SummaryIntracellular cargo transport requires microtubule-based motors, kinesin and cytoplasmic dynein, and the actin-based myosin motors to maneuver through the challenges presented by the filamentous meshwork that comprises the cytoskeleton. Recent in vitro single molecule biophysical studies have begun to explore this process by characterizing what occurs as these tiny molecular motors happen upon an intersection between two cytoskeletal filaments. These studies, in combination with in vivo work, define the mechanism by which molecular motors exchange cargo while traveling between filamentous tracks and deliver it to its destination when going from the cell center to the periphery and back again.

Certain types of intracellular organelle transport to the cell periphery are thought to involve long-range movement on microtubules by kinesin with subsequent handoff to vertebrate myosin Va (myoVa) for local delivery on actin tracks. This process may involve direct interactions between these two processive motors. Here we demonstrate using single molecule in vitro techniques that myoVa is flexible enough to effectively maneuver its way through actin filament intersections and Arp2/3 branches. In addition, myoVa surprisingly undergoes a one-dimensional diffusive search along microtubules, which may allow it to scan efficiently for kinesin and/or its cargo. These features of myoVa may help ensure efficient cargo delivery from the cell center to the periphery.cytoskeleton ͉ molecular motor ͉ motility ͉ processivity C oordinated organelle transport along certain secretory pathways starts with kinesin-powered movement on microtubules and finishes with vertebrate myosin Va (myoVa)-based motility on actin (1-4). This trip requires that both motors be present when a microtubule-actin intersection is encountered, but how these two motors find one another or the cargo they share is still unknown. Another challenge is to understand how cargo is delivered to its destination by way of the complex cytoskeletal network. Vertebrate myoVa is a processive motor that can travel long distances along its actin track (5). However, the cytoskeletal intersections that myoVa must encounter along its journey may present a physical barrier to forward motion or an alternate path to its final destination.To investigate this issue from myoVa's point of view, we observed single myoVa molecules labeled with highly photostable quantum dots (Qdots) in an objective-type total internal reflectance microscope (6) as they maneuvered through a model of cytoskeletal intersections created on a microscope coverslip coated with either actin filaments (Fig. 1A) or actin filaments and microtubules (Fig. 1B). In this model system, actin-actin intersections could inhibit or alter myoVa's direction of travel while actin-microtubule intersections should act as a hurdle to myoVa's processive movement. Given myoVa's ability to take 72-nm steps as it walks processively along actin filaments in a handover-hand fashion (6-9) and its inherent flexibility (10, 11), we have observed that myoVa can easily maneuver past actin filament intersections. In contrast, when encountering a microtubule, myoVa cannot step over this physical barrier, but surprisingly can step onto the microtubule and begin a onedimensional diffusive search. Thus, myoVa has evolved to handle the challenges of the cytoskeletal network and through its interaction with the microtubule can effectively scan for its transport partner, kinesin, and/or its cargo. Results and Discussion MyoVa Dynamics at Actin Filament Intersections.To determine what happens when myoVa encounters an overlapping actin filament, we adhered Alexa Fluor 660 phalloidin-labeled actin filaments to the coverslip, followed by TRITC...

Membrane proteins impose enormous challenges to cellular protein homeostasis during their post-translational targeting, and require chaperones to keep them soluble and translocation-competent. Here we show that a novel targeting factor in the chloroplast Signal Recognition Particle (cpSRP), cpSRP43, is a highly specific molecular chaperone that efficiently reverses the aggregation of its substrate proteins. In contrast to AAA+-chaperones, cpSRP43 utilizes specific binding interactions with its substrate to mediate its disaggregase activity. This ‘disaggregase’ capability can allow targeting machineries to more effectively capture their protein substrates, and emphasizes a close connection between protein folding and trafficking processes. Moreover, cpSRP43 provides the first example of an ATP-independent disaggregase, and demonstrates that efficient reversal of protein aggregation can be attained by specific binding interactions between a chaperone and its substrate.

Organelle transport to the periphery of the cell involves coordinated transport between the processive motors kinesin and myosin V. Long-range transport takes place on microtubule tracks, whereas final delivery involves shorter actin-based movements. The concept that motors only function on their appropriate track required further investigation with the recent observation that myosin V undergoes a diffusional search on microtubules. Here we show, using single-molecule techniques, that a functional consequence of myosin V's diffusion on microtubules is a significant enhancement of the processive run length of kinesin when both motors are present on the same cargo. The degree of run length enhancement correlated with the net positive charge in loop 2 of myosin V. On actin, myosin V also undergoes longer processive runs when kinesin is present on the same cargo. The process that causes run length enhancement on both cytoskeletal tracks is electrostatic. We propose that one motor acts as a tether for the other and prevents its diffusion away from the track, thus allowing more steps to be taken before dissociation. The resulting run length enhancement likely contributes to the successful delivery of cargo in the cell.actin ͉ microtubule ͉ molecular motor ͉ Qdot ͉ electrostatic M ovement of membrane-bound organelles from the center of the cell to its periphery and back involves the interplay between processive molecular motors on both microtubule (MT) and actin tracks. In amphibian melanophores, where pigment granules disperse and aggregate to cause skin color changes, long-range transport toward the plus-end of microtubules is carried out by kinesin-2, whereas myosin V (myoV) takes over in the actin-rich cell cortex to deliver melanosomes to the cell periphery during dispersion (1). This process requires switching between cytoskeletal tracks. We have shown, at the singlemolecule level, that myoV could effectively navigate actin-actin intersections that normally exist within cells, either by executing a turn, or by stepping over the crossing actin filament (2). The frequency of these two events appeared to be related to myoV's inherent flexibility and the availability of actin-binding sites on the intersecting filaments that were within reach of the leading head. Surprisingly, myoV could also undergo a diffusional search on MTs, resulting from an electrostatic interaction between the myoV head and the negatively charged tubulin E-hook. We hypothesized that this diffusive process helps myoV find cargo that is undergoing MT-based movement, and/or facilitates the binding of myoV to kinesin if the two motors directly interact (3).Can myoV's interaction with the MT affect kinesin's processivity when both motors are present on the same cargo? Here we show that this interaction enhances the processivity of kinesinbased cargo transport. We also provide evidence that the structural element on myoV that acts as an electrostatic tether is loop 2, a charged surface loop that also influences myoV's processive run length on actin (...

Myosin V is a two-headed, actin-based molecular motor implicated in organelle transport. Previously, a single myosin V molecule has been shown to move processively along an actin filament in discrete approximately 36 nm steps. However, 36 nm is the helical repeat length of actin, and the geometry of the previous experiments may have forced the heads to bind to, or halt at, sites on one side of actin that are separated by 36 nm. To observe unconstrained motion, we suspended an actin filament in solution and attached a single myosin V molecule carrying a bead duplex. The duplex moved as a left-handed spiral around the filament, disregarding the right-handed actin helix. Our results indicate a stepwise walking mechanism in which myosin V positions and orients the unbound head such that the head will land at the 11th or 13th actin subunit on the opposing strand of the actin double helix.

Intracellular cargo transport relies on myosin Va molecular motor ensembles to travel along the cell's three-dimensional (3D) highway of actin filaments. At actin filament intersections, the intersecting filament is a structural barrier to and an alternate track for directed cargo transport. Here we use 3D super-resolution fluorescence imaging to determine the directional outcome (that is, continues straight, turns or terminates) for an ∼10 motor ensemble transporting a 350 nm lipid-bound cargo that encounters a suspended 3D actin filament intersection in vitro. Motor–cargo complexes that interact with the intersecting filament go straight through the intersection 62% of the time, nearly twice that for turning. To explain this, we develop an in silico model, supported by optical trapping data, suggesting that the motors' diffusive movements on the vesicle surface and the extent of their engagement with the two intersecting actin tracks biases the motor–cargo complex on average to go straight through the intersection.

We investigated the role of binding partners of full-length Drosophila Bicaudal D (BicD) in the activation of dynein-dynactin motility for mRNA transport on microtubules. In single-molecule assays, full-length BicD robustly activated dynein-dynactin only when both the mRNA binding protein Egalitarian (Egl), and K10 mRNA cargo were present. Electron microscopy showed that both Egl and mRNA were needed to disrupt an auto-inhibited, looped BicD conformation that sterically prevents dynein-dynactin binding. In vitro reconstituted messenger ribonucleoprotein (mRNP) complexes with two Egl molecules showed faster speeds and longer run lengths than mRNPs with one Egl, suggesting that cargo binding enhances dynein recruitment. Labeled dynein showed that BicD can recruit two dimeric dyneins to the mRNP, resulting in faster speeds and longer run lengths than with one dynein. The fully reconstituted mRNP provides a model for understanding how adaptor proteins and cargo cooperate to confer optimal transport properties to a dynein-driven transport complex.

We investigated the role of full-length Drosophila Bicaudal D (BicD) binding partners in dynein-dynactin activation for mRNA transport on microtubules. Full-length BicD robustly activated dynein-dynactin motility only when both the mRNA binding protein Egalitarian (Egl) and K10 mRNA cargo were present, and electron microscopy showed that both Egl and mRNA were needed to disrupt a looped, auto-inhibited BicD conformation. BicD can recruit two dimeric dyneins, resulting in faster speeds and longer runs than with one dynein. Moving complexes predominantly contained two Egl molecules and one K10 mRNA. This mRNA-bound configuration makes Egl bivalent, likely enhancing its avidity for BicD and thus its ability to disrupt BicD auto-inhibition. Consistent with this idea, artificially dimerized Egl activates dynein-dynactin-BicD in the absence of mRNA. The ability of mRNA cargo to orchestrate the activation of the mRNP (messenger ribonucleotide protein) complex is an elegant way to ensure that only cargo-bound motors are motile.