Control of the Initiation and Termination of Kinesin-1-Driven Transport by Myosin-Ic and Nonmuscle Tropomyosin

Abstract

Summary Intracellular transport is largely driven by processive microtubule- and actin-based molecular motors. Non-processive motors have also been localized to trafficking cargos, but their roles are not well understood [1–7]. Myosin-Ic (Myo1c), a non-processive actin motor, functions in a variety of exocytic events, although the underlying mechanisms are not yet clear. To investigate the interplay between myosin-I and the canonical long distance transport motor kinesin-1, we attached both motor types to lipid membrane-coated bead (MCB) cargo, using an attachment strategy that allows motors to actively reorganize within the membrane in response to the local cytoskeletal environment. We compared the motility of kinesin-1-driven cargos in the absence and presence of Myo1c at engineered actin/microtubule intersections. We found that Myo1c significantly increases the frequency of kinesin-1-driven microtubule-based runs that begin at actin/microtubule intersections. Myo1c also regulates the termination of processive runs. Beads with both motors bound have a significantly higher probability of pausing at actin/microtubule intersections, remaining tethered for an average of 20 s, with some pauses lasting longer than 200 s. The actin-binding protein non-muscle tropomyosin (Tm) provides spatially-specific regulation of interactions between myosin motors and actin filaments in vivo [8, 9, 11, 13, 14]; in the crossed-filament in vitro assay, we found that Tm2-actin abolishes Myo1c-specific effects on both run initiation and run termination. Together these observations suggest Myo1c is important for the selective initiation and termination of kinesin-driven runs along microtubules at specific actin filament populations within the cell.

Tropomyosin – master regulator of actin filament function in the cytoskeleton

“…Subsequent work demonstrated that Tpm1.6 was able to account for the intracellular location of Myo1b by excluding its interaction with actin filaments that contain Tpm1.6 (Tm2) (Tang and Ostap, 2001). The ability of Tpms to also prevent Myo1c from interacting with actin filaments has been observed both in other cells and in in vitro systems McIntosh et al, 2015).…”

“…This suggests that specific Tpm isoforms impact on organelle trafficking, which might reflect their differential effect on myosin motors. For instance, Ostap and co-workers have shown that Myo1b, which is involved in organelle transport, does not recognize actin filaments that contain either Tpm1.6 (also known as Tm2) or Tpm3.1 (Tang and Ostap, 2001;Kee et al, 2015;McIntosh et al, 2015). In addition, Tpm1.8 (also known as Tm5a) and Tpm1.9 regulates the recycling of the cystic fibrosis transmembrane receptor (CFTR) (Dalby-Payne et al, 2003).…”

Myosin-I molecular motors at a glance

“…In vitro work suggests that Myo1c is in fact capable of halting processive microtubulebased transport at actin intersections, which not only implicates Myo1c in cargo docking, but also connects microtubule-and actinbased transport pathways (McIntosh et al, 2015). Additionally, in vitro and cellular studies have shown that cargo docking by Myo1c is regulated by the presence of non-muscle tropomyosin, which might spatially regulate the location of cargo docking to tropomyosin-free filaments just beneath the plasma membrane (Kee et al, 2015;McIntosh et al, 2015). Myo1c has also been implicated in the mechanical adaptation of signaling in inner ear hair cells (Batters et al, 2004;Gillespie et al, 1993;Holt et al, 2002;Lin et al, 2011), as well as in the regulation of a Na + channel after antidiuretic hormone stimulation in the kidney collection ducts (Wagner et al, 2005).…”

“…For example, Myo1c has been proposed to facilitate docking of GLUT4-containing vesicles at the plasma membrane prior to fusion in response to insulin stimulation (Boguslavsky et al, 2012;Bose et al, 2002Bose et al, , 2004Chen et al, 2007;Huang et al, 2005;Yip et al, 2008). In vitro work suggests that Myo1c is in fact capable of halting processive microtubulebased transport at actin intersections, which not only implicates Myo1c in cargo docking, but also connects microtubule-and actinbased transport pathways (McIntosh et al, 2015). Additionally, in vitro and cellular studies have shown that cargo docking by Myo1c is regulated by the presence of non-muscle tropomyosin, which might spatially regulate the location of cargo docking to tropomyosin-free filaments just beneath the plasma membrane (Kee et al, 2015;McIntosh et al, 2015).…”

“…In vitro and in vivo data suggests that myosin-I motors avoid tropomyosin-coated actin filaments (Collins et al, 1990;Kee et al, 2015;McIntosh et al, 2015;Tang and Ostap, 2001) and instead prefer Arp2/3-nucleated (Almeida et al, 2011) and non-tropomyosin-coated cytoskeletal actin. Although Myo1a, Myo1b, Myo1c and Myo1g have been found to preferentially bind to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ], all characterized myosin-I isoforms can bind to anionic phospholipids to some extent (Adams and Pollard, 1989;Dart et al, 2012;Doberstein and Pollard, 1992;Feeser et al, 2010;Hayden et al, 1990;Komaba and Coluccio, 2010;McKenna and Ostap, 2009;Miyata et al, 1989;Patino-Lopez et al, 2010).…”