Association of kinesin with the Golgi apparatus in rat hepatocytes

2417

Journal of Cell Science 107, 2417-2426 (1994) Printed in Great Britain © The Company of Biologists Limited 1994

Association of kinesin with the Golgi apparatus in rat hepatocytes David L. Marks, Janet M. Larkin and Mark A. McNiven* Center for Basic Research in Digestive Diseases, Mayo Clinic and Mayo Foundation, Rochester, MN 55905, USA *Author for correspondence

SUMMARY The Golgi apparatus is a dynamic membranous structure, which has been observed to alter its location and morphology during the cell cycle and after microtubule disruption. These dynamics are believed to be supported by a close structural interaction of the Golgi with the microtubule cytoskeleton and associated motor enzymes. One microtubule-dependent motor enzyme, kinesin, has been implicated in Golgi movement and function although direct evidence supporting this interaction is lacking. In this study, we utilized two well-characterized kinesin antibodies in conjunction with subcellular fractionation techniques, immunoblot analysis and immunofluorescence microscopy to conduct a detailed study on the association of kinesin with the Golgi and other membranous organelles in a polarized epithelial cell, the primary rat hepatocyte. We found that kinesin represents ~0.3% of total protein in rat liver homogenates, with ~30% membrane-associated and the remainder in the cytosol. Among membrane fractions,

kinesin was concentrated markedly in Golgi-enriched fractions, which were prepared using two independent techniques. Kinesin was also abundant in fractions enriched in transcytotic carriers and secretory vesicles, with lower levels detected on fractions enriched in endosomes, endoplasmic reticulum, lysosomes and mitochondria. Immunofluorescence microscopy showed that kinesin is concentrated on Golgi-like structures in both primary cultured hepatocytes and rat hepatocyte-derived clone 9 cells. Double-label immunofluorescence demonstrated that kinesin staining colocalizes with the Golgi marker, α-mannosidase II, in both cell types. These results provide compelling evidence showing that kinesin is associated with the Golgi complex in cells and implicate this motor enzyme in Golgi structure, function and dynamics.

INTRODUCTION

of tubulovesicular processes of the trans-Golgi outward from the centrosome along Mts, as has been seen by video fluorescence microscopy (Cooper et al., 1990), or the translocation of nascent secretory vesicles away from the Golgi. These observations suggest the association of the Golgi with an anterograde motor such as kinesin. Kinesin is a Mt-activated ATPase, originally isolated from neuronal tissues (Brady, 1985; Vale et al., 1985), which supports the translocation of vesicular organelles such as secretory vesicles (Ferreira et al., 1992; Leopold et al., 1992; Rothwell et al., 1993; Urrutia et al., 1991), pigment granules (Rodionov et al., 1991) and lysosomes (Hollenbeck and Swanson, 1990) outward from the cell centre to the periphery in various cell types. Despite detailed morphological and biochemical studies of kinesin distribution in neurons (Hirokawa et al., 1991; Leopold et al., 1992) and sea urchin coelomecytes (Henson et al., 1992), there is little evidence for the association of kinesin with the Golgi. In addition, there is no information on the distribution of kinesin in epithelial cells. To this end, we have utilized two well-characterized mono- and polyclonal kinesin antibodies to conduct a detailed morphological and biochemical study on the distribution of this enzyme in a polarized epithelial cell, the rat hepatocyte. Hepatocytes are highly polarized cells that maintain welldifferentiated apical (canalicular) and basolateral (sinusoidal)

It is well documented that the Golgi apparatus typically occupies a perinuclear position in cells at the centrosomal complex. This organizational scheme is highly conserved and is restored after the Golgi is disrupted or repositioned during centrosomal migration (Kupfer et al., 1982), cell division (Lucocq et al., 1989; Lucocq and Warren, 1987) and microtubule (Mt) depolymerization (Ho et al., 1989). Numerous authors have described an intimate structural interaction between the Golgi and the Mt cytoskeleton (Kronebusch and Singer, 1987; Rogalski and Singer, 1984; Thyberg and Moskalewski, 1985; Turner and Tartakoff, 1989). These studies have predicted that Mts may participate in the maintenance of Golgi structure and support vesicular trafficking pathways to and from this organelle. Because of its dynamic capacity, it is likely that the association of the Golgi with Mts is mediated, in part, by Mt-dependent motor enzymes. Recent functional studies have implicated the Mt-dependent enzyme, cytoplasmic dynein, in the positioning of the Golgi at the centrosome (Corthesy-Theulaz et al., 1992). While this function is consistent with the retrograde activity of a dynein motor, it is not known if dynein acts alone or with other motor enzymes to support the organization of the Golgi apparatus. Indeed, it is difficult to explain how dynein could support the extension

Key words: liver, microtubule motors, immunofluorescence, transcytotic carriers

2418 D. L. Marks, J. M. Larkin and M. A. McNiven domains via Mt-dependent vesicular trafficking processes (Bartles et al., 1985; Roman and Hubbard, 1983). These cells possess well-developed synthetic machinery and secrete over 30 different proteins into the basolateral sinusoid, while internalizing, transporting and degrading numerous plasma proteins (Donohue et al., 1990; Marks and LaRusso, 1993). In addition, hepatocytes utilize a transcytotic pathway in which nascent proteins destined for the canalicular domain are first transported to the sinusoidal plasmalemma, then vesiculated and transcytosed to the canalicular membrane (Bartles et al., 1985). Thus, the hepatocyte possesses both conserved and unique vesicular pathways and provides an exceptional model with which to examine the participation of kinesin in organelle transport and Golgi dynamics. In this study, we have conducted extensive purification and characterization of different vesicular organelles from rat liver and, by immunoblot analysis, provide strong evidence that kinesin is associated with secretory vesicles, transcytotic carriers and, most dramatically, the Golgi. Immunofluorescent staining of the Golgi in primary hepatocytes and a hepatocytederived cell line (clone 9) with several different kinesin antibodies supports this observation. These results suggest that kinesin is involved in multiple vesicular pathways in the hepatocyte and provide novel evidence for an interaction of kinesin with the Golgi apparatus. MATERIALS AND METHODS Materials Polyclonal antisera to α-mannosidase II (α-man II) and rab7 were gifts from Dr Marilyn Farquhar, University of California, San Diego, and Marino Zerial, EMBL, Heidelberg, respectively. Anti-α-man II monoclonal ascites fluid was purchased from Berkeley Antibody Co., Berkeley, CA. Hybridoma supernatant containing SUK-4, a previously described monoclonal antibody made against sea urchin kinesin heavy chain (Ingold et al., 1988), was obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA). Purified rat liver kinesin was prepared using a modification of a method used to purify kinesin from bovine adrenal medulla (Urrutia and Kachar, 1992). Leupeptin, pepstatin, tosyl arginine methyl ester, phenylmethylsulfonyl fluoride, benzamidine and soybean trypsin inhibitor were from Sigma (St Louis, MO). Preparation and purification of a polyclonal antibody to kinesin heavy chain (MMR44) Peptides (KKLSGKLYLVDLAGSEKVSKTGAEG and HIPYRDSKLTRILQESLGGNARTT), based on consensus regions of the microtubule-binding domain of the kinesin heavy chain conserved in squid, fly and sea urchin (Wright et al., 1991), were synthesized by the Mayo Clinic Peptide Core, Rochester, MN. The peptides were conjugated to keyhole limpet hemocyanin, suspended in Freud’s adjuvent, and injected into male New Zealand White rabbits. Serum samples were collected and screened for the ability to recognize kinesin from rat liver and rat brain on immunoblots. For immunofluorescence, the polyclonal anti-kinesin antibodies were affinity purified from serum using the above mentioned peptides immobilized on an Amino-Link column (Pierce Chemical, Rockford IL). Purified antibodies were eluted from the columns using the Immunopure buffer system (Pierce Chemical), and concentrated and equilibrated into Dulbecco’s phosphate buffered saline (DPBS: 8 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 0.5 mM MgCl2, 0.04% NaN3, pH 7.25) using Centricon-10 filters (Amicon Co., Beverly, MA). Isolation of rat liver subcellular fractions All liver fractionation steps were performed at 4°C. First, fractions

enriched in specific organelles were prepared from rat liver by combining the crude liver fractionation scheme of de Duve et al. (1955) with a method used to subfractionate total liver microsomes (Larkin and Palade, 1991). Rats were anesthetized with pentobarbital and the livers perfused with buffer A (100 mM Tris-base, 0.25 M sucrose plus multiple protease inhibitors (10 µg ml−1 leupeptin, 10 µg ml−1 tosyl arginine methyl ester, 1 µg ml −1 pepstatin, 10 mM benzamidine, 0.1 mg ml−1 soybean trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride) via the portal vein to remove blood. Livers were then homogenized in two volumes of buffer A by ten passes of a Potter-Elverjeim homogenizer at 2000 rpm. The homogenate was centrifuged at 800 g for 10 minutes and the supernatant was saved; the pellet was homogenized twice (single pass of the homogenizer at 2000 rpm) in buffer A and centrifuged as above to prepare a nuclearenriched (N) pellet (de Duve et al., 1955). The postnuclear supernatants were pooled and centrifuged at 36,000 g for 11 minutes; the pellet was twice homogenized (single pass at 2000 rpm) in buffer A and centrifuged at 36,000 g for 11 minutes to prepare a pellet enriched in mitochondria and lysosomes (ML). The remaining supernatants were pooled and centrifuged at 105,000 g for 90 minutes to separate a total microsome pellet (TM fraction) from the final supernatant or cytosol (S). The total microsome pellet was resuspended in buffer A and fractionated through two sucrose gradient centrifugation steps as detailed previously (Larkin and Palade, 1991) to prepare fractions enriched in Golgi membranes (Golgi light (GL) and Golgi heavy (GH)), transcytotic carriers (TC), secretory vesicles (SV) and crude endoplasmic reticulum (ER). In addition, we used separate techniques to prepare liver fractions enriched in specific organelles. Hepatic lysosomes were purified by the Ca2+ shift method of Yamada et al. (1984). Intact Golgi cisternae were isolated from rat liver by the method of Hamilton et al. (1991). Hepatic mitochondria were prepared from rat liver by sucrose gradient centrifugation as described previously (Rickwood et al., 1987). Fractions enriched in late endosomes were prepared as described (Mullock and Luzio, 1992). Each technique was carried out as described except that homogenization buffers contained protease inhibitors as listed above. Total protein contents in liver subcellular fractions were measured using the BCA method (Pierce Chemical, Rockford IL) after solubilization of fractions in 1% SDS. Enzymatic activities in subcellular fractions of β-N-acetylglucosaminidase (β-NAG), a lysosomal marker enzyme, and malate dehydrogenase (MDH), a mitochondrial enzyme, were measured as previously described (Dupourque and Kun, 1969; LaRusso and Fowler, 1979). Enrichment for α-man II in Golgi fractions was determined by quantitative immunoblotting as described below for kinesin, except that polyclonal anti-α-man II antibody (1:1000 dilution) was used as the primary antibody. SDS-PAGE and immunoblotting Kinesin-containing fractions were run on 8.5% acrylamide SDSPAGE gels with dithiothreitol, using the method of Laemmli (1970) with 20:1 (v/v), acrylamide:bis-acrylamide (Porter et al., 1987). Protein bands in gels were visualized by Coomassie Blue staining. For immunoblots, proteins were transferred from gels to polyvinyldifluoride by established methods (Towbin et al., 1979). Blots were blocked with 5% bovine serum albumin, incubated with polyclonal antikinesin antibodies, diluted to 1:1000 followed by 1:2000 goat antirabbit-alkaline phosphatase (Tago Immunochemicals, Burlingame CA) and then visualized as previously described (Dubreuil et al., 1985). Signals detected on blots were scanned using a UMAX ultravision scanner attached to a MacIntosh Quadra 700; data were quantified using the Image 1.47 program. Samples of purified rat liver kinesin were run as internal standards on all kinesin blots. Cell culture and immunofluorescence Clone 9 cells (a rat hepatocyte-derived cell line from the American Type Culture Collection, Rockville MD) were grown on glass cover-

Golgi-associated kinesin 2419 slips in DMEM with 10% FBS. Rat hepatocytes were isolated by the method of Seglen (1976) and cultured on type I rat tail collagencoated coverslips in DMEM supplemented with 10% FBS, 0.5 i.u./ml insulin, 20 ng/ml EGF, 7.5 µg/ml hydrocortisone, 200 µg/ml streptomycin, and 200 i.u./ml penicillin for 2-4 hours. Cells were fixed for 10 minutes with 2% formaldehyde, permeabilized with Triton X-100 (0.1% for 4 minutes for clone 9 cells; 0.2% for 10 minutes for hepatocytes), quenched with 0.01 M glycine (3× 5 minutes), washed with DPBS and incubated for 1 hour at room temperature with blocking buffer (5% normal goat serum, 5% glycerol in DPBS). Cells were then incubated with primary antibodies overnight at 4°C. Primary antibodies used were 1:400 diluted affinity-purified polyclonal antikinesin antibody, undiluted SUK4 hybridoma supernatant, 1:1000 polyclonal anti-α-man II and 1:1000 monoclonal anti-α-man II. After washing with DPBS, cells were incubated in appropriate secondary antibodies for 1 hour at room temperature. Secondary antibodies used were 1:500 diluted FITC-conjugated goat anti-rabbit, 1:500 FITCconjugated goat anti-mouse, 1:200 TRITC-conjugated goat anti-rabbit (TAGO Immunochemicals, Burlingame, CA), and 1:200 TRITC-conjugated goat anti-mouse (Kirkegarde and Perry, Gaithersburg, MD). The cells were then washed with DPBS and mounted on slides in Slowfade (Molecular Probes, Eugene, OR). In double-label immunofluorescence experiments, cells were incubated simultaneously with polyclonal and monoclonal primary antibodies, washed and then incubated simultaneously with two secondary antibodies conjugated to different fluorophores. All other procedures were as described above for single label immunofluorescence.

RESULTS Characterization of polyclonal kinesin antibodies To study the distribution of kinesin in hepatocytes, we prepared polyclonal antibodies to synthetic peptides representing two different conserved regions of the kinesin Mt-binding domain (KKLSGKLYLVDLAGSEKVSKTGAEG (MMR43 and 44) and HIPYRDSKLTRILQESLGGNARTT (MMR48)). Because all three of these antibodies gave identical results by immunoblotting and immunfluorescence, we only present data for MMR44. Characterization of these antibodies was performed by immunoblotting kinesin-containing homogenates from rat liver, rat brain and hepatocyte-derived clone 9 cells. As shown in Fig. 1, MMR44 recognizes a single band (molecular mass ~120 kDa) in homogenates from whole liver (L) and clone 9 cells (C9). In brain homogenate (Br), however, MMR44 recognizes three protein bands, including a prominent doublet at ~120 kDa and a single band at ~130 kDa. The usefulness of MMR44 for quantitative immunoblotting was established by blotting serial dilutions of purified liver kinesin. Kinesin signals detected on blots were linearly related to the amount of kinesin loaded between 50 and 300 ng (data not shown). In addition, MMR44 immunoprecipitates a single ~120 kDa protein band from hepatocyte homogenates that is recognized by multiple kinesin antibodies via immunoblotting (data not shown). Thus, from these biochemical criteria, we are confident that MMR44 recognizes, and is specific for, kinesins from different cells and tissues, making it a useful tool for the studies described below. Kinesin is associated with Golgi-enriched fractions from rat liver To determine the intracellular distribution of kinesin in rat liver, we isolated crude subcellular fractions and fractions

Kinesin heavy chain

Kinesin light chains Tubulin

L

Br

C9

K

K'

Fig. 1. The polyclonal antibody, MMR44, recognizes kinesin in tissue and cell homogenates. Homogenates of liver (L), brain (Br), clone 9 cells (C9), and purified liver kinesin (K) were run on SDSPAGE gels, transferred to polyvinyldifluoride membranes and probed with MMR44. For comparison, a Coomassie Blue-stained gel of purified liver kinesin is shown (K′). From left to right, protein loaded per lane was 50, 50, 15, 0.02 and 5 µg. In liver and clone 9 cells, a single kinesin heavy chain band (molecular mass ~120 kDa) is recognized; while in brain, a doublet (molecular mass ~120) and an upper band (molecular mass ~130 kDa) are detected.

Table 1. Relative enrichment of organelle marker enzymes in liver fractions Fold enrichment* Golgi Lysosomes Mitochondria (α-man II)† (β-NAG)‡ (MDH)§ Crude fractions Nuclear pellet (N) Lysosomal/mitochondrial pellet (LM) Total microsomes (TM) Cytosol (S) Organelle-enriched fractions Golgi heavy (GH) Golgi light (GL) Intact Golgi (IG) Transcytotic carriers (TC) Secretory vesicles (SV) Crude endoplasmic reticulum (ER) Mitochondria (M) Purified lysosomes (L) Endosomes (E)

0.25 3.00 0.16