© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs249938. doi:10.1242/jcs.249938

RESEARCH ARTICLE Stable tug-of-war between kinesin-1 and cytoplasmic dynein upon different ATP and roadblock concentrations Gina A. Monzon1, Lara Scharrel2, Ashwin DSouza2, Verena Henrichs2,3, Ludger Santen1,* and Stefan Diez2,4,*

ABSTRACT and dynein motors are known to be often simultaneously bound to The maintenance of intracellular processes, like organelle transport and the cargo (Gennerich and Schild, 2006; Welte, 2004; Soppina et al., cell division, depend on bidirectional movement along microtubules. 2009; Hendricks et al., 2010). Without any regulatory mechanism These processes typically require kinesin and dynein motor proteins, the cargo might be transported in the kinesin or dynein direction, which move with opposite directionality. Because both types of motors might randomly switch direction or might get stuck at a random are often simultaneously bound to the cargo, regulatory mechanisms are position. However, regulatory mechanisms ensuring targeted required to ensure controlled directional transport. Recently, it has transport remain poorly understood. been shown that parameters like mechanical motor activation, ATP In the past, different regulatory mechanisms have been proposed. concentration and roadblocks on the microtubule surface differentially One mechanism suggests coordinating the motor activity (Gross, influence the activity of kinesin and dynein motors in distinct manners. 2004). In this model, motors are assumed to be a priori in a passive However, how these parameters affect bidirectional transport state. By activating one motor team, targeted cargo transport occurs systems has not been studied. Here, we investigate the regulatory in the direction of the active team (Gross, 2004). One such activation influence of these three parameters using in vitro gliding motility mechanism involves adaptor proteins (McKenney et al., 2014; assays and stochastic simulations. We find that the number of active Schroeder and Vale, 2016; Blasius et al., 2007; Elshenawy et al., kinesin and dynein motors determines the transport direction and 2019). Another hypothesizes a mutual mechanical activation to velocity, but that variations in ATP concentration and roadblock trigger cargo transport (Monzon et al., 2019; Ally et al., 2009; De density have no significant effect. Thus, factors influencing the force Rossi et al., 2017). Mechanical dynein activation has been shown to balance between opposite motors appear to be important, whereas determine the velocity in unidirectional dynein-driven transport the detailed stepping kinetics and bypassing capabilities of the (Monzon et al., 2019). In that study, mechanical dynein activation motors only have a small effect. was shown to strongly depend on the number of involved dynein motors (Monzon et al., 2019) suggesting that in bidirectional KEY WORDS: Kinesin, Dynein, ATP concentration, Roadblocks, transport mechanical activation might also be linked to the number Gliding motility assay, Stochastic modeling, Tug-of-war of motors. The influence of varying the number of motors has been studied previously. Rezaul et al. (2016), for instance, reversed a INTRODUCTION dynein-driven membrane organelle in vivo by adding a large Intracellular transport is essential for cell division or organelle number of kinesin motors. Moreover, Vale et al. (1992) showed that transport (Lodish et al., 2000; Verhey and Hammond, 2009; the transport direction in bidirectional gliding assays depends on the Soppina et al., 2009), and dysfunction leads to neurodegenerative number of kinesin motors. However, for a complete understanding diseases like Alzheimer’s disease or amyotrophic lateral sclerosis of the role of mechanical dynein activation in bidirectional (ALS) (De Vos et al., 2008; Goldstein, 2001; Hurd and Saxton, transport, a systematic analysis is needed. 1996; Chen et al., 2014; Karki and Holzbaur, 1999). In particular, Another model proposes modifying the motor properties as a microtubule-based transport is carried out by teams of the opposite- regulatory mechanism. Müller et al. (2008) showed that modified directed motor proteins kinesin and dynein. By actively moving motor properties lead to different motility states. The motor velocity, cargo back and forth along microtubules, kinesin and dynein deliver for instance, is known to be modified by ATP concentration cargo to where it is needed. Mitochondria, for instance, are (Schnitzer et al., 2000; Ross et al., 2006; Torisawa et al., 2014; transported to locations of low ATP concentration (Morris and Nicholas et al., 2015a). If the ATP concentration asymmetrically Hollenbeck, 1993) and chromosomes are perfectly aligned on modified the kinesin and dynein velocities, a bidirectionally moved spindle microtubules during cell division (She and Yang, 2017; cargo would likely change its net direction. Another motor property Goshima and Vale, 2003). Importantly, however, teams of kinesin influenced by ATP concentration is the motor stall force (Mallik et al., 2004; Visscher et al., 1999). While the dynein stall force is known to 1Center for Biophysics, Department of Physics, Saarland University, D-66123, increase linearly with ATP concentration (Mallik et al., 2004), the Saarbrücken, Germany. 2B CUBE Center for Molecular Bioengineering and Cluster kinesin stall force is slightly reduced for very low ATP concentrations of Excellence Physics of Life, Technische Universität Dresden, D-01307 Dresden, Germany. 3Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, but invariant otherwise (Visscher et al., 1999). Thus, an increased CZ-25250 Prague West, Czech Republic. 4Max Planck Institute of Molecular Cell ATP concentration might strengthen the dynein team and might Biology and Genetics, D-01307 Dresden, Germany. reverse a cargo mainly transported in the kinesin direction. Consistent *Authors for correspondence ([email protected]; with this, a directional change as a function of ATP concentration is [email protected]) predicted by the theoretical work of Klein et al. (2014). However, whether the transport direction can be changed with a change in ATP L. Santen., 0000-0001-8478-9667; S.D., 0000-0002-0750-8515 concentration has not been tested experimentally.

Handling Editor: Michael Way Yet another regulatory mechanism is using hindering roadblocks

Received 13 June 2020; Accepted 18 October 2020 to control bidirectional transport. Single motor proteins have been Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249938. doi:10.1242/jcs.249938 observed to have different reactions when encountering a roadblock (Telley et al., 2009; Dixit et al., 2008; Schneider et al., 2015). While a single kinesin detaches, for instance, when encountering the microtubule-associated protein tau, dynein continues stepping (Vershinin et al., 2007; Siahaan et al., 2019; Tan et al., 2019). Moreover, different mechanisms to bypass roadblocks have been observed in single-molecule experiments (Ferro et al., 2019). While kinesin has to detach and reattach behind the roadblock, dynein uses its ability to take sidesteps (Ferro et al., 2019). However, how a cargo that is bidirectionally transported by many motors reacts when encountering a roadblock, and whether roadblocks change the transport direction of such cargo remains unclear. Moreover, parameters such as second messengers, viscosity, inhibitors or even size changes of the cargo could have an effect on bidirectional transport (Gennerich and Schild, 2006; Soppina et al., 2009; Hendricks et al., 2012; Coy et al., 1999; Firestone et al., 2012). However, here we focus on the relative contribution of number of motors, ATP concentration and roadblocks to regulate bidirectional transport. To fully understand the relative contribution of different parameters, the parameters need to be varied systematically, and the bidirectional transport needs to be measured without affecting the activity of the motors themselves. The use of microtubule gliding assays is one way to achieve this (see Fig. 1A for an illustration). To measure the collective behavior of motors without affecting the motors themselves, microtubules are labeled and tracked. The number of motors involved in the transport can be systematically changed by varying the motor density on the surface. Moreover, the ATP concentration in the surrounding medium can be systematically changed, and microtubules can be coated with Fig. 1. Varying the kinesin density regulates the transport direction in roadblocks at different concentrations. Consequently, microtubule bidirectional microtubule gliding assays. (A) Schematic diagram of a gliding assays are highly suitable for a systematic analysis of the bidirectional gliding assay. Kinesin-1 and cytoplasmic dynein are permanently effects of motor number, ATP concentration and hindering bound to the surface (coverslip) through their tail region. By stepping on a microtubule situated above them, they move the microtubule back and forth. roadblocks on bidirectional transport. Additionally, to get a detailed While attached kinesin steps towards the microtubule plus-end, dynein steps picture of the role of factors such as mechanical activation, we towards the microtubule minus-end. This results in a tug-of-war between the performed simulations of mathematical kinesin and dynein models, opposite directed motor teams. For a specific microtubule length, the density of which are based on all known single-molecule parameters. motors determines the number of motors involved in the transport. (B) Example trajectories of microtubule gliding for various kinesin densities and a constant μ −2 RESULTS dynein density of 64 m . The microtubules lengths were in the interval 5−10 μm. Kinesin-driven transport is defined to be in positive direction Regulating transport direction by varying the motor number (positive displacement) and dynein-driven transport in negative direction Previous studies have shown that changing the number of kinesin (negative displacement). For the ‘only kinesin’ case (red curve) and a high motors involved in bidirectional transport alters the transport kinesin density of 20 μm−2 (yellow curve), we see kinesin-driven transport. For direction (Rezaul et al., 2016; Vale et al., 1992). To test whether our a kinesin density of 1 μm−2 we see almost no net movement. Forces between set-up (Fig. 1A) shows a similar behavior, we varied the surface the kinesin and the dynein team are balanced. For a very low kinesin density of −2 density (0 to 100 μm−2)ofDrosophila kinesin heavy chain 0.1 μm (light blue curve) and the ‘only dynein’ case (purple curve), we (hereafter referred to as kinesin, see Materials and Methods for observe dynein-driven transport. Together, we see three motility states, the kinesin-driven state, the balanced state and the dynein-driven state. details) at a constant high density (64 μm−2) of recombinant human cytoplasmic dynein (referred to as dynein, see Materials and Methods for details). Because we only take into account three motility states could be distinguished depending on the kinesin microtubules with lengths in a certain range (see figure legends density: (1) the dynein-driven state, (2) the balanced state (almost no for values), the motor density directly translates into motor number. net movement) and (3) the kinesin-driven state. Thus, by varying the The particular dynein density was chosen because we previously kinesin density the transport direction can be changed. observed saturated velocities values in unidirectional dynein gliding When seeking to understand the regulation by the kinesin density assays under similar conditions (Monzon et al., 2019). To determine and the role of mechanical dynein activation, an insight into the state the transport direction, microtubules were polarity-marked and of the motors at the molecular level is required. Experimentally, it is gliding trajectories were measured. Example trajectories (Fig. 1B) difficult to determine the number of attached kinesin and dynein show dynein-driven transport for the ‘only dynein’ case and very motors, and it is not possible to know whether individual motors are low kinesin densities of 0.1 μm−2. For higher kinesin densities of active or passive. We therefore used the mathematical models of 1 μm−2 almost no net movement was observed. In this case, forces single kinesin and dynein molecules developed by Monzon et al. between the kinesin and dynein team are balanced and the transport (2019) and Klein et al. (2014). The models are based on all known is halted. For higher kinesin densities of 20 μm−2 and the ‘only kinesin and dynein single-molecule properties (see Table S1). In kinesin’ case, kinesin-driven transport was observed. In summary, Monzon et al. (2019), the models are used for unidirectional kinesin Journal of Cell Science

2 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249938. doi:10.1242/jcs.249938 and dynein gliding assay simulations. Here, we adjusted the models To be able to rely on the information provided by the bidirectional to the bidirectional set-up (see Fig. 2A for an illustration, and the gliding assay simulation, it has to be shown that the simulation Materials and Methods for detailed model descriptions). reproduces the experimental observations. Therefore, velocity

Fig. 2. Using mathematical kinesin and dynein models for bidirectional microtubule gliding assay simulations to give insights at the molecular level. (A) Scheme of the bidirectional gliding assay simulation. The microtubule gliding assay is implemented as a one-dimensional system. The microtubule position

XMT(t) is determined by the microtubule minus-end. The microtubule plus-end is therefore at XMT(t)+LMT, with LMT being the constant microtubule length. On the one-dimensional surface, kinesin and dynein motors are randomly distributed with mean distances δkin and δdyn, respectively. The permanent position of the ith i i motor on the surface is xs and, when attached, its position on the microtubule is xf ðtÞ (filament position). Detached kinesin motors are drawn as green squares and attached kinesin motors as green circles. Detached dynein motors are drawn as light blue squares, passive attached dynein as light blue circles and active i i attached dynein as dark blue circles. While motors attach with the constant rates ka,kin andka,dyn, the detachment [kd,kin(F ) for kinesin and kd,dyn(F ) for passive and i i i active dynein] and stepping [skin(F ) for kinesin, sdyn(F ) for active dynein and s±(F ) for passive dynein] of attached motors depend on its load force. Since motors D i i i are modeled as linear springs, the load force of the motors is proportional to the motor deflection ½ x ¼ XMTðtÞþxf ðtÞxs. For load forces smaller than the stall force, kinesin and active dynein step directionally towards the microtubule plus- and minus-end, respectively. For load forces greater than the stall force, kinesin and active dynein step backward. Passive dynein diffuses in the harmonic potential of the motors springs. (B) Example motor deflections for the ‘only dynein’ case. (C) Alignment of attached dynein motors by the activity of one kinesin motor. (D) Force-dependent kinesin stepping (upper panel) and detachment (lower panel). Under forward load (negative for kinesin) the kinesin stepping (red curve) is high, but constant and decreases for backward loads (positive for kinesin) until reaching stall (Fs,kin=6 pN). For a higher backward load than the stall force, kinesin steps backward with a small but constant rate. The kinesin detachment (green curve, lower panel) increases exponentially and symmetrically for forward and backward load. (E) Force-dependent dynein stepping (upper panel) and detachment (lower panel). Active dynein steps with a high but constant rate (red curve) under forward load (positive for dynein). Under backward load (negative for dynein) the directional stepping of active dynein decreases until reaching the stall force (Fs,dyn=1.25 pN). Beyond the stall force, active dynein steps backwards i i with a small but constant rate. The diffusive stepping of passive dynein (blue curve) increases exponentially for stepping towards the equilibrium position xeq (F =0) i of the motor and decreases exponentially for stepping away from it. Thus s±(F ) is mirrored at the y-axis. The dynein detachment (green curve) increases linearly but asymmetrically for backward and forward load. Under forward load, the detachment rate increases faster than under backward load. For more details regarding the model, see Materials and Methods and Monzon et al. (2019). Journal of Cell Science

3 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249938. doi:10.1242/jcs.249938 histograms from simulation and experiment were compared (see intermediate (13 μm−2) and high (64 μm−2 and 128 μm−2 for Fig. 3). Here again, the kinesin densities were varied at a constant simulation only) dynein densities, a transition from the dynein- high dynein density. For both simulation and experiment, kinesin- driven (negative velocities) to the kinesin-driven state (positive driven transport was observed for high and intermediate kinesin velocities) was observed with increasing kinesin density. densities, characterized by a narrow peak at high positive velocities. Comparing the kinesin densities of the balanced states, we find, At low kinesin density, a velocity distribution around zero is the lower the dynein density the more the balance was shifted to observed for simulation and experiment indicating the balanced lower kinesin densities. Thus, the dynein density also regulates the state. For the ‘only dynein’ case, simulation and experiment showed directionality of bidirectional transport. a broad distribution of negative velocities (see Fig. 3). Thus, the When varying the kinesin density at the lowest dynein density simulation reproduces the experiment, and we consider our model a (3 μm−2), no dynein-driven state was observed but rather the reliable tool to obtain a deeper insight into bidirectional transport at balanced state extended to very low kinesin densities (see Fig. 4B). the molecular level. For the non-existence of a dynein-driven state, two explanations are We observed that kinesin and dynein teams balanced each other at possible: (1) either no dynein is attached to the microtubules or high dynein density (64 μm−2) but low kinesin density (1 μm−2). (2) attached dynein is passive and therefore not transporting the This raises the question of why more dynein than kinesin motors are microtubule directionally. To understand which scenario is at play, needed to balance each other. To understand the mutual interplay the median number of attached dynein motors was determined as a between kinesin and dynein motors, and especially the influence of function of the kinesin density using the simulation (Fig. 4D, upper the number of dynein motors, we next considered different dynein panel). We see that one dynein motor is attached for the lowest densities. Different dynein densities have been shown to strongly dynein density and kinesin densities smaller than 0.5 μm−2. This influence the transport velocity of unidirectional dynein transport rules out the first possibility, namely, that no dynein motor is (Monzon et al., 2019). However, the influence of the number of attached at all. To test whether the second scenario holds, we need to dynein motors on bidirectional transport has not been studied. know the internal state of the attached dynein. Unlike kinesin, which Hence, we measured the median microtubule gliding velocities for is always active, dynein is known to have an active and a passive different constant dynein densities as a function of the kinesin state (Zhang et al., 2017; Torisawa et al., 2014; Monzon et al., density in experiment (Fig. 4A) and simulation (Fig. 4B). For 2019). In our previously published model (Monzon et al., 2019),

Fig. 3. Comparison of gliding assay simulations and experimental results. (A,B) Normalized histograms of microtubule gliding velocities from experiment (A) and from simulation (B). For both the experiments and simulation, the same distribution of lengths within the

interval LMT=10–15 μm were applied at a −2 constant dynein density of σdyn=64 μm . The kinesin density was varied as written in the plots, including the ‘only kinesin’ case with a kinesin density of −2 σkin=100 μm . At higher kinesin densities −2 (σkin=20–100 μm ), we observed fast kinesin-driven transport (positive velocities) with a peak at ∼800 nms−1 for simulation and experiment. At kinesin −2 densities around σkin=1-2 μm , experiment and simulation showed a balanced state with velocity distributions around zero. At even lower kinesin −2 densities (σkin=0.1 μm ) and for the ‘only dynein’ case, dynein-driven transport with a wide distribution of negative velocities was observed for simulation and experiment. Thus, the simulation reproduces the experiment. Numbers of microtubules (N) are given in each subplot. Journal of Cell Science

4 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249938. doi:10.1242/jcs.249938

Fig. 4. Dynein density regulates bidirectional transport and kinesin stabilizes the balanced state by activating passive dynein and increasing the number of attached dynein. (A,B) Bidirectional gliding assay experiment (A) and simulation (B) at different constant dynein densities. Median instantaneous velocities with interquartile range (IQR) are shown as a function of various kinesin densities. The microtubule length was LMT>12 μm for the −2 experiment and LMT=25 μm for the simulation. For all dynein densities, we found a kinesin-driven state for σkin≥20 μm and a dynein-driven state for −2 −2 σkin≤0.1 μm (σkin≤0.01 μm for intermediate dynein density in experiment). For experiment and simulation, the balanced states shifted towards lower kinesin densities the lower the dynein density was. (C) Median±IQR number of attached kinesin as a function of various kinesin densities at different constant dynein densities corresponding to A. The number of attached kinesin motors increased monotonically with increasing kinesin density and did not significantly depend on the dynein density. (D) Median±IQR number of total (active and passive) attached dynein (upper panel) and median±IQR number of active attached dynein motors (lower panel). Median numbers are depicted as a function of the various kinesin density for different constant dynein densities correspondingtoA. The horizontal dashed line represents the one. For the lowest dynein density, the total attached dynein number was one in the dynein-driven state and decreased −2 to zero as soon as a kinesin was attached (σkin≥0.3 μm , see C). For the intermediate dynein density, two dynein motors were attached in the dynein-driven and −2 the balanced state. As soon as a kinesin was attached (σkin≥0.1 μm , see C), one of the two attached dynein motors was active (see lower panel). For high −2 −2 dynein densities (σdyn=64 μm and σdyn=128 μm ), the total attached dynein number and the active attached dynein number reached their maxima at the kinesin density of the balanced state. We used at least n=657 data points to obtain each median velocity for the experiment, and n=3000 data points to obtain median velocities and number of attached kinesin and (active) dynein motors for the simulation. dynein is mechanically activated, in agreement with previous and a mechanically activated dynein deactivates when it is studies (Zhang et al., 2017; Torisawa et al., 2014; Belyy et al., unstretched again. In the gliding assay set-up, motors are tightly 2016). Mechanically activated dynein (from now on called active bound to the surface. When attaching to the microtubule, they are dynein) performs directional motion on the microtubule, and mechanically coupled via the microtubule. Owing to this coupling, a passive dynein diffuses in the harmonic potential of the motors passive dynein motor is stretched and activated when the microtubule modeled as linear springs (see also Materials and Methods). In our is transported by other attached motors. Consequently, when only one model, a passive dynein mechanically activates when it is stretched (aprioripassive) dynein is attached, like in the ‘only dynein’ case at Journal of Cell Science

5 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249938. doi:10.1242/jcs.249938 the lowest dynein density, it will never activate or walk directionally competed against six active dynein motors in the balanced state towards the microtubule minus-end. Thus, no net movement occurs. (Fig. 4C,D). Looking at the number of active dynein motors, we find This is in agreement with previous studies stating that single it reaches its maximum in the balanced state. This means kinesin mammalian dynein does not perform processive and directional activated more dynein and thereby stabilized the balanced state. motion towards the microtubule minus-end (Nicholas et al., 2015a; Moreover, the total number of attached dynein (active and passive) Brenner et al., 2020; McKenney et al., 2014). However, as soon as a also reached its maximum in the balanced state. This means that kinesin motor is attached, the kinesin motor transports the having kinesin pulling back dynein, reduces the overall dynein microtubule and thereby stretches and activates the passive attached detachment. This could be interpreted as an effect originating from a dynein motor. When determining the median number of active dynein catch-bond. However, rather than a catch-bond, a slowly attached dynein (Fig. 4D, lower panel) and kinesin motors (Fig. 4C) increasing detachment rate (slip-ideal bonding) has been observed using the simulation, it turns out that, for the lowest dynein density, previously (Cleary et al., 2014; Rao et al., 2019) and is implemented the median number of active dynein motors is always zero. Thus, the in our model for single dynein. Therefore, the reason for the reduced activated state has a very short life time before being pulled off by overall detachment has to be a collective effect. Imagine having kinesin. This means one dynein cannot resist one kinesin and, kinesin transporting the microtubule, then, the attached dynein therefore, no balanced state exists. motors will be aligned under backward load. Under backward load, To understand how many dyneins are needed to resist against the dynein detachment increases more slowly with the force than kinesin, we looked at a slightly higher dynein density. At the under forward load (see Fig. 2E, lower panel) (Nicholas et al., intermediate dynein density (13 μm−2), we observed a clearly 2015b; Cleary et al., 2014; Rao et al., 2019). Consequently, being separated balanced state (Fig. 4A,B) indicating that dynein is able to aligned under backward load, fewer dynein motors detach resist kinesin. Looking again at the number and internal states of the compared to the number of dynein motors that detach in the ‘only attached motors (Fig. 4C,D), we find two attached dynein motors in dynein’ case, where the deflections of attached dynein motors are in the dynein-driven state. Although the median number of active random directions (forward and backward), as the individual motors attached dynein motors (Fig. 4D, lower panel) is zero, the median step stochastically with different velocities (see Fig. 2B,C for an velocity in the dynein-driven state is greater than zero. Thus, illustration). In conclusion, kinesin stabilizes the balanced state first temporarily, a passive attached dynein motor activated (the mean by (1) activating passive dynein and (2) reducing the dynein number of active attached dynein is 0.43) and drove the microtubule. detachment when aligning dynein motors under backward load. In the balanced state, where additionally one kinesin is attached, one In summary, the directionality of bidirectional transport can be of the two dynein motors is active. This means that the kinesin regulated by varying the number of kinesin or the number of dynein activated one dynein motor, which then holds against the kinesin. motors. To understand how one active dynein can hold against one kinesin, we need to have a detailed look at the motor forces. Previously, a Stable balanced state upon different ATP concentrations motor was defined as ‘strong’ when having a large ‘stall force to Single-molecule velocities of dynein and kinesin strongly depend detachment force ratio’ and as ‘weak’ when this ratio is small on ATP concentration (Schnitzer et al., 2000; Ross et al., 2006; (Müller et al., 2008). For kinesin, the force ratio is one because the Torisawa et al., 2014). To see whether the ATP concentration could stall force is similar to the detachment force (see Fig. 2D,E; have an effect on bidirectional transport performed by many motors, Table S1). For dynein, we have to distinguish between forward and we first studied the influence of ATP on individual teams of kinesin backward loads because dynein detaches significantly faster under and dynein motors (intermediate motor densities of 18 μm−2)in forward loads (smaller detachment force) than under backward unidirectional gliding assays (Fig. 5A,B). We observed that the loads (higher detachment force) (Fig. 2D,E; Table S1) (Gennerich median velocities of kinesin- and dynein-driven transport increased et al., 2006; Cleary et al., 2014; Nicholas et al., 2015b; Rao et al., with increasing ATP concentration for simulation and experiment. 2019). When resisting against kinesin (backward load), the force While the median velocities of the kinesin assay could be fitted by a ratio is 0.3125. Therefore, dynein is the weaker motor in the Michaelis–Menten equation, velocities of the dynein assay did not competition with kinesin. Consequently, an active dynein is likely show such a behavior. In the dynein assay the velocity increased to be pulled off by a kinesin, coinciding with what we have seen for more in a linear manner and did not saturate at the highest applied the lowest dynein density where one dynein competed against one ATP concentrations. This means, kinesin and dynein, indeed, react kinesin. However, for the studied intermediate dynein density, there differently to ATP concentration, and changing the ATP is still the passive attached dynein, when the active dynein is pulled concentration might regulate the directionality of bidirectional off. The passive dynein is then activated. A balanced state can then transport. be reached when in the meantime, a new passive dynein attaches to To investigate the potential regulation of bidirectional transport the microtubule, ‘helping out’ when the active dynein is again by ATP concentration, we varied the kinesin density at a constant pulled off by kinesin. We conclude the role of the passive dynein is dynein density of 18 μm−2 (the same as for the unidirectional assay) helping out when active dynein is pulled off by kinesin. for ATP concentrations of 1000 μM and 5000 μM in experiment Consequently, an active dynein having a passive dynein as a (Fig. 5C) and simulation (Fig. 5D). When increasing the ATP substitute is able to temporarily hold against one kinesin. concentration from 1000 μM ATP to 5000 μM, we found that in the Our findings show that two dynein can temporarily hold against unidirectional kinesin assay (Fig. 5A), the gliding velocity remained one kinesin. However, this balance is not stable, because the active almost constant, while in the unidirectional dynein assay (Fig. 5B), dynein is continuously pulled off. Once a new passive dynein does the velocity nearly doubled. We reasoned that this behavior may give not attach fast enough to help out, kinesin will take over. Obviously, rise to a shift of the balanced state. For both ATP concentrations, we a higher number of dynein motors are expected to stabilize the observed the three formerly described motility states in the balanced state. Indeed, at high dynein densities of 64 μm−2 (same bidirectional assay. However, the balanced state was invariant to for 128 μm−2 in simulation) a stable balanced state with only small ATP concentration, indicating that the ATP concentration does not fluctuations was observed (Fig. 4A,B). Here, two kinesin motors change the transport direction. Journal of Cell Science

6 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249938. doi:10.1242/jcs.249938

Fig. 5. Although unidirectional kinesin and dynein assays react differently to ATP concentration, changes of ATP concentration cannot regulate the direction of bidirectional transport. (A) Median instantaneous gliding velocities (with IQR) as a function of ATP concentration for the unidirectional −2 kinesin assay. For simulation (red curve) and experiment (light blue curve) a constant kinesin density of σkin=18 μm was applied. Experiment and simulation results showed a Michaelis–Menten dependence on the ATP concentration. To the experimental data a Michaelis–Menten equation (v=vmax×[ATP]/(Km+[ATP])) −1 with vmax=914 nm s and Km=69 μM was fitted (solid fit, orange curve). We used at least n=31,169 data points to obtain each median velocity for the experiment and n=4000 data points for the simulation. (B) Median instantaneous gliding velocities with IQR as a function of ATP concentration for the unidirectional dynein −2 assay. For simulation (red curve) and experiment (light blue curve), a constant dynein density of σkin=18 μm was applied. In simulation and experiment, the same distribution of microtubules lengths bigger than 15 μm was used. Experimental and simulation results matched but could not be fitted with a meaningful Michaelis–Menten equation. Median instantaneous gliding velocities increased more in a linear manner with the ATP concentration and might not be saturated at the highest measured ATP concentration. The orange line shows the Michaelis–Menten fit found by Torisawa et al. (2014). We used at least n=12,504 data points to obtain each median velocity for the experiment and the simulation. (C,D) Bidirectional gliding assay experiment (C) and simulation (D) at different ATP concentrations. Median instantaneous velocities with IQR are presented as a function of the various kinesin densities and a constant dynein density of −2 σdyn=18 μm . The microtubule length was LMT>15 μm in the experiment and LMT=25 μm in the simulation. For experiment and simulation, the velocities in the dynein-driven state were reduced more strongly by the lower ATP concentration than in the kinesin-driven state. The balanced state, however, remained at the −2 −2 same kinesin density (σkin=0.1–1.0 μm for experiment and σkin=0.1–1.0 μm for simulation) for all ATP concentrations. We used at least n=4069 data points to obtain each median velocity for the experiment and n=4000 data points for the simulation.

To understand why the balanced state does not shift with ATP ATP concentration as long as the applied forces do not depend on the concentration, we again used the simulation for an insight into the ATP concentration (see Fig. S2 for more ATP concentrations). In molecular level. We found that, in the balanced state, mainly diffusive conclusion, ATP concentration cannot regulate the directionality of stepping of passive dynein occurs (Fig. S1). Although diffusive bidirectional transport. dynein stepping also depends on ATP concentration, passive dynein does not exert directed forces to the microtubule. Therefore, changing Stable balanced state in the presence of roadblocks the diffusive stepping of passive dynein does not shift the force Previous work has suggested that roadblocks might regulate balance. Consequently, the balanced state is stable upon changing bidirectional transport by asymmetrically inhibiting the motor Journal of Cell Science

7 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249938. doi:10.1242/jcs.249938 activity (Siahaan et al., 2019; Henrichs et al., 2020; Tan et al., 2019; whether roadblocks alter the transport behavior in bidirectional Monroy et al., 2018). Single kinesin and dynein motors indeed show gliding assays. different behavior upon encountering a roadblock (Telley et al., To know whether roadblocks could have an effect on 2009; Dixit et al., 2008; Schneider et al., 2015). A single kinesin is bidirectional transport by multiple motors, we first studied how known to either pause when encountering a roadblock or completely multiple dynein and kinesin motors react to roadblocks using detach from the microtubule (Dixit et al., 2008; Schneider et al., unidirectional gliding assays. We coated microtubules with 2015). A single dynein frequently side steps (Wang et al., 1995) and rKin430-T39N, a rigor mutant of rat kinesin-1 (hereafter referred circumvents roadblocks without detaching. Consequently, single to as roadblocks) (Schneider et al., 2015) at different concentrations dynein motors overcome roadblocks more successfully than single and measured the median gliding velocities at constant motor kinesin motors (Ferro et al., 2019). It is therefore of interest, to study densities of 50 μm−2 (see Fig. 6A for experiment and Fig. 6B for

Fig. 6. Although unidirectional kinesin and dynein assay are differently affected by roadblocks, roadblocks cannot change the direction of bidirectional transport. In this set-up, microtubules were coated with rigor-binding kinesin mutants referred to as roadblocks. While in the experiment a roadblock concentration is given, in the simulation a roadblock line density λRB was applied. (A,B) Unidirectional kinesin (purple curve) and dynein (light blue curve) assay results of experiments (A) and simulations (B) in the presence of roadblocks. Median instantaneous velocities with IQR relative to the velocity in the absence of roadblocks are depicted as a function of roadblock concentration (experiment) and roadblock line density (simulation). In both, the kinesin and the −2 dynein assay, motor densities of σkin=σdyn=50 μm were applied and the microtubule length was in the interval of LMT=25–30 μm for the experiment and LMT=25 μm for the simulation. In both assays the velocity decreased with increasing roadblock line density/concentration, whereby velocities of the dynein assay decreased faster than of the kinesin assay. We used at least n=5034 data points to obtain each median velocity for the experiment and n=4000 data points for the simulation. (C,D) Bidirectional gliding assay experiments (C) and simulations (D) at different roadblock line densities/concentrations. Median instantaneous −2 velocities with IQR are shown as a function of various kinesin densities at a constant dynein density of σdyn=50 μm . The microtubule length was LMT=25 μmin the simulation. We found that the dynein-driven state was affected more strongly by roadblocks than the kinesin-driven state. However, the balanced state −2 −2 remained at the same kinesin density (σkin=0.1–1.0 μm in the experiment and σkin=1.0 μm in the simulation) for all roadblock line densities /concentrations.

We used at least n=1749 data points to obtain each median velocity for the experiment and n=4000 data points for the simulation. Journal of Cell Science

8 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249938. doi:10.1242/jcs.249938 simulation). In both kinesin and dynein assays, the median velocity However, when the number of involved motors changes, the decreased with increasing roadblock concentration for experiment balanced state can be shifted towards the kinesin- or dynein-driven and simulation. Comparing kinesin and dynein gliding assays, we state. Unlike ATP and roadblock concentration, the number of find dyneins are more affected than kinesins. This is in contrast to motors influences the force balance between kinesin and dynein previous studies stating that, during cargo transport, multiple motors. Having more kinesin motors means the kinesin team is kinesins are as affected as multiple dyneins (Ferro et al., 2019). stronger and vice versa. Therefore, factors that influence the force Since dynein likely uses side steps to circumvent roadblocks (Reck- balance between the kinesin and dynein team would be expected to Peterson et al., 2006), we hypothesize that dynein might not be able play a key role in the regulation of bidirectional transport. to take side steps in the gliding assay set-up and is therefore more Our results show that the kinesin activity influences the force affected. To test this hypothesis, we used the simulation to balance. Using a mathematical model, we find that the kinesin implement multiple protofilaments and allowed dynein to take activity strengthens dynein. The kinesin activity, on one hand, side steps. Using this simulation, we observed that dynein was as activates passive attached dynein motors and on the other hand affected by roadblocks as kinesin (Fig. S3a) in agreement with increases the number of total attached dynein motors. As a first findings of Ferro et al. (2019). However, when not allowing dynein consequence, the number of attached dynein motors in the balanced to take side steps in the multiple protofilament simulation, dynein is state is higher than the number of kinesin motors. This is in again more affected than kinesin. Consequently, if dynein does not agreement with previous studies reporting that more dynein motors take side-steps, both motors have to detach and reattach to overcome than kinesin motors are involved in the tug-of-war (Soppina et al., roadblocks. Because the kinesin attachment rate is much higher than 2009; Hendricks et al., 2010). A second consequence is that kinesin the dynein attachment rate (see Table S1), multiple dyneins are stabilizes the balanced state by strengthening the weaker and partly affected more strongly by roadblocks in unidirectional gliding passive dynein motors. This explains why we find a stable force assays than multiple kinesins. balance between kinesin and dynein. Thus, in addition to We observed that multiple dynein and kinesin motors react antagonistic effects between kinesin and dynein teams, there are differently to roadblocks in unidirectional gliding assays. This raises also cooperative effects. The cooperative effect of kinesin the question of whether the different reaction to roadblocks stabilizing the balanced state might be involved in holding a influences bidirectional transport in gliding assays. Therefore, we cargo at a specific location inside the cell. applied the same constant dynein density as for the unidirectional We find that from the three tested regulation factors, number of assay with roadblocks and varied the kinesin densities at different motors, ATP concentration and roadblocks, that only the number of roadblock concentrations. In the simulation, for all roadblock motors can regulate bidirectional transport. However, the latter densities, clear motility states were observable (Fig. 6D). In the might not be the only way that the stable force balance could be experiment, the dynein-driven state ‘merged’ with the balanced regulated. Adding adaptor proteins that activate passive attached state at very high roadblock concentrations (Fig. 6C). However, for dynein could likely also change the force balance. Belyy et al. simulation and experiment, the balanced state stayed at the same (2016) showed that a single dynein with adaptor proteins (a dynein– kinesin density for all roadblock concentrations. Looking at the dynactin–BicD2 complex) can hold against a single kinesin, while a simulation results of the bidirectional gliding assays with multiple single dynein without adaptor proteins cannot. The latter is in protofilaments, we see that even though dynein takes side steps, the agreement with our work showing that a single dynein without balanced state stayed at the same kinesin density for all roadblock adaptor proteins cannot hold against a single kinesin. Moreover, concentrations (Fig. S3b). As a consequence, independently of the measuring the dynein stall force, Belyy et al. (2016) found that a side stepping ability of dynein, the balanced state remains at the dynein plus adaptor proteins has a stall force of 4.4 pN, which has to same kinesin density for all roadblock concentrations. be compared to the stall force of a single dynein without adaptor To obtain an explanation for the balanced state being stable in the proteins (1−2 pN) (Mallik et al., 2004; Kunwar et al., 2011; presence of roadblocks, we used again the simulation for insights at the McKenney et al., 2010; Ori-McKenney et al., 2010; Nicholas et al., molecular level. As stated before, kinesin and active dynein almost did 2015a; Brenner et al., 2020; Belyy et al., 2016), which we assume not move at all in the balanced state. In detail, the median moved for our mechanically activated dynein. Thus, dynein activated by distance is 65 nm for kinesin and 16 nm for dynein (see also Fig. S4), adaptor proteins is stronger than our mechanically activated dynein which is small compared to the mean distance between roadblocks motors. Based on our work showing that factors influencing the (∼166.67 nm at a density of 6 μm−1). This strong motor localization force balance are key regulators of bidirectional transport, we would reduces their interactions with the roadblocks and leads to a stable predict that adaptor proteins would be able to regulate bidirectional balanced state in the presence of roadblocks. In conclusion, transport by increasing the dynein stall force. This suggests that roadblocks do not regulate the directionality of bidirectional transport. even transport in the kinesin direction could be reversed by adding adaptor proteins. DISCUSSION Beside adaptor proteins changing the dynein stall force and In this work, we showed that the direction of bidirectional transport therefore affecting the force balance, it has been shown that ATP is robust (i.e. is not substantially altered) against variation of ATP concentration can also change the dynein stall force. Mallik et al. and roadblock concentration in microtubule gliding assays. We have (2004) showed that for ATP concentrations lower than 1000 μM, the seen that while ATP and roadblock concentrations influence the dynein stall force increases linearly with ATP concentration and dynein- and kinesin-driven states, the balanced state remains stable. remained constant above 1000 μM. In our work, we showed that for In the balanced state, active dynein and kinesin almost do not step at ATP concentrations equal to or greater than 1000 μM, the ATP all. This suggests that parameters influencing the stepping of the concentration does not influence the force balance. However, the motors cannot be used to change the balanced state and therefore force balance could be influenced by lower ATP concentrations. cannot regulate the directionality of bidirectional transport. When simulating the bidirectional gliding assay at lower ATP Consequently, the inability of dynein to take side steps in our concentration, we did in fact see a tendency of the balanced state to bidirectional gliding assays does likely not change our result. be moved towards lower kinesin densities (Fig. S2b). But the shift Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249938. doi:10.1242/jcs.249938 was as small, as shifts arose from fluctuations in the number of mechanisms, have little effect. Future work should now focus on the motors involved in the transport. However, in Klein et al. (2014), the influence of adaptor proteins in light of this or roadblocks that transport direction could be changed upon changes in ATP differentially influence the number of attached motors. concentration in cargo transport simulations. Unlike in microtubule gliding assays, in cargo transport simulations, the MATERIALS AND METHODS number of involved kinesin and dynein motors is constant and Materials and reagents dynein is always active. That is why fluctuations are smaller, and the All reagents unless otherwise stated were purchased from Sigma. cDNA effect of the ATP concentration altering the force balance is visible. encoding full-length D. melanogaster Kinesin Heavy Chain (KHC) was Thus, we predict that, in microtubule gliding assays, shifts of the sub-cloned into an in-house-generated insect cell expression vector in frame force balance stemming from altered ATP concentrations are small with a C-terminal 6×His tag. Codon optimized gene sequence of compared to shifts stemming from fluctuating numbers of motors. R. norvegicus Kif5c (GenArt, gene synthesis, Invitrogen) truncated to the Our study shows that the presence of roadblocks hindering both first 430 amino acids with a T39N rigor mutation was cloned upstream of a motors does not change the force balance. However, having 6×His affinity tag (with and without eGFP). Plasmids encoding H. sapiens roadblocks that act exclusively just on one kind of motor could have dynein subunits (DYNC1H1, DYNC1I2, DYNC1LI2, DYNLT1, DYNLL1 an effect. Tau islands, for instance, have an asymmetric effect on and DYNLRB1) were a kind gift from Max Schlager and Andrew Carter (MRC Laboratory of Molecular Biology, UK). motor proteins. While kinesin detaches when encountering a tau island, dynein continues walking (Siahaan et al., 2019; Henrichs Microtubules et al., 2020; Tan et al., 2019). This means tau islands change the Tubulin was purified from pig brains obtained from a local slaughter house motor number and therefore the force balance. Having a kinesin- by two cycles of polymerization and depolymerization in high concentration driven transport that encounters a tau island, for instance, would PIPES buffer (Castoldi and Popov, 2003), labeled with Alexa Fluor 488 dye, cause a shift to a dynein-driven state because kinesin motors would diluted to 4 mg/ml, aliquoted and stored at −80°C. detach and dynein could take over. Therefore, roadblocks that Polarity-marked microtubules were prepared by preferentially adding a asymmetrically detach one kind of motor can be understood to alter short brightly labeled seed to plus-ends of GMPCPP and taxol-polymerized ‘ ’ the number of attached motors and therefore shift the force balance. microtubules (referred to as double-stabilized microtubules) in the As a consequence, such roadblocks could be used to regulate presence of N-ethylmaleimide (NEM)-labeled tubulin. This was carried out as follows. First, 4.8 μM tubulin was freshly labeled with 1 mM NEM in bidirectional transport. the presence of 0.5 mM GTP for 20 min on ice. Then, excess NEM reagent Another mechanism that could alter the force balance by varying was quenched with 8 mMβ-mercaptoethanol. An elongation mix, consisting the number of motors, are temporarily changing binding affinities of of 1 mM GTP, 4 mM MgCl2, 1 mM Alexa-Fluor-488-labeled tubulin and the motors. Kinesin is known to alter the microtubule structure in a 0.64 μM NEM-tubulin in BRB80 buffer was incubated on ice for 5 min, way that following kinesin motors show a higher binding affinity shifted to 37°C for 40 s followed by the addition of 250 μM double stabilized (Peet et al., 2018; Shima et al., 2018). Using a floor field model, microtubules and incubated at 37°C for 1 h. Resultant microtubules were Jose and Santen (2020) show that this temporarily changed binding stabilized with 20 μM taxol. Polarity-marked microtubules were always affinity leads to the formation of kinesin and dynein lanes during prepared fresh every day at the beginning of an experiment. bidirectional axonal transport. This suggests that altered binding affinities of the motors shift the force balance and regulate Motor proteins bidirectional transport. Expression and purification of D. melanogaster KHC KHC was purified from SF9 cells as described in Korten et al. (2016). Our finding that dynein gliding assays do not show a Michaelis– Briefly frozen cell pellets infected with FlexiBAC baculoviral particles for Menten-like dependence on ATP concentration is in contrast to the 72 h were lysed via ultra-centrifugation at 4°C. Lysates were pre-clarified results from previous studies of Torisawa et al. (2014) and over a cation exchange column and the resultant elute was passed over an Nicholas et al. (2015a), which both found such a dependence. To His-tagged IMAC column. Column-bound KHC dimers were eluted with understand why we do not see such a dependence, we simulated the 300 mM imidazole, desalted and snap frozen. unidirectional gliding assay at a higher dynein density and various ATP concentrations. At a higher dynein density (Fig. S2a), the Expression and purification of H. sapiens dynein simulation indeed shows a Michaelis–Menten-like dependence on Recombinant cytoplasmic dynein expression and purification was ATP concentration. We know from our previous work (Monzon et al., performed based on a published protocol (Schlager et al., 2014). The 2019) that, at low dynein densities, passive dynein motors slow down MultiBac plasmid was a generous gift by Max Schlager and Andrew Carter. The MultiBac plasmid was integrated into the baculoviral genome of microtubule gliding. However, having a lower ATP concentration, the DH10EMBacY cells by using Tn7 transposition. 50 ng of MultiBac plasmid activity of passive motors is lower. This means, the lower the ATP was incubated with 100 μl of chemical competent DH10EMBacY cells for concentration, the more the passive motors slow down the 45 min on ice. The mix was heat shocked for 45 s at 42°C and incubated microtubule gliding. This explains why the increase in gliding with 600 μl LB medium for 6 h at 37°C. 150 μl cell suspension was streaked velocity with the ATP concentration at lower dynein density is slower out on agar plates containing 50 μgml−1 Kanamycin, 10 μgml−1 than predicted by a Michaelis–Menten equation. Consequently, we do Gentamycin, 10 μgml−1 Tetracycline, 20 μgml−1 X-gal and 1 μgml−1 not see a Michaelis–Menten-like dependence on ATP concentration at IPTG and incubated at 37°C for 2–3 days. Plasmids from white colonies low and intermediate dynein densities. At high dynein density, the were isolated and checked for presence of all subunits via PCR. SF9 insect slowing down by passive dynein motors is negligible and we see a cells were infected with bacmids at a 1:1000 virus-to-cell ratio and grown for Michaelis–Menten dependence on ATP concentration. This implies 4 days. Cells were harvested at 300 g for 15 min at 4°C and pellets were snap frozen in liquid nitrogen at stored at −80°C. the ATP dependence of dynein-driven transport depends on the For purification of recombinant dynein, a frozen pellet corresponding to number of dynein motors. 250 ml insect cell culture was thawed on ice and resuspended in lysis buffer In our study, we have provided key insights into the factors [50 mM HEPES pH 7.4, 100 mM NaCl, 1 mM DTT, 0.1 mM ATP, 10% influencing bidirectional transport in the absence of adaptor proteins. (v/v) glycerol] supplemented with 1× protease inhibitor cocktail (complete Here, the factors that influence the force balance seem to be most EDTA free, Roche) to a final volume of 25 ml. Cells were lysed in a Dounce important, whereas other factors, such as stepping or bypassing homogenizer with 20 strokes. The lysate was clarified at 504,000 g Journal of Cell Science

10 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249938. doi:10.1242/jcs.249938 for 45 min at 4°C and added to 3 ml equilibrated (in lysis buffer) were incubated for 1 min and excess unbound microtubules were washed IgG–Sepharose beads in a column and incubated on a rotary mixer for out with motility buffer. 6 h. Protein-bound beads were washed with 50 ml lysis buffer and 50 ml Gliding microtubules were imaged on an inverted fluorescence TEV buffer [50 mM Tris-HCl pH 7.4, 148 mM potassium acetate, 2 mM microscope (Axiovert 200M, Zeiss) with a 40×, NA 1.3 oil immersion magnesium acetate, 1 mM EGTA, 10% (v/v) glycerol, 0.1 mM ATP, 1 mM objective maintained at 27°C. Samples were illuminated with a metal arc DTT and 0.1% Tween 20]. The ZZ affinity tag was cleaved off with lamp (Lumen 200, Prior Scientific) with an excitation 534/30 and emission 25 μgml−1 TEV protease in TEV buffer at 4°C on a rotatory mixer 653/40 filter set in the optical path. A total of 50–200 frames were acquired overnight. After TEV cleavage, beads were removed and recombinant with an iXon Ultra EMCCD (Andor) camera with a 100 ms exposure time at dynein was concentrated in a 100 kDa cut-off filter (Amicon Ultracel, a frame rate of 1 Hz. Images were acquired with MetaMorph (Universal Merck-Millipore) to a final volume of 500 μl. The TEV protease was Imaging). A minimum of three independent sets of gliding assays removed by size-exclusion chromatography using a TSKgel G4000SWXL (per experimental condition) were performed to obtain statistically column equilibrated in GF150 buffer (25 mM HEPES pH 7.4, 150 mM significant results. KCl, 1 mM MgCl2, 5 mM DTT, 0.1 mM ATP and 0.1% Tween 20). Peak fractions were collected, pooled and concentrated to a maximum Data analysis concentration of 1 mg/ml with a 100 kDa cut-off filter. All purification Microtubules were tracked with Fluorescence Image Evaluation Software steps were performed at 4°C. Recombinant dynein was frozen in liquid for Tracking and Analysis (FIESTA; Ruhnow et al., 2011) which automates ∼ nitrogen in the presence of 10% (v/v) glycerol. Protein concentration was Gaussian fitting of fluorescence signals to extract position coordinates. All determined with Bradford reagent. tracks were manually curated to filter out tracks of microtubules with significant changes in length between successive frames as observed when Expression and purification of the rigor mutant rKin430-T39N two microtubules cross paths during gliding. Data analysis and visualization A preculture of rKin430-T39N-transformed E. coli BL21 pRARE cells was were carried out in MATLAB2014b (MathWorks, Natick, MA). Instantaneous grown overnight in LB medium at 37°C. 750 ml fresh LB medium, velocity was defined as the ratio of distance traversed by a microtubule and supplemented with 50 μg/ml Kanamycin, was inoculated with 5 ml difference in time between consecutive frames. Velocities were smoothed with preculture and incubated in a shaker at 180 rpm, 37°C till optical density a rolling frame average over a window of five frames. Mean, median and reached 0.6. Protein expression was induced with 0.5 mM IPTG and quantiles of velocity distributions were calculated with in-built MATLAB incubated at 18°C overnight. Cells were harvested at 7500 g for 10 min at functions. 4°C. Cell pellet was resuspended in PBS with 10% glycerol (volume Microtubule filament length was an important parameter that influenced equivalent to weight of cell pellet in grams) and either snap frozen in liquid gliding velocities (especially with dynein). Unless otherwise indicated, only nitrogen or used immediately. 10−15 μm long microtubules (filtered from length values acquired from Cells were resuspended in lysis buffer (50 mM sodium phosphate buffer, FIESTA) were used for analysis to obtain a more homogenous velocity β pH 7.4, 300 mM KCl, 5% glycerol, 1 mM MgCl2,10mM -mercaptoethanol distribution. and 0.1 mM ATP) supplemented with 30 mM Imidazole and protease inhibitor cocktail and lysed by 4–5 passages in an Emusiflux French press. Lysate was clarified at 186,000 g for 1 h at 4°C and passed through a 0.45 μm Surface density estimation membrane filter to further get rid of particulate matter. A His-trap column was The density of kinesin and dynein motors was estimated by taking into a μ equilibrated with 10 column volumes (CV) of lysis buffer, followed by lysate consideration the protein concentration of motors perfused (7 l) into a 2 μ −1 application at a flow rate of 1 ml min−1. The column was washed with 10 CV 2×18×3 mm flowcell; the highest dynein concentration of 55 gml
m 2 of lysis buffer supplemented with 60 mM imidazole and protein eluted in lysis yielded a density of 1280 m . Motility measurements performed on buffer supplemented with 300 mM imidazole. Protein was desalted, axonemal dynein-coated surfaces have shown that only 10% of the total aliquoted, snap frozen and stored at − 80°C. motor population contributes to active motion (Kotani et al., 2007). Applying dynein concentrations of 55, 28, 11, 6, 3 and 1.5 μgml−1 were μ −2 Gliding assay thus estimated to yield surface densities of 128, 64, 26, 13, 6 and 3 m , Glass coverslips (22×22 mm and 18×18 mm) were cleaned as follows: respectively (Monzon et al., 2019). Similar assumptions were also made for sonicated in 1:20 Mucasol for 15 min, rinsed in distilled water for 2 min, the estimated surface densities of kinesin. sonicated in 100% ethanol for 10 min, rinsed in distilled water for 2 min, The linear relationship between applied motor concentrations and surface rinsed in double distilled water for 2 min and blow-dried with nitrogen. density was confirmed by observing landing events of microtubules on Flow channels were prepared by placing 1.5 mm parafilm strips on a cleaned motor-coated surfaces (Katira et al., 2007; Agarwal et al., 2012). The 22×22 mm coverslip (∼3 mm apart; four strips to prepare three channels) number of microtubules landing on the surface were counted every 10 s for and covered with a 18×18 mm coverslip. This flow cell was placed on a heat 100 frames. The plot of microtubule number versus time was fit to the ðRt=NmaxÞ block maintained at 55°C to melt the parafilm thereby making channels curveNðtÞ¼Ninit þ Nmax½1 e , where N(t) is number of landed water tight. microtubules, Ninit is number of microtubules non-specifically adsorbed to Aliquots of dynein complexes were subjected to microtubule the surface, Nmax is maximum number of microtubules in the field of view, sedimentation to get rid of dead/rigor-binding motors before being used R is the landing rate and t is time elapsed. Landing rate R derived from fit, in gliding assays. Briefly, dynein complexes were incubated with unlabeled microtubule area A (=Length of microtubule × 25 nm) and maximal microtubules in dilution buffer (10 mM PIPES, pH 7.0, 50 mM potassium diffusion-limited landing rate Z (assumed to be equal to landing rate of μ microtubules on very high densly coated motor surfaces) were plugged into acetate, 4 mM MgSO4, 1 mM EGTA, 0.1% Tween20, 10 M taxol, 2 mM σ − σ Mg-ATP and 10 mM dithiothreitol) for 10 min followed by centrifugation at the equation = [ln(1-R=/Z)]/A to yield surface density . The surface density of kinesin measured by the landing rate method was 120,000 g for 10 min at room temperature. Active dynein complexes μ retained in the supernatant were kept on ice until further use. in agreement with the density estimated by the first method (7 lof μ −1 μ −2 Flow channels were perfused with 2.5 mg ml−1 Protein A in double 6 gml of kinesin solution yielded a density estimate of 102 m versus μ −2 distilled water and incubated for 5 min followed by washing with dilution 100 m by the landing rate method). buffer. Solutions with various concentrations (prepared in dilution buffer) of kinesin and dynein were incubated in these channels for 5 min and excess Simulation of kinesin and dynein models motors were washed out with dilution buffer. To block the rest of the surface, Here, we first describe details of the kinesin and dynein model and then motor-coated channels were incubated with 500 μg/ml casein in dilution present a simulation run. The dynein model was previously published in buffer for 5 min followed by washing with dilution buffer. Double stabilized Monzon et al. (2019) and the kinesin model in Monzon et al. (2019) and microtubules prepared in motility buffer (dilution buffer supplemented with Klein et al. (2014). Here, some parameter values were slightly changed and −1 −1 40 mM glucose, 110 μgml glucose oxidase and 10 μgml catalase) the models were slightly expanded. All expansions compared to the models Journal of Cell Science

11 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249938. doi:10.1242/jcs.249938 published in Monzon et al. (2019) are marked in the text. All parameter Attachment values are listed in Table S1 including references to the literature whenever If detached kinesin and dynein motors are under the microtubule (within the possible. attachment area), they bind to the microtubule with the constant rates ka,kin and ka,dyn, respectively. Gliding assay The gliding assay is modeled as a one-dimensional system. This means only Detachment one protofilament of the microtubule is taken into account. The microtubule The detachment rates of attached kinesin and dynein motors depend on the itself is described as a rigid one-dimensional object, which is situated at load force of the motors. The detachment rate of kinesin increases constant height above the one-dimensional surface (glass coverslip). exponentially with the load force and is symmetrical with respect to forward Attached motors move the microtubule back and forth. (assisting force, Fi≤0) and backward loads (resisting forces, Fi>0):

i Attachment area jF j For the simulation, the motor density given by the experiment has to be i 0 Fd;kin kd;kinðF Þ¼kd;kin e ð7Þ converted into a number of motors able to attach to the microtubule. Having a microtubule with length LMT, we assume that all motors in the area LMT×Lattach The detachment rate of dynein increases asymmetrically for forward are able to attach to the microtubule. Thus the number of motors is: (assisting force, Fi>0) and backward load (resisting forces, Fi≤0). It increases linearly with a steeper slope for forward load than for backward N ¼ L L ; s for dynein load: dyn MT attach dyn dyn ð1Þ Nkin ¼ LMT Lattach;kin skin for kinesin i i i 0:1F þ 0:4 for F 0 kd;dynðF Þ¼ ð8Þ The widths of the attachment areas Lattach,dyn and Lattach,kin are the reach 3:2Fi þ 0:4 for Fi . 0 length of dynein and kinesin. In total Nkin+Ndyn motors are involved in the transport. In the simulation, Nkin+Ndyn motors are randomly distributed on the The dynein detachment behavior is taken from Cleary et al. (2014), who one-dimensional surface. To randomly distribute the motors, first the type of shows the force dependence of the detachment behavior for yeast dynein. motor (kinesin or dynein) is thrown with probability Nkin/(Ndyn+Nkin) that the Recent studies from Rao et al. (2019) show similar results. motor is a kinesin motor and with probability Ndyn/(Ndyn+Nkin)thatitisa dynein motor. Next, the position of the motor is randomly chosen taking Stepping mutual spatial exclusion between the motors into account. If D=LMT/ Kinesin steps predominantly towards the microtubule plus-end and active last (Ndyn+Nkin) is the mean distance between motors, xs the position of the last dynein towards the microtubule minus-end. Kinesin steps in a force- and set motor and Ri the radii of the last, current and next set motors, the position of ATP-dependent manner, which is taken from Schnitzer et al. (2000). Under i the currently set motor is uniformly distributed in the interval backward load forces smaller than the kinesin stall force (0

i Force calculation i; Vmax½ATP kcatðF Þ½ATP skinðF ½ATPÞ ¼ ¼ i i ð9Þ Dynein and kinesin motors are modeled as linear springs. Therefore, the load ½ATPþKM ½ATPþkcatðF Þ=kbðF Þ force of a motor is calculated from the deflection of the motor. The deflection with k (Fi) being the catalytic turnover rate constant and k (Fi) the second- of a motor is the difference between the position of the head of the motor cat b i order rate constant for ATP binding. Schnitzer et al. (2000) suggested a on the microtubule [XMTðtÞþxf ðtÞ] and the position of the tail of the motor i i Boltzmann-type force dependence for the rate constants: on the surface ðxsÞ. xf ðtÞ is the motor position on the microtubule that is zero when the motor is at the microtubule minus-end and LMT when the motor is at 0 i km [ ; the microtubule plus-end. X (t) is the position of the microtubule minus-end kmðF Þ¼ id= with m fcat bgð10Þ MT p þ q eF kBT in the overall coordination system of the surface. Therefore, the position of the m m 0 = head of the motor in the overall coordination system of the surface (coverslip) With kcat ¼ vf;kin d. Thereby vf,kin is the maximal kinesin stepping rate and i 0 is XMTðtÞþxf ðtÞ.Thus,thedeflectionis: d the step size. qm, pmwith qm+pm=1 and kb are taken from Schnitzer et al. (2000) and δ is determined by setting the stepping rate at stall force equal to D i i i −1 x ðtÞ¼XMTðtÞþxf ðtÞxs ð2Þ 0.1 s : The load force of kinesin is divided into three regimes depending on its k F ; ATP ! ; catð s kinÞ½ : 1 untensioned length L0,kin: sðFs;kin ½ATPÞ ¼ = ¼ 0 1s ð11Þ 8 ½ATPþkcatðFs;kinÞ kbðFs;kinÞ < k D i D i . kin ð x ðtÞL0;kinÞ if x ðtÞ L0;kin We solved this equation using MATLAB. For forward load forces (Fi<0) i k D i D i , F ðtÞ¼: kin ð x ðtÞþL0;kinÞ if x ðtÞ L0;kin ð3Þ and the unloaded case (Fi=0), we apply Fi=0 to Eqn 9 and find the following 0else Michaelis–Menten equation: with κkin being the stiffness of kinesin. The load force of dynein is also 0 i kcat ½ATP divided into three regimes depending on L0,dyn (=width of the deactivation sðF ¼ 0; ½ATPÞ ¼ ð12Þ 0 = 0 region). However, unlike kinesin, dynein is not completely untensioned ½ATPþkcat kb within L0,dyn. Within L0,dyn the force is calculated using a smaller stiffness If kinesin is under very high backward loads that is bigger than the kinesin κ : i 1,dyn stall force (F >Fs,kin), kinesin steps backwards (towards the microtubule i i minus end) with a low constant rate F ðtÞ¼k1;dyn Dx ðtÞð4Þ vb;kin If dynein is stretched outside L the load force is: sðFiÞ¼ ð13Þ 0,dyn d i i F ðtÞ¼k1;dyn L0;dyn þ k2;dyn ðDx ðtÞL0;dynÞð5Þ For active dynein, the same force and ATP dependence of the stepping rate i as for kinesin are applied with different parameter values. For backward if Δx (t)>L0,dyn and i forces below the stall force (0>F >−Fs,dyn), we apply Eqn 9, and for forward i 0 δ i k k D i load (F >0) we apply Eqn 12 with kcat ¼ vf;mean;dyn d and determined by F ðtÞ¼ 1;dyn L0;dyn þ 2;dyn ð x ðtÞþL0;dynÞ, ð6Þ 0 Eqn 11 using Fs,dyn, qm, pm and kb , which have the same values as for

i i Journal of Cell Science if Δx (t)<−L0,dyn. Thereby, is κ2,dyn the dynein stiffness outside L0,dyn. kinesin. Under backward load greater than the stall force (F <−Fs,dyn) active

12 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249938. doi:10.1242/jcs.249938 dynein steps backward with rate: same protofilament, it cannot continue walking on this protofilament. However, when a motor encounters a roadblock or another motor protein v ; sðFiÞ¼ b dyn ð14Þ that is on another protofilament, it is not influenced by this obstacle and d continues walking normally. From previous single-motor experiments, it is Another difference to kinesin is that, for dynein, the maximal velocity, vf,dyn known that dynein frequently takes side steps (Reck-Peterson et al., 2006), is Gaussian distributed with the mean vf,mean,dyn, the standard deviation σv while kinesin stays on the same protofilament (Ray et al., 1993). Here, we and a cut at vf,high and vf,low. Moreover, Mallik et al. (2004) found that the implemented a side-stepping rate sside for dynein to change the stall force of dynein is ATP dependent for low ATP concentrations. Here, we protofilament. However, kinesin stays on the same protofilament as long added the findings of Mallik et al. (2004) to the previously published dynein as the kinesin motor is attached. Thus, to circumvent a roadblock, dynein model (Monzon et al., 2019) and set the dynein stall force to: can use its side-stepping ability, whereas kinesin has to detach and reattach after the roadblock. The results of the gliding assay with seven 3 pN F ; ¼ 0:95 10 ½ATPþ0:3pN ð15Þ protofilaments is shown in Fig. S3. s dyn mM if [ATP]≤1000 μM and to: Simulation details In the following, one run of the simulation is described. Fs;dyn ¼ 1:25 pN ð16Þ if [ATP]>1000 μM. Initialization Unlike the active dynein stepping predominately towards the microtubule As a first step the one-dimensional surface is coated with motors. At the minus end, passive dynein diffuses in the harmonic potential of its linear beginning the microtubule is situated at position XMT(t=0)=0 and no motor spring. Thus the stepping rate is: is attached to the microtubule.

Fi d + Update i 2k T ’ s+ðF ; ½ATPÞ ¼ s0ð½ATPÞ e B : ð17Þ Using Gillespie s first reaction sampling (Gillespie, 1977), the next event [attachment, detachment, stepping or (de)activation] is chosen. Then the For the stepping rate of passive dynein in the unloaded case (Fi=0), we chosen motor event is performed, meaning the chosen motor is updated. applied the same Michaelis–Menten equation as found for the unloaded case After the motor event, the microtubule is moved to its nearest equilibrium of directional stepping of active dynein (see Eqn 12): position using a bisection search algorithm. s ; ½ATP s ð½ATPÞ ¼ 0 max : ð18Þ 0 = 0 Measurement and output data ½ATPþs0;max kb After the relaxation time trelax, the microtubule position and instantaneous velocity are measured. Each second (like in the experiment) the time, the Mechanical dynein activation velocity and the position of the microtubule are measured and output as well When dynein attaches to the microtubule, it is first in the passive state. When as the number of attached motors (distinguishing between kinesin, active passive attached dynein is stretched more than L0,dyn, the deactivation and passive dynein motors). To mimic the measurement uncertainty of the i i σ region, it activates with rate ra(Δx ). The activation rate ra(Δx ) depends on experiment, a white noise with standard derivation pos is added to the actual the deflection of the motor in an Arrhenius-like manner: simulation position. 0 1 The microtubule instantaneous velocity is measured from the last and Ea current microtubule position (plus white noise) and the last and current time: B C D i 0@ kBT A; rað x Þ¼ra 1 e ð19Þ xn xn1 vn ¼ , ð22Þ tn tn1 with Ea being the energy of the harmonic potential of the motor spring: where xn−1 is the position at the last measurement time tn−1.

1 2 i 2 E ¼ ðk ; L þ k ; ðjDx jL ; Þ Þ: ð20Þ a 2 1 dyn 0;dyn 2 dyn 0 dyn Termination The complete simulation is terminated after N program runs or if N When active attached dynein is stretched less than L , the deactivation samples mes 0,dyn measurements were performed. A single program run is terminated either region, it deactivates with the constant rate rd. after a specific amount of measurements nmes or after the simulation time Tend, or when no motor is attached to the microtubule at a time point greater Roadblocks than zero. The reason for the last termination condition is that in the For the simulations shown in Fig. 6 and Fig. S3, we added roadblocks to the experiment a microtubule trajectory is also terminated once no motor is previously published gliding assay model (Monzon et al., 2019). Here, we attached anymore. If no motor is attached anymore in the experiment, the used rigor-binding kinesin motor mutants (from now on referred to as microtubule diffuses vertically away from the surface and cannot be roadblocks). The number of roadblocks on the microtubule is calculated as: measured.

NRB ¼ LMT lRB, ð21Þ Acknowledgements with λRB being the roadblock line density. The roadblocks are uniformly We thank Andrew Carter and Max Schlager for kindly providing the plasmids distributed on the one-dimensional microtubule (one protofilament), encoding genes for H. sapiens dynein subunits and the multiBac plasmid. whereby mutual spatial exclusion between the roadblocks were taken into account. Competing interests The authors declare no competing or financial interests. Gliding assay with seven protofilaments To compare our results to cargo transport by multiple motors on multiple Author contributions Conceptualization: G.A.M., L. Scharrel, L. Santen, S.D.; Formal analysis: G.A.M., protofilaments (Ferro et al., 2019), we implemented a gliding assay with L. Scharrel, L. Santen, S.D.; Investigation: G.A.M., L. Scharrel, A.D., V.H., L. Santen, seven protofilaments in the presence of roadblocks. In this modification, not S.D.; Resources: L. Scharrel, S.D.; Data curation: G.A.M., L. Scharrel, L. Santen, only one protofilament is considered, like above, but seven protofilaments. S.D.; Writing - original draft: G.A.M.; Writing - review & editing: A.D., L. Santen, S.D.; The roadblocks and motors are randomly distributed on the seven Supervision: L. Santen, S.D.; Project administration: L. Santen, S.D.; Funding protofilaments. If a motor encounters a roadblock or another motor on the acquisition: L. Santen, S.D. Journal of Cell Science

13 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249938. doi:10.1242/jcs.249938

Funding Gross, S. P. (2004). Hither and yon: a review of bi-directional microtubule-based This work was supported by Deutsche Forschungsgemeinschaft (SFB1027) and transport. Phys. Biol. 1, R1-R11. doi:10.1088/1478-3967/1/2/R01 Technische Universität Dresden. We also acknowledge funding from Boehringer Hendricks, A. G., Perlson, E., Ross, J. L., Schroeder, H. W., Tokito, M. and Ingelheim Fonds to A.D. (PhD stipend). Holzbaur, E. L. F. (2010). Motor coordination via a tug-of-war mechanism drives bidirectional vesicle transport. Curr. Biol. 20, 697-702. doi:10.1016/j.cub.2010.02. Supplementary information 058 Hendricks, A. G., Holzbaur, E. L. F. and Goldman, Y. E. (2012). Force Supplementary information available online at measurements on cargoes in living cells reveal collective dynamics of https://jcs.biologists.org/lookup/doi/10.1242/jcs.249938.supplemental microtubule motors. Proc. Natl. Acad. Sci. 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