Summary
Intracellular transport is largely driven by processive microtubule- and actin-based molecular motors. Non-processive motors have also been localized to trafficking cargos, but their roles are not well understood [1–7]. Myosin-Ic (Myo1c), a non-processive actin motor, functions in a variety of exocytic events, although the underlying mechanisms are not yet clear. To investigate the interplay between myosin-I and the canonical long distance transport motor kinesin-1, we attached both motor types to lipid membrane-coated bead (MCB) cargo, using an attachment strategy that allows motors to actively reorganize within the membrane in response to the local cytoskeletal environment. We compared the motility of kinesin-1-driven cargos in the absence and presence of Myo1c at engineered actin/microtubule intersections. We found that Myo1c significantly increases the frequency of kinesin-1-driven microtubule-based runs that begin at actin/microtubule intersections. Myo1c also regulates the termination of processive runs. Beads with both motors bound have a significantly higher probability of pausing at actin/microtubule intersections, remaining tethered for an average of 20 s, with some pauses lasting longer than 200 s. The actin-binding protein non-muscle tropomyosin (Tm) provides spatially-specific regulation of interactions between myosin motors and actin filaments in vivo [8, 9, 11, 13, 14]; in the crossed-filament in vitro assay, we found that Tm2-actin abolishes Myo1c-specific effects on both run initiation and run termination. Together these observations suggest Myo1c is important for the selective initiation and termination of kinesin-driven runs along microtubules at specific actin filament populations within the cell.
Results
Membrane-bound cargos are transported throughout the cell by molecular motors that move along microtubules and actin filaments. This transport is essential for normal cellular function, as mutations in either the motors or their adaptors contribute to diseases including neurodegeneration [15] and sensory and metabolic disorders [16, 17]. Organelles and vesicles undergoing active transport in the cell typically bind multiple types of microtubule- and actin filament-specific motors [18]. Most research in the field has focused on characterizing the cargo-associated motors that drive processive movement along cytoskeletal filaments [18, 19]. Non-processive motors, i.e. motors that take only a single step before detaching from their cytoskeletal track, also contribute to intracellular transport, yet their contributions to cargo dynamics during trafficking are not yet well defined [1–6, 20, 21].
Myosin-I proteins are single-headed, non-processive molecular motors that facilitate a variety of dynamic actin-membrane interactions [1, 2, 4, 20–22]. The widely expressed isoform, myosin-Ic (Myo1c), participates in exocytic trafficking [1], recycling of lipid raft cargos [1], and the final stages of GLUT4 transport beneath the plasma membrane [2, 3, 7], consistent with a possible role in cargo sorting to specific destinations [1, 3]. Throughout these transport events, Myo1c associates with cargos that bind a range of processive microtubule and actin motors, including kinesin-1 and myosin-V [3, 23–26]. It has been suggested that Myo1c acts as either a slow actin filament transporter [1, 6] or a molecular tether [4, 6, 8] during these processes [2, 3, 16]. Here, we use in vitro reconstitution assays to identify specific roles for Myo1c in both the initiation and the termination of long-distance kinesin-1-driven runs.
We examined the transport of synthetic membrane-bound cargos by directly observing fluorescently-tagged kinesin-1 in the presence and absence of Myo1c using engineered cytoskeletal intersections, in which coverslip-attached microtubules intersect with actin filament overpasses. We utilized a truncated, biotinylated, two-headed kinesin-1 construct and a biotinylated Myo1c construct truncated after the lever-arm domain (hereafter referred to as “kinesin-1” and “Myo1c” respectively, see Methods). Motors were specifically attached to synthetic cargos via a NeutrAvidin intermediate to biotinylated lipids incorporated into a DOPC lipid bilayer, surrounding 1 μm silica beads [27]. Biotin-mediated attachment to lipid membrane-coated beads (MCBs) permits control of the number of motors bound to the cargo by altering the mole-percent of biotin-PE in the DOPC membrane (see Methods and Figure S1), and allows the diffusion of motors around the cargo in response to local changes in cytoskeletal filament geometry during transport.
In flow chambers containing actin filaments (AF) and microtubules (MT), MCBs bound to both kinesin-1 and Myo1c preferentially initiate processive runs on microtubules at AF/MT intersections (Figures 1B and 1C, Movie S1). In contrast, MCBs bound only to kinesin-1 show no preference for run initiation at intersections. Rather, kinesin-1-only MCBs stochastically initiate runs along the length of the microtubule (Figures 1A and 1C, Movie S1). When Myo1c is present, the distribution of landing distances from the nearest actin intersection is significantly different from randomly generated points in the same fields of view, whereas kinesin-only landing distances mimic the random points (Figure 1D). These observations suggest that Myo1c facilitates the initiation of a microtubule-based run by recruiting cargo preferentially to cytoskeletal intersections.
Figure 1. Myo1c initiates kinesin-1-driven runs at engineered AF/MT intersections.
Lipid membrane-coated beads (MCBs) containing kinesin-1-only or kinesin-1 and Myo1c were observed as they initiated kinesin-driven motility along microtubules. Events were scored by the distance between the location of initiated processive motility and the nearest actin filament intersection.
(A) A sample interaction showing that kinesin-1-only cargo stochastically initiate microtubule-based runs with respect to the nearest actin intersection. In this time series, microtubules are pseudo-colored green, while actin filaments are pseudo-colored purple. The MCB was monitored as it approached a microtubule immobilized on the surface, with the initiation event denoted as the location at which processive motility begins on the kymograph (labeled “0 s” in the time series). The blue arrowhead next to the kymograph indicates the actin filament intersection. Data acquired at 10 frames per second (fps). Scale bars represent 1 μm in distance, 3 s in time. See Movie S1.
(B) Myo1c increases the frequency that kinesin-1-driven runs initiate at AF/MT intersections. This sample interaction shows a run beginning at an AF/MT intersection, “0 sec,” in which the center of the cargo initiates movement along the microtubule < 0.5 μm from the center of the intersection, in a time series and corresponding kymograph. The blue arrowhead next to the kymograph indicates the actin filament intersection. Scale bars represent 1 μm in distance, 3 s in time. See Movie S1.
(C) Myo1c induces a significant increase in run initiation events at AF/MT intersections. Significantly more cargo initiation events are observed at AF/MT intersections when Myo1c is present on kinesin-1-driven cargo. Events are designated as beginning at an AF/MT intersection if the centroid of the MCB is ≤ 0.5 μm from the center of the intersection at the time processive motility along the microtubule is initiated by kinesin-1. Kinesin-1-only: n = 33 events from 2 chambers. Kinesin-1+Myo1c: n = 36 events from 2 chambers. Acquired at 10 fps. *** p ≤ 0.001 Kruskal-Wallis with Dunn’s multiple comparisons test.
(D) Cumulative frequency distribution showing the increased number of run initiation events occurring in proximity to the nearest AF intersection when Myo1c is bound to the MCB. While run initiation distances for kinesin-only cargo have the same distribution as randomly chosen points in the same fields of view, kinesin-1 and Myo1c cargo preferentially initiate runs at AF/MT intersections. Kinesin-1-only cargo: n = 33 events from 2 chambers. Kinesin-1+Myo1c cargo: n = 36 events from 2 chambers. Acquired at 10 fps. *** p ≤ 0.001, ** p ≤ 0.01 Kruskal-Wallis with Dunn’s multiple comparisons test.
Also see Figure S1.
Next, we investigated the influence of Myo1c on kinesin-mediated processive motility at actin filament intersections encountered during motility along a microtubule. Kinesin-1-only MCBs tend to pass actin intersections, with only 33% of cargo pausing for greater than 0.5 s (Figure 2A and 2C, Movie S2). In contrast, 92% of MCBs bound to kinesin-1 and Myo1c paused at actin filament intersections (Figure 2B and 2C, Movie S2). The mean pause length was 20 s, with some pauses lasting longer than 200 s (Figure 2B). Kinesin cargo with or without Myo1c present tended to detach (without passing or pausing) at the actin intersection with similar low frequencies, 5% and 4% of the time, respectively (Figure 2C).
Figure 2. Myo1c halts kinesin-1-driven MCBs at engineered AF/MT intersections.
We observed the behavior of MCBs traveling along microtubules via kinesin-1-driven transport as they encountered actin filament intersections.
(A) MCBs with only kinesin-1-bound predominantly pass AF/MT intersections. Time series and kymograph showing a kinesin-only cargo passing an actin filament intersection. The blue arrowhead next to the kymograph denotes the actin intersection in the kymograph. Scale bars = 1 μm and 3 s; acquired at 2 fps. See Movie S2.
(B) MCBs with kinesin-1 and Myo1c primarily pause at actin filament intersections. Time series and kymographs depicting two example pauses. The top event illustrates a pause of average length, 20 s, while the bottom event shows a pause of > 220 s. Note the change in time scale between frames in both time series. The blue arrowhead next to the kymograph denotes the AF intersection. Scale bars = 1 μm and 3 s. See Movie S2.
(C) Significantly more cargo pause at actin intersections when Myo1c is present (92% vs. 33%). A halt in motility along the microtubule at an actin intersection for > 0.5 s is denoted as a “pause” event. In contrast, kinesin-1-only MCBs tend to pass actin intersections (63% of the time). Both kinesin-only and kinesin and Myo1c cargo detach at actin intersections (without a pause or pass) at approximately the same frequency (~4%). Kinesin-1-only: n = 92 observed events from 7 chambers. Kinesin-1+Myo1c: n = 61 observed events from 5 chambers. Error bars: bootstrapped SD. **** p ≤ 0.0001 Kruskal-Wallis.
Also see Figure S2.
After a pause, MCBs continued motility along a microtubule an average of 73% of the time, or released from the intersection and diffused away an average of 27% of the time. This was not significantly altered by the presence of Myo1c on the MCB (Figure S2a). Cumulatively, these in vitro data support the hypothesis that Myo1c can dock intracellular cargo at actin filament intersections within the cell. Following docking, MCB transport along actin filaments was not observed in our experiments. Myo1c has a 50-fold slower motility rate than kinesin-1 [28, 29], and is non-processive unless numerous motors are present [20, 29, 30], making it unlikely that we would observe substantial motility along, or recruitment to, actin filaments using these experimental parameters (see Discussion).
To determine whether the observed cargo docking at actin intersections is specific to Myo1c, we tested the ability of the non-motor actin filament-binding protein, α-actinin, to stall cargo at AF/MT intersections. We added 200 nM of a biotinylated actin-binding domain construct of α-actinin (hereafter referred to as “α-actinin”) to kinesin-1-coated MCBs, the same concentration as was used for Myo1c. We found that in the presence of α-actinin, MCBs were sequestered to actin filaments, resulting in approximately 80% fewer microtubule-based runs (Figure S2b). Within 5 min of addition to the flow cell, 75% of kinesin-1 and α-actinin-coated cargo were stably bound to actin filaments (Figure S2c). In comparison, only 18% of kinesin-only, and 29% of kinesin and Myo1c-containing MCBs were bound to actin. Of the few kinesin-1 and α-actinin-containing MCBs that resulted in kinesin-driven motility, 63% of MCBs passed the actin intersection (Figure S2d), demonstrating that α-actinin is not able to dynamically tether MCBs to AF/MT intersections during processive kinesin-1-driven runs, unlike the observed results with Myo1c.
Non-muscle tropomyosins have been reported to activate [8, 10, 12], inhibit [11, 31], or not affect myosin motility and ATPase activities when bound to actin filaments in an isoform-specific manner [9, 13, 32]. We focused on non-muscle tropomyosin-2 (Tm2) because this isoform is generally found throughout the cytoplasm, but is specifically excluded from the leading edge of the cell and other areas of highly dynamic actin filament populations [14, 33, 34]. To test the effects of full-length Tm2 on Myo1c motility, we examined Tm2-actin gliding over a bed of Myo1c. In the absence of tropomyosin, Myo1c robustly powers actin filament motility with a velocity of 16 nm/s at room temperature (Figures 3A and 3B, Movie S3). In contrast, tropomyosin-bound actin filaments were not directionally driven, and only transiently interacted with the surface of the coverslip (Figures 3A and 3B, Movie S3). Even these transient interactions disappeared when methylcellulose, used to concentrate actin at the surface of the coverslip, was omitted from the assay buffer. Meanwhile, non-tropomyosin actin filaments were still capable of directionally gliding. Taken together, these results show that Tm2 inhibits force-generating Myo1c-actin filament interactions.
Figure 3. Non-muscle Tm2 regulates Myo1c interactions at Tm2-AF/MT intersections.
(A) Actin filament gliding assays were performed in the presence or absence of Tm2. Representative images of the first frame, last frame, and maximum intensity projection from 5 min gliding assays in the presence and absence of Tm2, at 50 nM Myo1c. In the absence of Tm2, Myo1c powers continuous actin gliding. Tm2 disrupts gliding and only non-directional movement is observed. Magenta and cyan stars label two example actin filaments in each condition. Acquired at 0.5 fps. Scale bar = 1 μm. See Movie S3.
(B) Tm2 inhibits Myo1c actin filament gliding over a range of Myo1c concentrations (50–500 nM Myo1c). Actin filaments without Tm2 glide in directional tracks, whereas Tm2-actin interact transiently and non-directionally with Myo1c on the surface. n > 150 filaments per condition.
(C) Tm2-actin abolishes Myo1c-based cargo run initiation at Tm2-AF/MT intersections. Initiation of processive microtubule-based kinesin-1 motility was scored with respect to the nearest Tm2-actin intersection. Kinesin + Myo1c MCBs do not preferentially initiate runs at actin intersections when Tm2 is present. Acquired 10 fps; kinesin-1+Myo1c n = 37, kinesin-1-only n = 33, events from 27 fields-of-view from 2 chambers, each; Scale bar = 1μm. Error bars: bootstrapped SD. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01 Kruskal-Wallis with Dunn’s multiple comparison’s test. Also see Figure S3.
(D) Run initiation is not enhanced at MT/MT intersections. Initiation of processive microtubule-based kinesin-1 motility was scored with respect to the nearest MT/MT intersection. Both kinesin-1-only and kinesin-1+Myo1c MCBs initiate runs at MT/MT intersections with the same frequency as randomly generated points in the same fields of view. Data acquired at 10 fps from Tm2-actin movies. Kinesin-1+Myo1c n = 41, kinesin-1-only n = 13 events from 2 chambers, each. Error bars: bootstrapped SD. Initiation event distances from MT/MT intersections between groups are not significantly different based on Kruskal-Wallis with Dunn’s multiple comparison’s test.
(E) Tm2-actin prevents Myo1c-based cargo pausing at AF/MT intersections. MCB behavior was observed at AF/MT or Tm2-AF/MT intersections and scored as: “detach,” meaning the cargo detached at the intersection without a pause > 0.5 s; “pass,” where the MCB passed the intersection without a pause > 0.5 s; or “pause” where MCBs paused for > 0.5 s (limited by 2 fps acquisition rate). The behavior of kinesin-1-only (red) and kinesin-1+Myo1c (blue) MCBs at AF/MT intersections was previously described in Figure 2. Bars with grey stripes illustrate bead behavior at Tm2-AF/MT intersections. Significantly fewer kinesin-1+Myo1c MCBs pause at Tm2-AF/MT than AF/MT intersections, replicating kinesin-1-only cargo behavior. Kinesin-1, n = 92; kinesin-1+Myo1c, n = 64; kinesin-1 (+ Tm2-actin), n = 37; kinesin-1 +Myo1c (+ Tm2-actin), n = 61 observed events in 5 chambers. Error bars: bootstrapped SD. **** p ≤ 0.0001 Kruskal-Wallis with Dunn’s multiple comparison’s test.
(F) Representative time series and kymograph showing that kinesin-1-only MCBs pass through actin intersections when Tm2 is present. Microtubules are pseudo colored green and Tm2-actin is pseudo colored pink; the blue arrowhead denotes the Tm2-actin intersection. Scale bars = 1 μm and 3 s; acquired at 2 fps. See Movie S4.
(G) Representative time series and kymograph showing that cargo with kinesin-1+Myo1c pass Tm2-actin intersections. When Tm2 is bound to actin filaments, kinesin-1+Myo1c cargo do not pause at AF/MT intersections. Scale bars = 1 μm and 3 s; acquired at 2 fps. See Movie S4.
To determine if Tm2 can modulate the Myo1c-mediated effects of MCB behavior at AF/MT intersections, we added Tm2-coated actin to our in vitro crossed-filament assay. Strikingly, Tm2-actin inhibited Myo1c-specific cargo run initiation at Tm2-AF/MT intersections (Figure 3C). In contrast to our observations in the absence of Tm2, MCBs transported by kinesin-1 in the presence and absence of Myo1c initiate microtubule-based runs stochastically with respect to the nearest Tm2-actin intersection (Figure S3). We also found that kinesin-1 MCBs with or without Myo1c are no more likely to land at MT/MT intersections than randomly chosen points in the same fields of view (Figure 3D). If run initiation at AF/MT intersections was solely due to increased motor-cytoskeleton binding sites at a particular point, we would expect to see a similar enhancement in initiation at MT/MT intersections. Thus, Myo1c recruitment of cargo to AF/MT intersections is a specific property of Myo1c coordinating with kinesin-1 to initiate microtubule-based cargo runs, and is regulated by the presence of Tm2.
Finally, we found that Tm2-actin robustly prevents Myo1c-induced pausing of MCBs at Tm2-AF/MT intersections (Figures 3E–G, Movie S4). In the absence of Tm2, 33% of kinesin-only cargo and 92% of kinesin and Myo1c MCBs pause at AF/MT intersections (Figures 2D, 3E). When Tm2-actin is present, rather than pausing at actin intersections, kinesin-1 cargo with Myo1c-bound pass Tm2-actin intersections with frequencies comparable to those observed for kinesin-1-only cargo (66%) (Figure 3E).
Discussion
Intracellular transport is largely driven by processive motors, such as kinesin-1, which are capable of transporting cargos over long distances within the cell. Previous studies have shown that when different types of processive motors compete at cytoskeletal intersections, both the number of motors bound and the biophysical properties of these motors and their cargo adaptors determine which motor type will dominate [19, 35, 36]. Here, we use a similar strategy to investigate the role of non-processive motors in intracellular trafficking. Our observations suggest that non-processive motors such as Myo1c may be key in regulating the specificity of intracellular targeting of vesicular and organelle cargos.
Myosin-I motors participate in a variety of membrane-actin interactions, including dynamic membrane transformations [4, 7, 20–22, 37]. For example, Myo1c has been hypothesized to participate in the recycling of lipid raft cargo toward the plasma membrane from peri-nuclear recycling tubes [1], and the last steps of GLUT4 trafficking toward the plasma membrane [2, 3, 7, 37]. The localization of Myo1c to these specific cargos suggests that myosin-I motors could be used to actively sort and target cargo to particular destinations within the cell.
Here we used an in vitro reconstitution assay to demonstrate that the non-processive motor Myo1c plays important roles both at the beginning and end of kinesin-driven long distance transport (Figure 4). Myo1c facilitates cargo run initiation by selectively recruiting cargo to cytoskeletal intersections, where kinesin-1 can rapidly begin to transport cargo along a microtubule (Figures 1 and 4). Strikingly, the Myo1c-specific effects on cargo initiation and termination events are not reproduced by a different actin-binding protein, α-actinin, suggesting that the active motor activity of Myo1c, not just its actin binding capacity, is necessary for successful cargo tethering. Myo1c-specific cargo run initiation and termination is also abolished by the binding of Tm2 to actin filaments. This provides a mechanism to selectively regulate Myo1c activity, and thus cargo behavior in the cell, in a spatially-controlled manner.
Figure 4. Myo1c affects both the initiation and termination of kinesin-1-driven runs.
Myo1c facilitates kinesin-1 run initiation at AF/MT, but not Tm2-AF/MT intersections, and specifically delivers cargo to non-Tm2-actin filament intersections to terminate long distance microtubule-based transport. Also see Figure S4.
We note that while Myo1c motors induce the preferential binding of membrane-bound cargos at AF/MT intersections, these cargos do not become stably tethered at these junctions. This is likely due to the robust motor activity of kinesin-1 motors, as cargo that initiate runs at AF/MT intersections typically clear the intersection within 500 ms. In contrast, when cargo moving along a microtubule encounter an actin filament, Myo1c motors have over a second to bind to the actin intersection and form a more stable interaction. Further, kinesin-1 motors are not adept at navigating roadblocks (Figure 2) [38], so the physical obstruction of the actin intersection likely helps facilitate Myo1c-domination of the processive/non-processive motor interaction during run termination.
We know that multiple kinesins are driving the motility of the MCBs along microtubules, since the run lengths observed under our assay conditions ( > 5 μm) exceed those of single kinesin-1 motors (~1 μm; [28]). Myo1c is a non-processive motor that interacts only transiently with actin filaments, so the engagement of many Myo1c motors are likely to be necessary to stall a cargo at an AF/MT intersection over tens to hundreds of seconds [20, 30]. However, constraints dictated by the bead, motors, and cytoskeleton geometry limit the number of motors available to bind at the AF/MT intersection, so we predict that no more than 6 Myo1c and 11 kinesin-1 motors are capable of interacting at any given time (Figure S4). We estimate that only a few Myo1c motors are necessary to effectively stall kinesin-1 motility at AF/MT intersections.
Myosin-V, kinesin-1, and Myo1c have all been localized to the insulin-responsive membrane compartment that contains GLUT4 glucose transporters. Actin-based transport has been proposed to play an active role in the transport of this compartment to the plasma membrane in adipocytes and muscle cells; yet, how GLUT4-containing membranes are transferred from kinesin-1-dependent microtubule transport to actin-based transport in the cell periphery, and the role of Myo1c during this process, are poorly understood [23–26]. The data presented here support the previously proposed role for Myo1c as a motor necessary for docking or tethering GLUT4-containing vesicles, trapping these vesicles in the cortical actin network before GLUT4 vesicle fusion [2, 3]. Specifically, we now show that the intrinsic properties of kinesin-1 and Myo1c motors allow for cargo docking at actin filament intersections without any further regulators. Cumulatively, these results support a model in which kinesin motors drive long distance transport of GLUT4 cargo toward the cell periphery along microtubules. At the periphery, Myo1c halts microtubule-based transport, docking cargo at AF/MT intersections until they complete their transport to, and fusion with, the plasma membrane upon insulin-stimulated myosin-V transport. This finding is consistent with the observation of GLUT4 vesicle docking adjacent to microtubules prior to fusion with the plasma membrane [39]. Given its slow motility and non-processive nature, our data do not support a model in which Myo1c actively transports cargos along actin filaments in this geometry. Instead, our data support a model in which Myo1c is required as a dynamic tether between microtubule- and actin-based transport regimes.
Tropomyosin has been shown to both positively [8, 10, 12] and negatively [11, 31] regulate motor motility within the cell [9, 13, 32, 40]. For instance, S. cerevisiae myosin-V is selectively activated by tropomyosin-coated actin populations [8]. Alternatively, Myo1b and Myo1a preferentially localize to tropomyosin-free actin populations and show inhibited actin gliding of tropomyosin-coated filaments [11, 13, 32]. Here, we find that Myo1c activity is inhibited by Tm2-actin in vitro in both gliding and crossed-filament assays. Tm2 inhibits Myo1c-mediated cargo run initiation, as well as processive run termination, at actin intersections. Since Tm2 is located just behind the leading edge of the cell, prevention of Myo1c-mediated cargo docking would promote cargo passage through the dense cortical actin network, enhancing delivery to the plasma membrane for exocytic fusion. These results strengthen the argument that Myo1c is an important factor in cargo sorting, providing an underlying mechanism by which Myo1c-induced cargo run initiation and run termination occurs preferentially when encountering specific actin filament populations, such as the highly dynamic actin filament populations near the peri-nuclear region and at exocytic zones just beneath the plasma membrane (Figure 4) [14, 41].
There are a variety of actin-binding and microtubule-associated proteins and post-translational modifications that can modify the ability of motors to interact with specific cytoskeletal populations. While actin filament binding proteins may specify distinct subcellular domains, microtubules can be modified post-translationally to form differentially-localized cytoskeletal populations within the cell that may lead to either activation or inhibition of cargo transport [42]. Cumulatively, these observations suggest that the specific complement of processive and non-processive molecular motors bound to an intracellular cargo specifies delivery to subcellular domains via motor-specific deciphering of the cytoskeletal “code.”
Conclusion
Our in vitro reconstitution approach allowed us, for the first time, to directly observe the interactions between specific non-processive and processive molecular motors on a physiologically-relevant, yet simplified, cargo in unambiguous cytoskeletal environments. Our results suggest Myo1c-bound cargo can be loaded on to microtubules for kinesin-driven long distance transport and later docked, or tethered in peripheral actin-rich regions to facilitate the initiation and termination of long distance transport. Additionally, we find that both run initiation and run termination are regulated by the actin-binding protein tropomyosin, providing a mechanism to regulate the localized delivery of cargos to regions of tropomyosin-free actin filaments within the cell. Thus, the tropomyosin regulation of Myo1c may permit localized regulation of cargo behavior without direct cargo or motor modification, enabling effective sorting of exocytic cargo.
Experimental Procedures
See Supplemental Methods for detailed information about experimental reagents and procedures.
Supplementary Material
Lipid membrane-coated beads (MCBs) containing kinesin-1-only or kinesin-1+Myo1c were observed as they initiated kinesin-driven motility along microtubules. The movie on the left shows an example run of a kinesin-1-only MCB initiating a microtubule-based run stochastically in relation to the nearest actin filament intersection (See Figure 1A). The movie on the right shows a sample initiation event of a kinesin-1+Myo1c bead that begins at an AF/MT intersection (See Figure 1B). Movie is played back in real time (2 fps). Scale bar = 1 μm. Timestamp labels landing event at “0 s.”
We observed the behavior of MCBs traveling along microtubules via kinesin-1-driven transport as they encountered actin filament intersections. The movie on the left shows a sample kinesin-only MCB that passes the AF/MT intersection (See Figure 2A). The center and right movies show sample kinesin-1+Myo1c events that pause at the AF/MT intersection for 20 s (center) and 220 s (right), respectively (See Figure 2B). Movie is played back at 3x real time (6 fps). Scale bar = 1 μm.
Actin filament gliding assays were performed with 50 nM Myo1c in the presence or absence of Tm2 and assessed for directional gliding. The movie on the right shows a sample non-Tm2 gliding assay with directionally gliding actin filaments, while the movie on the right shows a sample Tm2-actin gliding assay with transient Myo1c-actin interactions (See Figures 3A and 3B). Movie is played back at 40x real time (20 fps). Scale bar = 1 μm.
We observed the behavior of kinesin-1 MCBs in the presence or absence of Myo1c at they approached Tm2-AF/MT intersections. Both kinesin-1-only (movie on left) and kinesin-1+Myo1c MCBs (right) pass Tm2-AF/MT intersections. Movie is played back at 3x real time (6 fps). Scale bar = 1 μm.
Acknowledgments
We thank Tianming Lin and Mariko Tokito for their excellent technical assistance, and Michael Greenberg and Serapion Pyrpassopoulos for help developing the experimental protocols. Additionally, we thank Michael Woody, Abbey Weith, Henry Shuman, and other members of the Ostap and Holzbaur labs as well as the Pennsylvania Muscle Institute for helpful suggestions and stimulating conversation. Finally, we thank Sarah Hitchcock-DeGregori for the cDNA encoding rat non-muscle tropomyosin-2 (Tm2). This work was supported by an American Heart Association Predoctoral Fellowship 12PRE12030164 to B.B.M. and by National Institute of Health grants T32 AR053461-08 to B.B.M. and P01 GM087253-10 to E.L.F.H. and E.M.O.
Footnotes
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
References
- 1.Brandstaetter H, Kendrick-Jones J, Buss F. Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion. J Cell Sci. 2012;125:1991–2003. doi: 10.1242/jcs.097212. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Bose A, Robida S, Furcinitti PS, Chawla A, Fogarty K, Corvera S, Czech MP. Unconventional Myosin Myo1c Promotes Membrane Fusion in a Regulated Exocytic Pathway. Mol Cell Biol. 2004;24:5447–5458. doi: 10.1128/MCB.24.12.5447-5458.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Boguslavsky S, Chiu T, Foley KP, Osorio-Fuentealba C, Antonescu CN, Bayer KU, Bilan PJ, Klip A. Myo1c binding to submembrane actin mediates insulin-induced tethering of GLUT4 vesicles. Mol Biol Cell. 2012;23:4065–4078. doi: 10.1091/mbc.E12-04-0263. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Almeida CG, Yamada A, Tenza D, Louvard D, Raposo G, Coudrier E. Myosin 1b promotes the formation of post-Golgi carriers by regulating actin assembly and membrane remodelling at the trans-Golgi network. Nat Cell Biol. 2011;13:779–789. doi: 10.1038/ncb2262. [DOI] [PubMed] [Google Scholar]
- 5.Santos-Argumedo L, Maravillas-Montero JL, López-Ortega O. Class I myosins in B-cell physiology: functions in spreading, immune synapses, motility, and vesicular traffic. Immunol Rev. 2013;256:190–202. doi: 10.1111/imr.12105. [DOI] [PubMed] [Google Scholar]
- 6.Tiwari A, Jung JJ, Inamdar SM, Nihalani D, Choudhury A. The myosin motor Myo1c is required for VEGFR2 delivery to the cell surface and for angiogenic signaling. Am J Physiol - Heart Circ Physiol. 2013;304:H687–H696. doi: 10.1152/ajpheart.00744.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Yip MF, Ramm G, Larance M, Hoehn KL, Wagner MC, Guilhaus M, James DE. CaMKII-Mediated Phosphorylation of the Myosin Motor Myo1c Is Required for Insulin-Stimulated GLUT4 Translocation in Adipocytes. Cell Metab. 2008;8:384–398. doi: 10.1016/j.cmet.2008.09.011. [DOI] [PubMed] [Google Scholar]
- 8.Clayton JE, Pollard LW, Sckolnick M, Bookwalter CS, Hodges AR, Trybus KM, Lord M. Fission yeast tropomyosin specifies directed transport of myosin-V along actin cables. Mol Biol Cell. 2014;25:66–75. doi: 10.1091/mbc.E13-04-0200. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Ostap EM. Tropomyosins as discriminators of myosin function. Adv Exp Med Biol. 2008;644:273–282. doi: 10.1007/978-0-387-85766-4_20. [DOI] [PubMed] [Google Scholar]
- 10.Chacko S. Effects of phosphorylation, calcium ion, and tropomyosin on actin-activated adenosine 5′-triphosphatase activity of mammalian smooth muscle myosin. Biochemistry (Mosc) 1981;20:702–707. doi: 10.1021/bi00507a005. [DOI] [PubMed] [Google Scholar]
- 11.Tang N, Ostap EM. Motor domain-dependent localization of myo1b (myr-1) Curr Biol. 2001;11:1131–1135. doi: 10.1016/s0960-9822(01)00320-7. [DOI] [PubMed] [Google Scholar]
- 12.Umemoto S, Bengur AR, Sellers JR. Effect of multiple phosphorylations of smooth muscle and cytoplasmic myosins on movement in an in vitro motility assay. J Biol Chem. 1989;264:1431–1436. [PubMed] [Google Scholar]
- 13.Fanning AS, Wolenski JS, Mooseker MS, Izant JG. Differential regulation of skeletal muscle myosin-II and brush border myosin-I enzymology and mechanochemistry by bacterially produced tropomyosin isoforms. Cell Motil Cytoskeleton. 1994;29:29–45. doi: 10.1002/cm.970290104. [DOI] [PubMed] [Google Scholar]
- 14.Gunning PW, Schevzov G, Kee AJ, Hardeman EC. Tropomyosin isoforms: divining rods for actin cytoskeleton function. Trends Cell Biol. 2005;15:333–341. doi: 10.1016/j.tcb.2005.04.007. [DOI] [PubMed] [Google Scholar]
- 15.Millecamps S, Julien JP. Axonal transport deficits and neurodegenerative diseases. Nat Rev Neurosci. 2013;14:161–176. doi: 10.1038/nrn3380. [DOI] [PubMed] [Google Scholar]
- 16.Batters C, Arthur CP, Lin A, Porter J, Geeves MA, Milligan RA, Molloy JE, Coluccio LM. Myo1c is designed for the adaptation response in the inner ear. EMBO J. 2004;23:1433–1440. doi: 10.1038/sj.emboj.7600169. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Mele C, Iatropoulos P, Donadelli R, Calabria A, Maranta R, Cassis P, Buelli S, Tomasoni S, Piras R, Krendel M, et al. MYO1E Mutations and Childhood Familial Focal Segmental Glomerulosclerosis. N Engl J Med. 2011;365:295–306. doi: 10.1056/NEJMoa1101273. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Holzbaur EL, Goldman YE. Coordination of molecular motors: from in vitro assays to intracellular dynamics. Curr Opin Cell Biol. 2010;22:4–13. doi: 10.1016/j.ceb.2009.12.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Ali MY, Kennedy GG, Safer D, Trybus KM, Sweeney HL, Warshaw DM. Myosin Va and myosin VI coordinate their steps while engaged in an in vitro tug of war during cargo transport. Proc Natl Acad Sci U S A. 2011;108:E535–541. doi: 10.1073/pnas.1104298108. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Greenberg MJ, Ostap EM. Regulation and control of myosin-I by the motor and light chain-binding domains. Trends Cell Biol. 2013;23:81–89. doi: 10.1016/j.tcb.2012.10.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.McConnell RE, Tyska MJ. Leveraging the membrane-cytoskeleton interface with myosin-1. Trends Cell Biol. 2010;20:418–426. doi: 10.1016/j.tcb.2010.04.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Gupta P, Gauthier NC, Cheng-Han Y, Zuanning Y, Pontes B, Ohmstede M, Martin R, Knölker HJ, Döbereiner HG, Krendel M, et al. Myosin 1E localizes to actin polymerization sites in lamellipodia, affecting actin dynamics and adhesion formation. Biol Open. 2013;2:1288–1299. doi: 10.1242/bio.20135827. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Semiz S, Park JG, Nicoloro SMC, Furcinitti P, Zhang C, Chawla A, Leszyk J, Czech MP. Conventional kinesin KIF5B mediates insulin-stimulated GLUT4 movements on microtubules. EMBO J. 2003;22:2387–2399. doi: 10.1093/emboj/cdg237. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Chen Y, Wang Y, Zhang J, Deng Y, Jiang L, Song E, Wu XS, Hammer JA, Xu T, Lippincott-Schwartz J. Rab10 and myosin-Va mediate insulin-stimulated GLUT4 storage vesicle translocation in adipocytes. J Cell Biol. 2012;198:545–560. doi: 10.1083/jcb.201111091. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Ishikura S, Klip A. Muscle cells engage Rab8A and myosin Vb in insulin-dependent GLUT4 translocation. Am J Physiol - Cell Physiol. 2008;295:C1016–C1025. doi: 10.1152/ajpcell.00277.2008. [DOI] [PubMed] [Google Scholar]
- 26.Yoshizaki T, Imamura T, Babendure JL, Lu JC, Sonoda N, Olefsky JM. Myosin 5a Is an Insulin-Stimulated Akt2 (Protein Kinase B?) Substrate Modulating GLUT4 Vesicle Translocation. Mol Cell Biol. 2007;27:5172–5183. doi: 10.1128/MCB.02298-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Pyrpassopoulos S, Shuman H, Ostap EM. Method for Measuring Single-Molecule Adhesion Forces and Attachment Lifetimes of Protein Membrane Interactions. In: Coutts AS, editor. Adhesion Protein Protocols Methods in Molecular Biology. Humana Press; 2013. [Accessed June 12, 2014]. pp. 389–403. Available at: http://link.springer.com/protocol/10.1007/978-1-62703-538-5_24. [DOI] [PubMed] [Google Scholar]
- 28.Walter WJ, Beránek V, Fischermeier E, Diez S. Tubulin Acetylation Alone Does Not Affect Kinesin-1 Velocity and Run Length In Vitro. PLoS ONE. 2012;7:e42218. doi: 10.1371/journal.pone.0042218. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Pyrpassopoulos S, Feeser EA, Mazerik JN, Tyska MJ, Ostap EM. Membrane-Bound Myo1c Powers Asymmetric Motility of Actin Filaments. Curr Biol. 2012;22:1688–1692. doi: 10.1016/j.cub.2012.06.069. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Greenberg MJ, Lin T, Goldman YE, Shuman H, Ostap EM. Myosin IC generates power over a range of loads via a new tension-sensing mechanism. Proc Natl Acad Sci. 2012;109:E2433–E2440. doi: 10.1073/pnas.1207811109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Huckaba TM, Lipkin T, Pon LA. Roles of type II myosin and a tropomyosin isoform in retrograde actin flow in budding yeast. J Cell Biol. 2006;175:957–969. doi: 10.1083/jcb.200609155. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Collins K, Matsudaira P. Differential regulation of vertebrate myosins I and II. J Cell Sci Suppl. 1991;14:11–16. doi: 10.1242/jcs.1991.supplement_14.3. [DOI] [PubMed] [Google Scholar]
- 33.Lin JJ, Hegmann TE, Lin JL. Differential localization of tropomyosin isoforms in cultured nonmuscle cells. J Cell Biol. 1988;107:563–572. doi: 10.1083/jcb.107.2.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Percival JM, Thomas G, Cock TA, Gardiner EM, Jeffrey PL, Lin JJC, Weinberger RP, Gunning P. Sorting of tropomyosin isoforms in synchronised NIH 3T3 fibroblasts: Evidence for distinct microfilament populations. Cell Motil Cytoskeleton. 2000;47:189–208. doi: 10.1002/1097-0169(200011)47:3<189::AID-CM3>3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
- 35.Schroeder HW, III, Mitchell C, Shuman H, Holzbaur ELF, Goldman YE. Motor Number Controls Cargo Switching at Actin-Microtubule Intersections In Vitro. Curr Biol. 2010;20:687–696. doi: 10.1016/j.cub.2010.03.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Schroeder HW, III, Hendricks AG, Ikeda K, Shuman H, Rodionov V, Ikebe M, Goldman YE, Holzbaur ELF. Force-Dependent Detachment of Kinesin-2 Biases Track Switching at Cytoskeletal Filament Intersections. Biophys J. 2012;103:48–58. doi: 10.1016/j.bpj.2012.05.037. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Bose A, Guilherme A, Robida SI, Nicoloro SMC, Zhou QL, Jiang ZY, Pomerleau DP, Czech MP. Glucose transporter recycling in response to insulin is facilitated by myosin Myo1c. Nature. 2002;420:821–824. doi: 10.1038/nature01246. [DOI] [PubMed] [Google Scholar]
- 38.Dixit R, Ross JL, Goldman YE, Holzbaur ELF. Differential Regulation of Dynein and Kinesin Motor Proteins by Tau. Science. 2008;319:1086–1089. doi: 10.1126/science.1152993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Dawicki-McKenna JM, Goldman YE, Ostap EM. Sites of Glucose Transporter-4 Vesicle Fusion with the Plasma Membrane Correlate Spatially with Microtubules. PLoS ONE. 2012;7:e43662. doi: 10.1371/journal.pone.0043662. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Lord M. Cytoskeletal Regulation: Sorting Out Stress Fibers with Tropomyosin. Curr Biol. 2011;21:R255–R257. doi: 10.1016/j.cub.2011.02.043. [DOI] [PubMed] [Google Scholar]
- 41.Vindin H, Gunning P. Cytoskeletal tropomyosins: choreographers of actin filament functional diversity. J Muscle Res Cell Motil. 2013;34:261–274. doi: 10.1007/s10974-013-9355-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Sirajuddin M, Rice LM, Vale RD. Regulation of microtubule motors by tubulin isotypes and post-translational modifications. Nat Cell Biol. 2014;16:335–344. doi: 10.1038/ncb2920. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Lipid membrane-coated beads (MCBs) containing kinesin-1-only or kinesin-1+Myo1c were observed as they initiated kinesin-driven motility along microtubules. The movie on the left shows an example run of a kinesin-1-only MCB initiating a microtubule-based run stochastically in relation to the nearest actin filament intersection (See Figure 1A). The movie on the right shows a sample initiation event of a kinesin-1+Myo1c bead that begins at an AF/MT intersection (See Figure 1B). Movie is played back in real time (2 fps). Scale bar = 1 μm. Timestamp labels landing event at “0 s.”
We observed the behavior of MCBs traveling along microtubules via kinesin-1-driven transport as they encountered actin filament intersections. The movie on the left shows a sample kinesin-only MCB that passes the AF/MT intersection (See Figure 2A). The center and right movies show sample kinesin-1+Myo1c events that pause at the AF/MT intersection for 20 s (center) and 220 s (right), respectively (See Figure 2B). Movie is played back at 3x real time (6 fps). Scale bar = 1 μm.
Actin filament gliding assays were performed with 50 nM Myo1c in the presence or absence of Tm2 and assessed for directional gliding. The movie on the right shows a sample non-Tm2 gliding assay with directionally gliding actin filaments, while the movie on the right shows a sample Tm2-actin gliding assay with transient Myo1c-actin interactions (See Figures 3A and 3B). Movie is played back at 40x real time (20 fps). Scale bar = 1 μm.
We observed the behavior of kinesin-1 MCBs in the presence or absence of Myo1c at they approached Tm2-AF/MT intersections. Both kinesin-1-only (movie on left) and kinesin-1+Myo1c MCBs (right) pass Tm2-AF/MT intersections. Movie is played back at 3x real time (6 fps). Scale bar = 1 μm.