Author response: Reconstitution of dynein transport to the microtubule plus end by kinesin (2014)

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Cytoplasmic dynein powers intracellular movement of cargo toward the microtubule minus end. The first step in a variety of dynein transport events is the targeting of dynein to the dynamic microtubule plus end, but the molecular mechanism underlying this spatial regulation is not understood. Here, we reconstitute dynein plus-end transport using purified proteins from S. cerevisiae and dissect the mechanism using single-molecule microscopy. We find that two proteins–homologs of Lis1 and Clip170–are sufficient to couple dynein to Kip2, a plus-end-directed kinesin. Dynein is transported to the plus end by Kip2, but is not a passive passenger, resisting its own plus-end-directed motion. Two microtubule-associated proteins, homologs of Clip170 and EB1, act as processivity factors for Kip2, helping it overcome dynein's intrinsic minus-end-directed motility. This reveals how a minimal system of proteins transports a molecular motor to the start of its track. https://doi.org/10.7554/eLife.02641.001 eLife digest Eukaryotic cells use transport systems to efficiently move materials from one location to another. Much transport in the cell interior is achieved using molecular motors, which carry cargoes along tracks called microtubules. Unlike roads of human construction, microtubules are very dynamic. One of their ends (the 'plus' end) explores the outskirts of the cell, growing and shrinking through the addition and loss of protein building blocks. The other microtubule end (the 'minus' end) typically lies in a hub near the center of the cell. There are two types of molecular motor that move on microtubules. Kinesin motors move toward the plus end of the microtubule, and dynein motors move in the opposite direction, toward the minus end. But if dynein only moves to the minus end of the microtubule, a problem arises: how would dynein initially reach the plus end of the microtubule and the outskirts of the cell, where it collects cargoes? Using purified yeast proteins, Roberts et al. reveal that a group of three proteins can solve this problem by transporting dynein to the plus end of the microtubule. The proteins comprise a kinesin motor, and two additional proteins that connect the dynein motor to the kinesin. Imaging the transport process shows that the dynein motor is not a passive passenger: it is able to resist against the kinesin. However, an additional microtubule-associated protein can help the kinesin motor to win this 'tug of war', and so the protein complex—including the dynein motor—moves toward the plus end of the microtubule. https://doi.org/10.7554/eLife.02641.002 Introduction Cytoskeletal motor proteins transport and position a variety of macromolecules, organelles and mRNAs in the cell interior (Vale, 2003), but how these motors are themselves targeted to specific locations within the cell is an important unsolved question. Cytoplasmic dynein, a large and complex AAA+ motor protein (Carter, 2013), uses the energy from ATP hydrolysis to move cargoes toward the minus end of microtubules (typically toward the cell center). In addition, dyneins can act while anchored at the cell cortex, where they pull the microtubule network toward them (Moore et al., 2009). Live-cell imaging in diverse organisms reveals that, surprisingly for a minus-end-directed motor, dynein accumulates at the plus ends of microtubules that grow and shrink near the cell periphery (Vaughan et al., 1999; Han et al., 2001; Ma and Chisholm, 2002; Lee et al., 2003; Sheeman et al., 2003; Lenz et al., 2006; Kobayashi and Murayama, 2009). By localizing to dynamic microtubule plus ends (Howard and Hyman, 2009), dynein is thought to 'search-and-capture' (Kirschner and Mitchison, 1986) cargo molecules, before transporting them toward the minus end (Wu et al., 2006). The targeting of the dynein machinery to the microtubule plus end is crucial for proper dynein function in budding yeast (Moore et al., 2009), filamentous fungi (Wu et al., 2006), and a subset of metazoan cells, including mammalian neurons (Lomakin et al., 2009; Lloyd et al., 2012; Moughamian and Holzbaur, 2012; Moughamian et al., 2013). However, the molecular mechanisms that target dynein to the microtubule plus end are poorly understood. Microtubule plus ends are dynamic binding platforms for a variety of proteins, collectively referred to as +TIPs (plus-end tracking proteins) (Akhmanova and Steinmetz, 2008). Previous studies have illuminated two general mechanisms by which proteins can be targeted to microtubule plus ends. First, a subset of +TIPs preferentially bind to unique structural features present at the plus end vs the body of the microtubule, and can recruit additional proteins to these sites. The prototypical example of this class of +TIP are EBs (end-binding proteins), which are involved in recruiting binding partners such as Clip170 and the dynein regulator dynactin to plus ends in metazoans (Watson and Stephens, 2006; Bieling et al., 2007; Dixit et al., 2009; Zanic et al., 2009; Maurer et al., 2012; Moughamian et al., 2013). Second, kinesin motor proteins can power the vectorial transport of proteins along the microtubule body to the plus end (Bieling et al., 2007; Subramanian et al., 2013). These two mechanisms (direct recruitment and vectorial transport) are not mutually exclusive, and evidence suggests that both may contribute to the plus-end targeting of dynein (Wu et al., 2006). In the yeast Saccharomyces cerevisiae, genetic studies implicate at least three proteins in targeting dynein to the microtubule plus end: Lis1, a regulator of dynein motility (Lee et al., 2003; Sheeman et al., 2003); Bik1, a homolog of Clip170 (Sheeman et al., 2003; Markus et al., 2009); and Kip2, a plus-end-directed kinesin motor (Carvalho et al., 2004; Caudron et al., 2008; Markus et al., 2009). Deletion of the EB homolog (Bim1) in S. cerevisiae appears to have little effect on the plus-end targeting of dynein and its co-factors (Carvalho et al., 2004; Caudron et al., 2008; Markus et al., 2011), despite (1) Bim1 binding directly to Bik1, an essential factor for dynein plus-end targeting (Sheeman et al., 2003; Blake-Hodek et al., 2010); and (2) The homologs of Kip2, Bik1 and Bim1 functioning together as a plus-end tracking system in fission yeast (Bieling et al., 2007). While dynein plus-end targeting can persist in the absence of Bim1, it is unknown if Bim1 is involved in the dynein pathway in the native situation, because +TIPs often interact in an interconnected and, in some cases, redundant manner (Caudron et al., 2008). More generally, how a system of molecules can function together to target dynein to the plus end is not clear in any system. This problem is particularly pressing for S. cerevisiae dynein: a forceful minus-end-directed motor with constitutive activity in motility assays (Reck-Peterson et al., 2006; Gennerich et al., 2007). In vitro reconstitution can provide powerful mechanistic insights into cellular processes, complementary to those derived from the complex in vivo environment (Liu and Fletcher, 2009). Here, we have reconstituted kinesin-driven transport of dynein to the microtubule plus end using purified proteins, allowing us to dissect the mechanism using single-molecule imaging, protein engineering and DNA origami. Results Dynein-Lis1 and Bik1-Bim1-Kip2 form interacting sub-complexes We began by purifying the S. cerevisiae proteins Lis1, Bik1, Bim1 and Kip2, in addition to a well-characterized dynein motor construct (GST-dynein331 kDa; referred to herein as 'dynein') (Reck-Peterson et al., 2006). This dynein construct lacks the cargo-binding tail, which is dispensable for plus-end targeting in vivo (Markus et al., 2009), and is dimerized by GST to yield a motor with highly-similar motile properties to full-length dynein (Reck-Peterson et al., 2006). Each purified protein migrated as a single band by SDS-PAGE (Figure 1A) with the exception of Kip2, which migrated as a doublet. The Kip2 bands correspond to differently phosphorylated forms of the protein; after treatment with λ phosphatase, the Kip2 doublet collapsed into a single band (Figure 1A, inset). Figure 1 with 1 supplement see all Download asset Open asset Purification and analysis of the putative dynein plus-end transport machinery. (A) SDS-PAGE and diagrams of purified proteins. Kip2 from S. cerevisiae migrates as a doublet of bands, corresponding to differently phosphorylated isoforms as verified by phosphatase treatment (right panel). (B) Analysis of sub-complex formation by size-exclusion chromatography. Elution volumes of the individual proteins are shown on the top axis. V0: void volume. Dynein and Lis1 co-elute as a complex (green trace), as do Bik1, Bim1 and Kip2 (magenta trace). A mixture of all five proteins elutes as dynein-Lis1 and Bik1-Bim1-Kip2 sub-complexes, rather than one stable assembly (blue trace). SDS-PAGE analysis of the fractions is shown below, pseudo-colored to match the traces. (C) TIRF microscopy microtubule recruitment assay. Fluorescently labeled wild-type (WT) dynein or a mutant with weak microtubule affinity (K3116A, K3117A, E3122A, R3124A: Weak) were incubated with microtubules in the presence of the indicated proteins. Representative microtubule images and the corresponding dynein channel are shown in each condition. The width of each panel corresponds to 5.4 μm. Mean dynein fluorescence intensity ± SEM is shown below (N = 30–50 microtubules). https://doi.org/10.7554/eLife.02641.003 To explore the interactions within this putative dynein plus-end transport machinery, we mixed the proteins in different combinations and analyzed their behavior by size-exclusion chromatography. Dynein and Lis1 co-elute in a complex, as verified by SDS-PAGE of the fractions (Figure 1B; Huang et al., 2012). We also found that Bik1, Bim1 and Kip2 co-elute in a ternary complex (Figure 1B), resembling the behavior of related proteins from Schizosaccharomyces pombe (Bieling et al., 2007; Table 1). A pair-wise mixture of Kip2 with Bim1 did not elute as a complex under the same conditions (Figure 1—figure supplement 1), suggesting that Bim1 binds with higher affinity to the Kip2-Bik1 complex. In summary, these first experiments with all five purified components demonstrate that dynein-Lis1 and Bik1-Bim1-Kip2 each form complexes that are sufficiently stable to co-elute by size-exclusion chromatography. Table 1 Protein homolog names https://doi.org/10.7554/eLife.02641.005 GenericS. cerevisiaeS. pombeKinesinKip2Tea2Clip170Bik1Tip1EB1Bim1Mal3Lis1Pac1– Given that the dynein-Lis1 and Bik1-Bim1-Kip2 sub-complexes did not stably associate in solution (Figure 1B), we hypothesized that these two assemblies might interact dynamically on the microtubule. To test this idea, we devised a microtubule recruitment assay. When wild-type dynein is mixed with microtubules in the absence of nucleotide, it binds strongly to the microtubule, as visualized by total internal reflection fluorescence (TIRF) microscopy in Figure 1C (lane 1). In order to monitor dynein recruitment to the microtubule by other proteins, we introduced four mutations into its microtubule-binding domain (Koonce and Tikhonenko, 2000; Carter et al., 2008; Redwine et al., 2012), which severely weakened its association with microtubules (Figure 1C, lane 2; see also Figure 2—figure supplement 2). However, the weak-binding dynein could be recruited efficiently to the microtubule by the combined presence of Lis1, Bik1, Kip2 and Bim1 (Figure 1C, lane 3). This dynein recruitment depended strictly on the presence of Lis1 and Bik1 (Figure 1C, lanes 4 and 5), consistent with the idea that Lis1 and Bik1/Clip170 interact directly (Coquelle et al., 2002; Sheeman et al., 2003; Lansbergen et al., 2004). To determine if the reported interaction between Lis1 and the C-terminal zinc-knuckle of Bik1/Clip170 (Coquelle et al., 2002; Sheeman et al., 2003; Lansbergen et al., 2004) is involved in connecting the dynein-Lis1 and Bik1-Bim1-Kip2 complexes, we purified a Bik1ΔC construct lacking the C-terminal region. The Bik1ΔC construct retained the ability to bind Kip2 and Bim1 (Figure 1—figure supplement 1). However, its ability to recruit the weak-dynein to the microtubule in the presence of Lis1, Kip2 and Bim1 was abolished (Figure 1C, lane 6). Together, these results suggest that dynein-Lis1 and Bik1-Bim1-Kip2 each form sub-complexes, which in turn interact in a manner dependent on Lis1 and the C-terminal region of Bik1. Reconstitution of dynein transport to the microtubule plus end with purified proteins Having established a potential chain of connection between dynein and Kip2 with purified proteins, we next sought to visualize the emergent motile behavior of these opposite polarity motors on dynamic microtubules. We first monitored the motility of dynein and Kip2 together, but in the absence of the other proteins, on dynamic microtubules grown from stabilized seeds using three-color TIRF microscopy (Figure 2A). Dynein and Kip2 were labeled with different-colored fluorophores (tetramethylrhodamine and Atto647, respectively), while microtubules contained a fraction of tubulin labeled with a third color (Alexa488). In the absence of Lis1, Bik1, and Bim1, dynein moved toward the minus end of the microtubule, and Kip2 moved toward the plus end, as expected (Figure 2B). Notably, upon reaching the minus end of the microtubule, dynein accumulated at, and moved with, the slowly growing end of the polymer (Figure 2B). Dynein showed the same behavior in the absence of Kip2, indicating that dynein has the intrinsic ability to track dynamic microtubule minus ends. Figure 2 with 3 supplements see all Download asset Open asset Reconstitution of dynein transport to the microtubule plus end. (A) Diagram of dynamic microtubule assay, after Bieling et al. (2007). A bright, biotinylated, GMP-CPP-stabilized microtubule seed is attached to the coverslip via streptavidin and biotin-PEG. Dimly labeled microtubule extensions grow from this seed in the presence of tubulin and GTP. Tubulin, dynein and Kip2 are visualized using TIRF microscopy, and motor protein movement on the dynamic extensions is analyzed. (B) Dynein and Kip2 motility on dynamic microtubules. Kymographs of the dynein and microtubule channels are overlaid, and the Kip2 channel is shown separately for clarity. Plus (+) and minus (−) denote microtubule polarity. Dynein moves to the minus end and Kip2 moves to the plus end. Scale bar, 5 μm. (C) In the presence of Lis1, Bik1, Bim1 and Kip2, dynein moves to the plus end of the microtubule (lane 1). Weakening dynein's microtubule affinity via mutagenesis (Figure 2—figure supplement 2) increases its plus-end velocity and accumulation (lane 2). In the absence of Lis1, Bik1 or Kip2, dynein resumes minus-end-directed motion (lanes 3–5). Arrows mark microtubule catastrophe events seen in the absence of Kip2 (lane 5). When Bim1 is omitted, dynein displays both plus- and minus-end-directed movements (lane 6). Mean velocities ± SD for dynein and free Kip2 (without dynein bound) are shown at bottom (N = 36–122). https://doi.org/10.7554/eLife.02641.006 Next, we explored the effect of adding the remaining factors. Strikingly, in the presence of Lis1, Bik1, Bim1 and Kip2, the large majority of dynein movements along the body of the microtubule were directed to the plus end (Figure 2C, lane 1) (90 ± 3%; proportion ± SE, N = 122). Moreover, on 63 ± 7% of microtubules, dynein was present at the growing plus end itself (Figure 2—figure supplement 1), reminiscent of dynein localization in living yeast cells (Lee et al., 2003; Sheeman et al., 2003; Markus et al., 2009). Minus-end-directed dynein movements were now rare (10 ± 3% of events). We observed similar behavior for the full-length dynein complex purified from S. cerevisiae (Reck-Peterson et al., 2006; Figure 2—figure supplement 1). Thus, we conclude that a system of four proteins (Lis1, Bik1, Bim1, and Kip2) is sufficient to strongly bias dynein's movement toward the microtubule plus end. Dynein can resist its transport to the plus end though its microtubule-binding domain Dynein's plus-end-directed motion depended strictly on Kip2 (Figure 2C, lane 5), but was slower than that of free Kip2 (Figure 2—figure supplement 2), leading us to hypothesize that dynein might have the capacity to resist its own transport to the plus end. To test this model, we repeated the reconstitution using the dynein mutant engineered to have weak affinity for the microtubule. Strikingly, the plus-end velocity of the weak-binding dynein was increased relative to the wild-type protein, becoming close to that of Kip2 (Figure 2C, Figure 2—figure supplement 2). Moreover, the weak-binding dynein also showed strong accumulation at the plus end of the microtubule (Figure 2C, lane 2). These results indicate that wild-type dynein is not a passive passenger during its plus-end-directed transport. Instead, the data support a model in which wild-type dynein and Kip2 have the potential to engage simultaneously with the microtubule in a 'tug-of-war'-like mechanism. If dynein's plus-end-directed movement is driven directly by Kip2, these two species should colocalize and move together on the microtubule. As expected from our biochemical data (Figure 1B), binding events between the dynein and Kip2 subcomplexes were extremely rare when both species were diluted to the sub-nanomolar concentrations required for single-molecule imaging. However, by exploiting the weak-binding dynein construct that does not bind appreciably to the microtubule at low nanomolar concentrations (Figure 1C, lane 2), we were able to observe clear co-localized movements of dynein with Kip2 in the presence of Bik1 and Lis1 (Figure 2—figure supplement 3). Notably, the dynein construct frequently localized with Kip2 midway through a run, and dissociated before its end, consistent with transient binding as indicated by our biochemical experiments (Figure 1B). These observations suggest that the movement of dynein toward the plus end is driven directly by Kip2. Lis1 and Bik1 couple dynein to Kip2, and Bim1 regulates the efficiency of dynein plus-end transport To probe the roles played by individual components in the plus-end-directed transport of dynein, we performed dropout experiments in which proteins were selectively omitted from the reconstitutions (Figure 2C). Lis1 and Bik1 were critical for dynein's plus-end-directed movement (Figure 2C, lanes 3 and 4). In their absence, dynein moved to the minus end of the microtubule, despite the continued movement of Kip2 to the plus end. These results indicate that Lis1 and Bik1 are required to connect dynein to Kip2. Omitting Kip2 abolished dynein's plus-end-directed movement, showing that the pathway we have reconstituted is kinesin dependent (Figure 2C, lane 5). Interestingly, removal of Kip2 also increased the frequency of microtubule catastrophes (the conversion from growth to shrinkage; Figure 2C, arrows), consistent with the short microtubule phenotype caused by Kip2 deletion in vivo (Cottingham and Hoyt, 1997). Finally, we observed an unexpected influence of Bim1 (Figure 2C, lane 6). In the absence of Bim1, dynein could still be transported to the plus end, but the fraction of plus- vs minus-end-directed dynein movements was roughly balanced (47 vs 53 ± 5%, respectively). Thus, Lis1, Bik1 and Kip2 constitute the minimal machinery for transporting dynein to the plus end, and Bim1 can regulate this machinery to increase the fraction of dynein movements that are directed to the plus end. Bik1 and Bim1 enhance Kip2 processivity Because Bim1 forms a ternary complex with Bik1 and Kip2 (Figure 1B), we suspected that the influence of Bim1 on dynein's plus-end transport might be exerted through these proteins. Furthermore, analysis of the Kip2 amino acid sequence revealed that there is an SxIP motif within an extension N-terminal to the kinesin motor domain (Figure 3A). The SxIP motif is a signature sequence that interacts with EB-family proteins such as Bim1 (Honnappa et al., 2009), suggesting that Bim1 may interact with Kip2 through this motif. Bim1 also binds to the Cap-Gly domain of Bik1 (Blake-Hodek et al., 2010) and the microtubule lattice (Zimniak et al., 2009), while Bik1's coiled coil is thought to interact with Kip2 (Newman et al., 2000; Carvalho et al., 2004). These observations suggest that a network of interactions exists within the Bik1-Bim1-Kip2 complex (Figure 3A), consistent with our pairwise binding analysis (Figure 1—figure supplement 1). Therefore, we wanted to test if Bik1 and Bim1 could promote Kip2's microtubule interactions by providing additional, transient microtubule-binding sites (Figure 3A). Figure 3 with 2 supplements see all Download asset Open asset Bik1 and Bim1 are Kip2 processivity factors. (A) Top: diagram of Kip2, Bik1 and Bim1 domain structure. Arrows indicate reported and putative interactions (see main text for details). Bottom: Kip2 contains an SxIP motif (orange) within an N-terminal extension to its kinesin motor The SxIP motif is by that can be phosphorylated et al., 2009; et al., (B) Top: showing the movement of along microtubules in the absence and presence of Bik1 and Scale 1 and 5 μm. Bottom: showing the of Kip2 in each condition. single on the function of each were from the were by with each (C) of ± of dynein and Kip2 toward the minus and plus end of the microtubule, in the indicated conditions (N = To directly visualize the of Bik1 and Bim1 on Kip2's we labeled Kip2 with and the movement of single molecules along microtubules (Figure In the absence of the by Kip2 microtubule was ± (Figure The addition of Bik1 or Bim1 on Kip2 However, in the presence of both Bik1 and Bim1, the of Kip2 increased to ± (Figure there was a in Kip2 while the Kip2 by fluorescence intensity was (Figure supplement 1). These were specific to Bik1 and Bim1 minimal on dynein motility (Figure supplement 1). Analysis of Kip2 events and revealed that Bik1 and Bim1 increase the and the of Kip2's microtubule (Figure supplement 2), indicating that these factors Kip2's microtubule In summary, we conclude that Bik1 and Bim1 together act as processivity factors for Kip2. The of a between dynein and Kip2 is by Bim1 and Bik1 Finally, we sought to if regulation of Kip2's processivity could its ability to overcome dynein in a reconstitutions that Bik1 is required to connect dynein to any additional of Bik1 in the was Therefore, we DNA and 2010) to couple dynein and Kip2 of any other proteins (Figure Dynein and Kip2 were attached at opposite ends of a DNA a which we the et al., 2012). this the large majority ± ± of movements on the microtubule were in the direction, as visualized by the of labeled DNA (Figure analysis indicated that the motors were in a both and plus-end-directed velocity to (Figure supplement 1). Figure 4 with 1 supplement see all Download asset Open asset Bik1 and Bim1 regulate the of a between dynein and Kip2. (A) dynein and Kip2 to a DNA them to be directly against each other in a DNA were attached to dynein and Kip2 via at their or et al., 2012). These DNA with complementary from the (B) Top: of motility on microtubules in the presence and absence of Bik1 and Bottom: that are minus-end-directed plus-end-directed (orange) or (C) of the different types of behavior in the indicated as the ± SEM (N = 3 assembly with analyzed in each The addition of Bik1 and Bim1 the fraction of plus-end-directed to increase relative to Scale bar, The DNA us to directly determine if Bik1 and Bim1 influence the of the The addition of Bik1 or Bim1 a effect on the motility of the the fraction of and plus-end-directed was little (Figure In of Bik1 and Bim1 together caused the fraction of plus-end-directed to increase (Figure these the also in during a (Figure ± of events). These results reveal that proteins can the of a between dynein and the kinesin Kip2. Discussion in vitro protein engineering and DNA we have a mechanism that dynein toward the microtubule plus start of its track (Figure 5). The of the mechanism dynein's motor domain to a plus-end-directed Kip2. By the between these motors for the first we that two proteins, Lis1 and Bik1, are sufficient to connect dynein and Kip2. This connection is of on the microtubule, but is not stable to be observed by size-exclusion chromatography. As is the for interactions found in other systems (Kirschner et al., a between dynein and Kip2 might for dynamic for example dynein to from Kip2 in the of dynein from the microtubule plus end its on the cell is on the order of (Markus and Figure 5 Download asset Open asset of the minimal machinery for transporting dynein to the microtubule plus end. on results from this and Dynein is to the plus-end-directed motor Kip2 by Lis1 and Bik1. domain binds to the of dynein's motor domain et al., 2012). The complex interacts dynamically with the C-terminal zinc-knuckle domain of Bik1/Clip170 (Coquelle et al., 2002; Sheeman et al., 2003; Lansbergen et al., 2004; Markus et al., as indicated by the Bik1's coiled coil is to interact with Kip2 (Newman et al., 2000; Carvalho et al., 2004). Bim1 is not strictly required for dynein's plus-end but together with Bik1, it can enhance Kip2's processivity and help Kip2 overcome dynein's intrinsic minus-end-directed motility. As with it is that the C-terminal of Bim1 interacts with the N-terminal Cap-Gly domain of Bik1 et al., and the cargo-binding domain of Bim1 interacts with the SxIP motif of Kip2 (Honnappa et al., 2009). This would the domain of Bim1 and free to interact with the microtubule and promote Kip2's microtubule A single of each is shown for with the other in S. cerevisiae is experiments that dynein has the potential to resist its plus-end transport through its microtubule-binding We also find that factors can the efficiency with which Kip2 a against Bik1 and Bim1 can promote Kip2's microtubule interactions and help this kinesin to overcome dynein's intrinsic minus-end-directed motility. The effect of these factors is when dynein and Kip2 are using DNA suggesting that it is in its