Nucleotide-Dependent Behavior of Single Molecules of Cytoplasmic Dynein on Microtubules in Vitro

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FEBS Letters 584 (2010) 2351–2355

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Nucleotide-dependent behavior of single molecules of cytoplasmic dynein on microtubules in vitro

Michi Miura 1, Aiko Matsubara, Takuya Kobayashi, Masaki Edamatsu, Yoko Y. Toyoshima *

Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan

article info abstract

Article history: We visualized the nucleotide-dependent behavior of single molecules of mammalian native cyto- Received 8 February 2010 plasmic dynein using fragments of dynactin p150 with or without its N-terminal microtubule bind- Revised 30 March 2010 ing domain. The results indicate that the binding afﬁnity of dynein for microtubules is high in AMP- Accepted 7 April 2010 PNP, middle in ADP or no nucleotide, and low in ADPPi conditions. It is also demonstrated that the Available online 13 April 2010 microtubule binding domain of dynactin p150 maintains the association of dynein with microtu- Edited by Dietmar J. Manstein bules without altering the motile property of dynein in the weak binding state. In addition, we observed bidirectional movement of dynein in the presence of ATP as well as in ADP/Vi condition, suggesting that the bidirectional movement is driven by diffusion rather than active transport. Keywords: Cytoplasmic dynein Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Dynactin p150 Single molecule Microtubule

1. Introduction conditions. We observed single molecules of native cytoplasmic dynein using the dynein binding protein, dynactin p150. Dynactin Cytoplasmic dynein is a minus-end directed microtubule motor, p150 is the major component of the dynactin complex, and is and functions as a large complex within the cell [1,2]. The N-termi- responsible for binding to dynein by the interaction with dynein nal region of the heavy chain forms the tail domain, which is intermediate chains via its coiled-coil region [12]. Its N-terminus responsible for dimerization and associating with intermediate contains a conserved CAP-Gly (cytoskeleton-associated protein, and light intermediate chains [3]. Cytoplasmic dynein hydrolyzes glycine-rich) motif, which binds to microtubules [13], and has been ATP at the ring-shaped motor domain located in the C-terminal shown to promote the motor processivity of cytoplasmic dynein two-thirds of the heavy chain [4–6]. Cytoplasmic dynein binds to [14,15]. We prepared two fragments of dynactin p150 fused to gel- microtubules via the stalk head, the distal tip of the anti-parallel solin. Molecules of the dynein/dynactin complex can be visualized coiled-coil protruding from the ring structure [7]. as fluorescent actin spots by epifluorescence microscopy according Results of previous studies on recombinant cytoplasmic dynein to the method of Yajima et al. [16]. This method is suitable for long suggest that the binding affinity for microtubules depends on the term observation, which is often prevented by fluorescence registry of the coiled-coil of the stalk and the nucleotide states at quenching in single molecule motility assays, because each short AAA (ATPases associated with diverse cellular activities) modules actin filament contains multiple fluorescent dyes. [8–10]. For native cytoplasmic dynein, the motile property of sin- By investigating the behavior of the dynein/dynactin complex gle dynein molecules associated with the dynactin complex was on microtubules, we will discuss the nucleotide dependence of investigated in the presence of ATP [11], but their behavior on binding affinity for microtubules and the effect of dynactin p150 microtubules in various nucleotide conditions has not been on the behavior of cytoplasmic dynein on microtubules. explored sufficiently. Here, we examined the behavior of single molecules of native 2. Materials and methods cytoplasmic dynein on microtubules under different nucleotide

2.1. Preparation of proteins Abbreviations: AMP-PNP, 50-adenylylimidodiphosphate; Vi, vanadate * Corresponding author. Fax: +81 3 5454 6722. E-mail address: [email protected] (Y.Y. Toyoshima). Expression plasmids of gelsolin-fused proteins (C1G and NC1G) 1 Present address: Laboratory of Virus Control, Institute for Virus Research, Kyoto were prepared by inserting the DNA fragments coding human dyn- University, Kyoto 606-8507, Japan. actin p150 into the 50 terminus of human plasma gelsolin. Proteins

0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.04.016 2352 M. Miura et al. / FEBS Letters 584 (2010) 2351–2355 were expressed bacterially and purified (for the details, see Supple- 1 mM, which was an excess amount compared with the trace mentary data). amount of ATP (1 lM) from the final purification step of native Cytoplasmic dynein was prepared from porcine brain [17].To cytoplasmic dynein. exclude the possible dynein fraction that binds to actin filaments We counted the numbers of dynein molecules per unit length of used as a fluorescent probe, we removed this fraction by co-precip- microtubules and tracked those individual molecules. As shown in itating dynein with F-actin. Fig. 2a, a relatively large amount of dynein molecules was associ- Tubulin was purified from porcine brain [18], and was labeled ated with microtubules in the presence of AMP-PNP, and they re- with BODIPY FL (Molecular Probes) [19]. Microtubules were poly- mained on microtubules for tens of seconds. Under the condition merized [19] and stabilized with 40 lM taxol. of ADP or no nucleotide, the number of molecules on microtubules Actin was prepared from rabbit skeletal muscle [20], and la- was 10–50% of that in AMP-PNP. In the presence of ADP/Vi, dynein beled with phalloidin-tetramethylrhodamine B isothiocyanate molecules rarely associated with microtubules, resulting in <1% of conjugate (Fluka) [21]. dynein molecules on microtubules compared with AMP-PNP, and dynein molecules dissociated from the microtubules within a few 2.2. Single molecule imaging of the gelsolin–actin complex seconds after binding to the microtubules. The histogram of the duration on microtubules in ADP/Vi was fitted to a single exponen- The procedures for preparing gelsolin-capped actin filaments tial decay, and its mean duration was calculated to be 1.34 s and epifluorescence microscopy were as described previously (Fig. 2b). Thus, the numbers of molecules on microtubules were [16]. In this study, dynein and gelsolin–actin mixture in motility correlated to the duration on microtubules. The trajectory data buffer (10 mM Pipes-KOH, pH 6.7/4 mM MgSO4/1 mM DTT) con- show that dynein did not move widely on microtubules under taining 20 mM K-acetate, 0.6mg ml1 casein, 40 lM taxol, 0.1% the condition of AMP-PNP. The center position of typical tracked Tween 20 (Nacalai Tesque) and the oxygen scavenger system molecules in the presence of AMP-PNP distributed within approx- [22] was loaded. This solution was supplemented with either of imately 60 nm, which is consistent with previous results of kinesin 1 mM AMP-PNP, 1 mM ADP/Vi, 1 mM ADP or no nucleotide. Each bound to microtubules in the AMP-PNP condition [16]. Under the fluorescent actin filament was severed by gelsolin shortly enough condition of ADP or no nucleotide, dynein fluctuated slightly. to be visualized as a spot rather than a rod-like structure. Analysis However, dynein fluctuated widely along the longitudinal axis of of the behavior of fluorescent molecules is described in Supple- microtubules in the presence of ADP/Vi (Fig. 2c and also see mentary data. Supplementary Movie S1 and S2). The dynein–C1G complex interacts with microtubules through 3. Results and discussion the dynein stalk heads because C1G lacks the N-terminal microtubule binding domain (Fig. 1), and the numbers of bound molecules 3.1. Nucleotide dependence of C1G-labeled cytoplasmic dynein on microtubules are considered to correspond to the affinity for microtubules. Thus, these results are comparable to those of the Fig. 1 shows the fusion proteins used in this study and sche- previous study by Imamula et al. [9] on the recombinant single- matic diagrams of the complex of cytoplasmic dynein and these headed cytoplasmic dynein. The dissociation constant was approx- proteins on a microtubule. C1G contains a dynein binding region imately 0.2 lM under the condition of AMP-PNP, ADP or no nucle- of 217–548 amino acids [12], and NC1G contains both dynein bind- otide as the strong binding state, whereas the constant was ing and microtubule binding regions. >10 lM in the presence of vanadate as the weak binding state. In First, we labeled dynein with C1G/rhodamine–actin and ob- our study, the number of dynein molecules associated with micro- served its behavior on microtubules under four nucleotide condi- tubules in the presence of AMP-PNP was distinct from the other tions (AMP-PNP, ADP/Vi, ADP, or no nucleotide). The presence of three conditions. Among these three conditions, dynein frequently vanadate inhibits ATPase activity of dynein and mimics the ADPPi associated with microtubules and did not move widely under the state [23]. Each nucleotide was added at a final concentration of condition of ADP or no nucleotide, whereas dynein rarely associated with microtubules and fluctuated largely once associated with microtubules in the presence of ADP/Vi, indicating that the microtubule binding property of dynein under these three conditions can be categorized further into two groups. Taken together, our results suggest that the affinity of native cytoplasmic dynein for microtubules is high in AMP-PNP, middle in ADP or no nucleotide, and low in ADPPi conditions. These results show that the binding affinity of dynein for microtubules under the condition of ADP or no nucleotide is distinguishable from that of AMP-PNP, whereas the binding affinity in these three conditions was similar in the previous study by Imamula et al. [9]. The difference might be attributed to the double-headed dynein in this study and the single-headed dynein in the study of Imamula et al. Namely, both heads of the double- headed molecule may bind to the microtubule in AMP-PNP, whereas only one head of the double-headed molecule may bind to the microtubule in ADP or no nucleotide as is the case for kinesin [24,25], although the relation between the two heads of dynein has not been elucidated.

Fig. 1. Schematic diagrams of the fusion proteins used in this study and the 3.2. The behavior of cytoplasmic dynein associated with NC1G complex of cytoplasmic dynein and these proteins. C1G has a dynein binding domain and NC1G contains both dynein binding and microtubule binding domains. Since NC1G contains the microtubule binding domain at its N- Gelsolin caps rhodamine-labeled short actin filament (red) and, thus, native cytoplasmic dynein is visualized as a fluorescent actin spot by epifluorescence terminus, we examined whether NC1G/rhodamine–actin binds to microscopy. microtubules by itself without dynein. NC1G alone bound to M. Miura et al. / FEBS Letters 584 (2010) 2351–2355 2353

Fig. 2. The behavior of the dynein–C1G complex. (a) Relative numbers of dynein molecules per unit length of microtubules in each nucleotide condition standardized by the numbers in the AMP-PNP condition. (b) The histogram of duration of the dynein–C1G complex on microtubules in the presence of ADP/Vi. The graph was ﬁtted to a single exponential decay. The mean duration was 1.34 s. (c) Displacement of the dynein–C1G complex along the longitudinal axis of microtubules in each nucleotide condition. Three typical examples are shown in three colors.

Fig. 3. The behavior of the dynein–NC1G complex. (a) The numbers of NClG molecules without dynein and the dynein–NC1G complex in each nucleotide condition per unit length of microtubules, standardized by the numbers in the AMP-PNP condition. (b) Displacement of NC1G along the longitudinal axis of microtubules. (c) Displacement of the dynein–NC1G complex along the longitudinal axis of microtubules in each nucleotide condition. Three typical examples are shown in three colors. microtubules (Fig. 3a), and fluctuated on the longitudinal axis of ments may stay on microtubules together with the dynein–NC1G microtubules (Fig. 3b), as observed previously [26]. At a high molar complexes, and it must be difficult to distinguish the dynein– ratio of NC1G/dynein in solution, unassociated free NC1G frag- NC1G complexes from free NC1G fragments. We determined that 2354 M. Miura et al. / FEBS Letters 584 (2010) 2351–2355 a NC1G/dynein molar ratio of 1/4 is appropriate, and confirmed ADP/Vi (Fig. 3c, ADP/Vi) and in ATP (Fig. 4) were bidirectional and that almost all the fluorescent actin spots represents NC1G-associ- indistinguishable, although we have not determined the direction- ated dynein, because all the molecules observed on microtubules ality of movement. We estimated the diffusion constant of the dy- did not fluctuate in the presence of AMP-PNP, which is not the nein–NC1G complex in the condition of ADP/Vi and ATP from the property of NC1G alone, but the property of dynein, as was ob- MSD (mean square displacement) plots, and found that the preli- served above. minary values for each condition were within a similar range When cytoplasmic dynein was associated with NC1G, the num- (approximately 6–10 1010 cm2 s1). These results suggest that bers of molecules on microtubules in the four nucleotide condi- the dynein–microtubule interaction is weak and the contribution tions became similar (Fig. 3a). In particular, the numbers of of diffusion is large in ATP, and that the observed movement in molecules associated with microtubules in ADP/Vi were signifi- ATP may be attributed mostly to diffusion, but not to energy trans- cantly increased, and its duration on microtubules became compa- duction from ATP hydrolysis. The motility of dynein molecules pre- rable to that of the other nucleotide conditions, but the dynein– pared in this study was confirmed to be active by the microtubule NC1G complex still fluctuated widely on microtubules (Fig. 3c). gliding assay (average velocity of 700nm s1), and the method of These results are consistent with the idea that the microtubule preparing dynein used in this study was identical to that in the sin- binding domain of dynactin p150 contributes to an increase in gle molecule bead assay [27]. Thus, the diffusive movement shown the duration of dynein on microtubules by maintaining dynein here is not because the dynein molecules are inactivated, but prob- association with microtubules [14,15]. Moreover, this tethering ably due to the nature of single molecules of mammalian cytoplas- force of dynactin p150 does not seem to alter the behavior that dy- mic dynein in vitro without the reducing effect of diffusion by the nein fluctuates widely on microtubules in the weak binding state. bead or the laser trap. Ross et al. [11] suggested that the bidirectional motility is attributed to an active energy transduction prop- 3.3. Motile property of cytoplasmic dynein and contribution of erty of dynein because the velocity in both direction is dependent dynactin p150 on the ATP concentration. Based on our observation that the dynein–NC1G complex in ADP/Vi (ATP non-consuming state) moves Next, we observed the behavior of the dynein–C1G complex and largely enough, the movement by Ross et al. may be explained the dynein–NC1G complex in the presence of 1 mM ATP. The dy- by the idea that the waiting time of ATP binding decreases as the nein–C1G complex rarely associated with microtubules, while ATP concentration increases, and, thus, the ratio of the ADPPi state the dynein–NC1G complex associated with microtubules for a (weak binding state) in the ATPase cycle increases, resulting in dy- while and moved in both directions along the longitudinal axis of nein being more susceptible to the driving force of diffusion. the microtubules (Fig. 4 and also see Supplementary Movie S3), This behavior of mammalian native dynein seems to be differ- demonstrating the effect of the microtubule binding region at the ent from that of Saccharomyces cerevisiae dynein, which moves N-terminus of NC1G, as discussed above. The behavior of the dy- on microtubules towards the minus-end of the microtubules with nein–NC1G complex in ATP is consistent to previous studies show- infrequent backward steps [28]. It is plausible that mammalian dy- ing that the microtubule binding region of dynactin increases the nein associates with microtubules more weakly than yeast dynein, duration of cytoplasmic dynein on microtubules [14,15], and that resulting in the frequent backward movement of the mammalian the mammalian native dynein/dynactin complex moves on micro- dynein/dynactin complex over a long distance as a diffusional tubules with a run length of hundreds of nanometers in both direc- movement. Recently, it was reported that the yeast dynactin com- tions [11]. However, the behaviors of the dynein–NC1G complex in plex increases the run length of native yeast dynein [29]. Notably,

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