Molecular Motors Objectives

• Regulation of molecular motors in smooth muscle • Other • based voters • • Regulating Blood Flow

• Increased oxygen extraction • Resting Skeletal Muscle: 20-40% • Exercising Skeletal Muscle: 70-80% • Cardiac Muscle: 70-80% • Kidneys: 10-15% Regulating Blood Flow • Blood flow is controlled by varying regional vascular resistance • Vascular Resistance is determined by the tone of the vascular smooth muscle cells • Systemic and local influences are integrated to maintain homeostasis Smooth Muscle Structure • Non-uniform organization • and myosin bundles connect contact points (dense bodies) • Tension develops slowly, but is maintained at very low energy consumption • Latch Mechanism • Property of smooth muscle myosin- has a very slow off rate • Contraction regulated at both the thin and thick filaments

http://s3.images.com/huge.101.506757.JPG Smooth Muscle Function • Regulation of Contraction • Thin-Filament Regulation • Caldesmon (CaD)-Blocks access to Actin • Thick Filament • Phosphorylation of Myosin Regulatory Light Chain Smooth Muscle Function • Calcium Entry Smooth Muscle Function • Myosin Phosphatase (aka Myosin light chain phosphatase) • Regulation • Inhibition allows for Ca2+ dependent contraction • Activated by cyclic nucleotides Endothelium • Ligand mediated vasodilation is often dependent on the endothelium Endothelium Feed-Forward Regulation Molecular Motors in Cell Physiology

• Limits of diffusion • D for a small in water is ≈0.1 µm2/msec • For this object to diffuse down a 1 meter axon would take ≈ 1.7 billion second or 53 years. x2 t = 6D kT D = 6⇡r⌘ The Myosin Family 80 T.A. Masters et al.

Motor Myo1a IQ Myo1b

TH1 Myosin Family Myo1c PH Myo1d

SH3 Myo1e TH2

Coiled-coil Homologus Motor Myo1f TH2 • MyTH Myo1g

domain Myo1h FERM

NMIIA/NMIIB/NMIIC

Myo3a Kinase Some are monomers 3THDI 3THDII Myo3b Kinase

• 3THDI

CBD (Class I) Myo5a PEST

CBD

Myo5b PEST Most are dimers Myo5c CBD • Myo6 SAH CBD

447 ( Class II, V, VI, etc.) Myo7a SAH Myo7b 447

Myo9a NTE L2I C1 RhoGAP

All bind actin NTE L2I C1 RhoGAP • Myo9b 3PH 4 SAH Myo10 PEST Most move toward Myo15a NTE 44 • Myo16 ARD NHM

the (+) end Myo18a KE PDZ

Myo18b

Myo19

Fig. 1 Domain organisation of the myosin motors expressed in humans. The most common domains are also illustrated in the key in the top right. The 11 muscle-expressed share domain homology with the three non-muscle myosin IIs Myh9/Myh10/Myh14. Full definitions of the domain labels are as follows: motor – actin-activated ATPase motor domain; IQ – IQ-binding motif; TH1 – tail homology 1; PH – pleckstrin homology domain; TH2 – tail homology 2; SH3 – Src homology 3; 3THDI – myosin 3 tail homology domain 1; 3THDII – myosin 3 tail homology domain 2 (contains an actin-binding motif); PEST – region rich in proline, glutamic acid, serine

• (+) end away from the nucleus Kinesin Kinesin REVIEWS Kinesin

KIF5 + KLC KIF5 Mitochondrion

Synaptotagmin mRNA APOER2 APP UNC51 Syntaxin JIP1 SNAP25 GluR2 Milton, FMRP Kinesin UNC76 Miro, FEZ1 GRIP1 light chain Syntabulin syntabulin Motor and RanBP2 domain Coiled coil

Microtubule

KIF1A or KIF1B KIF13B KIF16B KIF13A KIF3A or KIF3B + KAP3 KIF3A or KIF3B KIF17

Synaptotagmin PtdIns(3,4,5)P 3 APC NR2B PtdIns(4,5)P2 DENN/MADD ? RAB3 M6PR PAR3 RAB11 Kinesin AP1 Fodrin LIN10, PtdIns(3,4,5)P3 tail RIP11 (also known as LIN2 and FHA domain LIN7 Centaurin-1 RAB11 FIP5)

Microtubule Figure 3 | , cargos and molecules involved in cargo recognition. Major transport mechanisms that are based on Nature Reviews | Molecular Cell Biology kinesin superfamily (also known as KIFs). Microtubules consist of helically polymerized molecules. Kinesins associate with microtubules through their head motor domains, either as monomers (one sphere) or dimers (two spheres). Dimerization of KIFs occurs through the coiled-coil domains that are present in their stalks. KIFs associate with their cargos (as indicated) through their tails or occasionally through their light chains or adaptor or scaffold proteins. The kinesin 1 family KIF5 motors can bind to their cargo either through or independently of their kinesin light chains. The kinesin 3 family motors KIF1A and KIF1Bβ redundantly transport synaptic vesicle precursors and GTP-bound RAB3 through the Examplesadaptor of protein the mitogen-activated diversity protein kinase-activatingof Kinesin death domain cargos (MADD; also known as DENN and shown as • DENN/MADD). KIF13B (also known as guanylate kinase-associated kinesin (GAKIN)), KIF16B and KIF13A are also kinesin 3 family motors. The KIF3 motors and KIF17 (also known as OSM3) are kinesin 2 family motors that have dual functions in cilia, flagella and the cytoplasm. Post-translational modifications of the proteins involved and/or differential constitution of the adaptor complex might determine the motor–cargo pairing in each case. AP1, adaptor protein complex 1; APC, adenomatous polyposis coli; APP, β-Amyloid precursor protein; FEZ1, fasciculation and elongation protein- ζ1; FHA, Forkhead associated; FMRP, fragile X mental retardation protein (also known as FMR1); GluR2, AMPA (α-amino-3-hydroxy-5- methyl-4-isoxazole propionic acid)-type glutamate receptor 2; GRIP1, glutamate receptor-interacting protein 1; JIP1, JUN amino-terminal kinase (JNK)-interacting protein 1 (also known as MAPK8IP1); KAP3, kinesin superfamily-associated protein 3 (also known as KIFAP3); M6PR, mannose-6-phosphate receptor, cation dependent; NR2B, NMDA (N-methyl-d-aspartate)-

type glutamate receptor 2B; PAR3, partitioning defective 3; PtdIns(3,4,5)P3, phosphatidylinositol3,4,5-trisphosphate;

PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; RanBP2, Ran-binding protein 2; RIP11, RAB11 family-interacting protein 3 (also known as RAB11FIP5); SNAP25, soluble N-ethylmaleimide-sensitive factor attachment protein 25.

Regulation by Rab GTPases. Members of the Rab fam- RAB6 is thought to control intra-Golgi transport110. ily of small GTPases control localization in a The GTP-bound form of RAB6 localizes to the Golgi GTP–GDP-dependent manner109. The nucleotide states apparatus but the GDP-bound form does not. KIF20A of Rab GTPases are controlled by GAPs and GEFs. was identified as a kinesin that can specifically bind to When GAPs are activated, Rab GTPases exist in a GDP- the GTP-bound form of RAB6 (REF. 75). When RAB6 bound form. When GEFs are activated, Rab GTPases is converted to its GDP-bound form, KIF20A dissoci- exist in a GTP-bound form. Each Rab binds to a specific ates from the Golgi. This could control the motility and class of organelle (see below). The GTP-bound forms localization of the Golgi apparatus. Other work suggests of Rab proteins specifically bind to ‘Rab effector’ pro- a role for KIF20A in cytokinesis in mitotic cells, rather teins, whereas the GDP-bound forms do not. If Rab than in Golgi transport in interphase cells111,112. proteins on recruit motor proteins in the RAB5 controls endocytosis113. The GTP-bound GTP-bound form and release them in the GDP-bound form of RAB5 locally recruits human VPS34, a phospho- form, this could explain why the nucleotide switch of inositide 3-kinase, to endosomes. VPS34 locally syn- Rab GTPases can control the distribution of organelles. thesizes PtdIns-3-phosphate in endosomes and recruits Thus, Rab GTPases are also good candidates for a role KIF16B91. It has also been suggested that cytoplasmic in regulating the association and dissociation between dynein is recruited to endosomes by the GTP-bound kinesins and organelles. form of RAB5 (REF. 114). Thus, RAB5–GTP can recruit

690 | OCTOBER 2009 | VOLUME 10 www.nature.com/reviews/molcellbio Walking the walk: how kinesin and dynein coordinate their steps Gennerich and Vale 61

Figure 2 parked against the MT-bound head toward the MT plus- end and this interaction creates a ‘polymer gate’ by preventing the tethered head from binding to tubulin. This gate is opened when ATP binds to the MT-bound head (the ‘gatekeeper’ is ATP binding), which relieves the interaction between the two motor domains, thus allowing the tethered head to rebind to the MT and take a step. However, a new study suggests that the ‘stepping’ head is not parked in front of the MT-bound head [37]. Models of Nevertheless, one cannot rule out head–head interaction 62 Cell structure and dynamics contributing to gating, perhaps as a back-up mechanism. Direct interactions between the motor domains also is plausible for dynein, where the two AAA rings are likely Figure 3 to be very closetwo-head-bound to one another waiting state. in A the recent MT-bound single-molecule state Procession FRET study also supports a two-head-bound waiting (Figure 2b). state when kinesin moves processively at physiological ATP concentrations (at which the trailing head’s tran- Motor domainssition also into the might weakly influence MT binding each ADP state other is limited without by ATP hydrolysis and phosphate release) [37], which is being in directin agreement contact. with previous Such‘action-at-a-distance’ results [38]. However, the Intramolecular Tension models requirestudy simultaneous of Mori et al. also suggested MT-binding that there of is a both one-head- motor • bound waiting state at very low ATP concentrations, domains (Figurewhen the 3b– reard). head One can way enter in a which low MT this affinity could ADP be accomplishedstate is before through ATP binds intramolecular to the nucleotide-free tension, front head. which could developUnlike when the Alonso the motor’set al. model solution [36], their structure data indicates dis- Front head pulls back head off. that the ‘detached’ head is positioned behind the MT- • torts to allowbound the heads head. Even to separatethough ‘detached’, and to the simultaneously rear head might bind to the MT.weakly The interact resulting with the rearwardMT and might tension make briefon the transitions into the two-head-bound state [37]. MT front head andinteraction the forward of the rear tension head is on in the agreement rear head with bio- could Nucleotide Gating affect the nucleotidechemical data or from MT Hackney affinity [40 of]. bothThus, inheads summary, asym- • metrically (Figurethere is 3 evidencebandc),creatinggatingmechanisms for a two-head-bound waiting state when kinesin walks at physiological ATP concentrations. that will beHowever, discussed the in nature more of the detail waiting below. state at Two low ATP MT- bound motorrequires domains clarification, also particularly could communicate with regard to the on pos- the Average step is 8 nm ition and MT interactions of the rear head. • basis of the conformations of the mechanical elements that connectTension both sensing heads also rather has received than thesupport magnitude from exper- of tension appliediments to with these mutant elements kinesins that(Figure had flexible 3d) [37 spacers,38]. introduced between the neck linkers and the coiled-coil, Finally, thewhich MT isitself predicted might to reduce serve tension as a between communication the two pathway, sincemotors motor domains. binding In a study toby Hackney the MTet al.[ can41], it induce was structural changesfound that in the the chemical MT kinetic lattice processivity [39](notcovered (number of calculated ATP molecules hydrolyzed per MT encoun- further in thister) of review). such kinesin The mutants next was section greatly reduced. focuses In a on Potential head–head coordination mechanisms. (a) In this tension- more recent single-molecule study, Yildiz et al.[18] independent, ‘polymer gate’ pathway, the tethered headrecent is detached studies on kinesin and dynein, which provide from the MT and parked against the MT-bound head toward the MT showed that kinesins with neck-linker extensions display plus-end. This interaction prevents the tethered head fromsupport binding to for thesenormal processivity ‘action-at but-a-distance’ have slow velocities communication owing to an tubulin. The gate is opened when ATP binds to the MT-boundmechanisms. head (the impaired coupling of ATP hydrolysis forward stepping. ‘gatekeeper’ is ATP binding). (b) Internal tension might affect the MT- This coupling defect could be reversed by recovery of affinity of the head domains (‘polymer gating’). In this pathway, forward tension on the rear head is suggested to weaken its MT-affinity and tension by chemical crosslinking of the neck linkers or by promote its detachment. Acceleration of rear head detachmentCoordinating might be applying kinesin external tension movement with an optical trap. These enhanced by the initiation of neck-linker docking in theDoes front head intramolecular (the experiments tension strongly facilitate suggest that coordination tension plays an of ATP-driven conformation change). However, to be consistent with important role in the coordination of kinesin’s head models in which the front head cannot bind ATP until thekinesin’s rear head twodomains motor and domains? that the dimensions and flexibility of the detaches [50], the ADP-bound rear head must be sufficiently mobile to relieve the block on the front head. (c) Internal tension (theMost ‘gatekeeper’), modelsneck for linkers tension are sensing key to tension in head sensing.–head Where communi- does the Models for kinesin and dynein stepping.derived(a) fromConsensus the simultaneous stepping binding of both motor domainscation to the require MT, tension that come both from kinesin and what heads is its are magnitude? bound Thesimul- sequence of kinesin (for a detailed description,controls the binding/release see text). ofA nucleotides nucleotide- (‘nucleotide gating’). tension may derive simply from the MT-binding by Backward tension on the front head transmitted via thetaneously neck-linker tothe adjacent front head, tubulin since this binding alone would sites. stretch In out this both two- driven conformational change in the tightlyelements MT-interacting is suggested to prevent front ATP head binding, while the forward tension neck linkers (Figure 3b). The magnitude of this tension displaces the weakly MT-interacting rearon the head rear head toward is proposed the toMT inhibit plus-end, ADP release, thushead-bound helping to keep waiting state, the kinesin neck linker in the both heads out-of-phase. (d) Neck-linker conformation-based can be estimated from ‘worm-like chain’ models that treat biasing its diffusional search and rebindingmechanism. to Inthe this next pathway, available the neck-linker MT- elementsfront might act head as mustthe neck-linker extend backwardpeptide as an andentropic the spring, neck but linker calcu- in binding site in front of its partner head.‘control While levers’ the to rear regulate head the undergoes binding/release a of nucleotidesthe rear headlations must for extend the forces forward required (Figure to stretch 3b). out Does the neck such allosterically. The backward-pointing/backward-docked orientation in 16 nm displacement, kinesin’s center-of-mass advances 8 nm. (b) linkers differ considerably (between 4 pN [18] and 12– the front head might prevent ATP binding and the forward-pointing/a state exist? The alternating 16 and 0 nm steps observed Possible dynein stepping sequence. Dynein’sforward-docked head orientation domains in the aretrailing MT- head might inhibit ADP release. 15 pN [42]). The tension also might be enhanced by the bound with partially overlapping AAAAlthough rings, tensionaligned might parallel assist in to creating the long and/or maintainingby single-molecule these fluorescence microscopy at rate-limit- MT axis. A nucleotide-dependent conformationalconformations, the change neck-linker of conformation-based the linker mechanisming ATP concentration(b) and (c) of this figure, [20 in] which are the most neck-linker consistent elements serve as with a discussed here envisions the neck linkers as active regulatory elements, passive intermediaries that relay tension to the nucleotide and polymer element in the tightly MT-binding frontin contrast head displaces to the tension-based the weakly gating mechanisms MT- described in parts binding sites. interacting partner head toward the MT minus-end (opposite to the mass movement of 8 nm. Although dynein takes predominantly 8 nm direction of kinesin movement). The displaced head then undergoes a steps, it has a considerable diffusional component to its step, resulting in rapid diffusional search and rebinds toCurrent the MT,Opinion resulting in Cell Biology in a center-of-2009, 21:59–67 different sized center-of-mass steps (4–24 nm). www.sciencedirect.com

www.sciencedirect.com Current Opinion in Cell Biology 2009, 21:59–67 Cell Science at a Glance 4369

The dynein family at a functions. Although at least 14 classes of accessory subunits bind; and a ~380 kDa kinesin and 17 classes of myosin have motor domain. The motor domain glance been identified, the fall into contains six discernible AAA ATPase Peter Höök* and Richard B. only two major classes, axonemal and units, identifying the dynein HC as a Vallee cytoplasmic dyneins, based on both divergent member of the AAA+ family functional and structural criteria. of Cell ATPases Science (Neuwald at a Glance et al., 1999).4369 Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA. Axonemal dyneins are responsible for Members of the AAA+ family are *Author for correspondence (e-mail: functions.ciliary and Althoughflagellar beating; at least cytoplasmic 14 classes of involvedaccessory in subunits a very bind; wide and range a ~380 of kDa [email protected]) dynein family at a dyneins are involved in intracellular functions but have a common feature: the kinesin and 17 classes of myosin have motor domain. The motor domain transport, , cell polarization and formation of ring-shaped oligomeric glanceJournal of Cell Science 119, 4369-4371 been identified, the dyneins fall into contains six discernible AAA ATPase Published by The Company of Biologists 2006 directed cell movement complexes of the AAA ATPase module. Peterdoi:10.1242/jcs.03176 Höök* and Richard B. only two major classes, axonemal and Withinunits, the identifying AAA+ the proteins, dynein dynein HC as a Vallee cytoplasmicAll dynein dyneins, forms based that on have both occupiesdivergent a divergent member branchof the alongAAA+ with family functional and structural criteria. of ATPases (Neuwald et al., 1999). DepartmentThree familiesof Pathology of and cytoskeletal Cell Biology, motor been identified biochemically are midasin (Iyer et al., 2004). This branch Columbiaprotein University, – the New myosins, York, NY 10032, kinesins USA. and Axonemalmultisubunit dyneins proteins. are Each responsible has one to for is Memberscharacterized of by the the AAA+incorporation family of are *Authordyneins for correspondence – have evolved (e-mail: to mediate ciliarythree heavy and flagellar chains (HCs) beating; of >500cytoplasmic kDa; allinvolved six AAA in modules a very within wide a single range of [email protected])transport of cells and of structures and dyneinsthese correspond are involved to the in number intracellular of giantfunctions polypeptide. but have The a commonAAA family feature: has the

Journalmaterials of Cell Science within 119, 4369-4371 cells in eukaryotes. transport,morphologically mitosis, identifiable cell polarization heads and and membersformation in prokaryotes of ring-shaped and it oligomeric seems PublishedWhereas by The myosin Company uses of Biologists actin 2006polymers to directedcontain cell the movement motor domains of the likely,complexes therefore, of the that AAA the ATPase dyneins module. had doi:10.1242/jcs.03176carry out its tasks, kinesin and dynein are molecule. The dynein HC forms two theirWithin origin the very AAA+ early proteins, in evolution. dynein microtubule-associated motors. Dyneins Allprominent dynein structures: forms a ~160 that kDa have N- Inoccupies dynein, a energydivergent from branch nucleotide along with Threeuse energy families from of cytoskeletal ATP hydrolysis motor to beenterminal identified domain that biochemically forms the base of are hydrolysismidasin at(Iyer the AAAet al., units 2004). is conveyed This branch proteinpower – a the wide myosins, variety kinesins of cellular and multisubunitthe molecule, proteins. to which Each most has of one the to to is the characterized base of the moleculeby the incorporation and to the of dyneins – have evolved to mediate three heavy chains (HCs) of >500 kDa; all six AAA modules within a single transport of cells and of structures and these correspond to the number of giant polypeptide. The AAA family has materials within cells in eukaryotes.The Dyneinmorphologically Family identifiable at heads a andGlancemembers in prokaryotes and it seems Whereas myosin uses actin polymers to contain the motor domains of the likely, therefore, that the dyneins had carry out its tasks, kinesin and dynein are Petermolecule. Höök The a nd dynein Rich HCard formsB. Va llee two their origin very early in evolution. microtubule-associated motors. Dyneins prominent structures: a ~160 kDa N- In dynein, energy from nucleotide use energy from ATP hydrolysis to terminal domain that forms the base of hydrolysis at the AAA units is conveyed + S. pombe power a wide variety of cellular the molecule, to which most of the to the baseD. discoideum of the molecule and to the C. elegans - D. melanogaster D. rerio Dynein H. sapiens R. norvegicus M. musculus U. maydis E. nidulans + The+ Dynein Family at a GlanceA. fumigatus Dynein 1 I A. oryzae - + - + Family N. crassa - - N. haematococca - - Peter Höök and Richard B. Vallee P. tetraurelia + - +

T. thermophila Cytoplasmic

+ C. albicans E. gossypii S. cerevisiae G. lamblia + D. melanogasteS. pomber L. mexicanD. discoideuma T. thermophilC. elegana s C. elegans Dynein 2 - D. melanogaster II T. gratilla D. rerio R. norvegicus Microtubule Axoneme H. sapiens M. musculus R. norvegicus Dynein 1 Inner-arm dynein C. reinhardtii Outer-arm dynein M. musculus III D. melanogaster Dynein 2 U. maydis Outer-arm dynein D. melanogaster H. sapiens DHC9E. nidulans + + Clamp loader A. crassispina A. fumigatus Dynein 1 I DnaA/Cdc6/ORC A. oryzae - T. gratilla + - + H. sapiens DHC11N. crassa Outer-arm dynein IV - - Classical M. musculus DHC11N. haematococca - - AAA D. melanogasterP. tetraurelia + - +

AAA+ D. hydei DHC7T. thermophila Cytoplasmic HsIU/ClpX/Lon/Clp l + C. reinhardtii C. albicans jcs.biologists.org Dynein T. thermophila E. gossypii Helix 2 C. reinhardtii S. cerevisiae

insert M. musculus DHC5 G. lamblia Axonema Midasin H. sapiens DHC5 D. melanogasteOuter-armr dynein V M. musculus DHC8L. mexicana H. sapiens DHC8T. thermophila D. melanogaster Dynein 2 C. elegans Inner-arm dynein II C. reinhardtii DHCT.1 gratilla VI Stem/tail Linker AAA1 AAA2 AAA3 AAA4Stalk AAA5 AAA6 CT D. melanogasteR.r norvegicus Microtubule Axoneme C. reinhardtii DHC10 M. musculus Inner-arm dynein Dynein 1 Inner-arm dynein H. sapiens DHC7 VII H. sapiens C.DHC reinhardtii3 Outer-arm dynein III ...... D. melanogaster Dynein 2 C. reinhardtii DHC9 LIC Outer-arm dynein D. melanogaster IC HC Conserved residues H. sapiens DHC9 Clamp loader A. crassispina DnaA/Cdc6/ORC T. gratilla H. sapiens DHC11 Outer-arm dynein IV Classical M. musculus DHC11 AAA D. melanogaster AAA+ D. hydei DHC7 l HsIU/ClpX/Lon/Clp C. reinhardtii© Journal of Cell Science 2006 (119, pp. 4369-4371) jcs.biologists.org Dynein T. thermophila Helix 2 C. reinhardtii

insert M. musculus DHC5 Axonema Midasin H. sapiens DHC5 Outer-arm dynein V M. musculus DHC8 H. sapiens DHC8 (See poster insert) D. melanogaster Inner-arm dynein C. reinhardtii DHC1 VI Stem/tail Linker AAA1 AAA2 AAA3 AAA4Stalk AAA5 AAA6 CT D. melanogaster C. reinhardtii DHC10 H. sapiens DHC7 Inner-arm dynein VII H. sapiens DHC3 ...... C. reinhardtii DHC9 LIC IC HC Conserved residues

© Journal of Cell Science 2006 (119, pp. 4369-4371)

(See poster insert) LIU 337

TABLE 1 Composition of the dynactin complex66,67,169

Subunit p150 or its isoform p135 p62 p50/Dynamitin p27 p25 p24 Arp1 Arp11 CapZα CapZββ-actin Number 2 (homodimer)66,169 14 1121 8 1 1 1

and Entamoeba histolytica,aparasiticamoeba.15 In most of eukaryotic interacting/tracking proteins) EB1 and CLIP-170.31–37 Recently, the cells, dynein is responsible for the long-distance transport of organelles sequential recruitment of dynactin and dynein to MT plus ends by EB1 and vesicular structures including mitochondria, endoplasmic reticulum and CLIP-170 has been demonstrated by in vitro reconstitution with puri- (ER), peroxisomes, endosomes, lysosomes, phagosomes, autophagosomes, fied recombinant proteins.38 lipid droplets, pigment granules and a wide variety of organelle-derived AsecondmechanismforlocalizationofdyneintoMTplusendsis transport vesicles.16–27 In addition to cellular cargoes, many viruses that transport by kinesin. Studies in the budding yeast, filamentous fungi, undergo endocytosis and packaging into early endosomes make use of worm and mouse dorsal root ganglia (DRG) neurons reveal that targeting the MT-based transport machinery to reach the nucleus for replication.28 of dynein to MT plus ends requires kinesin.39–43 in vitro reconstitution To achieve spatiotemporal accuracy for efficient transport of different car- using proteins purified from yeast has demonstrated that CLIP-170 and goes, the motor activity of dynein is highly regulated by accessory factors. Lis1 (also called NudF, a member of the nuclear distribution (Nud) family), Among them is dynactin, a ~1.2 MDa multisubunit protein complex aregulatorofdyneinmotility,coupledyneintokinesinforitstransport (Table 1) that plays multiple roles in dynein-based motility, including towards the plus end, with EB1 and CLIP-170 acting as processivity fac- dynein recruitment to the plus end of MTs, cargo recognition, transport tors for kinesin to overcome the intrinsic minus-end-directed motility of initiation and processivity. As dynactin complexes with dynein via interac- dynein.44 In the worm ,UNC-16,ascaffoldingpro- tion between its subunit p150 (also known as p150Glued)anddyneinIC,29 tein of the JNK-interacting protein (JIP) family, binds to both dynein LIC and is needed for dynein to exert its motor function in vivo, I will regard and kinesin-1, linking kinesin-1 to dynein to drive its transport towards dynein-dynactin as a whole and discuss the process (Figure 1) and regula- MT plus ends in nerve processes.43 Most recent studies also demonstrate tion of vesicular transport driven by this motor complex in this review. that kinesin-1 drives slow axonal transport of dynein towards the MT plus ends in axonal terminals in mammalian neurons.45 It is worth noting that the mechanisms of direct recruitment and kinesin-driven transport are not 3 | THE PROCESS OF mutually exclusive. They might be exploited to fit the needs for dynein DYNEIN-DYNACTIN-DRIVENDynactin VESICULAR Motormolecules at different subcellular locations, for example, dynein distribu- TRANSPORT ALONG THE MT TRACKS ted far from the axonal terminal requires kinesin for active transport towards the plus ends distal to the neuronal cell body, and dynein located 3.1 | Anchoring and transportComplex of dynein to MT in the cell periphery, which is rich in MT plus ends, is recruited by p150 and +TIPs from the cytoplasm. Movesplus ends along microtubules toward the (-) end • As the major minus end-directed motor, dynein often needs to be 1.4recruited MDa to complex MT plus ends to containing: initiate cargo transport. Imaging of fluores- • 3.2 | Cargo recognition and membrane association centlyDynein labeled dyneinmotor in the domain fission yeast showed homodimer that single dynein mole- • 30 cules2- intermediate in the cytoplasm bindtoanddiffusealongMTs. chains In fly and Prior to transport along the MT track, dynein needs to recognize its • mammalian cells, direct recruitment of dynein from the cytoplasm to the various vesicular cargoes. It remains to be determined whether the • plus2- endlight requires intermediate interaction with p150 chains and the +TIPs (MT plus-end- dynamic MTs search and capture membranous cargoes to dynein- • 3 classes of light chains • Forms a complex with dynactin

FIGURE 1 A model for dynein-dynactin-driven vesicular transport along MT tracks. A, Dynein-dynactin localization to the MT plus ends. B, Cargo loading: dynamic MTs search and capture vesicular cargo from the cytoplasm to dynein-dynactin preloaded at the plus ends, or motor- associated cargo is directly recruited to the MT track. C, Dynein-dynactin-driven movement of vesicular cargo along the MT track. D, Cargo unloading from the motor complex. For clarity, all regulatory factors for MT recruitment of dynein-dynactin, transport initiation and activation, processivity along the MT track and motor-cargo interaction are omitted from the illustration. Walking the walk: how kinesin and dynein coordinate their steps Gennerich and Vale 61

60 Cell structure and dynamics Figure 2 parked against the MT-bound head toward the MT plus- end and this interaction creates a ‘polymer gate’ by preventing the tethered head from binding to tubulin. This gate is opened when ATP binds to the MT-bound Figure 1 Overview of the dynein mechanism head (the ‘gatekeeper’ is ATP binding), which relieves Unlike kinesin, relatively little is known about the mol- the interaction between the two motor domains, thus Dynein ecular mechanism of dyneinallowing (Figure the 1b). tethered Dynein, head which to rebind to the MT and take is a member of the AAA+ familya step. (AAA: However, ATPase a new associated study suggests that the ‘stepping’ with various cellular activities),head is is not involved parked in in front diverse of the MT-bound head [37]. processes in eukaryotic cells,Nevertheless, such as spindle one cannot formation, rule out head–head interaction segregation, andcontributing the trafficking to gating, of organelles perhaps as a back-up mechanism. Structure and mRNA [2]. Dynein isDirect a large interactions protein between complex the motor domains also is (1.2 MDa) composed of twoplausible identical for heavy dynein, chains where and the two AAA rings are likely several associated chains [1].to The be heavy very close chain to contains one another six in the MT-bound state AAA domains arranged in(Figure a ringStep 2 [b).27]. lengths The first four can vary domains (AAA1–AAA4) have• conserved ATP binding/ hydrolysis motifs. AAA1 is essentialMotorgreatly domains for dynein also (4-32 might motility, influence nm) each other without while the other sites may playbeing regulatory in direct roles contact. [28–30]. In Such‘action-at-a-distance’ contrast to kinesin and myosin,models whose require MT simultaneous interfaces are MT-binding of both motor located on the surface of theirdomains ATPase (Figure cores, 3b–d). dynein’s One way in which this could be MT-binding domain (MTBD)accomplished isWhen located isat through theloaded, end intramolecular of a average tension, which 15 nm long coiled-coil stalk•could [27,31 develop] that when emerges the motor’s be- solution structure dis- torts to allow the heads to separate and to simultaneously tween AAA4 and AAA5. step is ≈8 nm bind to the MT. The resulting rearward tension on the front head and the forward tension on the rear head could Single-molecule studies suggestaffect that the nucleotide dynein walks or MT in affinity a of both heads asym- hand-over-hand-like fashionmetrically (FigureCompact (Figure 2b), although 3bandc),creatinggatingmechanisms itsfor the stepping behavior and directionalitythat• will be are discussed more irregular in more detail below. Two MT- than kinesin’s [6 –8,9 ]. Evidencebound has motorsize shown domains of that dyneinthe also could stalk communicate and on the advances via coordination ofbasis its two of motor the conformations domains [8,32 of], the mechanical elements but the details of cytoplasmicthat dynein’sconnectring both step heads size, ratherstall than the magnitude of force, and directionality remaintension controversial applied to [6 these–8,9  elements,33]. (Figure 3d) [37,38]. Observations of variable stepFinally, sizes (4 the–32 MT nm) itself and might direc- serve as a communication tionality suggest a considerablepathway, diffusional since component motor binding to to the MT can induce its step, making dynein steppingstructural more changes akin in to the that MT of lattice [39](notcovered myosin VI (21–51 nm) [34] thanfurther kinesin. in this A review).10 nm The long next section focuses on ‘linker’ that connects the AAArecent ring studies to the on dimerization kinesin and dynein, which provide domain has been suggestedsupport to power for these dynein ‘action-at motion-a-distance’ communication (Figure 1b) [31]. The linkermechanisms. shifts its position relative to the catalytic ring during the ATPase cycle [31,35] and Schematic representations of kinesin and dynein and associated chains. Coordinating kinesin movement (a) Kinesin structure. Kinesin is composed of two identical heavy chains is believed to facilitate dynein motility (Figure 2b, steps 1 and two light chains. The heavy chain contains an N-terminal globular and 2) [8,32]. However, withDoes no intramolecular atomic structure tension for facilitate coordination of motor domain that possesses catalytic and MT-binding activity, a neck- dynein in hand, a structuralkinesin’s model for two dynein’s motor domains? motility linker element that connects the motor domain to the common coiled- – is less advanced than that forMost kinesin. models for tension sensing in head head communi- coil dimerization domain, and a C-terminalModels light for chain kinesin and and cargo-binding dynein stepping. (a) Consensus stepping cation require that both kinesin heads are bound simul- region. (b) Cartoon representation of dynein.sequence Dynein of kinesin is composed (for a detailed of two description, see text). A nucleotide- taneously to adjacent tubulin binding sites. In this two- identical heavy chains and several associateddriven conformational chains. The heavy change chain in the tightlyPotential MT-interacting pathways front head for head–head head-bound waiting state, the kinesin neck linker in the forms a C-terminal catalytic motor domaindisplaces that consists the weakly of multiple MT-interacting AAA rearcommunication head toward the MT plus-end, biasing its diffusional search and rebinding to the next available MT- front head must extend backward and the neck linker in ATP-binding sites (1–4) and a coiled-coil stalk with the microtubule- A gating mechanism requires that one motor domain can binding domain (MTBD) at its tip; AAA domainsbinding site 5 and in front 6 do of notits partner contain head. While the rear head undergoes a the rear head must extend forward (Figure 3b). Does such 16 nm displacement, kinesin’s center-of-mass advances 8 nm. (b) sequences associated with nucleotide binding and the C-terminus (C) influence the action of itsa partner. state exist? Before The alternating we discuss 16 and 0 nm steps observed Possible dynein stepping sequence. Dynein’s head domains are MT- does not contain sequences characteristic for AAA proteins. The motor detailed studies for kinesin and dynein, we consider some bound with partially overlapping AAA rings, aligned parallel to the long by single-molecule fluorescence microscopy at rate-limit- and dimerization domains are joined by a linker element. Multiple MT axis. A nucleotide-dependent conformationalgeneral change models of forthe linker how thising might ATP occur. concentration Possible models [20] are most consistent with a associated chains bind to dynein’s tailelement domain in(LIC, the light tightly intermediate MT-binding front headfor gating displaces are the discussed weakly MT- separately in this review. How- chain; IC, intermediate chain; Roadblock,interacting Tctex1 and partner LC8, head light toward chains). the MTever, minus-end many (opposite of these to the mechanismsmass movement are not of mutually 8 nm. Although exclu- dynein takes predominantly 8 nm direction of kinesin movement). The displacedsive and head it then is undergoes likely that a motorssteps, it employ has a considerable more than diffusional one component to its step, resulting in rapid diffusional search and rebinds to the MT, resulting in a center-of- different sized center-of-mass steps (4–24 nm). gating strategy. this docked conformation of the neck linker [22]. After a rapid diffusional search, the detached head rebinds to the One possibility is that the two motor domains are inter- MT in front of its partner. Evidencewww.sciencedirect.com is accumulating for a acting with one another directly during an intermediateCurrent Opinion in in Cell Biology 2009, 21:59–67 neck-linker conformational change [23–26 ], but how neck- the ATPase cycle. An example of such a model was linker docking is affected by load and the precise role of proposed by Alonso et al.[36 ](Figure 3a). In their neck-linker conformational changes in head–head coordi- model, kinesin waits in-between steps as a one-head- nation are still open questions. bound intermediate; the detached tethered head is

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