached (25,26), essentially replicating vesicular transport of microtubules in vivo. We measured a mean velocity of 1.09 pm/s, a value slightly slower than the reported value of 1.25 pm/s for bovine brain cytoplasmic dynein (5). This is the first report which measured a velocity of microtubule motility induced by hepatic cytoplasmic dynein, and it may suggest the existence of rapid intracellular transport by cytoplasmic dynein in the liver. Cytoplasmic dynein is thought to participate in the vesicular transport from cell surface to cell center (27, 28). As the size of the hepatocyte is 20-25 pm diameter, materials near the cell surface which bind to cytoplasmic dynein could be transported to the cell center in about 10 s. The ballooning of hepatocytes in patients with al-

coholic liver disease is thought to be the result of disturbed microtubule-dependent protein secretion caused by the toxic effect of acetaldehyde (29). We previously showed that acetaldehyde reversibly inhibited kinesindependent microtubule gliding (30). Acetaldehyde did not affect the polymerization of microtubules, suggesting that the toxic effects of acetaldehyde on hepatocytes included the disturbance of kinesin-microtubule interaction. The absence or functional deficit of axonemal dynein results in the immotile cilia syndrome (31). A patient with immotile cilia syndrome associated with hepatic steatosis was found to have retention of material in the cisternae of the endoplasmic reticulum and Golgi apparatus of hepatocytes (32). Although common components have not been reported between axonemal and cytoplasmic dynein, in this special case dysfunction of cytoplasmic dynein might be causing the failure of microtubule-dependent transport. Ballooning of hepatocytes, with multiple vesicle accumulation, is found in cholestasis. Ultrastructurally, the ballooning was reported to correspond with hypertrophy of Golgi apparatus, an increased number of lysosomes and dilatation of endoplasmic reticulum (33). Malfunction of the microtubule-dependent machinery might be involved in vesicular transport in cholestasis (34). Endoplasmic reticulum is reported to be kinesin-dependent (15), but Golgi apparatus and lysosome are thought to be cytoplasmic dynein dependent (35). Bile acids which are increased more than ten-fold in hepatocytes in cholestasis (36) would be the major cause of ballooning. A recent study showed that taurochenodeoxycholic acid, but not tauroursodeoxycholic acid, inhibited kinesin-dependent microtubule motility (37). This might explain the ballooning of endoplasmic reticulum. The effect of bile acids on cytoplasmic dynein will be tested.

Acknowledgements The authors thank Dr M. I? Sheetz for the gift of 70.1 anti-cytoplasmic dynein monoclonal antibody. This study was supported in part by grant-in-aid 05670545, 06670638 (I. Toyoshima) from the Ministry of Education, Science and Culture of Japan.

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