TY - JOUR
AU1 - Hirokawa,, Nobutaka
AB - Abstract Cells transport and sort various proteins and lipids following synthesis as distinct types of membranous organelles and protein complexes to the correct destination at appropriate velocities. This intracellular transport is fundamental for cell morphogenesis, survival and functioning not only in highly polarized neurons but also in all types of cells in general. By developing quick-freeze electron microscopy (EM), new filamentous structures associated with cytoskeletons are uncovered. The characterization of chemical structures and functions of these new filamentous structures led us to discover kinesin superfamily molecular motors, KIFs. In this review, I discuss the identification of these new structures and characterization of their functions using molecular cell biology and molecular genetics. KIFs not only play significant roles by transporting various cargoes along microtubule rails, but also play unexpected fundamental roles on various important physiological processes such as learning and memory, brain wiring, development of central nervous system and peripheral nervous system, activity-dependent neuronal survival, development of early embryo, left–right determination of our body and tumourigenesis. Furthermore, by combining single-molecule biophysics with structural biology such as cryo-electrom microscopy and X-ray crystallography, atomic structures of KIF1A motor protein of almost all states during ATP hydrolysis have been determined and a common mechanism of motility has been proposed. Thus, this type of studies could be a good example of really integrative multidisciplinary life science in the twenty-first century. quick-freeze electron microscopy, molecular cell biology, molecular genetics, structural biology, molecular motor, kinesin superfamily proteins 1. Introduction Cells transport and sort various proteins and lipids following synthesis as distinct types of membranous organelles and protein complexes to the correct destinations at appropriate velocities. The intracellular transports are fundamental for cell morphogenesis, functioning and survival not only in polarized cells such as neurons and epithelial cells but also in all other cells. Among the cells, neurons develop a highly polarized structure composed of dendrites and an axon along the direction of the impulse propagation. Most of the materials necessary for the axon and synaptic terminals must be transported down the axon after synthesis in the cell body. The proteins are conveyed in various types of membranous vesicles and protein complexes in the axon and dendrites. In dendrites, mRNAs such as CaMKIIa mRNA, Arc mRNA and Fmr1 mRNA are transported, and protein synthesis also occurs locally. Therefore, intracellular transport is fundamental for neuronal morphogenesis, function and survival. Here, in this review, I mainly follow a history of our research from the development of the quick-freeze electron microscopy (EM) to our current studies of kinesin superfamily proteins. More comprehensive reviews are found in [1–10]. 2. From rapid-freeze EM to identification of kinesin superfamily proteins, KIFs 2.1 From rapid-freeze to quick-freezedeep-etch EM There have been various efforts to preserve biological structures as naturally as they exist in situ. Although the usual chemical fixation is useful and convenient, it also has some shortcomings. For instance, it distorts the distribution of water [11,12] and cannot fix water soluble components in the tissues. Further, it is uncertain how long it takes to fix the biological samples by the chemical fixation so that very rapid phenomena of millisecond order cannot be captured by the conventional fixation. To overcome these shortcomings, physical fixations by freezing the tissues have been tried. Rapid freezing is also capable of fixing materials fast enough to capture some dynamic morphological changes. If the tissues are frozen at a rate of temperature loss of about >104 K/s, only a part of the available cell water is transformed into crystals which remain below 100 Å in size, vitrification results and the cells suffer little structural damage from ice crystals [13]. Many attempts have been made to obtain this critical-freezing rate [14–16]. Fernandez-Moran [17], for example, used liquid helium to bring about these high rates of freezing. In 1970s, I have studied the mechanism of synaptogenesis in the nervous system using EM and neurotoxins such as β-bungarotoxin and botulinum toxin as tools [18,19]. To capture the natural ultrastructure and rapidly occurring phenomena, we have developed rapid-freezing machines operated by liquid nitrogen and liquid helium in University of Tokyo. After trial and errors for several years, we have successfully developed rapid freezing and freeze substitution method and obtained new images of the ultrastructure of the central nervous system (CNS) [20–22]. In 1979, Prof J. Nakai, my supervisor retired at the University of Tokyo. Then as a tradition at that time I decided to go abroad to extend my research further. There were only limited numbers of laboratories that developed the rapid-freezing method. Actually, our group and Heuser and Reese's group were the only two groups in the world in 1978 who developed this method. Then it was natural for me to join John Heuser's laboratory. John Heuser just became independent from Tom Reese and started his laboratory in UCSF. I visited Heuser and Reese at the Marine Biology Institute in Woods whole in the summer of 1978 and showed the electron micrographs of the rapid freeze freeze-substituted CNS [20–22]. They were surprised to find that there is another group developing the rapid-freezing method. Heuser immediately invited me to his laboratory. I joined Heuser's laboratory in April 1979 by the support of NIH Fogarty Research Fellowship and subsequently the George Meany MDA postdoctoral fellowship. At that time, he has just finished a mile stone work proving exocytosis at the frog neuromuscular junction by the quick-freezing method using liquid helium [23]. I started my work in Heuser's laboratory by freeze substitution of the frog neuromuscular junction intoxicated with botulinum toxin [24]. Heuser has developed the quick-freeze deep-etch EM to reveal true surface of ACh receptors of Torpedo electric organ [25]. However, at the same time, we were disappointed by the fact that the structure of cytoplasm was not revealed well because of a lot of soluble proteins filling the cytoplasm. I was interested in visualizing the true outside and inside structures of specialized membranes such as neuromuscular junction and gap junction. I also tried to find a way to clearly expose the structures in the cytoplasm such as the cytoskeleton. Similar to former projects, I succeeded in uncovering the true outside of postsynaptic membrane structure of the neuromuscular junction filled with ACh receptors [26] and the true outside and inside structure of the gap junction [27]. Further, I succeeded in revealing structures inside the cells clearly by chemical permeabilization or physical permeabilization of the plasma membranes. These new procedures combined with quick-freeze deep-etch EM uncovered new structures associated with the true inside of the plasma membrane and cytoskeletons in various types of cells at a very high resolution that allows visualization of macromolecules. Using this approach, new structures associated with actin filaments in brush border of the intestinal epithelium (Fig. 1) [28–31], hair cells in the inner ear [32] and the axonal cytoskeleton in the peripheral nerve (Figs 2 and 3) [33] are visualized. Furthermore, application of the antibody decoration allowed us to identify chemical nature of the newly identified filamentous structures, such as fodrin and myosin in the brush border [29,30], neurofilament H, M and L proteins in the axon [34]. These studies led to focus on the cytoskeleton of the axon and dendrites in neurons and later discovery of the kinesin superfamily proteins. Fig. 1. Open in new tabDownload slide The terminal web of intestinal brush border observed by the quick-freeze deep-etch EM after physical breaking of the plasma membrane. Note the new fine filamentous structures associating rootelet actin filament bundles extending from microvilli. Myosin and fodrin were identified as components of these structures. ×97 000. Reproduced from Hirokawa et al. [29] with permission. Fig. 1. Open in new tabDownload slide The terminal web of intestinal brush border observed by the quick-freeze deep-etch EM after physical breaking of the plasma membrane. Note the new fine filamentous structures associating rootelet actin filament bundles extending from microvilli. Myosin and fodrin were identified as components of these structures. ×97 000. Reproduced from Hirokawa et al. [29] with permission. Fig. 2. Open in new tabDownload slide The cytoskeletal structure in the axon observed by the quick-freeze deep-etch EM after chemical permeabilization with saponin. Frequent short crossbridges are found between microtubules which tend to form bundles close with each other. Bar = 100 nm. Reproduced from Hirokawa et al. [39] with permission. Fig. 2. Open in new tabDownload slide The cytoskeletal structure in the axon observed by the quick-freeze deep-etch EM after chemical permeabilization with saponin. Frequent short crossbridges are found between microtubules which tend to form bundles close with each other. Bar = 100 nm. Reproduced from Hirokawa et al. [39] with permission. Fig. 3. Open in new tabDownload slide Quick-freeze deep-etch electron micrograph of an axon. A membranous organelle conveyed by fast transport is linked with a microtubule by a short crossbridge, which could be a motor molecule. Bar = 50 nm. Reproduced from Hirokawa [3] with permission. Fig. 3. Open in new tabDownload slide Quick-freeze deep-etch electron micrograph of an axon. A membranous organelle conveyed by fast transport is linked with a microtubule by a short crossbridge, which could be a motor molecule. Bar = 50 nm. Reproduced from Hirokawa [3] with permission. 2.2 Identification of the new filamentous structures associated with the cytoskeletonin the axon and dendrites in neurons In the axons and dendrites, microtubules and neurofilaments are the major longitudinal cytoskeletal filaments. The quick-freeze deep-etch EM uncovered three major classes of filamentaous structures associated with the neuronal cytoskeleton: (i) filamentous structures of different length associated with microtubules (Fig. 2), (ii) short sidearms between the neurofilaments and (iii) short crossbridges between membranous organelles and microtubules (Fig. 3). Axons contain many longitudinally oriented neurofilaments connected by numerous fine sidearms (20–50 nm in length) [33]. In this neurofilament domain, microtubules appear in groups and tend to form small bundles. In these bundles, microtubules are connected close to each other by short, ∼20 nm, sidearms (Fig. 2) [33,35,36]. Longer, ∼100 nm, filamentous structures are also observed. In contrast, microtubules are a prominent cytoskeletal element, whereas only a few neurofilaments are found in the dendrites (Fig. 4, lower panel) [37,38]. The distance between adjacent microtubules tends to be far apart compared with that in the axon and longer, ∼65–100 nm, filamentous structures are associated with microtubules (Fig. 4, lower panel). In both axons and dendrites, short, ∼20 nm, crossbridges are identified between membranous organelles and microtubules (Fig. 3). When I found these crossbridges between membranous organelles and microtubules, I immediately supposed that this structure could be composed of motor proteins transporting membranous organelles along microtubules in the axon, although at that time the chemical nature was not identified [33,35,36]. Fig. 4. Open in new tabDownload slide Upper panel: microtubules polymerized with Tau and observed by the quick-freeze deep-etch EM. Tau forms short crossbridges between microtubules which resemble the short filamentous structures associated with microtubules in the axon (see Fig. 2). Middle panel: microtubules polymerized with MAP2 and observed by the quick-freeze deep-etch EM. MAP2 forms long crossbridges between microtubules which resemble the long filamentous structures associated with microtubules in the dendrites (see lower panel). Lower panel: the cytoskeletal structures in the dendrite observed by the quick-freeze deep-etch EM. Microtubles are apart with each other, and longer filamentous structures are associating with them which look like microtubules polymerized with MAP2 in vitro (see middle panel). Bar = 100 nm. Fig. 4. Open in new tabDownload slide Upper panel: microtubules polymerized with Tau and observed by the quick-freeze deep-etch EM. Tau forms short crossbridges between microtubules which resemble the short filamentous structures associated with microtubules in the axon (see Fig. 2). Middle panel: microtubules polymerized with MAP2 and observed by the quick-freeze deep-etch EM. MAP2 forms long crossbridges between microtubules which resemble the long filamentous structures associated with microtubules in the dendrites (see lower panel). Lower panel: the cytoskeletal structures in the dendrite observed by the quick-freeze deep-etch EM. Microtubles are apart with each other, and longer filamentous structures are associating with them which look like microtubules polymerized with MAP2 in vitro (see middle panel). Bar = 100 nm. 2.3 Identification of microtubule-associated proteins (MAPs) as components associated with microtubules in the axons and dendrites In 1983, I came back to the Department of Cell Biology and Anatomy, Faculty of Medicine, University of Tokyo. I determined to focus my research on the neuronal cytoskeleton and wanted to characterize the chemical nature of the new filamentous structures associated with microtubules and neurofilaments. MAPs identified biochemically in the brain were good candidates. Major MAPs consisted of MAP1A, MAP1B, MAP2 and Tau. First, single molecular structure of these MAPs was revealed by low-angle rotary shadowing EM to be filamentous structures of different length dependent on the molecular weight. MAP1A, MAP1B, MAP2 and Tau were ∼185, ∼185, ∼100 and ∼50 nm in length, respectively [37–40]. If microtubules are polymerized in the presence of Tau and quick-frozen deep-etched, short ∼20 nm sidearms were observed between microtubule bundles resembling very much with the microtubules domain in the axon (Fig. 4, upper panel) [39]. On the other hand, microtubules polymerized with MAP2-formed bundles, which look like those in the dendrites. MAP2 formed 65- to 100 nm-long sidearms between the microtubules (Fig. 4, middle and lower panel). MAP1A and MAP1B also formed a longer ∼100 nm filamentous structure associating with microtubules [37,40]. These data together with electron microscopic immunocytochemistry using antibodies against these individual MAPs clearly revealed that, in the axon, Tau, MAP1A and MAP1B form microtubule-associated sidarms, while MAP1A, MAP1B and, specifically, MAP2 consist of filamentous structures associating with microtubules in dendrites [37,38,40,41]. As the next step, I tried to analyze the function of these MAPs in terms of neuronal morphogenesis. For this purpose, we used newly emerged molecular biology. First, we cloned Tau cDNA and transfected it into fibroblasts [42]. Interestingly, after transfection of Tau, cDNA microtubule organization in fibroblasts was remarkably changed. Microtubule bundles elongated in the thin processes which were extending from the cell bodies of fibroblasts. EM revealed that microtubules formed bundles resembling those in the axons, having ∼20 nm in distance. Next, as an extension of this direction, Tau and MAP2 cDNAs were transfected in the SF9 cells that are spherical in shape. Long neurite-like-processes were emerged from round SF9 cells in both cases, whereas Tau induced microtubule bundles close with each other and ∼20 nm sidearms were observed between adjacent microtubules. In contrast, MAP2 cDNA also extended microtubule bundles in the elongated processes, whereas the microtubules were ∼65 to 100 nm apart with each other and longer filamentous structures were formed between adjacent microtubules resembling those in dendrites [43]. This suggests that Tau and MAP2 elongate microtubules and form microtubule domains characteristic in the axons versus dendrites, respectively, thus play an important role for neuronal polarized morphogenesis. However, till this step, all data came from in vitro studies. We really wanted to know the functions of these sidearm structures composed of MAPs in vivo. To do this, molecular genetics, knockout mouse approach, was introduced. Tau knockout mice were generated. Surprisingly, the phenotype of Tau single knockout mice was subtle while microtubule stability in the axon was affected [44]. MAP2 single knockout affected dendrite elongation; the phenotype was also affected little [45]. The phenotype of MAP1B single knockout mice was also subtle [46]. Then because we assumed that the functions of MAPs are redundant we generated double knockout mice with tau−/−, MAP1B−/−. The axonal elongation was severely affected in vivo and also in vitro [47]. This was also true in MAP2−/−, MAP1B−/− double knockout mice. In this case, the dendrite elongation was remarkably suppressed [48]. From these results, it is concluded that Tau/MAP1B and MAP2/MAP1B synergistically play fundamental roles for axonal and dendritic morphogenesis, respectively, by elongating microtubules and forming microtubule domains with filamentous structures characteristic in the axons and dendrites. Parallel to the studies of microtubule-associated filamentous structures, we figured out that the C-terminal domains of neurofilament protein-H (NF-H) and neurofilament protein-M(NF-M) mainly form the sidearms between neurofilament bundles by the quick freeze deep etch EM combined with antibody decoration and molecular biology [34,49,50]. 2.4 Studies of dynamics of neuronal cytoskeleton Immunofluorescence microscopy and EM reveal cytoskeletal structures at the static state. In living cells, the cytoskeletal structures should be dynamic. The tubulin in microtubules, actin in actin filaments and even neurofilament triplet proteins, such as NF-L, NF-M and NF-H, could be dynamically turned over and cytoskeletal filaments could repeat polymerization and depolymerization. Therefore, it is important to analyse the dynamics of microtubules, actin and neurofilaments, and their associated proteins to know the functions of the cytoskeleton in terms of neuronal morphogenesis. We approached these questions by microinjection of fluorescence-labelled tubulin, actin and neurofilament proteins and subsequent laser photobleaching, microinjection of caged fluorescence-labelled cytoskeletal proteins and subsequent laser photoactivation, and microinjection of biotin-labelled cytoskeletal proteins and subsequent antibiotin immunocytochemistry at light microscopy and EM levels [51–60]. These studies, for the first time, uncovered the dynamics of microtubules, actin filaments and neurofilaments in living neurons. These lines of studies led us later to the analysis of slow axonal transport of cytoskeletal proteins [61]. 2.5 From the identification of short crossbridges between membranous organelles and microtubules to the discovery of kinesin superfamily proteins, KIFs When I found the third-class crossbridges between membranous organelles and microtubules, I immediately thought that this might be composed of motor proteins transporting membranous organelles along microtubule rails [33,35,36]. Then, I wanted to identify the chemical nature of these structures. For this purpose, we took two approaches, one was biochemistry and another was molecular biology. We searched proteins that change their binding ability with microtubules in the presence and the absence of ATP using biochemistry. This approach ended up the identification of GTPase dynamin [62]. For the molecular biological approach, we tried to identify cDNAs that contain consensus sequences of both Walker-type nucleotidase and microtubule binding. This approach successfully identified the first 10 members of KIFs from mouse brain cDNA library (Fig. 5) [63]. At this time, our new study on molecular cell biology, molecular genetics, biophysics and structural biology of KIFs have been started. The foundation of this new direction of our research was the discovery of the short crossbridge structures between membranous organelle and microtubules by the EM (Fig. 3) [33]. Fig. 5. Open in new tabDownload slide The structure and phylogeny of major mouse KIFs. (a) A phylogenetic tree of all 45 KIF genes in the mouse genome, which are classified into 15 families. (b) The domain structure of the major KIFs. In general, kinesins comprise a kinesin motor domain and a coiled-coil domain. Fig. 5. Open in new tabDownload slide The structure and phylogeny of major mouse KIFs. (a) A phylogenetic tree of all 45 KIF genes in the mouse genome, which are classified into 15 families. (b) The domain structure of the major KIFs. In general, kinesins comprise a kinesin motor domain and a coiled-coil domain. Based on the position of the motor domain within the molecule, KIFs can be classified into three major groups: N-terminal motor domain KIFs (N-KIFs), middle motor domain KIFs (M-KIFs) and C-terminal motor domain KIFs (C-KIFs). In mammals such as humans and mouse, the total number of Kif genes is 45, including three M-Kifs (Kif2a, Kif2b, Kif2c) and three C-KIFs (Kifc1, Kifc2, Kifc3). Kif genes have been classified into 14 classes (Figs 5 and 6) [63–66]. Fig. 6. Open in new tabDownload slide Principal members of KIFs observed by low-angle rotary shadowing. Diagrams, constructed on the basis of EM or predicted from the analysis of their primary structures, are shown on the right (the large red ovals in each diagram indicate motor domains). Reproduced with permission from Hirokawa [3]. Scale bar, 100 nm. Fig. 6. Open in new tabDownload slide Principal members of KIFs observed by low-angle rotary shadowing. Diagrams, constructed on the basis of EM or predicted from the analysis of their primary structures, are shown on the right (the large red ovals in each diagram indicate motor domains). Reproduced with permission from Hirokawa [3]. Scale bar, 100 nm. N-terminal KIFs generally move towards microtubule plus ends, whereas C-terminal KIFs move towards minus ends. Both KIF2A and 2C are unique KIFs that depolymerize microtubules in an ATP-dependent manner. Both N-KIFs and C-KIFs are composed of a motor domain, a stalk domain and a tail region. The overall homology of the amino acid sequence among the motor domains is 30–60%, while the other parts exhibit a significant divergence. The motor domain binds to microtubules and moves on them by hydrolyzing ATP, whereas, in general, the tail regions, and less frequently the stalk regions, recognize and bind to the cargo(s) (Figs 5 and 6) [3,4,6,7,10]. Various cargos including membranous vesicles, protein complexes and mRNAs with large protein complexes are transported in the axons and dendrites. Now, I introduce recent progress regarding the following questions: (i) What kind of cargo(s) does each motor transport? (ii) How does the motor recognize and bind its cargo(s)? (iii) How does the motor regulate loading and unloading of the cargo(s)? (iv) How is bidirectional transport regulated? (v) How is the fast transport of membranous organelles and the slow transport of cytoplasmic proteins regulated? (vi) How is the direction of transport in axons versus dendrites determined? (vii) What is the biological significance of the motor function at the whole-body level? (viii) To what diseases does disturbance in motor function relate, and how is the motor involved in the pathogenesis of those diseases? The primary mechanisms of intracellular transport in neurons have been elucidated for certain motors, such as their cargos, direction and velocity of transport and the mechanisms of cargo recognition and cargo loading/unloading. In addition, the mechanisms of directional transport in the axons versus dendrites are beginning to be unravelled. Furthermore, molecular genetic studies have revealed quite unexpected roles for motors in higher brain function, brain wiring, activity-dependent neuronal survival, suppression of tumourigenesis and central nervous system (CNS) and peripheral nervous system (PNS) development. In particular, I focus on the roles of KIF1A, KIF1Bα and KIF1Bβ, KIF2A, KIF3, KIF4, KIF5s, KIF17, KIF16B, KIF26A and KIFC2. 3. The mechanisms of intracellular transport: motors, cargos, recognition, binding and unloading of cargos 3.1 KIFs and axonal transport There are two types of transport in the axons: fast transport of membranous organelles and slow transport of cytosolic and cytoskeletal proteins. In terms of the fast transport, various cargo vesicles are conveyed by distinct KIFs, although the functions of each KIF are sometimes redundant. Cargos transported down the axon include synaptic vesicle precursors (KIF1A/KIF1Bβ), presynaptic membrane or active zone vesicles (KIF5), mitochondria (KIF1Bα/KIF5), APP-containing vesicles (KIF5), APOER2 vesicles (KIF5), TrkB vesicles (KIF5) and plasma membrane precursors (KIF3). KIF5 has also been identified as a slow transport motor (Fig. 7). Fig. 7. Open in new tabDownload slide Intracellular transport in neurons various KIFs transport membranous organelles anterogradely in axons and dendrites, whereas cytoplasmic dynein 1 and KIFC2 transport retrograde cargos. In neuronal cilia, anterograde transport is performed by KIF3 and KIF17, while retrograde transport is performed by cytoplasmic dynein 2. In short-range transport, such as transport in the pre- and postsynapses and growth cone filopodia, the myosin family proteins function as the molecular motors. Reproduced from Hirokawa et al. [10] with permission. Fig. 7. Open in new tabDownload slide Intracellular transport in neurons various KIFs transport membranous organelles anterogradely in axons and dendrites, whereas cytoplasmic dynein 1 and KIFC2 transport retrograde cargos. In neuronal cilia, anterograde transport is performed by KIF3 and KIF17, while retrograde transport is performed by cytoplasmic dynein 2. In short-range transport, such as transport in the pre- and postsynapses and growth cone filopodia, the myosin family proteins function as the molecular motors. Reproduced from Hirokawa et al. [10] with permission. 3.1.1 KIF1A and KIF1Bβ The kinesin-3 family members KIF1A and KIF1Bβ are similar molecular motors that transport components of synaptic vesicles, which are called synaptic vesicle precursors, and contain synaptic vesicle proteins such as synaptophysin, synaptotagmin and Rab3A [67,68]. Synaptic transmission is an important feature of neurons, which propagates nerve impulses between neurons and target cells. Functionally mature synaptic vesicles are generated by endocytosis at the synaptic plasma membrane. Prior to that, synaptic vesicle precursors must be transported from cell bodies to the synapse. The specificity for recognition of cargo, synaptic vesicle precursor is provided by adaptor proteins binding to the stalk domain. DENN/MADD has recently been identified as such an adaptor protein (Fig. 7) [69]. The death domain of DENN/MADD binds to the stalk region of KIF1A and KIF1Bβ, and the MADD domain interacts with the small molecular GTPase Rab3 on the cargo membrane. DENN/MADD binds to the GTP-bound form of Rab3, a synaptic vesicle protein, while it does not bind to the GDP-bound form of Rab3. Vesicles containing GTP–Rab3 can be transported down the axon, but those containing GDP–Rab3 cannot. Thus, conformational change in this small G-protein due to GTP hydrolysis could be a mechanism for cargo unloading. Liprin-α (SYD-2 in Caenorhabditis elegans) is also suggested to function as an adaptor [70]. Liprin-α regulates the motility of KIF1A [71]. KIF1A is able to move processively in the monomeric state [9,72–74]. However, the formation of clusters of monomers enhances its motility [74]. It has been suggested that binding of Liprin-α enhances the cluster formation of KIF1A and augments the motility [71]. 3.1.2 KIF1Bα KIF1Bα transports mitochondria [75]. Interestingly, this motor is derived from the same gene as KIF1Bβ by alternative splicing of mRNA, even though their tail domains are completely distinct from each other. Kinesin-binding protein (KBP) has been identified as a KIF1Bα-associated protein [76]. Both KIF1Bα and KBP are localized to mitochondria. However, KIF1Bα is able to bind to mitochondria in the absence of KBP. KBP augments the motility of KIF1Bα in vivo and in vitro by an unknown mechanism. Knockdown of KBP causes mitochondrial aggregation [76]. This may be due to the lowered activity of KIF1Bα in the absence of KBP. 3.1.3 KIF5 KIF5 (kinesin-1 family) forms a complex with kinesin light chains (KLCs) that bind to the tail domains of KIF5s [77–79]. Mammals have three Kif5 genes: Kif5a, Kif5b and Kif5c [65,80]. All three KIF5 isoforms are expressed in neurons, but their expression levels vary among different cell types [80]. KIF5A, KIF5B and KIF5C form homodimers and heterodimers. Thus, they are thought to have the similar function. By binding to distinct adaptor proteins, KIF5 transports many different cargos, including various vesicles, lysosome and mitochondria [81,82]. Among them, the axonal transport of APP has been studied intensively, because it may be involved in the progression of Alzheimer's disease [83–86]. Syntabulin has also been identified as an adaptor for vesicle transport mediated by KIF5 [87,88]. Syntaxin is an active zone protein essential for exocytosis of synaptic vesicles. Syntabulin serves as an adaptor for the KIF5 motor, whereas syntaxin acts as a receptor of presynaptic transport cargos. The syntaxin–syntabulin–KIF5 complex is the motor–adaptor transport machinery critical for assembling presynaptic boutons in developing hippocampal neurons [88]. It is known that neuronal activity enhances synaptic formation. Syntabulin is suggested to be essential for the activity-dependent formation of the active zone. KIF5 is responsible for the transport of retrograde motor proteins. Because cytoplasmic dynein transport cargos from the axon terminals to the cell bodies, all the components of the dynein–dynactin complex need to be first transported to axon terminals [89,90]. LIS1 and NDEL1 are dynein-associated proteins that function in neuronal migration which are also involved in axonal transport of the dynein–dynactin complex. KIF5 directly associates with LIS1, NDEL1 and mNUDC [91,92]. This association is reported to be required for the anterograde axonal transport of the dynein–dynactin complex. 3.1.4 Switching between fast and slow axonal transport There are two kinds of axonal transport: fast transport and slow transport. In axons, vesicles move quickly (50–400 mm/day) while soluble proteins move slowly (<8 mm/day). The transport of cytoplasmic proteins by slow transport is essential for neuronal homeostasis. KIF5 transports both fast and slow cargos [93–95]. How can the same motor protein implement both fast and slow axonal transport? A recent study has shown that slow transport depends on the interaction between the DnaJ-like domain in a tetratricopeptide repeat of KLCs in the KIF5 motor complex and Hsc70, which forms scaffolding between the cytoplasmic proteins and the KIF5 motor complex. This domain can bind to membranous organelles and competitive perturbation of it in squid giant axons disrupted cytoplasmic protein transport and strengthened membranous organelle transport. This indicates that this domain might function as a switch between slow and fast transport involving Hsc70. Transgenic mice overexpressing a dominant-negative form of this domain showed delayed slow transport, accelerated fast transport and optic axonopathy [96]. These findings provide a basis for the regulatory mechanism of fast and slow transport and its intriguing implication in neuronal dysfunction. 3.2 KIFs and dendritic transport In dendrites, various cargos are conveyed by KIFs, including NMDA receptor vesicles by KIF17, AMPA receptor vesicles by KIF5, GABA receptor vesicles by KIF5 and mRNAs with large protein complexes by KIF5 (Fig. 7). 3.2.1 KIF5 In addition to axonal cargos, several dendritic cargos are transported by KIF5. KIF5 associates with GRIP, a scaffolding protein that binds to AMPARs and transports them in dendrites [97]. Interestingly, the binding of GRIP to AMPARs drives KIF5 into dendrites, although the KIF5 motor domain preferentially moves into axons [98]. Another dendritic receptor species, GABA receptors, are transported by KIF5 via huntingtin-associated protein 1 (HAP1) [99]. Recent studies have suggested that local protein synthesis is important for neuronal function. KIF5 binds to mRNA-containing complexes and transports them in dendrites [100]. KIF5 binds to a large mRNP complex comprising at least 42 proteins, including Purα, Purβ and hnRNP-U, and mRNAs such as CaMKIIα mRNA and Arc mRNA [100]. 3.2.2 KIF17 NMDARs are thought to play an important role in synaptic plasticity, learning and memory. NMDARs are transported selectively to dendrites. KIF17 is a motor protein that transports NR2B (a subunit of NMDARs)-carrying vesicles. Because KIF17 is dendrite specific, it guarantees the dendritic localization of NMDARs [101]. KIF17 recognizes NR2B vesicles through the Mint1 (LIN10) scaffold protein complex (Fig. 7) [101]. When NR2B vesicles approach the postsynaptic region, KIF17 must release the vesicles. It has been revealed that CaMKIIα, which is active near the postsynapse, phosphorylates KIF17. CaMKIIα binds to the tail region of KIF17 and phosphorylates it at Ser1029, which dissociates Mint1 from the KIF17 tail domain and releases the cargos. Thus, phosphorylation of KIFs is an important mechanism for cargo unloading [102,103], besides the small G-protein-dependent unloading mechanism mentioned earlier for KIF1A/KIF1Bβ [69]. 3.2.3 KIFC2 KIFC2 is a C-terminal motor domain KIF that is predominantly localized in the neuronal dendrites in mice [104]. Uniquely, KIFC2 moves on microtubules towards their minus ends, similar to cytoplasmic dynein [104,105]. KIFC2 has been shown to transport multivesicular body-like organelles in dendrites [104], but the cargo molecules transported by this motor protein and the physiological relevance of this transport remain to be clarified. 3.3 Regulation of the direction of transport towards the axon versus the dendrites Neuronal polarity is fundamental for brain wiring. KIFs are involved in the generation of this neuronal polarity. Interestingly, the KIF5 motor domain preferentially localizes to axonal tips rather than dendrites [98]. The KIF5 motor domain specifically recognizes axonal microtubules, which somehow relates to microtubule dynamics. When neurons are treated with a low concentration of paclitaxel, axonal microtubules lose these characteristics and the KIF5 motor domain localizes in both the axon and the dendrites [98]. Consistent with this finding, the KIF5 motor domain accumulated in nascent axons during neuronal maturation [106]. What molecular structure does the KIF5 motor domain recognize? Several post-translational modifications of tubulin are suggested to augment the affinity of KIF5 for microtubules. For example, KIF5 preferentially associates with acetylated tubulin in Cos-7 cells [107]. Another study suggests that tubulin tyrosination steers KIF5 to axons [108]. However, a previous study has shown that the distribution of neither acetylated nor tyrosinated tubulins is biased towards axons [109]. Consistent with this, recent papers have shown that known tubulin modifications are not critical for the KIF5 motor domain to selectively localize to axons [110]. It thus remains an interesting question how the KIF5 motor domain discriminates axons from dendrites. The minus end-directed motor dynein is involved in cell body-to-dendrite sorting of cargos [111]. The mixed polarity of microtubules in dendrites is suggested to guide dynein into dendrites. The dendrite-specific KIF17 also sorts cargos into dendrites [101]. From another point of view, it has been proposed that in cell body-to-axon sorting, the initial segment of the axon has an ankyrin G- and F-actin-containing filter that functions as a diffusion barrier [112]. This selective axon initial segment-filtering has been proposed to contribute to preferential trafficking and segregation of cellular components in polarized neurons [112]. 4. Physiological relevance of intracellular transport and Unexp functions of KIFs 4.1 Functions Recent advances in molecular genetics in mice and humans have revealed that molecular motor-mediated axonal transport is relevant to neurogenesis and various neurological disorders. As we have seen in this review, molecular motors transport housekeeping molecules and signalling molecules, and sometimes even have non-motor functions in the regulation of signal transduction cascades. The delivery of healthy mitochondria, trophic factor receptors and neurotransmitters, cytoskeletal proteins, membrane lipids and mRNP complexes to the nerve terminals is indispensable for proper neuronal function. In addition, molecular motors can participate in signalling themselves: by transporting signalling molecules or their associated factors from one location to another within a neuron, they can modulate cellular behaviours such as fate decisions towards survival or apoptosis. In this way, molecular motors can be regarded as a new type of modifier of signal transduction molecules, not by changing their posttranslational modifications but merely by changing their locations. In the next section, I explore the genetic evidence for the physiological relevance of molecular motors in development, neuronal function and disease. 4.2 Genetic models relevant to the role of molecular motors in axonal and dendritic transport Molecular motors are essential for synapse generation and maintaining synaptic transmission. The importance of molecular motors at synapses has been revealed by both reverse and forward genetic studies in animal models and in human pedigrees carrying inherited diseases. 4.2.1 KIF1A and KIF1Bβ A major role of the KIF1A/KIF1Bβ kinesin-3 motors is to transport synaptic vesicle precursors [67–69]. These two motors share high similarity and both bind to DENN/MADD, which is a new effector of Rab3 GTPase that tethers the motor to the synaptic vesicle precursor membrane only when it is in a GTP-bound form [69]. Knockout mice for the Kif1a or Kif1b genes are similarly lethal during the perinatal period, because of severe neurological disorders [68,113]. Kif1b knockout pups do not start breathing because of defects in the respiratory centres in the brain stem [68]. The number and density of synaptic vesicles were decreased in the presynaptic area. In addition to defects in synaptic vesicle transport, Kif1a−/− and Kif1b−/− mice exhibited neuronal cell death [68,113]. It is also possible that KIF1A and KIF1Bβ can transport other cargos besides synaptic vesicle precursors. This will be the subject of future research. Haploinsufficiency in the Kif1b gene revealed a late-onset neuropathy. One-year-old animals exhibited significant deficits in behavioural tests, including the rotarod, and showed a staggering gait. Although the amplitude of the muscular potential was decreased, motor nerve conduction velocity was preserved. Because this excludes the presence of major demyelination phenotype, the failure was considered to be mainly of an axonal origin. The identification of a functional mutation in the motor domain in a family with Charcot–Marie–Tooth disease Type 2A1 (CMT2A1) neuropathy suggested that KIF1B is involved in normal neuronal function in humans [68]. 4.2.2 KIF5s The kinesin 1 motors KIF5A/B/C have multiple roles in neurons. In particular, primary or secondary axonal transport defects can result in neuronal degeneration in several neurological diseases [114–116]. Mice deficient for the Kif5b gene die during early embryonic development because of impaired transport of multiple essential organelles, including mitochondria and endosomes/lysosomes [82]. Kif5c knockout mice are viable and fertile, but their brain sizes were smaller than those of controls, with accompanying loss of neurons in the brain motor nuclei [80]. Because KIF5A, 5B or 5C were similarly able to rescue the mitochondrial phenotype of Kif5b-deficient extraembryonic cells, their functions were considered partially redundant. A mutation in KIF5A is responsible for human hereditary spastic paraplegia, which causes a dying-back neuropathy characterized by progressive weakness and spasticity of the legs [94]. Mutant mouse models revealed the loss of large calibre axons and neurofilament accumulation in neuronal cell bodies. 4.3 KIF17 controls learning and memory KIF17 transports vesicles containing NMDA-type glutamate receptors to dendrites [101]. Transgenic mice overexpressing KIF17 demonstrated greatly enhanced spatial and working memory [117]. Interestingly, the phosphorylation level of the cAMP response element-binding (CREB) transcription factor was enhanced and mRNAs of NR2B subunit of NMDAR and KIF17 were increased in amount. Furthermore, disruptions of the murine kif17 gene inhibits NR2B transport, accompanied by decreased transcription of nr2b, resulting in a loss of synaptic NR2B [118]. In kif17−/− hippocampal neurons, the NR2A level is also decreased because of accelerated ubiquitin–proteasome system-dependent degradation. Accordingly, NMDA receptor-mediated synaptic currents, early and late long-term potentiation, long-term depression, and CREB responses are attenuated in kif17−/− neurons, concomitant with a hippocampus-dependent memory impairment in knockout mice. In wild-type neurons, CREB is activated by synaptic inputs, which increase the levels of KIF17 and NR2B. Thus, KIF17 differentially maintains the levels of NR2A and NR2B, and, when synapses are stimulated, the NR2B/KIF17 complex is upregulated on demand through CREB activity [118]. These KIF17-based mechanisms for maintaining NR2A/2B levels could underlie multiple phases of memory processes in vivo. 4.4 KIF3 control left–right determinationof our body KIF3 motors act in both cilia and cytoplasm [119–121]. The heterotrimeric KIF3 complex (KIF3A/KIF3B/KAP3) bi-modally functions in intraflagellar transport (IFT) of ciliary components [122] and in axonal/intracellular transport of plasma membrane precursors [123] and the N-cadherin/β-catenin complex [124]. Knockout mice for the Kif3a or Kif3b genes exhibit randomized left–right determination of the body axis. Approximately half of knockout mouse embryos develop an abnormal heart loop [125–127]. The molecular mechanisms of this striking body axis formation were approached microscopically by observing the ventral node of mouse embryos, which is a ciliated organ transiently exposed on the surface of the ventral midline [5,8,125]. Scanning EM revealed the absence of ciliogenesis, consistent with KIF3 being an essential IFT motor. Interestingly, the cilia were clockwise-rotating on an axis tilted to the posterior, which could generate a leftward flow of extraembryonic fluid according to the shear stress of the embryonic surface [128]. This flow appeared to concentrate essential signalling molecules on extracellular particles towards the left side, to break the bilateral symmetry of the embryos [129]. This ‘nodal flow hypothesis’ could be an evolutionarily conserved mechanism for left–right determination. 4.5 KIF3 suppresses tumourigenesis In addition to its role in IFT, the KIF3 motor also serves in cytoplasmic transport [123]. Conditional gene targeting in mice of a non-motor subunit of the KIF3 complex, Kap3, results in disorganization of the proliferative zone of the neuroepithelium and transforms these cells into tumours [124]. In the knockout cells, N-cadherin and β-catenin failed to be transported to the plasma membrane and abnormally accumulated in the cytoplasm and nucleus. Accordingly, they enhanced canonical Wnt signalling in the nucleus to facilitate cell proliferation, and reduce cell–cell adhesion by N-cadherin. Thus, KIF3 seems to exclude oncoproteins from the perinuclear region. 4.6 Relevance of molecular motors in brain wiring and the development of the CNS and PNS The nervous system mainly develops from the neural tube and neural crest cells that arise from neuroectoderm on the dorsal side of the embryo. Generally, the neural progenitors for the CNS are located innermost in the neural tube, where they asymmetrically divide and produce neural cell populations that undergo differentiation. These postmitotic neurons migrate towards the cortex of the neural tube and form the cortical layers. Recent results from gene targeting have revealed that molecular motors and their associated proteins are responsible for many essential steps of this neurogenesis cascade. Their roles are quite divergent and many more will likely be revealed in future studies. 4.6.1 KIF2A is essential for brain wiring The motor domains of the kinesin-13 family, including KIF2A and KIF2C, have a special function to depolymerize microtubules by hydrolyzing ATP. Knockout mice for the Kif2a gene are perinatally lethal, without sucking milk [130–132]. Their brains showed multiple abnormalities, including ventricle enlargement, laminar defects and disorganization of nerve nuclei. Kif2a−/− neurons exhibited migratory defects both in vivo and in vitro, and abnormally elongated collateral branches of axons. This suggests that KIF2A contributes to brain wiring through suppressing unnecessary elongation of the collateral branches by microtubule depolymerization in growth cones. 4.6.2 KIF26A suppresses GDNF/Ret signalling and control development of enteric nervous system The motor domain of the kinesin-11 family, including KIF26A/B, has significantly diverged and lost the microtubule-activated ATPase activity and the microtubule-dependent motility. Knockout mice for the Kif26a gene exhibited severe megacolon, due to hyperplasia of enteric neurons and defects in myenteric neurite outgrowth, especially in the distal colon [133]. Kif26a−/− enteric neurons are hypersensitive to glial cell-derived neurotrophic factor (GDNF)-Ret signalling, because the KIF26A tail domain directly binds to Grb2 to negatively regulate the formation of the SHC-Grb2, Ret-SOS and Ret-Gab1 adaptor complexes. Thus, KIF26A plays a significant role as a suppressor of the GDNF/Ret signalling system and as a controller of development of the enteric nervous system. 4.6.3 KIF4A is a key factor controlling activity-dependent neuronal survival during brain development During brain development, activity-dependent neuronal survival is programmed to eliminate unnecessary neurons. The kinesin-4 motor KIF4A is expressed predominantly in juvenile neurons and is localized both in the nucleus and in the cytoplasm [134]. Unexpectedly, the survival rate of neurons lacking KIF4A was higher than that of control neurons [135]. The C-terminal domain of KIF4A could bind to and suppress the activity of the poly(ADP-ribose) polymerase 1 (PARP1) enzyme, which maintains cell homeostasis by repairing DNA and serving as a transcriptional regulator. When neurons are stimulated by membrane depolarization, CaMKII-mediated calcium signalling phosphorylates PARP1 and induces dissociation of KIF4A from PARP1. This activates PARP1 and the neuron escapes apoptosis. The dissociated KIF4A leaves the nucleus and functions as a transport motor. Thus, KIF4A has dual functions: one as a key regulator of activity-dependent neuronal survival and the other as a motor to transport cargos in the cytoplasm [135]. Because KIF17b also participates in transcriptional regulation through interacting with a transcriptional coactivator in male germ cells [136], KIFs may occasionally act as transcription factors in the nucleus. 4.7 KIF16B/Rab 14 complex is criticalfor embryonic development by transportingFGF receptor Kinesin-3 motor KIF16B/Rab14 complex acts in biosynthetic Golgi-to-endosome traffic of the fibroblast growth factor receptor (FGFR) during early embryonic development. Kif16B−/− mouse embryos failed in developing epiblast and primitive endoderm lineages and died in the peri-implantation stage, similar to previously reported FGFR2 knockout embryos [137]. KIF16B associated directly with the Rab14–GTP adaptor on FGFR-containing vesicles and transported them towards the plasma membrane. To examine whether the nucleotide state of Rab14 serves as a switch for transport, Rab14-GDP was overexpressed. This dominant-negative approach reproduced the whole putative sequence of KIF16B or FGFR2 deficiency: impairment in FGFR transport, FGF signalling, basement membrane assembly by the primitive endoderm lineage, and epiblast development [137]. These data provide a genetic evidence that microtubule-based membrane trafficking directly promotes early development. 4.8 Concluding remarks Thus, KIFs play a number of significant roles not only on various cell functioning by transporting important membranous organelles, protein complexes, and mRNAs but also play unique and important functions on fundamental events for life such as early embryonic development, left-right asymmetry, brain wiring, activity-dependent neuronal survival, control of PNS development, suppression of tumourigenesis and higher brain functions such as memory and learning. Furthermore, it has become increasingly evident that molecular motors are deeply related to the pathogenesis of certain neuronal diseases. In the near future, molecular genetics will deepen our understanding of not only neuronal and brain function but also functions of cells in general, and at the same time is likely to reveal more surprising roles of molecular motors. In summary, molecular motor research is one of the new frontiers of neuroscience, and general molecular cell biology. Although certain questions have been answered, a number of problems remain and new questions have arisen. Using a wide variety of approaches in molecular cell biology, new imaging techniques, electrophysiology, biophysics, molecular genetics and structural biology, molecular motor research will contribute to the further development of neuroscience and molecular cell biology in many directions. 5. How the molecular motor moveson the microtubules processively The mechanism how KIFs move on the microtubule rails is another important fundamental question. We approached this question by combining single-molecule biophysics, cryo-EM and X-ray crystallography. For this approach, it was very fortunate for us that we identified the simplest monomeric motor, KIF1A [63,67]. In mammalians such as humans and mouse, 45 genes were identified in Kinesin superfamily [65]. They all share globular heads or catalytic core domains (motor domains). Its crystal structure is solved for different classes of KIFs, and they all show highly conserved atomic structures, which suggests conserved motility mechanism. In this section, I mainly focus on two KIFs, KIF1A and KIF5, though I believe that essentially same mechanism would be applied to other N-terminal motor domain-type KIFs. The first identified kinesin KIF5 (conventional kinesin) belongs to the kinesin-1 family and transports various kinds of cargoes such as mitochondria, mRNA and AMPA-type glutamate receptor, GluR2-containing vesicles [77,78,82,97,100]. On the other hand, KIF1A transports synaptic vesicle precursor and plays a significant role on neuronal function and survival [67,113]. KIF1A recognizes and binds the synaptic vesicle precursor through the interaction among KIF1A stalk, an adaptor protein, DENN/MADD and Rab3 on the vesicle and this binding is regulated by GTP hydrolysis by Rab3 [69]. These two KIFs have been extensively studied for their motility mechanism. Single-molecule biophysics studies on the motility mechanism have been rapidly advanced with conventional kinesin since it was shown that a single molecule of kinesin can move along a microtubule over distances of 1 µm even under load up to several piconewtons [138–142]. To explain the processive movement, ‘hand-over-hand’ model was proposed [138,143]. The electron micrographs of conventional kinesin showed that two heads are closely positioned on one end of a coiled-coil stalk (Fig. 6) [79]. Thus, kinesin can use two heads for the binding to the microtubules. The hand-over-hand model postulates a built-in gating mechanism so that the release of the bound head is contingent on the binding of the other head. Then, kinesin is always attached to the microtubule via at least one head during the movement. Lines of recent experiments support this model [144,145]. However, the mechanism of the gating, the key assumption of this model, has been unclear. With single-molecule biophysics studies of KIF1A, it was shown that a single motor domain of KIF1A monomer can move diffusively along the microtubule (Figs 8 and 9) [72]. Thus, the hand-over-hand model cannot explain the motility of KIF1A monomers, as these do not have a second head to support the moving head while searching for the next binding site (Fig. 8). However, single-molecule studies of KIF1A show that its moving monomeric head is weakly supported by an electrostatic interaction between its K-loop (positively charged) and the C-terminal E-hook of the microtubules (negatively charged) [73,74]. This enables KIF1A to move by one-dimensional diffusion to next binding site without fully detaching from the microtubule (Figs 9 and 10) [73]. This diffusive monomeric movement was initially thought to be unique to KIF1A. However, KIF5 motor domains have the potential to move diffusively in monomeric state, as monomeric KIF5 constructs move diffusively when the K-loop of KIF1A is introduced [73]. Other N-kinesin, such as kinesin 5 family members, shows both hand-over-hand-type processive movement and diffusive movement [146]. Fig. 8. Open in new tabDownload slide Processivity of movement of monomeric motor along microtubules. Conventional kinesin is dimeric in its native form. It moves processively along microtubules in the dimeric forms, but not in the monomeric form. In contrast, KIF1A is monomeric in its native form and moves processively in the monomeric form. Fig. 8. Open in new tabDownload slide Processivity of movement of monomeric motor along microtubules. Conventional kinesin is dimeric in its native form. It moves processively along microtubules in the dimeric forms, but not in the monomeric form. In contrast, KIF1A is monomeric in its native form and moves processively in the monomeric form. Fig. 9. Open in new tabDownload slide Processive movement of monomeric motor KIF1A. (a) Movement of fluorescently labelled single C351 molecules (red) along microtubule (green). Sometimes C351 moves backward (arrowheads), but it usually moves in one direction (arrows). Scale bar, 2 μm; frame interval, 0.5 s. (b) Analysis of movement. Distribution of duration of movement of C351 (A) and K381 (B). Distribution of run length of C351 (shaded bars) and K381 (open bars) (C). Typical traces of displacement of C351 (D) and K381 (E). Distribution of displacement of C351 (F) and K381 (G). MSD of C351 (circles) and K381 (squares) plotted against time (H). Diffusion term of C351 (circles) and K381 (squares) plotted against time (I). (c) Distribution of step size of KIF1A beads measured by optical trapping. (d) Flush ratchet model of KIF1A movement along microtubule. Reproduced with permission from Okada and Hirokawa [72]. Fig. 9. Open in new tabDownload slide Processive movement of monomeric motor KIF1A. (a) Movement of fluorescently labelled single C351 molecules (red) along microtubule (green). Sometimes C351 moves backward (arrowheads), but it usually moves in one direction (arrows). Scale bar, 2 μm; frame interval, 0.5 s. (b) Analysis of movement. Distribution of duration of movement of C351 (A) and K381 (B). Distribution of run length of C351 (shaded bars) and K381 (open bars) (C). Typical traces of displacement of C351 (D) and K381 (E). Distribution of displacement of C351 (F) and K381 (G). MSD of C351 (circles) and K381 (squares) plotted against time (H). Diffusion term of C351 (circles) and K381 (squares) plotted against time (I). (c) Distribution of step size of KIF1A beads measured by optical trapping. (d) Flush ratchet model of KIF1A movement along microtubule. Reproduced with permission from Okada and Hirokawa [72]. Fig. 10. Open in new tabDownload slide (a–c) Docking of atomic model of KIF1A and tubulin. Surface representation from outside (c) and top view from plus end (a) and superposition of image on atomic model (b). (d) The tarzan model of biased Brownian movement of KIF1A. Panels (a–c) reproduced with permission from Kikkawa et al. [155]. Fig. 10. Open in new tabDownload slide (a–c) Docking of atomic model of KIF1A and tubulin. Surface representation from outside (c) and top view from plus end (a) and superposition of image on atomic model (b). (d) The tarzan model of biased Brownian movement of KIF1A. Panels (a–c) reproduced with permission from Kikkawa et al. [155]. M-kinesins also use a diffusive search along microtubules to reach the ends, where they cause microtubule depolyemerization [147]. The K-loop of KIF1A can also restore activity of an M-kinesin mutant [148]. Furthermore, a forced dimer of KIF1A moves processively like KIF5, possibly by the hand-over-hand mechanism [149]. It is proposed that KIF1A might dimerize in vivo through weak coiled-coil motif and move processively by the hand-over-hand mechanism [149]. Thus, this evidence suggests that kinesins move by a conserved mechanism, rather than by mechanism that are unique to each kinesin. Single-molecule studies of KIF1A clarified that the alternation between the tight-binding state and the loose-binding state to microtubule is essential for the motility. In the following sections, I first focus on the conformation change in the microtubule-binding interface of KIF1A during ATPase cycle. Then, I discuss on the putative regulatory mechanism for the reaction cycle. A key proposal is that the neck-linker, which is widely assumed to be the lever arm of kinesin, has another key role as the sensor for the load. Based on this model, we propose a gating mechanism required for the hand-over-hand movement. 5.1 The structure of the motor domain The X-ray crystal structure of kinesin motor domain was first solved with conventional kinesin [150] and ncd [151]. Then, two different conformations, AMP-PCP, ATP-like state, and Mg2+-ADP state, were solved for KIF1A [152]. After that, structures of intermediate states for ATP hydrolysis and ADP release were solved by using analogues and varying the timing to harvest crystals [153,154]. The overall structure was quite similar among different states of KIF1A and among other kinesins. A layer of central β-sheets is sandwiched between two layers of α-helices like a hamburger. The top bun contains the ATP hydrolysis catalytic centre, or nucleotide-binding pocket. Its minor conformation change during the ATPase cycle is conveyed to the bottom bun, which faces to the microtubule. Thus, a series of large conformation changes occur on the microtubule-binding surface. KIFs move along microtubules by alternating between two mechanical states: a strong-binding states in which the KIFs tightly binds to the microtubule, and one-dimensional diffusional binding state in which the KIF weakly binds to (or is detached from) the microtubule (Figs 11 and 12). KIFs with Mg2+-ADP are in the one-dimensional diffusional binding state, whereas KIFs with Mg2+-ATP, or without a nucleotide, are in the strong-binding state. Concerning the interface of KIF1A (Mg2+-ADP) and the microtubule in the one-dimensional diffusional binding state seen from the minus end of the microtubule, the basic centre line of a KIF loopL8 and the α5, α4 and α6 helices faces the acidic ridge of the microtubule (H12 and H5) and this is well conserved among most of the KIFs. However, monomeric motors, such as KIF1A, have an additional class-specific K-loop which interacts with the flexible E-hook at the C-terminal of α and β tubulin [73,74,155] (Figs 10, 12 and 13). These non-localized electrostatic interactions allow Mg2+-ADP-bound KIF1A to diffuse freely along the microtubule protofilament without full detachment. The conformation change during the Mg2+-ADP to Mg2+-ATP nucleotide exchange rearranges the microtubule-binding surface so that its motor domain rotates three-dimensionally about 20°. This rotation brings a cluster of acidic residues at the N-terminus of the α6 helix to the minus end of the basic centre line. This repulsive element at the minus end of KIF1A might bias the binding of KIF1A towards the plus end [74]. This rotation also brings loop L11 towards the microtubule. This loop, which is well conserved among KIFs, specifically contributes to the strong microtubule-binding state [74,152]. KIF1A therefore uses two microtubule-binding loops, the K-loop and loop L11, alternately for diffusion and strong binding, respectively [152,153] (Fig. 12). Fig. 11. Open in new tabDownload slide (a and b) Crystal structures of KIF1A. (a) The AMP-PNP form. The switch I, switch II and neck-linker regions are highlighted in red. (b) Superposition of AMP-PNP (red), ADP-AlFx (blue), ADP-Vi (green) and ADP (yellow) forms. (c–e) Conformational changes in two switch regions during ATP hydrolysis. AMP-PCP(c), AMP-PNP (d), ADP-AlFx (e) and ADP-Vi (f) forms. Reproduced with permission from Nitta et al. [153]. Fig. 11. Open in new tabDownload slide (a and b) Crystal structures of KIF1A. (a) The AMP-PNP form. The switch I, switch II and neck-linker regions are highlighted in red. (b) Superposition of AMP-PNP (red), ADP-AlFx (blue), ADP-Vi (green) and ADP (yellow) forms. (c–e) Conformational changes in two switch regions during ATP hydrolysis. AMP-PCP(c), AMP-PNP (d), ADP-AlFx (e) and ADP-Vi (f) forms. Reproduced with permission from Nitta et al. [153]. Fig. 12. Open in new tabDownload slide Structural models of active detachment of KIF1A and G proteins. (a–d) KIF1A (pink)–microtubule (grey) complex observed from minus end of microtubule in different states of ATP hydrolysis. (a) Prehydrolysis state. (b) Early ADP-Pi state. (c) Late ADP-Pi state, that is, detaching state (indicated by arrows). (d) ADP state. L11, α4 and L12 are shown as coloured ribbon model (red, yellow and green). H11′ and E-hook of tubulin are shown in purple. ATP, ADP and Pi are shown as red, green and orange spheres, respectively. Asterisks refer to the second ATP or ADP-Pi state of KIF1A. (e–h) The α-subunit of G proteins and region corresponding to switch II are colour-coded as in (a–d). Adenylyl cyclase and β and γ subunits of G proteins are shown in grey. (e–h) States during GTP hydrolysis corresponding to those described in (a–d). The energy derived from GTP hydrolysis is used for dissociation of G α from adenylyl cyclase as indicated by arrows in (g). Fig. 12. Open in new tabDownload slide Structural models of active detachment of KIF1A and G proteins. (a–d) KIF1A (pink)–microtubule (grey) complex observed from minus end of microtubule in different states of ATP hydrolysis. (a) Prehydrolysis state. (b) Early ADP-Pi state. (c) Late ADP-Pi state, that is, detaching state (indicated by arrows). (d) ADP state. L11, α4 and L12 are shown as coloured ribbon model (red, yellow and green). H11′ and E-hook of tubulin are shown in purple. ATP, ADP and Pi are shown as red, green and orange spheres, respectively. Asterisks refer to the second ATP or ADP-Pi state of KIF1A. (e–h) The α-subunit of G proteins and region corresponding to switch II are colour-coded as in (a–d). Adenylyl cyclase and β and γ subunits of G proteins are shown in grey. (e–h) States during GTP hydrolysis corresponding to those described in (a–d). The energy derived from GTP hydrolysis is used for dissociation of G α from adenylyl cyclase as indicated by arrows in (g). Fig. 13. Open in new tabDownload slide Cryo-EM maps of KIF1A–MT complexes in the Mg2+-ADP(a, c and e) and AMP-PNP states (b, d and f). (a and b) Isosurface representation of KIF1A–MT complexes. (c–f) Fitting of the X-ray crystal structures into cryo-EM maps. MTs are shown with their plus-end up and assignment of α- and β-tubulin is based on that of Kreb et al. [170]. The blue chicken wires are contoured at 0.7σ of the density map, with a mesh size of 1 Å. A 20.6° change of the kinesin core from the Mg2+-ADP state to the AMPPNP states. Reproduced with permission from Kikkawa and Hirokawa [158]. Fig. 13. Open in new tabDownload slide Cryo-EM maps of KIF1A–MT complexes in the Mg2+-ADP(a, c and e) and AMP-PNP states (b, d and f). (a and b) Isosurface representation of KIF1A–MT complexes. (c–f) Fitting of the X-ray crystal structures into cryo-EM maps. MTs are shown with their plus-end up and assignment of α- and β-tubulin is based on that of Kreb et al. [170]. The blue chicken wires are contoured at 0.7σ of the density map, with a mesh size of 1 Å. A 20.6° change of the kinesin core from the Mg2+-ADP state to the AMPPNP states. Reproduced with permission from Kikkawa and Hirokawa [158]. 5.2 Regulation of microtubule affinity with helix α4, L11 and L12 The key conformation change during the ATPase cycle is the up–down movement of the helix α4. This large movement is readily noticed when viewed from the microtubule-binding surface. This up–down movement of α4 is coupled to the bound nucleotide state, and also corresponds to the change in the microtubule affinity. In the ATP-bound state, KIF1A takes ‘up’ conformation, and binds strongly to the microtubule, in a so-called ‘rigour’ state. In this conformation, α4 helix is protruded from the surface with its N-terminal ends melted to elongate loop L11 (Figs 11 and 12). In the Mg2+-ADP state, KIF1A takes ‘down’ conformation, and diffuses one-dimensionally along the microtubule, which corresponds to the weak-binding state of conventional kinesin. In this conformation, α4 helix is embedded into the surface with its N-terminal side wound up with shortened L11. C-terminal end of α4 is partially melted to relax L12 (Figs 11 and 12). The implications of this conformation change in the microtubule affinity were elucidated by docking these crystal structures into the cryo-EM structures of KIF1A-microtubule complex (Fig. 13). In the Mg2+-ADP state, the ‘down’ form, L12, a positively charged loop with hexa-lysine insert (K-loop), is elongated so that it can interact with the flexible acidic C-terminus of tubulin (E-hook). This interaction was shown to be responsible for the one-dimensional diffusion [72,73]. Furthermore, α4 is buried to make the microtubule-binding surface flat and smooth. An array of basic residues forms a centre line of this binding surface. This basic centre line faces to the surface ridge of microtubule protofilament, along which acidic residues are aligned. The non-localized electrostatic interaction among the centre line residues will restrict KIF1A to follow along the protofilament axis and to support, which would enable the one-dimensional diffusion along the microtubule track like a monorail train. Similar mechanism will explain the recently reported diffusion of myosin along microtubule and kinesin along F-actin [156,157]. Here, it should be noted that this is based on the speculation from the structure at the end of the diffusion state. The cryo-EM structure of KIF1A–microtubule complex with excess ADP does not represent the true ‘down’ conformation of KIF1A. Albeit the α4 helix is in the ‘down’ position, it partially melts at the L11 side so that the loop L11 is elongated to interact with α-tubulin. This is the characteristic feature of the early transition from ‘down’ to ‘up’, represented by the Mg-releasing transition-1 state [154]. With excess KIF1A to microtubule, KIF1A cannot diffuse along the microtubule, but would stay on the rigour-binding site of microtubule, where KIF1A is in the dynamic equilibrium between Mg2+–ADP state and Mg2+-releasing transition-1 state. In the ‘up’ conformation, the helix α4 shifts and rotates three dimensionally. The end of α4 is lifted up by the neck-linker, and α4 now protrudes on the microtubule-binding surface. With this protrusion and rotation, the microtubule-binding surface of KIF1A takes the zigzag shape, which perfectly fits to the surface profile of microtubule protofilament. The protrusion of α4 fits into the intradimer groove. With this up conformation, the beginning of α4 helix is melted to elongate the loop L11, which interacts with the H11′ and H3′ helix of α-tubulin at the bottom of the intra-tubulin groove. Thus, α4 and L11 serves as the anchor to fix KIF1A on microtubule. Mutation studies confirmed that this interaction is responsible for the rigour binding [153]. Recent improvements in the resolution of the cryo-EM map (Fig. 13) [158] enabled to identify another structural component. At the outer left side of the loop L11, another loop L9 interacts with the H3′ helix of α-tubulin. L9 is the major component of the nucleotide-binding pocket. Thus, the direct interaction with tubulin might stabilize the conformation of the catalytic loop L9, which might explain the acceleration ATP hydrolysis by microtubule. 5.3 Mechanisms of nucleotide exchange The ATPase reaction fuels the energy for the movement of kinesins. The binding of ATP and the release of ADP is therefore important for the regulation of kinesin motility. For example, ADP release is very slow (0.002–0.009 s−1) in the absence of microtubules, but is accelerated by >104-fold after kinesin binds to β-tubulin in the microtubule. This microtubule-dependent regulation can act as a chemical checkpoint to suppress futile ATP consumption by free kinesins, and it is also essential to enable the diffusional search for the next binding site during movement (Figs 9 and 10). The atomic structures of KIF1A during Mg2+–ADP release first explained this well-known biochemical property [154]. 5.3.1 KIF1A structure during Mg2+–ADP release A high-resolution crystal structure of KIF1A with bound Mg2+–ADP explains the atomic mechanism of slow ADP release [154]. ADP on the surface shallow groove formed by the P-loop is stably covered with a Mg2+–water cap – a layer of crystal ice formed by the hydrogen bond network around the essential cofactor Mg2+ (Fig. 14a). This Mg2+ –water cap fills the gap between the β-phosphate of ADP and switches I and II. The nucleotide-binding pocket is therefore closed in the absence of microtubules. Structures of Mg2+-release intermediates of KIF1A explain the mechanism of microtubule-induced kinesin activation [154]. Switch I changes its conformation mainly during Mg2+ release. After ATP hydrolysis, switch I is linked to loop L7 and to switch II by two sets of hydrogen bonds (Fig. 14a). One set of hydrogen bonds forms near the N-terminus of the switch II α4 helix and stabilizes the conformation of the microtubule-binding surface in the post-hydrolysis position, which is referred to as the ‘latch’. Fig. 14. Open in new tabDownload slide Atomic mechanism of KIF1A nucleotide exchange. (a) Structures of the KIF1A motor domain before Mg2+ release. An oblique left-side view (left panel; a schematic in the dotted box shows the orientation), a left-side view to show the key structural elements surrounding loop L7 (middle panel) and a schematic model for the action of loop L7 as the microtubule sensor (right panel). The tri-residue interaction among Glu148 (in loop L7), Arg203 (in switch I) and Asp248 (in switch II) – the Mg2+ stabilizer – anchors the Mg2+–water cap, which keeps ADP in the nucleotide-binding pocket. Another tri-residue interaction among Tyr150 (in loop L7), Arg216 (in switch I) and Glu267 (in loop L11) – the ‘latch’ – pulls up loop L11 and stabilizes switch II in the Mg2+-ADP conformation. Loop L7 is the pivot for these two sets of interactions. The charged tip residues Glu152 and Arg153 of loop L7 are exposed to the microtubule surface (Arg158 and Glu159 in helix H4 of β-tubulin). The attractive interaction between these dipole pairs pulls loop L7 towards the β-tubulin. Loop L7 can thereby sense the binding to the microtubule, hence it is named microtubule sensor. (b) Structures of the KIF1A motor domain after Mg2+ release. An oblique left-side view (left panel), a left-side view to show the key structural elements surrounding loop L7 (middle panel) and a schematic model for the action of loop L7 as the microtubule sensor (right panel). The downwards movement of loop L7 (white arrow, middle panel) breaks both the Mg2+ stabilizer and the latch. The nucleotide-binding pocket is thereby opened to allow the immediate exchange of ADP for ATP, and loop L11 extends to the groove between α- and β-tubulin to make a strong interaction. The amino acids in the right panels are depicted in the same colour as their corresponding structures in the left and middle panels. Reproduced from Hirokawa et al. [9]. Fig. 14. Open in new tabDownload slide Atomic mechanism of KIF1A nucleotide exchange. (a) Structures of the KIF1A motor domain before Mg2+ release. An oblique left-side view (left panel; a schematic in the dotted box shows the orientation), a left-side view to show the key structural elements surrounding loop L7 (middle panel) and a schematic model for the action of loop L7 as the microtubule sensor (right panel). The tri-residue interaction among Glu148 (in loop L7), Arg203 (in switch I) and Asp248 (in switch II) – the Mg2+ stabilizer – anchors the Mg2+–water cap, which keeps ADP in the nucleotide-binding pocket. Another tri-residue interaction among Tyr150 (in loop L7), Arg216 (in switch I) and Glu267 (in loop L11) – the ‘latch’ – pulls up loop L11 and stabilizes switch II in the Mg2+-ADP conformation. Loop L7 is the pivot for these two sets of interactions. The charged tip residues Glu152 and Arg153 of loop L7 are exposed to the microtubule surface (Arg158 and Glu159 in helix H4 of β-tubulin). The attractive interaction between these dipole pairs pulls loop L7 towards the β-tubulin. Loop L7 can thereby sense the binding to the microtubule, hence it is named microtubule sensor. (b) Structures of the KIF1A motor domain after Mg2+ release. An oblique left-side view (left panel), a left-side view to show the key structural elements surrounding loop L7 (middle panel) and a schematic model for the action of loop L7 as the microtubule sensor (right panel). The downwards movement of loop L7 (white arrow, middle panel) breaks both the Mg2+ stabilizer and the latch. The nucleotide-binding pocket is thereby opened to allow the immediate exchange of ADP for ATP, and loop L11 extends to the groove between α- and β-tubulin to make a strong interaction. The amino acids in the right panels are depicted in the same colour as their corresponding structures in the left and middle panels. Reproduced from Hirokawa et al. [9]. The other set of hydrogen bonds forms at the root of loop L7. Glu148 in loop L7 makes a hydrogen bond with Arg203 in switch I, which interacts with Asp248 in switch II. These three residues not only link switches I and II to loop L7, but also make a hydrogen bond with the Mg2+–water cap, thus serving as the Mg2+ stabilizer – an anchoring point for the Mg2+–water cap. The release of Mg2+ is accompanied by breakage of the two sets of hydrogen bonds between loop L7 and switches I and II. The intermediate structures of Mg2+ release suggest that the downwards movement (towards the microtubule) of loopL7 triggers the breakage of its hydrogen bonds to switches I and II (Fig. 14b). This movement of loop L7 is only about 1 Å in the crystal structure, but re-inspection of recent cryo-EM structures of KIF5 and Kar3 (yeast kinesin 14), as well as KIF1A, support the idea that this conformation change actually takes place on the microtubule [9,158]. Moreover, these structures suggest that loop L7 interacts directly with β-tubulin (Fig. 14b). The tip of loop L7 has conserved residues (Glu152 and Arg153) that face oppositely charged residues (Arg158 and Glu159) in helix H4 of β-tubulin, which is located at the plus end of the microtubule. These residues are well conserved in β-tubulins but not in α-tubulins. This interaction, therefore, could contribute to the ability of kinesins to discriminate between β- and α-tubulin, and help to explain the directional bias for the diffusional movement of kinesins (Figs 9 and 10). Experiments in which KIF1A is mutated at these sites support the importance of these residues in the microtubule-induced activation of Mg2+–ADP release [154]. 5.3.2 Loop L7 as the trigger for Mg2+–ADP release Based on the above structural and mutational studies, we propose that loop L7 might act as a triggering lever for Mg2+ release and the nucleotide exchange that follows (Fig. 14). The root of loop L7 might have an essential role in bringing switch I, switch II and Mg2+–ADP together to close the nucleotide-binding pocket. Its tip might be the microtubule sensor that is selective for β-tubulin. If it is, the binding of β-tubulin would pull this sensor downwards to trigger breakage of the Mg2+ stabilizer and loss of the Mg2+–water cap. The nucleotide-binding pocket would then be fully open and the nucleotide readily exchanged without conformational changes (Fig. 14b). Consistent with this idea, the atomic structure of KIF1A immediately before and immediately after ADP–ATP exchange is essentially the same (with a root mean square deviation of ∼0.1 A) [152,154], but the atomic structure of KIF1A without a bound nucleotide is still missing. After nucleotide exchange, the nucleotide-binding pocket closes as a result of the formation of a salt bridge between loop L9 of switch I and loop L11 of switch II [9,153]. This closure is enabled by conformation changes in the microtubule-binding surface and the neck-linker (discussed below), which would correspond to the ‘isomerization’ step immediately before ATP hydrolysis – a step identified by transient ATPase kinetic experiments [9,153]. 5.4 Dual roles of neck-linker The crystal structures in the transition from the ADP-bound state to the ATP (AMP-PNP)-bound state revealed the contribution of the neck-linker docking for the down-to-up conformation change of switch II [154]. The neck-linker is located at the C-terminus of the motor core next to the helix α6, and connects the catalytic core domain to the coiled-coil stalk in the dimeric kinesins. Previous cryo-EM study suggested that the neck-linker has two conformations (Fig. 13) [158,159]. The neck-linker is fully detached from the catalytic core in the ADP state. Upon nucleotide exchange from ADP to ATP, the neck-linker docks to the core, and this docking is considered as the power stroke for the hand-over-hand movement of dimeric kinesin [160,161]. Our crystal structures [154] revealed the details of this docking, and suggested that it has two different functional roles. The neck-linker docks to the core in three steps. During the Mg release that precedes the nucleotide exchange, the initial segment of the neck-linker (NIS: neck initial segment) docks to the groove between the N-terminus of the catalytic core and the helix α4, and supports halfway up conformation of α4 from back side through the hydrophobic interaction between conserved Ile354 (NIS) and Leu285 (α4). Deletion mutant of NIS severely abrogated the ATPase activity, and NIS alone without the following neck-linker elements could restore most of the ATPase activity [153,159]. This indicates that the docking of NIS is required for the ATPase reaction in the pocket, putatively through supporting the down-to-up conformation change of switch II that is coupled to the opening of the pocket for the release of ADP (Fig. 15). The docking of the following segments (β9 and 10) is not essential for the ATPase reaction, but will accelerate it moderately (Fig. 15). Then, at the nucleotide exchange step, the next neck-linker element β9 docks to the core and forms a rigid triple β-strand structure with the N-terminal strand β0 and the loop L13 (switch II). This second docking is followed by the third docking of the third neck-linker element β10 at the isomerization step, when the gamma phosphate of Mg–ATP is recognized by the closure of the back door. Mutations to these β sheets did not significantly affect to the ATPase activity, but reduced the velocity in the microtubule gliding assay [74,153,159,160]. The reduction was much severer in β 9 mutant, suggesting that β9 docking is the major power stroke event, and that β10 docking is the secondary event that might contribute more to the force production rather than the stroke size (velocity) (Fig. 15). Fig. 15. Open in new tabDownload slide Three-step docking of the neck-linker. The docking of the neck-linker to the motor domain of KIF1A (white). An atomic model of a microtubule (grey) shows the orientation. The docked neck-linker is red in the space-filling models and the undocked neck-linker is shown as red-dotted lines. Key structural elements surrounding the neck-linker are shown as ribbon models in the blow-out boxes. The N-terminal β-strands β0 and β1 (blue) and the C-terminal α4 (yellow), α5 (orange) and α6 (brown) helices are shown. (a) After ATP hydrolysis (Mg2+-ADP state), the neck-linker is fully detached from the motor domain. (b) The NIS of the neck-linker then docks to the groove between the N-terminal β-strands (β0 and β1) and the α4 helix during Mg2+ release. The docking of NIS supports the protruded conformation of the α4 helix from the back. This elevated conformation of the α4 helix matches the zigzag surface of the microtubule, such that KIF1A binds strongly to a microtubule with this conformation. (c) Then, the next neck-linker element, β9, docks to the motor domain and forms a rigid triple β-strand structure with the N-terminal strand β0 and loop L13 during the ADP–ATP exchange. (d) Finally, the last neck-linker element, β10, docks to the motor domain after the closure of the nucleotide binding pocket (which is designated by an asterisk). Reproduced from Hirokawa et al. [9]. Fig. 15. Open in new tabDownload slide Three-step docking of the neck-linker. The docking of the neck-linker to the motor domain of KIF1A (white). An atomic model of a microtubule (grey) shows the orientation. The docked neck-linker is red in the space-filling models and the undocked neck-linker is shown as red-dotted lines. Key structural elements surrounding the neck-linker are shown as ribbon models in the blow-out boxes. The N-terminal β-strands β0 and β1 (blue) and the C-terminal α4 (yellow), α5 (orange) and α6 (brown) helices are shown. (a) After ATP hydrolysis (Mg2+-ADP state), the neck-linker is fully detached from the motor domain. (b) The NIS of the neck-linker then docks to the groove between the N-terminal β-strands (β0 and β1) and the α4 helix during Mg2+ release. The docking of NIS supports the protruded conformation of the α4 helix from the back. This elevated conformation of the α4 helix matches the zigzag surface of the microtubule, such that KIF1A binds strongly to a microtubule with this conformation. (c) Then, the next neck-linker element, β9, docks to the motor domain and forms a rigid triple β-strand structure with the N-terminal strand β0 and loop L13 during the ADP–ATP exchange. (d) Finally, the last neck-linker element, β10, docks to the motor domain after the closure of the nucleotide binding pocket (which is designated by an asterisk). Reproduced from Hirokawa et al. [9]. 5.5 Structural model of neck-linker mediated regulation of kinesin The neck-linker is well conserved among N-terminal motor domain-type motile kinesins. Recent structural studies revealed the conservation of not only the three-dimensional structure but also the docking conformation to the catalytic core. Thus, it is natural to assume that the neck-linker has conserved functions required for N-terminal-type kinesins. In fact, our chimaera construct of KIF1A catalytic core with conventional kinesin KIF5C neck-linker was fully functional [74,153]. Considering that the neck-linker is the sole element that links the motor domain to the cargo or the other motor domain in case of dimer, the catalytic core can receive feedbacks from outside through the neck-linker. Our structures suggest the mechanism of this feedback. The initial docking of NIS is required for the ATPase activity, putatively for the Mg release that precedes the ADP to ATP exchange. External load to pull the neck-linker backward (toward minus end) will disfavour the docking of NIS, which will explain the load dependence of the ATPase turnover rate. Kinesin's affinity for nucleotide is dependent on the directionality of an external load [162], and the apparent KD of a kinesin [163] head for ADP is weakened up to 7-fold for rearward versus forward load. The load dependence of the ATP binding to kinesin would reflect the coupling of the docking of β10 to the closure of the pocket to capture Mg–ATP (isomerization). Thus, in short, the backward load (pull to the minus end) will disfavour both ADP release and ATP binding (Fig. 15). This load dependence will enable kinesin to work cooperatively. Some kinesins especially those involved in mitosis are expected to work in clusters, where multiple kinesins are bound to the same cargo and to the same microtubule. KIF1A is also supposed to form clusters by the C-terminal PH domain that binds to the lipid microdomain on the cargo vesicles [149]. It was experimentally demonstrated that formation of cluster on the liposome [149] or on the plastic beads [74] significantly improves the motility of KIF1A, both in velocity and in force. The regulatory function of the neck-linker would contribute to this cooperative behaviour. When the neck-linker is pulled forward (toward plus end) by the other kinesin or by the movement of cargo, ATPase reaction and the detachment from the microtubule are facilitated. When pulled backward (towards minus end), ATPase reaction is retarded and kinesin binds tightly to the microtubule. Thus, the forward movement of each head would be synchronized by this neck-linker-mediated regulation. We would also like to point out here that KIF1A is a low duty-ratio motor like muscle myosin. In physiological conditions with abundant ATP, KIF1A is in the diffusional binding state (ADP state) for >90% of the cycle time [73]. Thus, each KIF1A motor domain does not produce much drag force to other motor domains, but still it keeps on the track of the microtubule. This is very suitable to work cooperatively in a cluster. KIF1A might have evolved to work efficiently in a cluster on the vesicle surface. 5.6 Neck-liner-based structural model for the gating in dimeric kinesin The same neck-linker is conserved in dimeric conventional kinesin, which is a high duty-ratio motor and moves by the hand-over-hand mechanism. As discussed above, the sequence and the structure are well conserved. Chimaera constructs also guarantee the functional conservation. Therefore, we would like to apply our structural model to the hand-over-hand movement (Fig. 15). The hand-over-hand processive movement of dimeric kinesin requires communication between the heads to synchronize their cycles to maintain them out of phase. In fact, kinetic data support the importance of inter-head communication for the processive run [163–168]. The mechanical strain that develops between heads during stepping is the most plausible mechanism for this inter-head communication. The regulatory aspect of the neck-linker docking discussed above is thus a good candidate. When one tries to put a dimeric kinesin on microtubule protofilament, one will immediately notice that the length of the neck-linker of dimeric conventional kinesin of animals is just long enough to allow both motor domains to bind to β tubulins 8 nm apart. The length limitation is too tight to allow the full docking of the neck-linker to the leading head without detaching the lagging head from the microtubule. Thus, the leading head should wait for the lagging head to hydrolyze ATP to detach from microtubule, which explains the strain gating model [164–166]. As exemplified here, the length limitation of the neck-linker allows only three pairs of neck-linker conformations: (i) NIS and β9 docked in the lagging head, undocked in the leading head, (ii) NIS to β10 docked, undocked and (iii) NIS to β10 docked, NIS docked. As discussed above, we propose that the neck-linker conformation is coupled with the nucleotide state in the pocket. Then, the possible nucleotide states of the lagging and the leading head in the two-head-binding state are (Apo, ADP), (ATP, ADP) and (ATP, ADP-release). This limitation explains that the ADP release at the leading head waits for the binding of ATP to the lagging head, the gating behaviour called ‘ATP-gating’ [163,167,168]. Thus, this simple model explains the inter-head communication, which should be tested with the neck-length mutants [169]. 6. Conclusion Thus, the structures of KIF1A kinesin give us much insight on the mechanisms of kinesins, not restricted to this specific kinesin but applicable to other kinesins in general. Several putative structural models are suggested from these structures, which will be waiting for the experimental challenge. Recent experiments revealed family-specific features that will enable each kinesin to undertake its specific physiological functions. Comparisons of their structures will enable us to understand the basis of such functional divergence, and will also allow us to design therapeutics to regulate specific kinesin functions involved in pathophysiology of various diseases such as cancer and neurological disorders. References 1 Hirokawa N . Quick-freeze, deep-etch electron microscopy , J. Electron Microsc. , 1989 , vol. 38 suppl.) (pg. s123 - s128 ) WorldCat 2 Hirokawa N . Organelle transport along microtubule – the role of KIFs (Kinesin superfamily proteins) , Trend Cell Biol. , 1996 , vol. 6 (pg. 135 - 141 ) Google Scholar Crossref Search ADS WorldCat Crossref 3 Hirokawa N . Kinesin and dynein superfamily proteins and the mechanism of organelle transport , Science , 1998 , vol. 279 (pg. 519 - 526 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 4 Hirokawa N , Takemura R . Molecular motors and mechanisms of directional transport in neurons , Nat. Rev. Neurosci. , 2005 , vol. 6 (pg. 201 - 214 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 5 Hirokawa N , Tanaka Y , Okada Y , Takeda S . Nodal flow and the generation of left–right asymmetry , Cell , 2006 , vol. 125 (pg. 33 - 45 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 6 Hirokawa N , Noda Y . Intracellular transport and kinesin superfamily proteins, KIFs: structure, function, and dynamics , Physiol. Rev. , 2008 , vol. 88 (pg. 1089 - 1118 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 7 Hirokawa N , Noda Y , Tanaka Y , Niwa S . Kinesin superfamily motor proteins and intracellular transport , Nat. Rev. Mol. Cell Biol. , 2009 , vol. 10 (pg. 682 - 696 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 8 Hirokawa N , Tanaka Y , Okada Y . Left-right determination: involvement of molecular motor KIF3, cilia, and nodal flow , Cold Spring Harbor Perspect. Biol. , 2009 , vol. 1 pg. a000802 Google Scholar Crossref Search ADS WorldCat Crossref 9 Hirokawa N , Nitta R , Okada Y . The mechanisms of kinesin motor motility: lessons from the monomeric motor KIF1A , Nat. Rev. Mol. Cell Biol. , 2009 , vol. 10 (pg. 877 - 884 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 10 Hirokawa N , Niwa S , Tanaka Y . Molecular motors in neurons: transport mechanisms and roles in brain function, development and disease , Neuron , 2010 , vol. 18 (pg. 610 - 638 ) Google Scholar Crossref Search ADS WorldCat Crossref 11 Van Harreveld A , Khattab F I . Perfusion fixation with glutaraldehyde and post-fixation with osmium tetroxide for electron microscopy , J. Cell Sci. , 1968 , vol. 3 (pg. 579 - 584 ) Google Scholar PubMed WorldCat 12 Van Harreveld A , Khattab F I . Changes in extracellular space of the mouse cerebral cortex during hydroxyadipaldehyde fixation and osmium tetroxide postfixation , J. Cell Sci. , 1969 , vol. 4 (pg. 437 - 453 ) Google Scholar PubMed WorldCat 13 Sandri C , Van Buren J M , Akert K . Membrane morphology of the vertebrate nervous system , Prog. Brain Res. , 1977 , vol. 46 (pg. 1 - 11 ) Google Scholar PubMed WorldCat Crossref 14 Bullivant S . Freeze substitution and supporting techniques , Lab. Invest. , 1965 , vol. 14 (pg. 440 - 457 ) WorldCat 15 Rebhun L I . Freeze-substitution: fine structure as a function of water concentration in cells , Feder. Proc. , 1965 , vol. 24 Suppl. 15) (pg. s217 - s236 ) WorldCat 16 Dempsey G P , Bullivant S . A copper block method for freezing non-cryoprotected tissue to produce ice-crystal-free regions for electron microscopy. I. Evaluation using freeze-substitution , J. Microscopy , 1976 , vol. 106 (pg. 251 - 260 ) Google Scholar Crossref Search ADS WorldCat 17 Fernandez-Moran H . Low-temperature preparation techniques for electron microscopy of biological specimens based on rapid freezing with liquid helium II , Ann. N. Y. Acad. Sci. , 1960 , vol. 85 (pg. 689 - 713 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 18 Hirokawa N . Disappearance of afferent and efferent nerve terminals in the inner ear of the chick embryo after chronic treatment with beta-bungarotoxin , J. Cell Biol. , 1977 , vol. 73 (pg. 27 - 46 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 19 Hirokawa N , Kitamura M . Binding of Clostridium botulinum neurotoxin to the presynaptic membrane in the central nervous system , J. Cell Biol. , 1979 , vol. 81 (pg. 43 - 49 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 20 Kirino T , Hirokawa N . Electron microscopic observation of the central nervous system observed by freeze-substitution method , J. Electron Microsc. , 1978 , vol. 27 pg. 339 WorldCat 21 Hirokawa N , Kirino T . An ultrastructural study of nerve and glial cells by freeze-substitution , J. Neurocytol. , 1980 , vol. 9 (pg. 243 - 254 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 22 Hirokawa N . Quick freeze, deep etch of the cytoskeleton , Method Enzymol. , 1986 , vol. 134 (pg. 598 - 612 ) WorldCat Crossref 23 Heuser J E , Reese T S , Dennis M J , Jan Y , Jan L , Evans L . Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release , J. Cell Biol. , 1979 , vol. 81 (pg. 275 - 300 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 24 Hirokawa N , Heuser J E . Structural evidence that botulinum toxin blocks neuromuscular transmission by impairing the calcium influx that normally accompanies nerve depolarization , J. Cell Biol. , 1981 , vol. 88 (pg. 160 - 171 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 25 Heuser J E , Salpeter S R . Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane , J. Cell Biol. , 1979 , vol. 82 (pg. 150 - 173 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 26 Hirokawa N , Heuser J E . Internal and external differentiations of the postsynaptic membrane at the neuromuscular junction , J. Neurocyt. , 1982 , vol. 11 (pg. 487 - 510 ) Google Scholar Crossref Search ADS WorldCat Crossref 27 Hirokawa N , Heuser J E . The inside and outside of gap-junction membranes visualized by deep etching , Cell , 1982 , vol. 30 (pg. 395 - 406 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 28 Hirokawa N , Heuser J E . Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells , J. Cell Biol. , 1981 , vol. 91 (pg. 399 - 409 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 29 Hirokawa N , Tilney L G , Fujiwara K , Heuser J E . Organization of actin, myosin, and intermediate filaments in the brush border of intestinal epithelial cells , J. Cell Biol. , 1982 , vol. 94 (pg. 425 - 443 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 30 Hirokawa N , Cheney R E , Willard M . Location of a protein of the fodrin-spectrin-TW260/240 family in the mouse intestinal brush border , Cell , 1983 , vol. 32 (pg. 953 - 965 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 31 Hirokawa N , Tilney L G . Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear , J. Cell Biol. , 1982 , vol. 95 (pg. 249 - 261 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 32 Hirokawa N , Keller T C III , Chasan R , Mooseker M S . Mechanism of brush border contractility studied by the quick-freeze, deep-etch method , J. Cell Biol. , 1983 , vol. 96 (pg. 1325 - 1336 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 33 Hirokawa N . Cross-linker system between neurofilaments, microtubules, and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method , J. Cell Biol. , 1982 , vol. 94 (pg. 129 - 142 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 34 Hirokawa N , Glicksman M A , Willard M B . Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton , J. Cell Biol. , 1984 , vol. 98 (pg. 1523 - 1536 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 35 Hirokawa N . Quick-freeze, deep-etch visualization of the axonal cytoskeleton , Trend. Neurosci. , 1986 , vol. 9 (pg. 67 - 70 ) Google Scholar Crossref Search ADS WorldCat Crossref 36 Hirokawa N , Yorifuji H . Cytoskeletal architecture in reactivated crayfish axons, with special reference to crossbridges among microtubules and between microtubule membrane organelles , Cell Mot. Cytoskel. , 1986 , vol. 6 (pg. 458 - 468 ) Google Scholar Crossref Search ADS WorldCat Crossref 37 Shiomura Y , Hirokawa N . The molecular structure of microtubule-associated protein 1A (MAP1A) in vivo and in vitro. An immunoelectron microscopy and quick-freeze, deep-etch study , J. Neurosci. , 1987 , vol. 7 (pg. 1461 - 1469 ) Google Scholar PubMed WorldCat 38 Hirokawa N , Hisanaga S , Shiomura Y . MAP2 is a component of crossbridges between microtubules and neurofilaments in the neuronal cytoskeleton: quick-freeze, deep-etch immunoelectron microscopy and reconstitution studies , J. Neurosci. , 1988 , vol. 8 (pg. 2769 - 2779 ) Google Scholar PubMed WorldCat 39 Hirokawa N , Shiomura Y , Okabe S . Tau proteins: the molecular structure and mode of binding on microtubules , J. Cell Biol. , 1988 , vol. 107 (pg. 1449 - 1459 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 40 Sato-Yoshitake R , Shiomura Y , Miyasaka H , Hirokawa N . Microtubule-associated protein 1B: molecular structure, localization, and phosphorylation-dependent expression in developing neurons , Neuron , 1989 , vol. 3 (pg. 229 - 238 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 41 Shiomura Y , Hirokawa N . Colocalization of microtubule-associated protein 1A and microtubule-associated protein 2 on neuronal microtubules in situ revealed with double-label immunoelectron microscopy , J. Cell Biol. , 1987 , vol. 104 (pg. 1575 - 1578 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 42 Kanai Y , Takemura R , Oshima T , Mori H , Ihara Y , Yanagisawa M , Masaki T , Hirokawa N . Expression of multiple tau isoforms and microtubule bundle formation in fibroblasts transfected with a single tau cDNA , J. Cell Biol. , 1989 , vol. 109 (pg. 1173 - 1184 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 43 Chen J , Kanai Y , Cowan N J , Hirokawa N . Projection domains of MAP2 and tau determine spacings between microtubules in dendrites and axons , Nature , 1992 , vol. 360 (pg. 674 - 677 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 44 Harada A , Oguchi K , Okabe S , Kuno J , Terada S , Ohshima T , Sato-Yoshitake R , Takei Y , Noda T , Hirokawa N . Altered microtubule organization in small-calibre axons of mice lacking tau protein , Nature , 1994 , vol. 369 (pg. 488 - 491 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 45 Harada A , Teng J , Takei Y , Oguchi K , Hirokawa N . MAP2 is required for dendrite elongation, PKA anchoring in dendrites, and proper PKA signal transduction , J. Cell Biol. , 2002 , vol. 158 (pg. 541 - 549 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 46 Takei Y , Kondo S , Harada A , Inomata S , Noda T , Hirokawa N . Delayed development of nervous system in mice homozygous for disrupted microtubule-associated protein 1B (MAP1B) gene , J. Cell Biol. , 1997 , vol. 137 (pg. 1615 - 1626 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 47 Takei Y , Teng J , Harada A , Hirokawa N . Defects in axonal elongation and neuronal migration in mice with disrupted tau and map1b genes , J. Cell Biol. , 2000 , vol. 150 (pg. 989 - 1000 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 48 Teng J , Takei Y , Harada A , Nakata T , Chen J , Hirokawa N . Synergistic effects of MAP2 and MAP1B knockout in neuronal migration, dendritic outgrowth, and microtubule organization , J. Cell Biol. , 2001 , vol. 155 (pg. 65 - 76 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 49 Nakagawa T , Chen J , Zhang Z , Kanai Y , Hirokawa N . Two distinct functions of the carboxyl-terminal tail domain of NF-M upon neurofilament assembly: cross-bridge formation and longitudinal elongation of filaments , J. Cell Biol. , 1995 , vol. 129 (pg. 411 - 429 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 50 Chen J , Nakata T , Zhang Z , Hirokawa N . The C-terminal tail domain of neurofilament protein-H (NF-H) forms the crossbridges and regulates neurofilament bundle formation , J. Cell Sci. , 2000 , vol. 113 Pt 21 (pg. 3861 - 3869 ) Google Scholar PubMed WorldCat 51 Okabe S , Hirokawa N . Rapid turnover of microtubule-associated protein MAP2 in the axon revealed by microinjection of biotinylated MAP2 into cultured neurons , Proc. Natl Acad. Sci. USA , 1989 , vol. 86 (pg. 4127 - 4131 ) Google Scholar Crossref Search ADS WorldCat Crossref 52 Okabe S , Hirokawa N . Incorporation and turnover of biotin-labeled actin microinjected into fibroblastic cells: an immunoelectron microscopic study , J. Cell Biol. , 1989 , vol. 109 Pt 1 (pg. 1581 - 1595 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 53 Okabe S , Hirokawa N . Turnover of fluorescently labelled tubulin and actin in the axon , Nature , 1990 , vol. 343 (pg. 479 - 482 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 54 Okabe S , Hirokawa N . Actin dynamics in growth cones , J. Neurosci. , 1991 , vol. 11 (pg. 1918 - 1929 ) Google Scholar PubMed WorldCat 55 Okabe S , Hirokawa N . Differential behavior of photoactivated microtubules in growing axons of mouse and frog neurons , J. Cell Biol. , 1992 , vol. 117 (pg. 105 - 120 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 56 Okabe S , Hirokawa N . Do photobleached fluorescent microtubules move?: re-evaluation of fluorescence laser photobleaching both in vitro and in growing Xenopus axon , J. Cell Biol. , 1993 , vol. 120 (pg. 1177 - 1186 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 57 Okabe S , Miyasaka H , Hirokawa N . Dynamics of the neuronal intermediate filaments , J. Cell Biol. , 1993 , vol. 121 (pg. 375 - 386 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 58 Takeda S , Okabe S , Funakoshi T , Hirokawa N . Differential dynamics of neurofilament-H protein and neurofilament-L protein in neurons , J. Cell Biol. , 1994 , vol. 127 (pg. 173 - 185 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 59 Takeda S , Funakoshi T , Hirokawa N . Tubulin dynamics in neuronal axons of living zebrafish embryos , Neuron , 1995 , vol. 14 (pg. 1257 - 1264 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 60 Funakoshi T , Takeda S , Hirokawa N . Active transport of photoactivated tubulin molecules in growing axons revealed by a new electron microscopic analysis , J. Cell Biol. , 1996 , vol. 133 (pg. 1347 - 1353 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 61 Terada S , Nakata T , Peterson A C , Hirokawa N . Visualization of slow axonal transport in vivo , Science , 1996 , vol. 273 (pg. 784 - 788 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 62 Nakata T , Iwamoto A , Noda Y , Takemura R , Yoshikura H , Hirokawa N . Predominant and developmentally regulated expression of dynamin in neurons , Neuron , 1991 , vol. 7 (pg. 461 - 469 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 63 Aizawa H , Sekine Y , Takemura R , Zhang Z , Nangaku M , Hirokawa N . Kinesin family in murine central nervous system , J. Cell Biol. , 1992 , vol. 119 (pg. 1287 - 1296 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 64 Nakagawa T , Tanaka Y , Matsuoka E , Kondo S , Okada Y , Noda Y , Kanai Y , Hirokawa N . Identification and classification of 16 new kinesin superfamily (KIF) proteins in mouse genome , Proc. Natl Acad. Sci. USA , 1997 , vol. 94 (pg. 9654 - 9659 ) Google Scholar Crossref Search ADS WorldCat Crossref 65 Miki H , Setou M , Kaneshiro K , Hirokawa N . All kinesin superfamily protein, KIF, genes in mouse and human , Proc. Natl Acad. Sci. USA , 2001 , vol. 98 (pg. 7004 - 7011 ) Google Scholar Crossref Search ADS WorldCat Crossref 66 Lawrence C J , Dawe R K , Christie K R , Cleveland D W , Dawson S C , Endow S A , Goldstein L S B , Goodson H V , Hirokawa N , Howard J , Malmberg R L , McIntosh J R , Miki H , Mitchison T J , Okada Y , Reddy A S N , Saxton W M , Schliwa M , Scholey J M , Vale R D , Walczak C E , Wordeman L . A standardized kinesin nomenclature , J. Cell Biol. , 2004 , vol. 167 (pg. 19 - 22 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 67 Okada Y , Yamazaki H , Sekine-Aizawa Y , Hirokawa N . The neuron-specific kinesin superfamily protein KIF1A is a unique monomeric motor for anterograde axonal transport of synaptic vesicle precursors , Cell , 1995 , vol. 81 (pg. 769 - 780 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 68 Zhao C , Takita J , Tanaka Y , Setou M , Nakagawa T , Takeda S , Yang H W , Terada S , Nakata T , Takei Y , Saito M , Tsuji S , Hayashi Y , Hirokawa N . Charcot–Marie–Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bbeta , Cell , 2001 , vol. 105 (pg. 587 - 597 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 69 Niwa S , Tanaka Y , Hirokawa N . KIF1Bbeta- and KIF1A-mediated axonal transport of presynaptic regulator Rab3 occurs in a GTP-dependent manner through DENN/MADD , Nat. Cell Biol. , 2008 , vol. 11 (pg. 1270 - 1276 ) WorldCat 70 Shin H , Wyszynski M , Huh K , Valtschanoff J , Lee J , Ko J , Streuli M , Weinberg R , Sheng M , Kim E . Association of the kinesin motor KIF1A with the multimodular protein liprin-alpha , J. Biol. Chem. , 2003 , vol. 278 (pg. 11393 - 11401 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 71 Wagner O , Esposito A , Köhler B , Chen C , Shen C , Wu G , Butkevich E , Mandalapu S , Wenzel D , Wouters F , Kloptenstein D . Synaptic scaffolding protein SYD-2 clusters and activates kinesin-3 UNC-104 in C. elegans , Proc. Natl Acad. Sci. USA , 2009 , vol. 106 (pg. 19605 - 19610 ) Google Scholar Crossref Search ADS WorldCat Crossref 72 Okada Y , Hirokawa N . A processive single-headed motor: kinesin superfamily protein KIF1A , Science , 1999 , vol. 283 (pg. 1152 - 1157 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 73 Okada Y , Hirokawa N . Mechanism of the single-headed processivity: diffusional anchoring between the K-loop of kinesin and the C terminus of tubulin , Proc. Natl Acad. Sci. USA , 2000 , vol. 97 (pg. 640 - 645 ) Google Scholar Crossref Search ADS WorldCat Crossref 74 Okada Y , Higuchi H , Hirokawa N . Processivity of the single-headed kinesin KIF1A through biased binding to tubulin , Nature , 2003 , vol. 424 (pg. 574 - 577 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 75 Nangaku M , Sato-Yoshitake R , Okada Y , Noda Y , Takemura R , Yamazaki H , Hirokawa N . KIF1B, a novel microtubule plus end-directed monomeric motor protein for transport of mitochondria , Cell , 1994 , vol. 79 (pg. 1209 - 1220 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 76 Wozniak M J , Melzer M , Dorner C , Haring H-U , Lammers R . The novel protein KBP regulates mitochondria localization by interaction with a kinesin-like protein , BMC Cell Biol. , 2005 , vol. 6 pg. 35 Google Scholar Crossref Search ADS PubMed WorldCat Crossref 77 Brady S T . A novel brain ATPase with properties expected for the fast axonal transport motor , Nature , 1985 , vol. 317 (pg. 73 - 75 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 78 Vale R , Reese T , Sheetz M . Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility , Cell , 1985 , vol. 42 (pg. 39 - 50 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 79 Hirokawa N , Pfister K K , Yorifuji H , Wagner M C , Brady S T , Bloom G S . Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration , Cell , 1989 , vol. 56 (pg. 867 - 878 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 80 Kanai Y , Okada Y , Tanaka Y , Harada A , Terada S , Hirokawa N . KIF5C, a novel neuronal kinesin enriched in motor neurons , J. Neurosci. , 2000 , vol. 20 (pg. 6374 - 6384 ) Google Scholar PubMed WorldCat 81 Nakata T , Terada S , Hirokawa N . Visualization of the dynamics of synaptic vesicle and plasma membrane proteins in living axons , J. Cell Biol. , 1998 , vol. 140 (pg. 659 - 674 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 82 Tanaka Y , Kanai Y , Okada Y , Nonaka S , Takeda S , Harada A , Hirokawa N . Targeted disruption of mouse conventional kinesin heavy chain, kif5B, results in abnormal perinuclear clustering of mitochondria , Cell , 1998 , vol. 93 (pg. 1147 - 1158 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 83 Bowman A , Kamal A , Ritchings B , Philp A , McGrail M , Gindhart J , Goldstein L . Kinesin-dependent axonal transport is mediated by the sunday driver (SYD) protein , Cell , 2000 , vol. 103 (pg. 583 - 594 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 84 Kamal A , Almenar-Queralt A , LeBlanc J , Roberts E , Goldstein L . Kinesin-mediated axonal transport of a membrane compartment containing beta-secretase and presenilin-1 requires APP , Nature , 2001 , vol. 414 (pg. 643 - 648 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 85 Muresan Z , Muresan V . Coordinated transport of phosphorylated amyloid-beta precursor protein and c-Jun NH2-terminal kinase-interacting protein-1 , J. Cell Biol. , 2005 , vol. 171 (pg. 615 - 625 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 86 Muresan V , Varvel N , Lamb B , Muresan Z . The cleavage products of amyloid-beta precursor protein are sorted to distinct carrier vesicles that are independently transported within neurites , J. Neurosci. , 2009 , vol. 29 (pg. 3565 - 3578 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 87 Cai Q , Gerwin C , Sheng Z . Syntabulin-mediated anterograde transport of mitochondria along neuronal processes , J. Cell Biol. , 2005 , vol. 170 (pg. 959 - 969 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 88 Cai Q , Pan P , Sheng Z . Syntabulin-kinesin-1 family member 5B-mediated axonal transport contributes to activity-dependent presynaptic assembly , J. Neurosci. , 2007 , vol. 27 (pg. 7284 - 7296 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 89 Hirokawa N , Sato-Yoshitake R , Yoshida T , Kawashima T . Brain dynein (MAP1C) localizes on both anterogradely and retrogradely transported membranous organelles in vivo , J. Cell Biol. , 1990 , vol. 111 (pg. 1027 - 1037 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 90 Hirokawa N , Sato-Yoshitake R , Kobayashi N , Pfister K K , Bloom G S , Brady S T . Kinesin associates with anterogradely transported membranous organelles in vivo , J. Cell Biol. , 1991 , vol. 114 (pg. 295 - 302 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 91 Yamada M , Toba S , Takitoh T , Yoshida Y , Mori D , Nakamura T , Iwane A , Yanagida T , Imai H , Yu-Lee L , et al. mNUDC is required for plus-end-directed transport of cytoplasmic dynein and dynactins by kinesin-1 , EMBO J. , 2010 , vol. 29 (pg. 517 - 531 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 92 Yamada M , Toba S , Yoshida Y , Haratani K , Mori D , Yano Y , Mimori-Kiyosue Y , Nakamura T , Itoh K , Fushiki S , et al. LIS1 and NDEL1 coordinate the plus-end-directed transport of cytoplasmic dynein , EMBO J. , 2008 , vol. 27 (pg. 2471 - 2483 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 93 Terada S , Kinjo M , Hirokawa N . Oligomeric tubulin in large transporting complex is transported via kinesin in squid giant axons , Cell , 2000 , vol. 103 (pg. 141 - 155 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 94 Xia C , Roberts E , Her L , Liu X , Williams D , Cleveland D , Goldstein L . Abnormal neurofilament transport caused by targeted disruption of neuronal kinesin heavy chain KIF5A , J. Cell Biol. , 2003 , vol. 161 (pg. 55 - 66 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 95 Roy S , Winton M , Black M , Trojanowski J , Lee V . Cytoskeletal requirements in axonal transport of slow component-b , J. Neurosci. , 2008 , vol. 28 (pg. 5248 - 5256 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 96 Terada S , Kinjo M , Aihara M , Takei Y , Hirokawa N . Kinesin-1 Hsc/70-dependent mechanism of slow axonal and its relation to fast axonal transport , EMBO J. , 2010 , vol. 29 (pg. 843 - 854 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 97 Setou M , Seog D-H , Tanaka Y , Kanai Y , Takei Y , Kawagishi M , Hirokawa N . Glutamate-receptor-interacting protein GRIP1 directly steers kinesin to dendrites , Nature , 2002 , vol. 417 (pg. 83 - 87 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 98 Nakata T , Hirokawa N . Microtubules provide directional cues for polarized axonal transport through interaction with kinesin motor head , J. Cell Biol. , 2003 , vol. 162 (pg. 1045 - 1055 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 99 Twelvetrees A , Yuen E , Arancibia-Carcamo I , MacAskill A , Rostaing P , Lumb M , Humbert S , Triller A , Saudou F , Yan Z , et al. Delivery of GABAARs to synapses is mediated by HAP1-KIF5 and disrupted by mutant huntingtin , Neuron , 2010 , vol. 65 (pg. 53 - 65 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 100 Kanai Y , Dohmae N , Hirokawa N . Kinesin transports RNA: isolation and characterization of an RNA-transporting granule , Neuron , 2004 , vol. 43 (pg. 513 - 525 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 101 Setou M , Nakagawa T , Seog D-H , Hirokawa N . Kinesin superfamily motor protein KIF17 and mlin-10 in NMDA receptor-containing vesicle transport , Science , 2000 , vol. 288 (pg. 1693 - 1920 ) Google Scholar Crossref Search ADS WorldCat 102 Guillaud L , Wong R , Hirokawa N . Disruption of KIF17-Mint1 interation by CamKII-dependent phosphorylation: a molecular model of kinesin-cargo release , Nat. Cell Biol. , 2008 , vol. 10 (pg. 19 - 29 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 103 Sato-Yoshitake R , Yorifuji H , Inagaki M , Hirokawa N . The phosphorylation of kinesin regulates its binding to synaptic vesicles , J. Cell Biol. , 1992 , vol. 267 (pg. 23930 - 23936 ) WorldCat 104 Saito N , Okada Y , Noda Y , Kinoshita Y , Kondo S , Hirokawa N . KIFC2 is a novel neuron-specific C-terminal type kinesin superfamily motor for dendritic transport of multivesicular body-like organelles , Neuron , 1997 , vol. 18 (pg. 425 - 438 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 105 Hanlon D , Yang Z , Goldstein L . Characterization of KIFC2, a neuronal kinesin superfamily member in mouse , Neuron , 1997 , vol. 18 (pg. 439 - 451 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 106 Jacobson C , Schnapp B , Banker G . A change in the selective translocation of the kinesin-1 motor domain marks the initial specification of the axon , Neuron , 2006 , vol. 49 (pg. 797 - 804 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 107 Reed N , Cai D , Blasius T , Jih G , Meyhofer E , Gaertig J , Verhey K . Microtubule acetylation promotes kinesin-1 binding and transport , Curr. Biol. , 2006 , vol. 16 (pg. 2166 - 2172 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 108 Konishi Y , Setou M . Tubulin tyrosination navigates the kinesin-1 motor domain to axons , Nat. Neurosci. , 2009 , vol. 12 (pg. 559 - 567 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 109 Dotti C , Banker G . Intracellular organization of hippocampal neurons during the development of neuronal polarity , J. Cell Sci. , 1991 , vol. 15 (pg. 75 - 84 ) Google Scholar Crossref Search ADS WorldCat 110 Hammond J , Huang C , Kaech S , Jacobson C , Banker G , Verhey K . Posttranslational modifications of tubulin and the polarized transport of kinesin-1 in neurons , Mol. Biol. Cell , 2010 , vol. 21 (pg. 572 - 583 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 111 Kapitein L , Schlager M , Kuijpers M , Wulf P , van Spronsen M , MacKintosh F , Hoogenraad C . Mixed microtubules steer dynein-driven cargo transport into dendrites , Curr. Biol. , 2010 , vol. 20 (pg. 290 - 299 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 112 Song A , Wang D , Chen G , Li Y , Luo J , Duan S , Poo M . A selective filter for cytoplasmic transport at the axon initial segment , Cell , 2009 , vol. 136 (pg. 1148 - 1160 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 113 Yonekawa Y , Harada A , Okada Y , Funakoshi T , Kanai Y , Takei Y , Terada S , Noda T , Hirokawa N . Defect in synaptic vesicle precursor transport and neuronal cell death in KIF1A motor protein-deficient mice , J. Cell Biol. , 1998 , vol. 141 (pg. 431 - 441 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 114 Chevalier-Larsen E , Holzbaur E L . Axonal transport and neurodegenerative disease , Biochim. Biophys. Acta , 2006 , vol. 1762 (pg. 1094 - 1108 ) Google Scholar Crossref Search ADS PubMed WorldCat 115 De Vos K J , Grierson A J , Ackerley S , Miller C C . Role of axonal transport in neurodegenerative diseases , Ann. Rev. Neurosci. , 2008 , vol. 31 (pg. 151 - 173 ) Google Scholar Crossref Search ADS WorldCat Crossref 116 Roy S , Zhang B , Lee V M , Trojanowski J Q . Axonal transport defects: a common theme in neurodegenerative diseases , Acta Neuropathol. , 2005 , vol. 109 (pg. 5 - 13 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 117 Wong R W-C , Setou M , Teng J , Takei Y , Hirokawa N . Overexpression of motor protein KIF17 enhances spatial and working memory in transgenic mice , Proc. Natl Acad. Sci. USA , 2002 , vol. 99 (pg. 14500 - 14505 ) Google Scholar Crossref Search ADS WorldCat Crossref 118 Yin X , Takei Y , Kido M , Hirokawa N . Molecular motor KIF17 is fundamental for memory and learning via differential support of synaptic NR2A.2B levels , Neuron , 2011 , vol. 70 (pg. 310 - 325 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 119 Kondo S , Sato-Yoshitake R , Noda Y , Aizawa H , Nakata T , Matsuura Y , Hirokawa N . KIF3A is a new microtubule-based anterograde motor in the nerve axon , J. Cell Biol. , 1994 , vol. 125 (pg. 1095 - 1107 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 120 Yamazaki H , Nakata T , Okada Y , Hirokawa N . KIF3A/B: a heterodimeric kinesin superfamily protein that works as a microtubule plus end-directed motor for membrane organelle transport , J. Cell Biol. , 1995 , vol. 130 (pg. 1387 - 1399 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 121 Yamazaki H , Nakata T , Okada Y , Hirokawa N . Cloning and characterization of KAP3: a novel kinesin superfamily-associated protein of KIF3A/3B , Proc. Natl Acad. Sci. USA , 1996 , vol. 93 (pg. 8443 - 8448 ) Google Scholar Crossref Search ADS WorldCat Crossref 122 Rosenbaum J , Witman G . Intraflagellar transport , Nat. Rev. Mol. Cell Biol. , 2002 , vol. 3 (pg. 813 - 825 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 123 Takeda S , Yamazaki H , Seog D-H , Kanai Y , Terada S , Hirokawa N . Kinesin superfamily protein 3 (KIF3) motor transports fodrin-associating vesicles important for neurite building , J. Cell Biol. , 2000 , vol. 148 (pg. 1255 - 1265 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 124 Teng J , Rai T , Tanaka Y , Takei Y , Nakata T , Hirasawa M , Kulkarni A B , Hirokawa N . The KIF3 motor transports N-cadherin and organizes the developing neuroepithelium , Nat. Cell Biol. , 2005 , vol. 7 (pg. 474 - 482 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 125 Nonaka S , Tanaka Y , Okada Y , Takeda S , Harada A , Kanai Y , Kido M , Hirokawa N . Randomization of left–right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein , Cell , 1998 , vol. 95 (pg. 829 - 837 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 126 Takeda S , Yonekawa Y , Tanaka Y , Okada Y , Nonaka S , Hirokawa N . Left-right asymmetry and kinesin superfamily protein KIF3A: new insights in determination of laterality and mesoderm induction by kif3a-/- mice analysis , J. Cell Biol. , 1999 , vol. 145 (pg. 825 - 836 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 127 Marszalek J R , Ruiz-Lozano P , Roberts E , Chien K R , Goldstein L S . Situs inversus and embryonic ciliary morphogenesis defects in mouse mutants lacking the KIF3A subunit of kinesin-II , Proc. Natl Acad. Sci. USA , 1999 , vol. 96 (pg. 5043 - 5048 ) Google Scholar Crossref Search ADS WorldCat Crossref 128 Okada Y , Takeda S , Tanaka Y , Belmonte J-C I , Hirokawa N . Mechanism of nodal flow: a conserved symmetry breaking event in left–right axis determination , Cell , 2005 , vol. 121 (pg. 633 - 644 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 129 Tanaka Y , Okada Y , Hirokawa N . FGF-induced vesicular release of Sonic hedgehog and retinoic acid in leftward nodal flow is critical for left–right determination , Nature , 2005 , vol. 435 (pg. 172 - 177 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 130 Noda Y , Sato-Yoshitake R , Kondo S , Nangaku M , Hirokawa N . KIF2 is a new microtubule-based anterograde motor that transports membranous organelles distinct from those carried by kinesin heavy chain or KIF3A/B , J. Cell Biol. , 1995 , vol. 129 (pg. 157 - 167 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 131 Homma N , Takei Y , Tanaka Y , Nakata T , Terada S , Kikkawa M , Noda Y , Hirokawa N . Kinesin superfamily protein 2A (KIF2A) functions in suppression of collateral branch extension , Cell , 2003 , vol. 114 (pg. 229 - 239 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 132 Ogawa T , Nitta R , Okada Y , Hirokawa N . A common mechanism for microtubule destabilizers—M-type kinesins stabilize curling of the protofilament using the class-specific neck and loops , Cell , 2004 , vol. 116 (pg. 591 - 602 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 133 Zhou R , Niwa S , Homma N , Takei Y , Hirokawa N . KIF26A is an unconventional kinesin and regulates GDNF-Ret signaling in enteric neuronal development , Cell , 2009 , vol. 139 (pg. 802 - 813 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 134 Sekine Y , Okada Y , Noda Y , Kondo S , Aizawa H , Takemura R , Hirokawa N . A novel microtubule-based motor protein (KIF4) for organelle transports, whose expression is regulated developmentally , J. Cell Biol. , 1994 , vol. 127 (pg. 187 - 201 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 135 Midorikawa R , Takei Y , Hirokawa N . KIF4 motor regulates activity-dependent neuronal survival by suppressing PARP-1 enzymatic activity , Cell , 2006 , vol. 125 (pg. 371 - 383 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 136 Macho B , Brancorsini S , Fimia G M , Setou M , Hirokawa N , Sassone-Corsi P . CREM-dependent transcription in male germ cells controlled by a kinesin , Science , 2002 , vol. 298 (pg. 2388 - 2390 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 137 Ueno H , Huang X , Tanaka Y , Hirokawa N . KIF16B/Rab14 molecular motor complex is critical for early embryonic development by transporting FGF receptor , Develop. Cell , 2011 , vol. 20 (pg. 60 - 71 ) Google Scholar Crossref Search ADS WorldCat Crossref 138 Howard J , Hudspeth A J , Vale R D . Movement of microtubules by single kinesin molecules , Nature , 1989 , vol. 342 (pg. 154 - 158 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 139 Block S M , Goldstein L S , Schnapp B J . Bead movement by single kinesin molecules studied with optical tweezers , Nature , 1990 , vol. 348 (pg. 348 - 352 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 140 Svoboda K , Schmidt C F , Schnapp B J , Block S M . Direct observation of kinesin stepping by optical trapping interferometry , Nature , 1993 , vol. 365 (pg. 721 - 727 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 141 Hackney D D . Highly processive microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains , Nature , 1995 , vol. 377 (pg. 448 - 50 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 142 Vale R D , Funatsu T , Pierce D W , Romberg L , Harada Y , Yanagida T . Direct observation of single kinesin molecules moving along microtubules , Nature , 1996 , vol. 380 (pg. 451 - 453 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 143 Hackney D D . Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis , Proc. Natl Acad. Sci. USA , 1994 , vol. 91 (pg. 6865 - 6869 ) Google Scholar Crossref Search ADS WorldCat Crossref 144 Asbury C L , Fehr A N , Block S M . Kinesin moves by an asymmetric hand-over-hand mechanism , Science , 2003 , vol. 302 (pg. 2130 - 2134 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 145 Kaseda K , Higuchi H , Hirose K . Alternate fast and slow stepping of a heterodimeric kinesin molecule , Nat. Cell Biol. , 2003 , vol. 5 (pg. 1079 - 1082 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 146 Kapitein L C , Kwok B H , Weinger J S , Schmidt C F , Kapoor T M , Peterman E J G . Microtubule cross-linking triggers the directional motility of kinesin-5 , J. Cell Biol. , 2008 , vol. 182 (pg. 421 - 428 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 147 Helenius J , Brouhard G , Kalaidzidis Y , Diez S , Howard J . The depolymerizing kinesin MCAK uses lattice diffusion to rapidly target microtubule ends , Nature , 2002 , vol. 441 (pg. 115 - 119 ) Google Scholar Crossref Search ADS WorldCat Crossref 148 Ovechkina Y , Wagenbach M , Wordeman L . K-loop insertion restores microtubule depolymerizing activity of a “neckless” MCAK mutant , J. Cell Biol. , 2002 , vol. 159 (pg. 557 - 562 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 149 Tomishige M , Klopfenstein D R , Vale R D . Conversion of Unc104/KIF1A kinesin into a processive motor after dimerization , Science , 2002 , vol. 297 (pg. 2263 - 2267 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 150 Kull F J , Sablin E P , Lau R , Fletterick R J , Vale R D . Crystal structure of the kinesin motor domain reveals a structural similarity to myosin , Nature , 1996 , vol. 380 (pg. 550 - 555 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 151 Sablin E P , Kull F J , Cooke R , Vale R D , Fletterick R J . Crystal structure of the motor domain of the kinesin-related motor ncd , Nature , 1996 , vol. 380 (pg. 555 - 559 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 152 Kikkawa M , Sablin E P , Okada Y , Yajima H , Fletterick R J , Hirokawa N . Switch-based mechanism of kinesin motors , Nature , 2001 , vol. 411 (pg. 439 - 445 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 153 Nitta R , Kikkawa M , Okada Y , Hirokawa N . KIF1A alternately uses two loops to bind microtubules , Science , 2004 , vol. 305 (pg. 678 - 683 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 154 Nitta R , Okada Y , Hirokawa N . Structural model for strain-dependent microtubule activation of Mg–ADP release from kinesin , Nat. Struct. Mol. Biol. , 2008 , vol. 15 (pg. 1067 - 1075 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 155 Kikkawa M , Okada Y , Hirokawa N . 15 Angstrom resolution model of the monomeric kinesin motor, KIFIA , Cell , 2000 , vol. 100 (pg. 241 - 252 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 156 Ali M Y , Krementsova E B , Kennedy G G , Mahaffy R , Pollard T D , Trybus K M , Warshaw D M . Myosin Va maneuvers through actin intersections and diffuses along microtubules , Proc. Natl Acad. Sci. USA , 2007 , vol. 104 (pg. 4332 - 4336 ) Google Scholar Crossref Search ADS WorldCat Crossref 157 Ali M Y , Lu H , Bookwalter C S , Warshaw D M , Trybus K M . Myosin V and kinesin act as tethers to enhance each others' processivity , Proc. Natl Acad. Sci. USA , 2008 , vol. 105 (pg. 4691 - 4696 ) Google Scholar Crossref Search ADS WorldCat Crossref 158 Kikkawa M , Hirokawa N . High-resolution cryo-EM maps show the nucleotide binding pocket of KIF1A in open and closed conformations , EMBO J. , 2006 , vol. 25 (pg. 4187 - 4194 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 159 Rice S , et al. A structural change in the kinesin motor protein that drives motility , Nature , 1999 , vol. 402 (pg. 778 - 784 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 160 Case R B , Rice S , Hart C L , Ly B , Vale R D . Role of the kinesin neck linker and catalytic core in microtubule-based motility , Curr. Biol. , 2000 , vol. 10 (pg. 157 - 160 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 161 Rice S , Cui Y , Sindelar C , Naber N , Matuska M , Vale R , Cooke R . Thermodynamic properties of the kinesin neck region docking to the catalytic core , Biophys. J. , 2003 , vol. 84 (pg. 1844 - 1845 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 162 Uemura S , Ishiwata S . Loading direction regulates the affinity of ADP for kinesin , Nat. Struct. Biol. , 2003 , vol. 4 (pg. 308 - 311 ) Google Scholar Crossref Search ADS WorldCat Crossref 163 Rosenfeld S S , Fordyce P M , Jefferson G M , King P H , Block S M . Stepping and stretching. How kinesin uses internal strain to walk processively , J. Biol. Chem. , 2003 , vol. 278 (pg. 18550 - 18556 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 164 Hancock W O , Howard J . Kinesin's processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains , Proc. Natl Acad. Sci. USA , 1999 , vol. 96 (pg. 13147 - 13152 ) Google Scholar Crossref Search ADS WorldCat Crossref 165 Crevel I M , et al. What kinesin does at roadblocks: the coordination mechanism for molecular walking , EMBO J. , 2004 , vol. 23 (pg. 23 - 32 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 166 Schief W R , Clark R H , Crevenna A H , Howard J . Inhibition of kinesin motility by ADP and phosphate supports a hand-over-hand mechanism , Proc. Natl Acad. Sci. USA , 2004 , vol. 101 (pg. 1183 - 1188 ) Google Scholar Crossref Search ADS WorldCat Crossref 167 Klumpp L M , Hoenger A , Gilbert S P . Kinesin's second step , Proc. Natl Acad. Sci. USA , 2004 , vol. 101 (pg. 3444 - 3449 ) Google Scholar Crossref Search ADS WorldCat Crossref 168 Alonso M C , et al. An ATP gate controls tubulin binding by the tethered head of kinesin-1 , Science , 2007 , vol. 316 (pg. 120 - 123 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 169 Yildiz A , Tomishige M , Gennerich A , Vale R D . Intramolecular strain coordinates kinesin stepping behavior along microtubules , Cell , 2008 , vol. 134 (pg. 1030 - 1041 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref 170 Krebs A , Goldie K N , Hoenger A . Complex formation with kinesin motor domains affects the structure of microtubules , J. Mol Biol. , 2004 , vol. 335 (pg. 139 - 153 ) Google Scholar Crossref Search ADS PubMed WorldCat Crossref © The Author 2011. Published by Oxford University Press [on behalf of Japanese Society of Microscopy]. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
TI - From electron microscopy to molecular cell biology, molecular genetics and structural biology: intracellular transport and kinesin superfamily proteins, KIFs: genes, structure, dynamics and functions
JF - Journal of Electron Microscopy
DO - 10.1093/jmicro/dfr051
DA - 2011-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/from-electron-microscopy-to-molecular-cell-biology-molecular-genetics-tGTACXXu0c
SP - S63
EP - S92
VL - 60
IS - suppl_1
DP - DeepDyve
ER -