Samara L. Reck-Peterson

Single-Molecule Analysis of Dynein Processivity and Stepping Behavior

Cytoplasmic dynein, the 1.2 MDa motor driving minus-end-directed motility, has been reported to move processively along microtubules, but its mechanism of motility remains poorly understood. Here, using S. cerevisiae to produce recombinant dynein with a chemically controlled dimerization switch, we show by structural and single-molecule analysis that processivity requires two dynein motor domains but not dyneins tail domain or any associated subunits. Dynein advances most frequently in 8 nm steps, although longer as well as side and backward steps are observed. Individual motor domains show a different stepping pattern, which is best explained by the two motor domains shuffling in an alternating manner between rear and forward positions. Our results suggest that cytoplasmic dynein moves processively through the coordination of its two motor domains, but its variable step size and direction suggest a considerable diffusional component to its step, which differs from Kinesin-1 and is more akin to myosin VI.

Class V myosins

1. IntroductionThe myosin family of actin-based molecular mo-tors consists of 15 known classes that are structurallydistinct based on comparisons of the primary struc-ture of the motor domains of the known myosinheavy chain genes [1^3]. Information regarding thefunction and/or biochemical properties of most ofthese myosin classes is sparse relative to the well-characterized class II and class I myosins; neverthe-less, the range of proposed functions for these myo-sins is already remarkably broad [1]. There are anumber of recent reviews that provide an overviewof the rapidly growing myosin gene family [1,4^8].Among the best characterized and functionally di-verse of the recently discovered myosin classes arethe class V myosins, the focus of this review (forother reviews see [9^11]).Myosin-V was initially characterized as an unusualcalmodulin binding protein from brain with a num-ber of myosin-like biochemical properties [12^14].Subsequently, myosin-V heavy chain genes werecloned from mouse, yeast and chicken, thus de¢ningthe ¢fth class of actin-based motors [15^19]. Studiesof the mouse and yeast class V myosins provided the¢rst insights regarding the cellular function of myo-sin-V. Phenotypes of the mutant dilute mouse andthe temperature-sensitive yeast mutant, myo2-66 ledto the hypothesis that class V myosins may functionin cell polarity and membrane tra⁄cking [15,16].Moreover, mutations in the human ortholog of thedilute heavy chain gene cause Griscelli syndrome, arare recessive disease characterized by pigmentarydilution and in most, but not all cases immunode¢-ciency [20^22]. Neurological disorders have also beenreported in Griscelli syndrome patients [22,23]. Sincethese ¢rst studies, a great deal has been learnedabout the biochemistry, biophysics and cellular func-tion of the class V myosins. This review will discussthe emerging evidence that myosin-V is a processiveactin-based motor that has multiple functions in thecell ranging from mRNA transport, cell polarity andmembrane tra⁄cking.There are currently nine complete myosin-V heavychain sequences known (Fig. 1). Analysis of the twocompleted eukaryotic genomes of Saccharomyces ce-revisiae and Caenorhabditis elegans reveals that yeasthave two class V myosins, while C. elegans has asingle class V myosin heavy chain gene. In verte-brates, there are at least three distinct subclasses ofmyosin-V. The three most closely related heavy chainsequences, that of chicken brain myosin-V, the

Force-Induced Bidirectional Stepping of Cytoplasmic Dynein

Cytoplasmic dynein is a minus-end-directed microtubule motor whose mechanism of movement remains poorly understood. Here, we use optical tweezers to examine the force-dependent stepping behavior of yeast cytoplasmic dynein. We find that dynein primarily advances in 8 nm increments but takes other sized steps (4-24 nm) as well. An opposing force induces more frequent backward stepping by dynein, and the motor walks backward toward the microtubule plus end at loads above its stall force of 7 pN. Remarkably, in the absence of ATP, dynein steps processively along microtubules under an external load, with less force required for minus-end- than for plus-end-directed movement. This nucleotide-independent walking reveals that force alone can drive repetitive microtubule detachment-attachment cycles of dyneins motor domains. These results suggest a model for how dyneins two motor domains coordinate their activities during normal processive motility and provide new clues for understanding dynein-based motility in living cells.

Tug-of-War in Motor Protein Ensembles Revealed with a Programmable DNA Origami Scaffold

Push Me, Release, Pull You In eukaryotic cells, nearly all long-distance transport of cargos is carried out by the microtubule-based motors kinesin and dynein. These opposite-polarity motors move cargos bidirectionally so that they reach their cellular destinations with spatial and temporal specificity. To understand transport by motor ensembles, Derr et al. (p. 662, published online 11 October; see the Persective by Diehl) used a DNA scaffold for building an artificial cargo that could be programmed to bind different numbers and types of molecular motors with defined geometry. A cargo with multiple copies of the same motor was transported with minimal interference, suggesting that similar-polarity motors can coordinate without the need for additional cellular factors. However, ensembles of opposite-polarity motors frequently engaged in a sort of “tug of war,” which could only be resolved by releasing one motor from the microtubule track. Thus, within the cell, it is likely that regulation is required for bidirectional transport. Two microtubule motors attached to the same cargo cooperate or compete, depending on relative directionality. Cytoplasmic dynein and kinesin-1 are microtubule-based motors with opposite polarity that transport a wide variety of cargo in eukaryotic cells. Many cellular cargos demonstrate bidirectional movement due to the presence of ensembles of dynein and kinesin, but are ultimately sorted with spatial and temporal precision. To investigate the mechanisms that coordinate motor ensemble behavior, we built a programmable synthetic cargo using three-dimensional DNA origami to which varying numbers of DNA oligonucleotide-linked motors could be attached, allowing for control of motor type, number, spacing, and orientation in vitro. In ensembles of one to seven identical-polarity motors, motor number had minimal affect on directional velocity, whereas ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one motor species.

Lis1 Acts as a ''Clutch'' between the ATPase and Microtubule-Binding Domains of the Dynein Motor

Summary The lissencephaly protein Lis1 has been reported to regulate the mechanical behavior of cytoplasmic dynein, the primary minus-end-directed microtubule motor. However, the regulatory mechanism remains poorly understood. Here, we address this issue using purified proteins from Saccharomyces cerevisiae and a combination of techniques, including single-molecule imaging and single-particle electron microscopy. We show that rather than binding to the main ATPase site within dyneins AAA+ ring or its microtubule-binding stalk directly, Lis1 engages the interface between these elements. Lis1 causes individual dynein motors to remain attached to microtubules for extended periods, even during cycles of ATP hydrolysis that would canonically induce detachment. Thus, Lis1 operates like a “clutch” that prevents dyneins ATPase domain from transmitting a detachment signal to its track-binding domain. We discuss how these findings provide a conserved mechanism for dynein functions in living cells that require prolonged microtubule attachments.

The Affinity of the Dynein Microtubule-Binding Domain is Modulated by the Conformation of its Coiled-Coil Stalk*

The microtubule-binding domain (MTBD) of dynein is separated from the AAA (ATPase with any other activity) core of the motor by an ∼15-nm stalk that is predicted to consist of an antiparallel coiled coil. However, the structure of this coiled coil and the mechanism it uses to mediate communication between the MTBD and ATP-binding core are unknown. Here, we sought to identify the optimal alignment between the hydrophobic heptad repeats in the two strands of the stalk coiled coil. To do this, we fused the MTBD of mouse cytoplasmic dynein, together with 12-36 residues of its stalk, onto a stable coiled-coil base provided by Thermus thermophilus seryl-tRNA synthetase and tested these chimeric constructs for microtubule binding in vitro. The results identified one alignment that yielded a protein displaying high affinity for microtubules (2.2 μm). The effects of mutations applied to the MTBD of this construct paralleled those previously reported (Koonce, M. P., and Tikhonenko, I. (2000) Mol. Biol. Cell 11, 523-529) for an intact dynein motor unit in the absence of ATP, suggesting that it resembles the tight binding state of native intact dynein. All other alignments showed at least 10-fold lower affinity for microtubules with the exception of one, which had an intermediate affinity. Based on these results and on amino acid sequence analysis, we hypothesize that dynein utilizes small amounts of sliding displacement between the two strands of its coiled-coil stalk as a means of communication between the AAA core of the motor and the MTBD during the mechanochemical cycle.

Regulation of the processivity and intracellular localization of Saccharomyces cerevisiae dynein by dynactin

Dynactin, a large multisubunit complex, is required for intracellular transport by dynein; however, its cellular functions and mechanism of action are not clear. Prior studies suggested that dynactin increases dynein processivity by tethering the motor to the microtubule through its own microtubule binding domains. However, this hypothesis could not be tested without a recombinant source of dynactin. Here, we have produced recombinant dynactin and dynein in Saccharomyces cerevisiae, and examined the effect of dynactin on dynein in single-molecule motility assays. We show that dynactin increases the run length of single dynein motors, but does not alter the directionality of dynein movement. Enhancement of dynein processivity by dynactin does not require the microtubule (MT) binding domains of Nip100 (the yeast p150Glued homolog). Dynactin lacking these MT binding domains also supports the proper localization and function of dynein during nuclear segregation in vivo. Instead, a segment of the coiled-coil of Nip100 is required for these activities. Our results directly demonstrate that dynactin increases the processivity of dynein through a mechanism independent of microtubule tethering.

Lis1 is an initiation factor for dynein-driven organelle transport

The dynein-associated protein Lis1 may be a ubiquitous determinant of dynein-dependent transport required primarily at the stage of motility initiation.

Structural Basis for Microtubule Binding and Release by Dynein

Motoring Along Dyneins are large and complex molecular motors that transport cargo along cellular microtubules and power the movement of cilia. An enigma is how microtubule binding and nucleotide hydrolysis are coordinated between sites separated by 25 nm. Redwine et al. (p. 1532) report an electron microscopy structure of the dynein microtubule-binding domain bound to microtubules in a high-affinity state and combined this with molecular dynamics and existing x-ray structures to provide a model for how dynein couples its affinity for microtubules with the nucleotide-bound state of the motor domain. The molecular motor dynein uses conformational changes within its microtubule-binding domain to modulate track affinity. Cytoplasmic dynein is a microtubule-based motor required for intracellular transport and cell division. Its movement involves coupling cycles of track binding and release with cycles of force-generating nucleotide hydrolysis. How this is accomplished given the ~25 nanometers separating dynein’s track- and nucleotide-binding sites is not understood. Here, we present a subnanometer-resolution structure of dynein’s microtubule-binding domain bound to microtubules by cryo–electron microscopy that was used to generate a pseudo-atomic model of the complex with molecular dynamics. We identified large rearrangements triggered by track binding and specific interactions, confirmed by mutagenesis and single-molecule motility assays, which tune dynein’s affinity for microtubules. Our results provide a molecular model for how dynein’s binding to microtubules is communicated to the rest of the motor.

Regulatory ATPase Sites of Cytoplasmic Dynein Affect Processivity and Force Generation

The heavy chain of cytoplasmic dynein contains four nucleotide-binding domains referred to as AAA1–AAA4, with the first domain (AAA1) being the main ATP hydrolytic site. Although previous studies have proposed regulatory roles for AAA3 and AAA4, the role of ATP hydrolysis at these sites remains elusive. Here, we have analyzed the single molecule motility properties of yeast cytoplasmic dynein mutants bearing mutations that prevent ATP hydrolysis at AAA3 or AAA4. Both mutants remain processive, but the AAA4 mutant exhibits a surprising increase in processivity due to its tighter affinity for microtubules. In addition to changes in motility characteristics, AAA3 and AAA4 mutants produce less maximal force than wild-type dynein. These results indicate that the nucleotide binding state at AAA3 and AAA4 can allosterically modulate microtubule binding affinity and affect dynein processivity and force production.