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Activation of Cytoplasmic Dynein Motility by Dynactin-Cargo Adapter Complexes Richard J

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Activation of Cytoplasmic Dynein Motility by Dynactin-Cargo Adapter Complexes Richard J RESEARCH | REPORTS MOLECULAR MOTORS with its tail bound to a single dynactin, iden- tifiable by its 37 nm Arp1 filament (Fig. 1C and fig.S3,AtoC).TheDDBcomplexappearedhigh- ly flexible, as evidenced by the variable orienta- Activation of cytoplasmic dynein tions of dynactin’s Arp1 filament (fig. S3B) and variable separation of the two motor domains motility by dynactin-cargo (fig. S3D). Next, we examined the motility of single DDB adapter complexes complexes by TIRF microscopy, using the GFP- tag on BicD2. In contrast to brain dynein, the Richard J. McKenney, Walter Huynh, Marvin E. Tanenbaum, DDB complexes moved robustly and processive- ly along MTs (Fig. 1D and movie S3), although a Gira Bhabha, Ronald D. Vale* small fraction (~15%) exhibited back-and-forth diffusive motion (fig. S2D). The DDB complexes Cytoplasmic dynein is a molecular motor that transports a large variety of cargoes also accumulated at MT minus-ends (Fig. 1E), (e.g., organelles, messenger RNAs, and viruses) along microtubules over long intracellular indicating tenacious binding upon reaching the distances. The dynactin protein complex is important for dynein activity in vivo, but its end of the MT track. Similar processive motility precise role has been unclear. Here, we found that purified mammalian dynein did not move and minus-end accumulation was observed re- processively on microtubules in vitro. However, when dynein formed a complex with cently for dynein-driven transport of purified dynactin and one of four different cargo-specific adapter proteins, the motor became mRNA particles in vitro (21). By kymograph anal- ultraprocessive, moving for distances similar to those of native cargoes in living cells. Thus, Downloaded from ysis, DDB processive movement appeared as ex- we propose that dynein is largely inactive in the cytoplasm and that a variety of adapter tended diagonal lines (Fig. 1F). At 2 mM ATP, DDB proteins activate processive motility by linking dynactin to dynein only when the motor is moved at a mean velocity of 376 nm/s (Fig. 1G) bound to its proper cargo. and run length of 8.7 mm (Fig. 1H), considerably longer than yeast dynein (8). When measured at ytoplasmic dynein 1 (dynein), a member of ment to surfaces. Brain dynein, which exhibited physiological temperature (37°C), the velocity was the AAA adenosine triphosphatase (ATPase) a characteristic two-headed shape by electron 892 nm/s (fig. S2E), very close to speeds of retro- http://science.sciencemag.org/ family, is the major minus-end–directed microscopy (EM) (Fig. 1A), produces fast motility grade transport in vivo (12). Processive motility microtubule (MT) motor in most eukaryotic (~0.6 mm/s) of MTs in a multiple motor gliding wascompletelyabolishedbytheATPaseinhib- C 15 cells (1). Several adapter proteins control assay ( ). However, individual Cy3-labeled na- itor vanadate (fig. S2F). The addition of BicD2 to the recruitment of a soluble pool of cytoplasmic tive dynein molecules, examined by total inter- purified brain dynein did not stimulate processive dynein to transport cargos at the right place and nal reflection (TIRF) microscopy in the presence motility, indicating a requirement for dynactin time in the cell (2–4). Dynein motor activity also of 1 mM adenosine 5´-triphosphate (ATP), most- (fig. S2G). To further confirm that motile DDB appears to be governed by two general regu- ly either bound statically to MTs or exhibited short complexes contained single BicD2 dimers and latory factors—the Lis1-NudEL complex and the back-and-forth movements (Fig. 1A, fig. S1A, and not oligomers or aggregates, we added two flu- dynactin complex (5). Lis1 may act as a “clutch” movie S1), which are likely due to thermal-driven orescently labeled BicD2 preparations (TMR- and that suppresses dynein motility and causes it to diffusion, as they persist after addition of the 505-Star–labeled) to pig brain lysates followed 6 7 16 form a tight binding complex with the MT ( , ). ATPase inhibitor vanadate (fig. S1B) ( ). Direc- by DDB complex purification. Two-color kymo- on November 23, 2017 Whether dynein motility requires just the detach- tional movements were only occasionally observed graphs revealed that moving DDB complexes con- ment of Lis1 or needs an additional activation (<1% of MT-bound dynein); those movements tained either TMR- or 505-Star-BicD2 but not a process has been unclear. were very slow (~90 nm/s, fig. S1A) and inhib- mixture (fig. S2H and movie S4), confirming that Yeast cytoplasmic dynein, the best charac- ited by vanadate (fig. S1B). A recombinant, gluta- each DDB complex contains only a single BicD2 terized dynein in terms of its motility, moves thione S-transferase (GST)–dimerized human molecule. Thus, BicD2 links dynein and dynactin processively on its own (run length of 1 to 2 mm) motor domain construct (fig. S1C) also did not to form a stable complex that moves toward the (8), and dynactin only increases its run length show fast, processive motion (Fig. 1B and movie MT minus-end with ultraprocessive run-lengths. by ~twofold (9). Mammalian cytoplasmic dynein S2), consistent with recent studies of a recom- ToexamineeachcomponentoftheDDBcom- is also generally thought to be constitutively ac- binant human cytoplasmic dynein holoenzyme plex during motility, we purified a triple-colored tive for motility, as it produces movement when (17). Thus, in contrast to yeast dynein, purified DDB complex consisting of Alexa647-labeled, attached to solid surfaces such as glass slides (10), mammalian dynein rarely displays processive SNAPf-tagged BicD2 (expressed in Escherichia plastic beads (11), or quantum dots (12). However, motility. coli); GFP-tagged dynein intermediate chain surface binding of kinesin activates this normally The poor single-molecule motility by mam- (GFP-IC); and a tetramethylrhodamine (TMR)– autoinhibited motor (13). Without direct visual- malian dynein might be due to the absence of labeled, Halo-tagged p62 dynactin subunit ization of the motor itself, it also can be difficult an activator in our purified preparations. BicD2 [tagged IC and p62 were coexpressed in human to determine whether one or multiple motors is a conserved, dimeric adapter protein that links RPE-1 cells (22); fig. S4, A and B)]. This triple- are contributing to movement. Prior studies of dynein to Rab6 GTPase on membrane organelles color–labeled DDB moved processively, and many fluorescently labeled mammalian dynactin, but (2, 18); the BicD N-terminal coiled coil (19)fa- moving GFP-dyneins colocalized with a TMR- with unlabeled dynein, have reported processive cilitates an interaction between dynein and dy- dynactin and an Alexa647-BicD2 molecule (Fig. 2A run lengths of <2 mm in both directions on the nactin, forming a stable dynein-dynactin-BicD2 and movie S5); the mean velocity and run length MT (14). ternary complex (DDB) that can be purified (20). of human DDB was 379 nm/s (Fig. 2B, left) and Here, we examined the motility of purified rat Using this well-characterized green fluorescence 8.84 mm (Fig. 2B, right), respectively. The flu- brain cytoplasmic dynein (termed “brain dynein”) protein (GFP)–tagged, N-terminal construct of orescence intensities of the motile GFP-labeled by single-molecule fluorescence without attach- BicD2 (20), we isolated the DDB complex from dynein molecules were similar to those of the pig brain (Fig. 1C and fig. S2A); this prepara- well-characterized dimeric protein, GFP-kinesin tion did not contain detectable kinesin-1, Lis1, K560 (fig. S4C). Additionally, the fluorescence Department of Cellular and Molecular Pharmacology and the or Nudel (fig. S2B). The DDB complex eluted as a intensities of isolated TMR-dynactin and Alexa647- Howard Hughes Medical Institute, University of California, San Francisco, CA 94158, USA. single peak by size-exclusion chromatography BicD2 were similar to the intensities of these *Corresponding author. E-mail: [email protected] (fig. S2C), and EM revealed a single dynein dimer components within the motile DDB complex SCIENCE sciencemag.org 18 JULY 2014 • VOL 345 ISSUE 6194 337 RESEARCH | REPORTS (fig. S4, D and E). Thus, consistent with the EM factors (9, 23), we next examined the single- MTs, but MT binding by human TMR-dynactin data (Fig. 1B), a ternary complex, containing single molecule behavior of dynein and dynactin in the was not observed (Fig. 2C), as was also reported copies of dynein, dynactin, and BicD2, is the active absence of BicD2. GFP-dynein and TMR-dynactin for yeast dynactin (9). The lack of MT binding entity that moves processively along MTs. could be separated from BicD2 by salt elution by dynactin was surprising, because the recom- Because dynactin has been suggested to ac- from a BicD2 affinity column (fig. S4F). Without binant N-terminal half of p150Glued (a subunit in tivate dynein motility in the absence of other ATP, human GFP-dynein molecules decorated the dynactin complex, Fig. 1B; herein termed A B Recombinant Brain Dynein GST-hDynein HC HC IC LIC 25 nm 25 nm Tail GST 60 sec 10 sec Downloaded from Motor Motor Domains Domains 5 µm 5 µm C Brain Dynein-Dynactin-BicD2 (DDB) D E GFP-BicD2 MT GFP-BicD2 MT HC http://science.sciencemag.org/ 0:00 * p150 * 0:11 Dynactin GFP- 5 µm BicD2 0:22 on November 23, 2017 p150 5 µm 25 nm 2 µm F G H GFP-DDB Complex 80 376 ± 218 nm/sec 1.0 0.8 60 = 8.69 µm R2 = 0.998 0.6 40 Counts 0.4 20 0.2 0.0 60 sec 0 0 300 600 900 1200 1500 1-cumulative frequency 020406080100 5 µm Velocity (nm/sec) Run Length (µm) Fig. 1. Formation of a dynein-dynactin-BicD2 complex induces processive Movie frames showing a single GFP-labeled DDB complex (yellow arrow- dynein motility. (A)SDS–polyacrylamide gel electrophoresis (SDS-PAGE) of head) moving processively along a MT. (E) DDB complexes accumulate at purified rat brain dynein and negative-stain EM.
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