©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of the Microtubule Cytoskeleton with Endocytic Vesicles and Cytoplasmic Dynein in Cultured Rat Hepatocytes (*)

Hitoshi Oda (1), Richard J. Stockert (1), Christine Collins (3), Hali Wang (2), Phyllis M. Novikoff (1), Peter Satir (2), Allan W. Wolkoff (1) (2)(§)

From the (1)Marion Bessin Liver Research Center and (2)Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461 and the (3)Department of Cell, Molecular, and Structural Biology, Northwestern University Medical School, Chicago, Illinois 60611

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In a recent study (Goltz, J. S., Wolkoff, A. W., Novikoff, P. M., Stockert, R. J., and Satir, P.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7026-7030), we found that ligand- and receptor-containing endocytic vesicles bind to endogenous microtubules in vitro after 60 min of receptor-mediated endocytosis of asialo-orosomucoid. In the presence of ATP, ligand-containing endocytic vesicles are released from microtubules, while those containing receptor are not. We hypothesized that cytoplasmic dynein may associate with ligand-containing, but not receptor-containing, domains of endocytic vesicles and might be involved in the movement of ligand-containing vesicles along microtubules during sorting of ligand from receptor. Direct evidence in support of this hypothesis has been obtained in the present study. Binding of ligand-containing vesicles to microtubules correlates highly (p < 0.001) with binding of dynein, but not kinesin, under a variety of conditions. Binding of receptor-containing vesicles to microtubules is independent of both cytoplasmic dynein and kinesin binding. Tight association of cytoplasmic dynein with a population of ligand-containing vesicles is seen directly by immunoprecipitation. These results support the view that in receptor-mediated endocytosis, ligand-containing vesicles become bound to microtubules by cytoplasmic dynein. While receptor domains of endosomes remain attached to microtubules in an ATP-independent manner, ligand-containing domains might be moved away toward pericentrosomal lysosomes by this motor molecule.


INTRODUCTION

Receptor-mediated endocytosis (RME)()of asialo-orosomucoid (ASOR) in hepatocytes is initiated by binding of ligand to specific cell-surface receptors. Following internalization, the ligand-receptor complex dissociates within endosomes, and segregation of ligand and receptor into distinct cellular compartments occurs. While receptor returns to the cell surface where it is reutilized, ligand is subsequently delivered to lysosomes for degradation(1, 2, 3, 4, 5, 6) . Treatment of cells with cytoskeletal inhibitors, such as nocodazole or colchicine, inhibits segregation (4) and degradation of ligand(7, 8, 9, 10) . In particular, it was found that microtubule (MT) inhibitors markedly delayed segregation of ligand and receptor into functionally discrete endosomal compartments. Based on these initial observations, we hypothesized that interaction of specific populations of late endocytic vesicles with a polarized network of microtubules in hepatocytes might be important in establishing their vectorial movement(11) . Although cytoplasmic dynein, a MT-activated ATPase that has been implicated in centripetal (minus end-directed) movement of vesicles and organelles along MTs (12-21), was suggested as a possible linking protein that could provide a vectorial force sufficient for ligand-receptor segregation, evidence for its association with components of the endocytic pathway, based largely on light microscopy immunofluorescent studies, was tenuous.

In a recent study, we found that ligand- and receptor-containing endocytic vesicles bind to stabilized endogenous MTs in vitro after 60 min of RME of ASOR(11) . In the presence of ATP, ligand-containing endocytic vesicles are released from MTs, while those containing receptor are not(11) . Several proteins, including cytoplasmic dynein, associate with MTs and are released by ATP addition, as determined by SDS-PAGE analysis. Based on this preliminary information, we hypothesized that cytoplasmic dynein may associate with ligand-containing, but not receptor-containing, domains of endocytic vesicles and might be involved in the movement of ligand-containing vesicles along MTs during sorting of ligand from receptor. Direct evidence in support of this hypothesis has been obtained in the present study. Earlier methods (11) have been modified to quantify endocytic vesicles and cytoplasmic dynein that bind to MTs in the presence of nucleotides. In some experiments, we have also quantified MT-bound kinesin, the plus-directed motor protein that may mediate anterograde organelle transport(18, 19, 22, 23, 24, 25, 26) . Binding of ligand-containing vesicles correlates highly with binding of dynein, but not kinesin, to MTs under a variety of conditions. Binding of receptor-containing vesicles is independent of both cytoplasmic dynein and kinesin binding. Most importantly, tight association of cytoplasmic dynein with a population of ligand-containing vesicles derived from a defined initiation of RME can now be demonstrated directly by immunoprecipitation.


EXPERIMENTAL PROCEDURES

Chemicals

ASOR was prepared from human orosomucoid (Sigma) by neuraminidase digestion (4) and was labeled with I by a chloramine-T method to a specific activity of 10,000-13,000 cpm/ng(4) . Rabbit anti-chicken embryo tubulin antibody, mouse monoclonal anti-74-kDa cytoplasmic dynein intermediate chain (IC) antibody, mouse monoclonal anti-130-kDa kinesin head chain (HC) antibody, mouse monoclonal anti--tubulin antibody, goat anti-rabbit IgG conjugated to peroxidase, goat anti-mouse IgM conjugated to peroxidase, goat anti-mouse IgM-agarose, and mouse IgM (MOPC-104E) were purchased from Sigma. Goat anti-mouse IgG conjugated to either fluorescein isothiocyanate (FITC) or Cy3 and goat anti-mouse IgM conjugated to FITC, Cy3, or Cy5 were obtained from Jackson ImmunoResearch Lab. Inc. (West Grove, PA). Rabbit anti-rat asialoglycoprotein receptor antibody (5) and rabbit anti-calf brain cytoplasmic dynein antibody (27) were prepared as previously described. ATP, ADP, AMP, AMP-PCP, AMP-PNP, GTP, CTP, adenosine, and vanadate were purchased from Sigma or Boehringer Mannheim. Taxol (Molecular Probes Inc., Eugene, OR) was dissolved in dimethyl sulfoxide and stored as a 1 mM stock solution at -20 °C.

Preparation of Short-term Cultured Rat Hepatocytes

Rat hepatocytes were isolated from 200-250-g male Sprague-Dawley rats (Taconic Farms, Germantown, NY) after perfusion of the liver with collagenase (Type IV, Sigma) and were cultured overnight as we have previously described(3, 4, 11, 28) .

Confocal Microscopy

For confocal microscopy studies, hepatocytes were cultured on collagen-coated coverslips as previously described(28) . At various times, hepatocytes were permeabilized in MEPS buffer (5 mM MgSO, 5 mM EGTA, 35 mM K PIPES, and 0.2 M sucrose, pH 7.1), containing 0.1 M GTP and 0.1% Triton X-100 for 1 min at 37 °C, and subsequently fixed in a 0.25% glutaraldehyde/MEPS mixture for 5 min at 37 °C. After washing, the permeabilized cells were exposed either to mouse monoclonal anti--tubulin antibody or anti-74-kDa dynein IC overnight at 0 °C and then rinsed and exposed to either FITC- or Cy3- labeled secondary antibody against mouse IgG for localization of microtubules or either FITC- or Cy3-labeled secondary antibody against mouse IgM for localization of dynein. Controls for nonspecific fluorescence binding of secondary antibodies were performed. In some experiments, continuous wave endocytosis of Texas Red-ASOR was established, and fluorescent endosomes and MTs were colocalized. The preparations were examined with a confocal fluorescent microscope (Bio-Rad 600) equipped with a krypton/argon laser at the appropriate wavelengths. Several optical sections of permeabilized cells were scanned for three-dimensional reconstruction.

Preparation of Endocytic Vesicles Bound to MTs during RME of I-ASOR

Cells were washed with serum-free medium (SFM), preincubated for 60 min at 37 °C, and then incubated for 60 min at 4 °C with I-ASOR (1.25 µg) in 3 ml of SFM to saturate cell surface receptors(4, 11) . Cells were washed with SFM at 4 °C to remove unbound ligand and were incubated at 37 °C for 60 min to initiate a synchronous single wave of RME. Cells were harvested into MEPS buffer (1.5 ml per 5 dishes) containing protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 20 µg/ml N--benzoyl-L-arginine methyl ester, 20 µg/ml soybean trypsin inhibitor, 20 µg/ml p-tosyl-L-arginine methyl ester, 2 µg/ml leupeptin) and 1 mM dithiothreitol(29) . After cells were homogenized by 20 strokes in a tight Dounce on ice, the homogenate was centrifuged at 1,000 g for 10 min at 4 °C. This supernatant was centrifuged at 40,000 g for 20 min at 4 °C(11) . The supernatant (crude cytosolic extract) was incubated at 37 °C for 30 min with 20 µM taxol to polymerize endogenous tubulin. Following an additional timed incubation at 37 °C with or without various concentrations of ATP, the incubation mixture was centrifuged at 16,000 g for 30 min at 4 °C to sediment MTs and associated endocytic vesicles and proteins. In some experiments, 40,000 g supernatants were kept at 4 °C for 60 min without taxol addition prior to centrifugation at 16,000 g. In other experiments, various nucleotides, adenosine, and vanadate were used instead of ATP. The pellet was dissolved in 0.5 ml of sample buffer, consisting of 2.3% SDS, 10% glycerol, 5% -mercaptoethanol, and 62.5 mM Tris, pH 6.8(28) . All nucleotides and adenosine were dissolved in MEPS buffer and adjusted to pH 7.1 with NaOH just before use. A small aliquot of the supernatant was used for protein determination according to Lowry et al.(30) .

Quantitative Analysis of Ligand-containing Vesicles Bound to MTs

The pellet dissolved in sample buffer was subjected to 10% SDS-PAGE. The dried gel was exposed to X-OMAT AR film (Eastman Kodak Co.) for 6-18 h at -70 °C. The amount of I-ASOR bound to MTs was estimated by densitometric analysis of autoradiograms using an UltraScan XL scanner (Pharmacia LKB Biotechnology, Uppsala, Sweden).

Quantitative Immunoblot Detection of Tubulin, Receptor, Cytoplasmic Dynein, and Kinesin in the MT Pellet

Following 5, 7.5, or 10% SDS-PAGE, proteins were transferred to 0.45-µm pore size nitrocellulose paper (Micron Separations Inc., Westboro, MA) in a Trans-Blot SD Cell (Bio-Rad) at 200 mA for 1 h. After transfer, the nitrocellulose sheet was soaked for 1 h in 3% nonfat dry milk in 0.15 M NaCl containing 50 mM Tris, pH 7.4 (TBS), followed by 1 h of shaking in rabbit anti-chicken embryo tubulin antibody (diluted 1:50 in 3.5% bovine serum albumin and 0.1% NaN in TBS), rabbit anti-rat asialoglycoprotein receptor antibody (diluted 1:100), rabbit anti-calf brain cytoplasmic dynein antibody (diluted 1:500), or mouse anti-130-kDa kinesin HC antibody (diluted 1:500). Each sheet was washed for 1 h in 1% Tween 20 in TBS and was agitated gently for 1 h in goat anti-rabbit IgG conjugated to peroxidase (diluted 1:1,000 in TBS) or anti-mouse IgM conjugated to peroxidase (diluted 1:1,000), as appropriate, followed by washing three times in 1% Tween 20 in TBS. IBI Enzygraphic Web (International Biotechnologies, Inc., New Haven, CT) was used for colorimetric detection of peroxidase-linked probes. All procedures were performed at room temperature. The amount of tubulin, receptor, cytoplasmic dynein, or kinesin in the MT pellet was estimated by densitometric analysis of each immunoblot using an UltraScan XL scanner.

Immunoprecipitation of Endocytic Vesicles with Dynein Antibody

Following a 60-min single wave of RME of I-ASOR, the 40,000 g supernatant of hepatocytes was incubated at 37 °C for 30 min with 20 µM taxol for MT polymerization. After an additional 30-min incubation at 37 °C with 10 mM ATP to release cytoplasmic dynein and endocytic vesicles from MTs, the incubation mixture was centrifuged at 16,000 g for 30 min at 4 °C. This supernatant (tubulin free) was collected and incubated overnight at 4 °C with monoclonal anti-74-kDa cytoplasmic dynein IC antibody, 130-kDa anti-kinesin HC antibody, or non-immune IgM. After addition of secondary antibody (anti-mouse IgM)-agarose beads, each mixture was incubated at 4 °C for 4 h. After low speed centrifugation, the pellet was washed four times in 50 mM Tris, pH 8.0, containing 150 mM NaCl and 2.5 mM EDTA. The pellet was heated at 90 °C for 10 min in 200 µl of sample buffer and was subjected to SDS-PAGE and radioautography to detect ligand or immunoblot to detect receptor.

Statistical Methods

Results are expressed as means ± S.E. Significance was evaluated by one-way analysis of variance. Correlations between either cytoplasmic dynein or kinesin and endocytic vesicles containing either ligand or receptor bound to MTs in the presence of various nucleotides were tested by linear regression analysis.


RESULTS

Immunolocalization of Dynein, Tubulin, and Ligand

Using confocal microscopy, we have confirmed earlier observations (11) that following RME, ligand-containing endosomes are found in linear arrays that lie in close association parallel to hepatocyte MTs in situ (data not shown). We have now shown that cytoplasmic dynein colocalizes with MTs in these cells (Fig. 1). Cytoplasmic dynein, as seen in Fig. 1b, is abundant in hepatocytes. Under the conditions of permeabilization in the absence of ATP, the major localization is clearly coincident (Fig. 1c) with the microtubular cytoskeleton (Fig. 1a). When primary antibody against cytoplasmic dynein is omitted, only background fluorescence that is not coincident (Fig. 1f) with MTs (Fig. 1d) is still seen (Fig. 1e).


Figure 1: Colocalization by confocal microscopy of cytoplasmic dynein and MTs in short-term cultured rat hepatocytes. Cultured rat hepatocytes were prepared, permeabilized, and fixed as described under ``Experimental Procedures.'' Panelsa-c represent cells incubated with antibodies to tubulin and dynein visualized with FITC- (a) or Cy3- (b) conjugated second antibodies, respectively. Overlay of both channels is seen in panelc and demonstrates coincidence of cytoplasmic dynein with microtubules (yellow). Panelsd-f represent another group of cells exposed to antibody to tubulin but not to cytoplasmic dynein. These cells were exposed to both secondary antibodies and serve as a control. When primary antibody against cytoplasmic dynein is omitted, a small amount of background fluorescence is seen (e), but this is not coincident with MTs (f).



Time Dependence of in Vitro Polymerization of Endogenous Tubulin

We have modified our previous qualitative assay (11) to develop an assay to quantify the association of cytoplasmic dynein and ligand-containing endosomes in vitro. The zero time points in each panel in Fig. 2represent aliquots of 40,000 g supernatant, which were kept at 4 °C for 60 min without taxol addition prior to centrifugation at 16,000 g. Under this condition, there was no recovery of tubulin, dynein, ligand, receptor, or kinesin (Fig. 2). This indicates that in the absence of microtubules, there is no sedimentation of endocytic vesicles at 16,000 g. Although GTP hydrolysis is needed to promote the assembly of tubulin(32, 33) , there was little difference in tubulin recovery in the MT pellets from crude cytosolic extracts incubated in 20 µM taxol at 37 °C (34) with or without 1 mM GTP (data not shown). We therefore polymerized MTs in the presence of taxol but not GTP as described above. Formation of polymerized MTs from endogenous tubulin during incubation at 37 °C with 20 µM taxol was linear for 30 min, after which a plateau was reached (Fig. 2a). Virtually all initially soluble tubulin was recovered in this pellet. In a preliminary experiment, immediate addition of 5 mM ATP to the incubation mixture reduced the amount of tubulin in the MT pellet (data not shown). However, addition of ATP after a 30-min preincubation with taxol had no effect on the recovery of MTs in the pellet (Fig. 2a). The amount of tubulin in the MT pellet increased linearly with the protein concentration of hepatic crude cytosolic extract from 1.8 to 7.2 mg/ml. We therefore chose 30 min of preincubation with 20 µM taxol, 30 min of additional incubation with or without 5 mM ATP, and 4-6 mg/ml of hepatic crude extract protein concentration as the optimal assay conditions.


Figure 2: Effects of incubation time on the polymerization of tubulin and on binding to MTs of cytoplasmic dynein, kinesin, and endocytic vesicles containing ligand or receptor. Rat hepatocytes were cultured overnight as previously described (3, 4, 11, 28). Cells were then washed with SFM and incubated for 60 min at 4 °C with I-ASOR (1.25 µg/3 ml) to saturate surface receptors. Unbound ligand was removed, and cells were incubated for 60 min at 37 °C to initiate a synchronous single wave of RME. Cells were then harvested into MEPS buffer containing protease inhibitors. They were homogenized by 20 strokes in a tight Dounce on ice, and the homogenate was centrifuged at 1000 g for 10 min at 4 °C. The supernatant was centrifuged at 40,000 g for 20 min at 4 °C. This supernatant was incubated at 37 °C for various times with 20 µM taxol to polymerize endogenous tubulin. After 30 min of incubation, some tubes were made 5 mM in ATP, as indicated. For all samples at the indicated time,, the mixture was centrifuged at 16,000 g for 30 min at 4 °C to sediment MTs and associated endocytic vesicles and proteins. The zero time point in these studies represents 40,000 g supernatants that were incubated at 4 °C for 60 min without taxol addition prior to centrifugation at 16,000 g. The 16,000 g pellets were analyzed for contents of tubulin (a), cytoplasmic dynein (b), ligand (c), receptor (d), and kinesin (e). Values are expressed as a percent of the total amount of the corresponding molecule quantified after SDS-PAGE of the 40,000 g supernatant. It should be emphasized that in the absence of MT polymerization (zero time), none of these constituents are pelleted at 16,000 g.



Time Dependence of in Vitro Binding of Cytoplasmic Dynein and Endocytic Vesicles to Polymerized MTs

During incubation at 37 °C, binding of cytoplasmic dynein to MTs increased for 90 min and then plateaued when approximately 50% of initially soluble dynein pelleted with MTs (Fig. 2b). Binding of ligand-containing endocytic vesicles to MTs paralleled that of cytoplasmic dynein (Fig. 2c). In contrast, binding of receptor-containing vesicles to MTs reached a plateau within 30 min when 80% or more of receptor pelleted with MTs (Fig. 2d), similar to results for MT polymerization. Only a small percent of total soluble kinesin associated with MTs prepared under these conditions (Fig. 2e). Addition of 5 mM ATP after a 30-min preincubation with taxol resulted in inhibition of binding of cytoplasmic dynein and kinesin to MTs. Binding of ligand-containing endocytic vesicles was also inhibited, but there was no effect of ATP on binding of receptor-containing vesicles (Fig. 2, b-e).

ATP Concentration Dependence of Binding of Cytoplasmic Dynein, Kinesin, and Endocytic Vesicles to MTs

Upon addition for 30 min of increasing concentrations of ATP (0.5-10 mM), binding of ligand-containing vesicles to MTs was reduced in parallel to binding of cytoplasmic dynein and also to kinesin, while binding of receptor-containing vesicles to MTs was unchanged (Fig. 3a). Under these conditions, there was no effect on recovery of tubulin in the MT pellet (Fig. 3a). Double-reciprocal plots of the fractional inhibition of binding of cytoplasmic dynein, kinesin, and ligand-containing vesicles versus [ATP] to MTs were linear (Fig. 4). The apparent values for K and maximal inhibition of binding to MTs are shown in .


Figure 3: Relative content of tubulin (), cytoplasmic dynein (▾), kinesin (), ligand (), and receptor () in the MT pellet as a function of nucleotide concentration. Taxol-treated supernatants, prepared as in the legend to Fig. 2, were incubated at 37 °C for 30 min prior to addition of various concentrations of ATP (a) or AMP-PNP (b) for 30 min before a 16,000 g MT pellet was prepared. The amount of tubulin (n = 6), cytoplasmic dynein (n = 5), ligand (n = 6), receptor (n = 6), and kinesin (n = 1) are expressed as a percentage of control values (incubation without nucleotide). Data represent means ± S.E.




Figure 4: Inhibition by ATP of binding of cytoplasmic dynein, kinesin, and ligand-containing vesicles to MTs. Data here correspond to data in Fig. 3 and are presented as a double reciprocal plot of the percent inhibition of MT binding versus [ATP]. The apparent values for K and maximal inhibition of binding to MTs are shown in Table I.



Effect of AMP-PNP on Binding of Cytoplasmic Dynein, Kinesin, and Endocytic Vesicles to MTs

The non-hydrolyzable ATP analog AMP-PNP was substituted for ATP in the previous experiment (Fig. 3b). With increasing concentrations of AMP-PNP, binding of cytoplasmic dynein and ligand-containing vesicles to MTs still decreased in parallel, while binding of receptor-containing vesicles to MTs and recovery of tubulin in the MT pellet remained unchanged. In contrast to results with ATP, binding of kinesin to MTs did not change in the presence of AMP-PNP.

Nucleotide Specificity

The effects of 5 mM concentrations of nucleotides and adenosine and 50 µM vanadate on binding of cytoplasmic dynein, kinesin, and endocytic vesicles to MTs are summarized in . None of the agents affected either receptor-containing vesicles bound to MTs or MT polymerization as measured by percent recovered in the pellet. GTP reduced binding of cytoplasmic dynein, kinesin, and ligand-containing vesicles to MTs only slightly less effectively than did ATP. CTP also reduced binding of cytoplasmic dynein and ligand-containing vesicles to MTs but had no significant effect on binding of kinesin. ADP, and to a lesser extent AMP, reduced binding of cytoplasmic dynein and ligand-containing vesicles to MTs. However, utilizing P nuclear magnetic resonance(35) , we found that ADP was converted rapidly to ATP in the hepatic extract, probably by myokinase activity. AMP-PCP, adenosine, and vanadate had no effect on binding of ligand-containing vesicles or cytoplasmic dynein.

Correlation between MT Binding of Cytoplasmic Dynein or Kinesin and Endocytic Vesicles

When data for all nucleotides were considered, high correlation was found between MT binding of ligand-containing vesicles and cytoplasmic dynein (r = 0.77, n = 62, p < 0.001) (Fig. 5a). In contrast, MT binding of cytoplasmic dynein did not correlate with binding of receptor-containing vesicles (r = 0.06, n = 62, p > 0.6) (Fig. 5b). There was no correlation between MT binding of kinesin and that of endocytic vesicles containing either ligand (r = 0.25, n = 13, p > 0.4) or receptor (r = 0.15, n = 13, p > 0.6) (Fig. 5, c and d).


Figure 5: Correlation between cytoplasmic dynein or kinesin and ligand or receptor in the MT pellet. Individual data points used to generate the means in Table II are shown here. Significant correlation is seen only for ligand-containing vesicles and dynein (r = 0.77, n = 62, p < 0.001). MT binding of cytoplasmic dynein did not correlate with binding of receptor-containing vesicles (p > 0.6). There was no correlation between MT binding of kinesin and that of endocytic vesicles containing either ligand or receptor. ATP, ; GTP, ; CTP, ; AMP-PNP, ; AMP-PCP, ; ADP, ; AMP, ; adenosine, ; and vanadate, .



Immunoprecipitation of Endocytic Vesicles with Dynein Antibody

To demonstrate directly that cytoplasmic dynein is bound to ligand-containing vesicles after RME, MTs were polymerized, and 10 mM ATP was added for 30 min as above. Following centrifugation, the supernatant was subjected to immunoprecipitation using monoclonal antibodies directed against the 74-kDa cytoplasmic dynein intermediate chain coupled to anti-IgM-agarose beads. Controls utilized isotype-specific non-immune IgM. In additional studies, immunoprecipitation was performed utilizing a monoclonal antibody to the 130-kDa kinesin heavy chain. Immunoprecipitates were analyzed for content of receptor and dynein by immunoblot. Ligand in the immunoprecipitate was quantified by radioautography. As seen in the toppanel of Fig. 6, cytoplasmic dynein, which is present in the initial ATP-containing supernatant, is not immunoprecipitated with non-immune IgM. However, dynein is quantitatively immunoprecipitated by the anti-dynein antibody. Both heavy chains and intermediate chains of cytoplasmic dynein have been identified in the immunoprecipitate. As anticipated, ligand-containing vesicles are also found in the supernatant. Immunoprecipitation with anti-dynein, but not control antibodies, precipitated approximately 15% of the total ligand from the supernatant. Immunoprecipitation of ligand-containing vesicles by kinesin antibodies was <1%. In confirmation of our results showing that receptor is not released from the MT pellet in the presence of ATP, there was no detectable receptor in the initial ATP-containing supernatant, nor was any seen in the dynein or kinesin immunoprecipitates.


Figure 6: Immunoprecipitation of ligand-containing vesicles by antibody to cytoplasmic dynein. Following a 60-min single wave of RME of I-ASOR, taxol-treated 40,000 g supernatants were incubated at 37 °C for 30 min with 10 mM ATP. The mixture was centrifuged at 16,000 g. The 16,000 g supernatant (tubulin free) was subjected to immunoprecipitation with IgM (non-immune (Non-Imm.), anti-dynein, or anti-kinesin) coupled to anti-IgM-agarose beads. Upper panel, immunoblot of 74-kDa cytoplasmic dynein intermediate chain. Lower panel, I-labeled ligand visualized by autoradiography. Lane 1, supernatant before immunoprecipitation. Lane 2, precipitate with isotype-specific non-immune IgM. Lane 3, precipitate with anti-74-kDa dynein IC antibody. Lane 4, precipitate with anti-130-kDa kinesin HC antibody.




DISCUSSION

A number of investigators have described a requirement for MTs in receptor-mediated endocytosis(4, 7, 8, 9, 10) , but the molecular basis for this requirement has been unclear. In an earlier study(11) , we presented evidence that ligand- and receptor-containing endocytic vesicles bind to stabilized endogenous MTs in vitro. In the presence of ATP, ligand-containing but not receptor-containing vesicles were released from MTs. We suggested that cytoplasmic dynein might functionally link the ligand-containing vesicles to the MTs so as to produce the firm attachment in the absence of ATP and release when ATP was added. Preliminary evidence was presented that ATP simultaneously released cytoplasmic dynein and ligand-containing vesicles from MTs. The present study quantitates and extends our previous findings and, further, presents direct evidence that cytoplasmic dynein binds to ligand-containing vesicles after RME. As previously suggested, this motor molecule could provide the force that is necessary for ligand-receptor segregation and/or for transport of ligand-containing vesicles to lysosomes. MTs are polarized with a plus (fast growing) end oriented toward the cell periphery and a minus (slow growing) end oriented toward a MT-organizing center or centrosome(36, 37, 38, 39) , which is perinuclear in hepatocytes. Lysosomes are typically abundant near the centrosome. Previous immunolocalization studies demonstrated association of endosomes and lysosomes with MTs (40) and colocalization of cytoplasmic dynein with lysosomes(27) . We have confirmed by electron and confocal microscopy that ligand-containing endocytic vesicles are associated with hepatocyte MTs. We now demonstrate by co-immunolocalization that in the absence of ATP, MTs in these hepatocytes become abundantly coated with cytoplasmic dynein. Some minor, nonspecific background fluorescence was present, but the procedure that we used for fixation and permeabilization of cells removed cytosolic proteins, including virtually all dynein not bound to MTs. The hepatocyte is known to have a high concentration of cytoplasmic dynein. The abundant distribution of cytoplasmic dynein along MTs in the absence of ATP before homogenization of the cells, the demonstration of its presence in the supernatant after homogenization and ATP addition, and the demonstration by immunoprecipitation that it is also bound to ligand-containing endocytic vesicles strongly support our hypothesis that cytoplasmic dynein is the ATP-sensitive link between ligand-containing endosomes and MTs that could function as the motor that moves endocytic vesicles toward lysosomes.

Several different hypotheses of how sorting occurs have been proposed (11, 41-43). Maxfield and colleagues(41) , in studies of the transferrin receptor performed in Chinese hamster ovary cells, described a sorting endosome with which early endosomes fuse and from which receptor is recycled. Eventually, this sorting endosome, enriched in ligand and depleted of receptors, ``matures'' and fuses with lysosomes. These findings are similar to those described in video microscopic studies of A431 cells following endocytosis of gold-labeled antibodies to the transferrin receptor(42) . Net centripetal movement of endosomes toward the juxtanuclear area was seen. From this area, small gold-containing vesicles were observed to pinch off and migrate toward the cell periphery. Disruption of MTs with nocodazole reduced both the juxtanuclear accumulation of endocytic vesicles and separation of recycling vesicles. Alternatively, Hopkins and colleagues (43) have studied Hep-2 cells, where the sorting endosome forms an extensive array of tubular cisternae. In this case, receptor may remain tacked to microtubules, while multivesicular bodies containing dissociated ligand move along the cisterns in a polar manner. We have suggested that the early endosome itself may be a sorting compartment in cultured hepatocytes from which ligand-containing vesicles bud in succession, while receptor domains cap and are carried back to the cell surface (11). Although the morphological details and perhaps the timing of sorting vary with cell types, all of the models are consistent with our current observations that suggest that endosomal maturation is MT-based and that ligand movement toward lysosomes is driven by cytoplasmic dynein.

The binding of dynein and ligand-containing endosomes to MTs is reduced in parallel in the presence of ATP and other nucleotides (). Consistent with these results are previous observations of Collins and Vallee(31) , who compared the rates of hydrolysis of several different nucleotides by cytoplasmic dynein that had been prepared from rat liver and testis. CTP, TTP, and UTP were the most rapidly hydrolyzed substrates, followed by GTP, ITP, and ATP(31) , although the relative affinity of cytoplasmic dynein for CTP was much lower than that for ATP (31). Only ATP, however, supports force production(14) , and only ATP would be expected to be present in cytosol in physiologically sufficient concentrations. The persistence of ATP in our incubation mixtures for up to 2 h is probably due to the presence of regenerating enzymes, perhaps myokinase, in the hepatic cytosolic extract.

Although the ligand-containing vesicles that have been released from MTs by ATP can be immunoprecipitated from the supernatant using antibodies to cytoplasmic dynein, these vesicles were not precipitated in control studies using non-immune antibodies or antibodies against kinesin. As seen in Fig. 6, although most dynein is immunoprecipitated, only approximately 15% of ligand was recovered in the immunoprecipitate. This may be due to the low probability that cytoplasmic dynein attached to MTs is associated with a specific ligand-containing vesicle population, to dissociation of dynein upon release from vesicles to which it had been bound, or to the possibility that cytoplasmic dynein associates with ligand-containing vesicles only through part of the endocytic pathway. It would be interesting to know whether the percentage of dynein associated with ligand-containing vesicles changes with different assay times and conditions. We know little as yet regarding the mechanism of binding of cytoplasmic dynein to ligand-containing vesicles. The dynactin complex (44, 45, 46) copurifies with liver cytoplasmic dynein and might serve as a ``bridge'' between vesicles and dynein. This possibility is being investigated.

In our studies, only a relatively small proportion (less than 10%) of available kinesin bound to MTs. This binding was reduced significantly in the presence of ATP or GTP. However, binding was slightly increased in the presence of AMP-PNP, although both cytoplasmic dynein and ligand binding fell (). These results are consistent with previous reports that kinesin has both ATPase and GTPase activities, is released from MTs in the presence of ATP (47, 48) or GTP(24, 25, 26) , but remains bound to MTs strongly in the presence of AMP-PNP(49) . This suggests that the kinesin binds to MTs independently of ligand-containing vesicles, such that the vesicles do not attach to MTs by kinesin, a conclusion supported by our immunoprecipitation results. Although it has been hypothesized that kinesin may be involved in the recycling of receptor to the cell surface after segregation, we found no association of kinesin with receptor-containing endocytic vesicles. However, since the amount of available kinesin associated with stabilized endogenous MTs is so small, it may be that factors necessary for this binding (23) are lacking in the assay conditions used in our studies.

Our current observations are consistent with two models, as diagrammed in Fig. 7. In these models, either before (a) or after (b) segregation of receptor-containing domains, the mature ligand-containing endosome is attached to and moves along a MT toward the centrosomally placed lysosomes via cytoplasmic dynein. Whether cytoplasmic dynein actually provides the force necessary for fission of the segregating endosomal vesicle is an important issue that is currently under investigation.


Figure 7: Hypothetical association of endocytic vesicles and cytoplasmic dynein with MTs during RME of ASOR in hepatocytes. Two potential models of the role of cytoplasmic dynein in endosomal sorting are shown. Both models are consistent with the data presented. a, cytoplasmic dynein becomes attached to the sorting endosome, pulling the endosome apart and moving the mature ligand-containing endosome toward the lysosome. b, cytoplasmic dynein attaches only to the mature ligand-containing endosome after segregation of receptor and ligand. Ligand, ; receptor, ; receptor-MT attachment molecule, ; vesicle-dynein attachment complex, possibly dynactin, ; dynein, .



  
Table: Analysis of inhibition by ATP of binding of cytoplasmic dynein and ligand-containing endocytic vesicles to MTs

Data, corresponding to Fig. 4, represent means ± S.E. (number of studies).


  
Table: The effects of nucleotides on binding to microtubules of cytoplasmic dynein, kinesin, and ligand- or receptor-containing endocytic vesicles and on the recovery of tubulin in the microtubule pellet

Following 30 min of incubation at 37 °C with 20 µM taxol to polymerize endogenous tubulin, 5 mM of each nucleotide or 50 µM vanadate was added. After an additional 30 min of incubation at 37 °C, the incubation mixture was centrifuged to sediment microtubules. Content of ligand- or receptor-containing endocytic vesicles, cytoplasmic dynein, kinesin, and tubulin in the microtubule-pellet was quantified as described under ``Experimental Procedures.'' The values are expressed as a percent of results obtained in the absence of nucleotides. Data represent means ± S.E.



FOOTNOTES

*
This work was supported by Program Project Grant DK-41918 and National Institutes of Health Grants CA-06576 and DK-41296. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Liver Research Ctr., Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2584. Fax: 718-918-0857.

The abbreviations used are: RME, receptor-mediated endocytosis; ASOR, asialo-orosomucoid; MT, microtubule; IC, intermediate chain; HC, head chain; FITC, fluorescein isothiocyanate; AMP-PCP, adenosine 5`-(,-methylene)triphosphate; AMP-PNP, adenosine -5`(,-imino)triphosphate; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; TBS, Tris-buffered saline; SFM, serum-free medium.


ACKNOWLEDGEMENTS

We thank Michael Cammer for expert assistance with the confocal microscopy studies. The Bio-Rad MRC 600 confocal microscope used in this study is part of the Image Analysis Facility of the Department of Anatomy.


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