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Stable Tug-Of-War Between Kinesin-1 and Cytoplasmic Dynein Upon Different ATP and Roadblock Concentrations Gina A

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Stable Tug-Of-War Between Kinesin-1 and Cytoplasmic Dynein Upon Different ATP and Roadblock Concentrations Gina A © 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
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