©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Microtubule-independent Phospholipid Stimulation of Cytoplasmic Dynein ATPase Activity (*)

(Received for publication, September 27, 1994; and in revised form, December 27, 1994)

Kristina L. Ferro Christine A. Collins

From the Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611-3008

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In this study we report that phospholipid vesicles activate ATP hydrolysis by cytoplasmic dynein but not kinesin, consistent with reported differences in the organelle/vesicle binding of these motor proteins. Dynein activation by phospholipids was comparable with that seen in the presence of microtubules but was not sensitive to moderate salt concentrations and was independent of the net charge of the phospholipid, suggesting that the means of interaction between dynein and the lipid vesicle was not strictly ionic in nature. Based on this result, previous data that show that the interaction between dynein and vesicles is not ATP sensitive, and the concentration dependence observed for lipid activation of cytoplasmic dynein, it is likely that the binding interaction between dynein and liposomes is a stable one. In contrast to a previous report, microtubules increased the hydrolysis rate of all naturally occurring nucleotides tested, whereas only ATPase activity was stimulated by phospholipids. As ATP is the physiologically relevant substrate and is the only nucleotide to promote motility, the activation of only the ATPase by phospholipids may represent a means of discriminating between coupled and uncoupled nucleotide hydrolysis in vitro.


INTRODUCTION

One of the defining characteristics of a cytoskeleton-associated motor protein is the enhancement of nucleotidase turnover rates of the enzyme in the presence of the appropriate cytoskeletal element, microtubules, or filamentous actin. It has been shown for both axonemal dynein and kinesin that interactions of the motor protein with the microtubule enhance ADP release, the rate-limiting step in the enzymatic cycle(1, 2, 3) . Although similarly detailed analyses have not been performed for cytoplasmic dynein, several laboratories have examined other enzymatic properties of its microtubule-activated ATPase (4, 5, 6, 7, 8) . The effect of microtubules is to enhance the V(max) of the hydrolysis reaction, with a corresponding modest increase in the K for ATP(8) . Cytoplasmic dynein hydrolyzes nucleotides other than ATP at basal turnover rates much higher than that observed for ATP(6, 7, 9) . This property is characteristic of cytoplasmic dyneins from such diverse sources as mammalian tissues(6, 7, 9) , Dictyostelium(10) , Paramecium(11) , and sea urchin eggs (12) and has been used as a criterion to distinguish cytoplasmic from axonemal dyneins(7, 9, 11, 12) . The effect of microtubules on hydrolysis of other nucleotides by calf brain cytoplasmic dynein has been reported(6) , with GTPase and TTPase stimulated 1.3-fold but no effect on CTPase activity in the presence of 2.1 mg/ml microtubules.

We have been interested in the means of regulation of organelle and microtubule binding and motility activity of the microtubule motors in vivo and in vitro(13, 14, 15, 16) . Dynein and kinesin appear to behave differently in their binding interactions with organelles and in how their ATPase and force transduction cycles proceed. Cytoplasmic dynein has been found to bind saturably and with high affinity to NaOH-extracted and trypsin-digested organelles, and it binds with similar characteristics to phospholipid vesicles(17, 18) . Kinesin exhibits tight binding to organelles in the presence of moderate ionic strength buffers (19) and may require a vesicle receptor protein, kinectin, in order to bind to membrane fractions(20, 21) . Although the rate-limiting step for kinesin and dynein may be the same, the duty cycle and force transduction mechanism may be quite distinct (22, 23) . There is some suggestion that binding of kinesin to its target organelle (or inert latex particle or glass) may alter its ATPase activity(24) . It is an intriguing hypothesis that the ``cargo'' for the motor molecule may be involved in the regulation of its motility activity. We were interested in determining whether binding interactions between dynein and its target organelle lead to alterations in the activity or regulation of dynein ATPase. We report here that phospholipid vesicles stimulate dynein ATPase activity to levels comparable with those measured in the presence of microtubules, and that this effect is selective for ATP over other nucleotides.


EXPERIMENTAL PROCEDURES

Materials

Phosphatidylserine (PS) (^1)and phosphatidylcholine (PC) were obtained from Avanti Polar Lipids (Alabaster, AL). A crude phosphoinositide preparation (15-20% phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-diphosphate, with the remainder as phosphatidylinositol and PS) was obtained from Sigma. Nucleotides and most other reagents were obtained from Sigma. Male Sprague-Dawley rats between 150 and 250 g were used as the source of tissue. Taxol was generously provided by the Drug Synthesis and Chemistry Branch, Division of Cancer Research, National Cancer Institute.

Protein Preparation

Cytoplasmic dynein was purified from rat testis as described previously (7) and used for assays within 2 days of isolation. Microtubules were assembled from pure bovine brain tubulin (25) and equimolar taxol by incubation at 37 °C for 5 min and kept at room temperature prior to dilution for use in nucleotidase assays. Kinesin was purified from rat testis using the same procedure as for cytoplasmic dynein except that 2 mM 5`-adenylylimidodiphosphate was included during the microtubule assembly step to promote kinesin binding. Kinesin was recovered in the 10 S region of the sucrose gradient constituting the last step in the purification procedure. Protein content was determined using either BCA reagents (Pierce) or by quantitative densitometry of Coomassie-stained polyacrylamide gel lanes compared with standards of known protein concentration.

Liposome Preparation

Liposomes (small unilamellar vesicles) were prepared from pure phospholipids or mixtures of phospholipids by drying from chloroform solution, resuspension into Tris/KCl buffer (20 mM Tris-HCl, pH 7.6, containing 50 mM KCl, 5 mM MgCl(2), and 0.5 mMEDTA) and sonication with a probe sonicator until the solution clarified.

Nucleotidase Assays

Nucleotidase assays were conducted in Tris/KCl buffer with a final substrate concentration of 0.25 mM (for ATP) or 1 mM (for other nucleotides). Phosphate release was determined colorimetrically after incubation of the complete reaction mixture for 10-30 min at 37 °C(7) . Liposomes or taxol-stabilized microtubules were preincubated with dynein or kinesin samples for 5-10 min at room temperature prior to initiating the assay by the addition of nucleotide substrate.


RESULTS

Phospholipid Vesicles Stimulate Cytoplasmic Dynein ATPase Activity

The identification and isolation of cytoplasmic dynein from rat testis have been previously described(5, 7, 8) . Fig. 1A illustrates the final purification step of the procedure consisting of sucrose density gradient sedimentation of material eluted from the microtubules with ATP. Fractions in the 20 S density region are shown. The composition of these fractions has been analyzed(7, 8) , and they consist almost exclusively of polypeptides identified as subunits of dynein. A small amount of co-sedimenting proteins at 45, 50 and 150 kDa, components of the dynactin complex(26, 27) , are also present. These fractions were assayed for ATPase activity in the presence and absence of small unilamellar phospholipid vesicles prepared from a mixture of PC, a zwitterionic phospholipid, and PS, a phospholipid with a net negative charge. As shown in Fig. 1B, there was a lipid concentration-dependent increase in ATPase activity across the gradient, peaking in the same position as the basal ATPase activity and the dynein protein content. The maximal lipid effect observed in this experiment was an increase in ATPase activity of 2.4-fold over basal levels. The apparent K(m) for phospholipid in ATPase activation varied with dynein concentration in the assay. Analysis of the data from Fig. 1gave an apparent K(m) = 0.09 mg/ml phospholipid when assayed with 0.032 mg/ml dynein, and 0.055 mg/ml phospholipid at 0.013 mg/ml dynein.


Figure 1: The ATPase activity of sucrose gradient-purified cytoplasmic dynein is stimulated by phospholipids. A, sucrose gradient fractions in the 20 S region were analyzed by SDS-polyacrylamide gel electrophoresis and stained with Coomassie Blue. The positions of molecular weight standards, dynein heavy chain (HC), and intermediate chain (74K) are indicated. B, the corresponding gradient fractions were assayed for ATPase activity in the absence or presence of the indicated amounts of phospholipid, a 25:75 (w/w) mixture of PS and PC, in the form of small unilamellar vesicles.



To determine whether the ionic charge of the liposomes had an effect on the ATPase activity level, liposomes were prepared using varying mixtures of PS and at a total lipid concentration of 0.2 mg/ml. As shown in Fig. 2, increasing the PS content of the liposomes from 0 to 100% in a background of PC had little effect on the extent of enzyme activation (average increase of 2.5-fold over basal activity of 1.28 nmol/min/ml). The effect of other acidic phospholipids was tested by using a mixture of phosphatidylinositol phosphates, phosphatidylinositol, and PS, in combination with PC. The average activation of dynein ATPase in the presence of saturating amounts (0.2-0.4 mg/ml) of phosphoinositide-containing liposomes was 2.8 ± 0.2-fold (n = 4), compared with an average of 2.4 ± 0.2-fold using a mixture of PS and PC in the same experiments (data not shown). Varying the concentration of liposomes prepared from pure PS, pure PC, and a mixture of the two lipids did not reveal differences in the concentration dependence or the degree of dynein activation within a given experiment (Fig. 3).


Figure 2: The effect of acidic phospholipid content on dynein ATPase activation. Dynein ATPase was measured in the presence of liposomes at 0.2 mg/ml total lipid. The PS content was varied from 0 to 100 percent of total lipid, with the remaining fraction PC. The mean and the range of data obtained using one dynein preparation are shown.




Figure 3: Dose dependence of varying lipid mixtures on dynein ATPase activation. ATPase activity was measured with increasing concentrations of liposomes. Data from two experiments (two different dynein preparations) are shown, with a best fit line calculated from the average value for each of the data sets. (circle, bullet) 25:75 (w/w) mixture of PS and PC; (box, ) 100% PS; (up triangle, ) 100% PC.



Since dynein ATPase can be stimulated severalfold by microtubules, we examined whether the effects of microtubules and phospholipid vesicles were additive. In Table 1are shown the ATPase activities of dynein, measured in the presence of phospholipids and microtubules, alone, or in combination. The enhancement of ATPase rates observed in this experiment was slightly higher for microtubules than for phospholipid vesicles. When microtubules and lipids were combined, the resulting activation levels were intermediate between that of either agent added alone.



Cytoplasmic Dynein Exhibits Stable Interactions with Liposomes

The stimulation of cytoplasmic dynein ATPase by both neutral and acidic phospholipids suggested that the interaction between the protein and lipid was not strictly electrostatic. To test this further, we analyzed the response of basal, lipid-activated, and microtubule-activated dynein ATPase to increasing salt concentrations. It had been previously shown that microtubule stimulation of dynein ATPase was very sensitive to ionic strength, with a 37-fold decrease in the affinity of dynein for microtubules when the KCl concentration of the assay buffer was raised from 32 to 62 mM(6) . We found similar results for microtubule-stimulated dynein ATPase (data not shown) but only slight inhibition of the phospholipid-stimulated dynein ATPase at moderate ionic strengths (Fig. 4), with 90% activity remaining at 160 mM added NaCl. This suggests that the interaction between dynein and phospholipid vesicles is distinct from the ionic interactions between dynein and microtubules.


Figure 4: Ionic strength effects on activation by phospholipid. Dynein ATPase was measured in the absence or presence of liposomes as indicated, with increasing concentrations of NaCl added to the assay mixture. Average values from one representative experiment are shown.



The GTP-binding protein, dynamin, has recently been shown to be activated by an association with acidic phospholipids(28) . The lipid effect appeared to be due, at least in part, to the promotion of interactions between dynamin molecules, with concomitant increases in the specific activity of GTP hydrolysis with increasing enzyme concentration(29) . To test whether similar interactions between dynein molecules give rise to the lipid activation observed, the phospholipid requirement for maximal stimulation of cytoplasmic dynein at two different enzyme concentrations was analyzed (Fig. 5). The specific activity reached at both concentrations was the same, suggesting no interacting effects between dynein molecules. However, as was shown in Fig. 1, the apparent K(m) for lipid in the activation of dynein ATPase depended on the enzyme concentration. Dynein at 0.015 mg/ml was fully activated at 0.05 mg/ml phospholipid, whereas dynein at 0.03 mg/ml required 2-3 times as much phospholipid to reach the same specific activity. At the lower enzyme concentrations tested, a reproducible decrease in activity with increasing phospholipid concentration was also observed (Fig. 5, filledcircles).


Figure 5: Lipid activation is not enzyme concentration dependent. Dynein at two different concentrations (0.015 mg/ml (filledcircles) and 0.03 mg/ml (opencircles)) was assayed for phospholipid activation using a 25:75 mixture of PS and PC.



To determine whether lipid stimulation is a general property of microtubule-activated nucleotidases, we assayed the effect of phospholipid vesicles on kinesin ATPase. Kinesin was partially purified by 5`-adenylylimidodiphosphate-induced microtubule affinity followed by ATP extraction and sucrose density gradient centrifugation. The kinesin fractions contained other minor protein species but had no detectable dynamin or dynein polypeptides (data not shown). The activity of the kinesin preparation (basal ATPase = 55 nmol/min/mg of protein) was increased approximately 10-fold (to 538 nmol/min/mg) in the presence of microtubules at 0.6 mg/ml, but phospholipid vesicles had little effect on the ATP hydrolysis rate (58 nmol/min/mg) when added at 0.2 mg/ml. Concentrations of lipid mixtures (PS and PC) from 0.05 to 0.4 mg/ml had no effect when added alone, and phospholipid did not enhance or inhibit microtubule activation of kinesin (data not shown).

Phospholipid Stimulation of Dynein Is Specific for ATP Hydrolysis

Dynein is capable of hydrolyzing many other nucleotides, at higher velocity but with lower affinity than for ATP (6, 7) . However, either no microtubule activation or only slight increases in the hydrolysis of these alternative substrates with microtubule addition was reported(6) . In Table 2are shown the results of microtubule and phospholipid additions on nucleotidase activities of cytoplasmic dynein. As found previously, the hydrolysis rates for naturally occurring nucleotides other than ATP were substantially higher than for the presumed physiological substrate. The hydrolysis rates of all the substrates tested were increased in the presence of microtubules, ranging from an average of 2-fold activation of UTPase to 3.8-fold effects on ATPase. However, the hydrolysis of only ATP was increased in the presence of phospholipid vesicles, with an average of 2.0-fold activation in these experiments (range of 1.4-2.6-fold). Hydrolysis of GTP, UTP, and TTP was not stimulated by the presence of PS/PC liposomes, as all experimental hydrolysis rates were equal to or lower than the basal activites. In some experiments there was some enhancement of CTPase activity. The average CTPase in the presence of lipid was 1.1-fold compared with basal activity, with a range of values from 0.7- to 1.5-fold. As CTP has generally been found to be the most rapidly hydrolyzed substrate in other studies and CTPase activity has been used as a diagnostic marker for cytoplasmic dynein(6, 7, 9, 11, 12) , we compared the effect of microtubules and phospholipid vesicles on CTPase activity of cytoplasmic dynein in more detail (Fig. 6). CTP hydrolysis was activated 2.2-fold at the highest microtubule concentration tested, but only slight inhibitory effects of phospholipid addition on CTPase were observed.




Figure 6: Phospholipids do not activate dynein CTPase activity. Dynein CTPase activity was assayed in the presence of increasing concentration of liposomes (25:75 mixture of PS and PC, filledcircles) or taxol-stabilized microtubules (opencircles).




DISCUSSION

The motor proteins dynein and kinesin exert their force-producing activity along a microtubule polymer, with net translocation of organelles with which they are associated. For in vitro motility assays, this motion takes the form of microtubules gliding across a surface coated with motor protein or the translocation of inert beads to which the motor molecule has been bound along stationary microtubules. In the cell, it is expected that cytoplasmic dynein and kinesin are associated in some specific and regulated fashion with various membranous organelles and that directed transport of these vesicles leads to the phenomenon of fast axonal transport and corresponding intracellular movements in nonneuronal cells. There have been reported differences in the ability of extracts and purified motor molecules to translocate microtubules and membrane-bounded vesicles under the same assay conditions, suggesting that vesicle components or accessory proteins are involved in regulation of the organelle motility process(30, 31) . Alterations in the state of the motor molecule may also be involved, as both kinesin and cytoplasmic dynein are phosphorylated in vivo(16, 32, 33, 34) , and phosphorylation changes are correlated with differences in properties of the motor molecule(16, 32, 34, 35) . There is also a suggestion that kinesin undergoes a conformational change when bound to glass or organelle, leading to changes in enzymatic properties(24) . According to this model, a folded conformation of the kinesin ``tail'' (24, 36) is inhibitory for ATPase activity. The interaction between the carboxyl-terminal domain of kinesin (37) and its cargo relieves the inhibition by allowing the protein to take on an extended conformation. In support of this model, enhanced ATPase activity has been measured for a 45-kDa proteolytic fragment of kinesin, which lacks the carboxyl-terminal domain(38) . However, this fragment was not capable of promoting microtubule motility, and it is not known whether the observed conformational changes in the intact kinesin molecule are required for coupled force production.

Due to the size and complexity of the cytoplasmic dynein molecule, similarly detailed analyses of force transduction and enzymology have not yet been carried out. However, in models for cytoplasmic dynein function, it has been assumed that the association of dynein with its target organelle is a passive one, i.e. that this ``end'' of the molecule is not directly involved in the force transduction process. We and others have been investigating the means by which dynein associates in a specific and functional manner with different populations of intracellular organelles(13, 14, 15, 16, 17, 18, 19, 20, 21) . Purified dynein is competent to bind to a variety of isolated organelles, including membrane preparations salt-extracted or alkaline-treated to remove peripherally associated proteins(17, 21) . Protease treatment of organelles had little effect on the saturability or affinity of dynein binding, and dynein was found to bind to pure phospholipid vesicles with similar characteristics(17, 18) . In another study, however, binding of cytoplasmic dynein and kinesin to microsomal membranes was reduced following protease treatment, suggesting that transmembrane or tightly associated proteins may function as receptors or act to stabilize the bound motor complex(21) .

We have shown in this report that cytoplasmic dynein binding to phospholipid vesicles increases the hydrolysis rates of ATP but not other nucleotides. Our data showing that the concentration of lipid required to activate dynein ATPase increases with enzyme concentration suggests that the dynein is forming a relatively stable association with the liposome. The mechanism of activation is therefore unlikely to involve cyclical binding and release as proposed for microtubule effects on dynein and kinesin ATPase. Compared with activation by microtubules, phospholipid stimulation of dynein was less affected by ionic strength and was specific toward ATP hydrolysis. It has been suggested that in general the rates and regulation of ATP hydrolysis are a reflection of motility activity (e.g. 39, 40), but cytoskeletal motor proteins appear to be rather promiscuous in their substrate utilization for in vitro hydrolysis assays. In particular, Shimizu and co-workers (41, 42) found that there was no correlation with the ability of microtubules to activate hydrolysis of various ATP analogs by axonemal dynein and kinesin and the ability of those nucleotides to promote motility by those motor proteins. We showed here that, unlike data previously reported, microtubules stimulate dynein-mediated hydrolysis of all nucleotides tested. The reason for the discrepancy between these data and the previous study is unknown, but as microtubule activation is very sensitive to ionic strength, slight differences in assay conditions between the studies could account for the differences observed. However, unlike the microtubule activation found for all nucleotidases, only ATPase activity appears to be increased by phospholipids. This latter result is interesting in that only ATP hydrolysis is coupled to force production(40, 43) . These results also suggest that rather than acting as a passive player in intracellular motility, the vesicle membrane may participate in the modulation of dynein nucleotide hydrolysis and force production.

The membrane receptor for dynein remains speculative. Like myosin I, which exhibits specific binding interactions with phospholipids and lipid modulation of actin-activated ATPase(44, 45, 46, 47) , cytoplasmic dynein shows high affinity saturable binding to lipid vesicles(18) , and this interaction was found in the present study to stimulate dynein ATPase. It is possible that a conformational change upon organelle (phospholipid) binding is required for dynein ATPase to be maximally active, as has been suggested for kinesin. It remains to be determined whether dynein bound to phospholipid vesicles is functional in motility assays, and structure/function analysis must be conducted to determine the domains of the dynein molecule involved in liposome and organellar membrane attachment. In addition to dynein/lipid binding interactions, membrane receptors or accessory proteins are likely involved in regulating binding specificity and additional modulation of force production.


FOOTNOTES

*
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.

(^1)
The abbreviations used are: PS, phosphatidylserine; PC, phosphatidylcholine.


ACKNOWLEDGEMENTS

We thank the Division of Cancer Research, National Cancer Institute for providing taxol and Dr. Russell Kohnken for critically reading the manuscript.


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