The influence of dynein processivity control, MAPs, and microtubule ends on directional movement of a localising mRNA

RESEARCH ARTICLE elifesciences.org The influence of dynein processivity control, MAPs, and microtubule ends on directional movement of a localising mRNA Harish Chandra Soundararajan†, Simon L Bullock* Division of Cell Biology, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom Abstract Many cellular constituents travel along microtubules in association with multiple *For correspondence: sbullock@ mrc-lmb.cam.ac.uk Present address: †Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, United States Competing interests: The authors declare that no competing interests exist. Funding: See page 23 Received: 25 September 2013 Accepted: 07 March 2014 Published: 15 April 2014 Reviewing editor: Randy Schekman, Howard Hughes Medical Institute, University of California, Berkeley, United States Copyright Soundararajan and Bullock. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. copies of motor proteins. How the activity of these motors is regulated during cargo sorting is poorly understood. In this study, we address this issue using a novel in vitro assay for the motility of localising Drosophila mRNAs bound to native dynein-dynactin complexes. High precision tracking reveals that individual RNPs within a population undergo either diffusive, or highly processive, minus end-directed movements along microtubules. RNA localisation signals stimulate the processive movements, with regulation of dynein-dynactin’s activity rather than its total copy number per RNP, responsible for this effect. Our data support a novel mechanism for multi-motor translocation based on the regulation of dynein processivity by discrete cargo-associated features. Studying the in vitro responses of RNPs to microtubule-associated proteins (MAPs) and microtubule ends provides insights into how an RNA population could navigate the cytoskeletal network and become anchored at its destination in cells. DOI: 10.7554/eLife.01596.001 Introduction Microtubule-based motility plays a major role in the distribution and sorting of organelles, vesicles, and macromolecules within cells. The significance of this process is underscored by the association of mutations in microtubule motor proteins and their co-factors with several neurological disorders (Hirokawa et al., 2010; Schiavo et al., 2013). Furthermore, many pathogens exploit cellular microtubule motors during infection (Grieshaber et al., 2003; Greber and Way, 2006; Ramsden et al., 2007). Despite the fundamental importance of the process, the mechanisms by which cargos are trafficked along microtubules are poorly understood. There is substantial evidence that individual cargos simultaneously associate with multiple microtubule motors (Gross et al., 2007). Not only can a single cargo associate with several copies of the same kind of motor (e.g., Leopold et al., 1992; Welte et al., 1998; Hendricks et al., 2010; Encalada et al., 2011; Rai et al., 2013), but opposite polarity motors are often stably bound (Ling et al., 2004; Pilling et al., 2006; Shubeita et al., 2008; Soppina et al., 2009; Hendricks et al., 2010; Encalada et al., 2011). Thus, in order to understand cargo trafficking in vivo it is essential to learn how the activity of multiple motors is orchestrated. The mechanisms governing translocation of cargos by teams of microtubule motors have predominantly been tackled in two ways. One set of studies has analysed motility of cargo populations within cells (e.g., Kural et al., 2005; Shubeita et al., 2008; Reis et al., 2012; Rai et al., 2013). Although physiologically relevant, the mechanistic insights that can be derived from this approach are limited by the complex in vivo environment. The second set of studies have analysed the in vitro behaviours of Soundararajan and Bullock. eLife 2014;3:e01596. DOI: 10.7554/eLife.01596 1 of 26 Research article Biophysics and structural biology | Cell biology eLife digest For a cell to do its job, the different components inside it need to be moved to different locations. This is achieved by an elaborate cellular transport system. To move a component to where it needs to be, motor proteins bind to it, often with the assistance of other ‘accessory’ proteins. This cargo-motor complex then moves along a network of tracks within the cell. Viruses also exploit this transport system in order to be trafficked to specific parts of the cell during their life cycles. Many cargos are moved along microtubule tracks. Multiple microtubule motor proteins often attach to the same cargo, but it is unclear how they work together during transport. Previous studies have attempted to address this issue by attaching motor proteins to artificial cargoes, such as synthetic beads. However, these experiments did not include some of the accessory proteins that are thought to play a role during transport within the living cell. Soundararajan and Bullock have now examined how complexes containing multiple motors bound to accessory proteins move molecules of messenger RNA to specific sites within cells. By visualising fruit fly mRNA moving along microtubules attached to a glass surface, the transport process can be studied in detail. It appears that the complexes travel using one of two methods: they either diffuse along the microtubules, which they can do in either direction, or they power themselves along the microtubules, which they can only do in one direction. Although previous experiments with artificial cargos suggested that the number of motors in the complex determines the likelihood of one-way traffic, it appears that one or more accessory proteins are actually in control during mRNA transport. Soundararajan and Bullock also documented how the mRNA-motor complexes react to roadblocks and dead-ends on the microtubule highway. Rather than letting go of the microtubule upon such an encounter, the complexes can reverse back down the track. This behaviour may help them to find a new route to their destination. DOI: 10.7554/eLife.01596.002 artificial cargos, such as beads or DNA origami, coupled to isolated motor proteins or motor domains (e.g., Vale et al., 1985; Block et al., 1990; Mallik et al., 2005; Diehl et al., 2006; Ross et al., 2006; Vershinin et al., 2007; Derr et al., 2012; Furuta et al., 2013). These in vitro studies have provided evidence that small increases in the number of purified motors of the same polarity strongly augment the average travel distance in that direction (Block et al., 1990; Mallik et al., 2005; Vershinin et al., 2007; Derr et al., 2012; Furuta et al., 2013). However, these experiments did not include potential regulatory co-factors that associate with motors in vivo. Thus, substantial debate persists over whether net movement of physiological cargo-motor complexes is dominated by motor copy number or by higher order mec (...truncated)