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Microtubules are core components of the eukaryotic cytoskeleton with essential roles in cell division, shaping, motility and intracellular transport. Despite their functional heterogeneity, microtubules have a highly conserved structure made from almost identical molecular building blocks: the tubulin proteins. Alternative tubulin isotypes and a variety of post-translational modifications control the properties and functions of the microtubule cytoskeleton, a concept known as the ‘tubulin code’. Here we review the current understanding of the molecular components of the tubulin code and how they impact microtubule properties and functions. We discuss how tubulin isotypes and post-translational modifications control microtubule behaviour at the molecular level and how this translates into physiological functions at the cellular and organism levels. We then go on to show how fine-tuning of microtubule function by some tubulin modifications can affect homeostasis and how perturbation of this fine-tuning can lead to a range of dysfunctions, many of which are linked to human disease.The mechanical and dynamic properties of microtubules are determined by their complement of subunits, known as tubulin isotypes, and the post-translational modifications found on these isotypes. This concept is known as the ‘tubulin code’. The regulation of microtubules and microtubule-associated proteins by this code is critical for the correct function of a range of tissues. Consequently, recent studies have linked perturbation of the tubulin code to disease, including neurodegenerative diseases.

The active transport of organelles and other cargos along the axon is required to maintain neuronal health and function, but we are just beginning to understand the complex regulatory mechanisms involved. The molecular motors, cytoplasmic dynein and kinesins, transport cargos along microtubules; this transport is tightly regulated by adaptors and effectors. Here we review our current understanding of motor regulation in axonal transport. We discuss the mechanisms by which regulatory proteins induce or repress the activity of dynein or kinesin motors, and explore how this regulation plays out during organelle trafficking in the axon, where motor activity is both cargo specific and dependent on subaxonal location. We survey several well-characterized examples of membranous organelles subject to axonal transport — including autophagosomes, endolysosomes, signalling endosomes, mitochondria and synaptic vesicle precursors — and highlight the specific mechanisms that regulate motor activity to provide localized trafficking within the neuron. Defects in axonal transport have been implicated in conditions ranging from developmental defects in the brain to neurodegenerative disease. Better understanding of the underlying mechanisms will be essential to develop more-effective treatment options.Homeostasis and function of neurons rely on long-distance, bidirectional microtubule-based transport along the axon, which is driven by both dynein and kinesin motors. How these motors are regulated by a plethora of adaptors and effectors to ensure appropriate and robust distribution of cargos is an area of intense study.

Key Points Diverse intracellular cargos are transported along microtubules by the actions of kinesin and dynein molecular motors. The 'tug-of-war' model describes bidirectional movement as a mechanical competition between kinesins and dyneins. This model is supported by both theoretical and experimental studies. There are several studies in which inhibiting one motor was found to suppress transport in both directions, contrary to predictions from the tug-of-war model. This phenomenon is termed the 'paradox of co-dependence' of antagonistic motors. Three hypothetical mechanisms are proposed to reconcile this paradox: microtubule tethering, mechanical activation and steric disinhibition. Each makes specific predictions regarding directional switching and the nature of the pause states. A more complete understanding of bidirectional transport will require mathematical modelling to both frame the best experiments and interpret quantitative data. The three hypothetical mechanisms, and other potential mechanisms, can be incorporated into a common modelling framework. Bidirectional movement by oppositely directed motors attached to the same cargo is frequently described as a 'tug of war'. However, some studies suggest that inhibiting one motor diminishes motility in both directions. To resolve this paradox, three bidirectional transport models, termed microtubule tethering, mechanical activation and steric disinhibition, are proposed, and a general mathematical modelling framework for bidirectional cargo transport is described. Vesicles, organelles and other intracellular cargo are transported by kinesin and dynein motors, which move in opposite directions along microtubules. This bidirectional cargo movement is frequently described as a 'tug of war' between oppositely directed molecular motors attached to the same cargo. However, although many experimental and modelling studies support the tug-of-war paradigm, numerous knockout and inhibition studies in various systems have found that inhibiting one motor leads to diminished motility in both directions, which is a 'paradox of co-dependence' that challenges the paradigm. In an effort to resolve this paradox, three classes of bidirectional transport models — microtubule tethering, mechanical activation and steric disinhibition — are proposed, and a general mathematical modelling framework for bidirectional cargo transport is put forward to guide future experiments.

The main organelles of the secretory and endocytic pathways—the endoplasmic reticulum (ER) and endosomes, respectively—are connected through contact sites whose numbers increase as endosomes mature 1 , 2 , 3 . One function of such sites is to enable dephosphorylation of the cytosolic tails of endosomal signalling receptors by an ER-associated phosphatase 4 , whereas others serve to negatively control the association of endosomes with the minus-end-directed microtubule motor dynein 5 or mediate endosome fission 6 . Cholesterol transfer and Ca 2+ exchange have been proposed as additional functions of such sites 2 , 3 . However, the compositions, activities and regulations of ER–endosome contact sites remain incompletely understood. Here we show in human and rat cell lines that protrudin, an ER protein that promotes protrusion and neurite outgrowth 7 , forms contact sites with late endosomes (LEs) via coincident detection of the small GTPase RAB7 and phosphatidylinositol 3-phosphate (PtdIns(3)P). These contact sites mediate transfer of the microtubule motor kinesin 1 from protrudin to the motor adaptor FYCO1 on LEs. Repeated LE–ER contacts promote microtubule-dependent translocation of LEs to the cell periphery and subsequent synaptotagmin-VII-dependent fusion with the plasma membrane. Such fusion induces outgrowth of protrusions and neurites, which requires the abilities of protrudin and FYCO1 to interact with LEs and kinesin 1. Thus, protrudin-containing ER–LE contact sites are platforms for kinesin-1 loading onto LEs, and kinesin-1-mediated translocation of LEs to the plasma membrane, fuelled by repeated ER contacts, promotes protrusion and neurite outgrowth. Repeated contacts between the endoplasmic reticulum (ER) and a subset of endosomes called late endosomes (LEs) is shown to promote microtubule-dependent translocation of LEs to the cell periphery and their subsequent fusion with the plasma membrane to induce outgrowth of neuronal protrusions. Role of endosomes in neuronal protrusion outgrowth The endoplasmic reticulum (ER) makes contact with various other cellular organelles including endosomes. Although the functional significance of ER–endosome contact sites is known, the composition, activity and regulation of these sites is poorly explored. Harald Stenmark and co-workers provide a glimpse into these enigmatic aspects. They show that the ER protein protrudin makes contact with the small GTPase RAB7 and phosphatidylinositol 3-phosphate (PtdIns(3)P) on a subset of endosomes called late endosomes (LEs). This allows transfer of the microtubule motor protein kinesin-1 from protrudin to the motor adaptor FYCO1 on LEs. Thus repeated ER–LE contacts promote microtubule-dependent translocation of LEs to the cell periphery and their subsequent fusion with the plasma membrane to induce neurite outgrowth of neuronal protrusions.

The ‘tubulin-code’ hypothesis proposes that different tubulin genes or post-translational modifications (PTMs), which mainly confer variation in the carboxy-terminal tail (CTT), result in unique interactions with microtubule-associated proteins for specific cellular functions. However, the inability to isolate distinct and homogeneous tubulin species has hindered biochemical testing of this hypothesis. Here, we have engineered 25 α/β-tubulin heterodimers with distinct CTTs and PTMs and tested their interactions with four different molecular motors using single-molecule assays. Our results show that tubulin isotypes and PTMs can govern motor velocity, processivity and microtubule depolymerization rates, with substantial changes conferred by even single amino acid variation. Revealing the importance and specificity of PTMs, we show that kinesin-1 motility on neuronal β-tubulin (TUBB3) is increased by polyglutamylation and that robust kinesin-2 motility requires detyrosination of α-tubulin. Our results also show that different molecular motors recognize distinctive tubulin ‘signatures’, which supports the premise of the tubulin-code hypothesis. Vale and colleagues report the distinct abilities of different tubulin isotypes and post-translational modifications to regulate different microtubule motors and their properties.

Chromosomal instability (CIN) is a hallmark of tumor cells caused by changes in the dynamics and control of microtubules that compromise the mitotic spindle. Thus, CIN cells may respond differently than diploid cells to treatments that target mitotic spindle regulation. Here, we test this idea by inhibiting a subset of kinesin motor proteins involved in mitotic spindle control. KIF18A is required for proliferation of CIN cells derived from triple negative breast cancer or colorectal cancer tumors but is not required in near-diploid cells. Following KIF18A inhibition, CIN tumor cells exhibit mitotic delays, multipolar spindles, and increased cell death. Sensitivity to KIF18A knockdown is strongly correlated with centrosome fragmentation, which requires dynamic microtubules but does not depend on bipolar spindle formation or mitotic arrest. Our results indicate the altered spindle microtubule dynamics characteristic of CIN tumor cells can be exploited to reduce the proliferative capacity of CIN cells. Kinesin motor proteins are critical for maintaining mitotic spindle integrity, which is important for chromosome stability. Here, the authors show that the kinesin motor protein, KIF18A, permits the proliferation of chromosomally unstable cells and knockdown of KIF18A induces centrosome fragmentation.

Accurately regulated ciliary beating in time and space is critical for diverse cellular activities, which impact the survival and development of nearly all eukaryotic species. An essential beating regulator is the conserved central apparatus (CA) of motile cilia, composed of a pair of microtubules (C1 and C2) associated with hundreds of protein subunits per repeating unit. It is largely unclear how the CA plays its regulatory roles in ciliary motility. Here, we present high-resolution structures of Chlamydomonas reinhardtii CA by cryo-electron microscopy (cryo-EM) and its dynamic conformational behavior at multiple scales. The structures show how functionally related projection proteins of CA are clustered onto a spring-shaped scaffold of armadillo-repeat proteins, facilitated by elongated rachis-like proteins. The two halves of the CA are brought together by elastic chain-like bridge proteins to achieve coordinated activities. We captured an array of kinesin-like protein (KLP1) in two different stepping states, which are actively correlated with beating wave propagation of cilia. These findings establish a structural framework for understanding the role of the CA in cilia. Here, authors solve cryo-EM structures of the central apparatus of motile cilia and analyze its dynamic conformations to elucidate the mechanism of ciliary beating.

Intracellular vesicular transport along cytoskeletal filaments ensures targeted cargo delivery. Such transport is rarely unidirectional but rather bidirectional, with frequent directional reversals owing to the simultaneous presence of opposite-polarity motors. So far, it has been unclear whether such complex motility pattern results from the sole mechanical interplay between opposite-polarity motors or requires regulators. Here, we demonstrate that a minimal system, comprising purified Dynein-Dynactin-BICD2 (DDB) and kinesin-3 (KIF16B) attached to large unilamellar vesicles, faithfully reproduces in vivo cargo motility, including runs, pauses, and reversals. Remarkably, opposing motors do not affect vesicle velocity during runs. Our computational model reveals that the engagement of a small number of motors is pivotal for transitioning between runs and pauses. Taken together, our results suggest that motors bound to vesicular cargo transiently engage in a tug-of-war during pauses. Subsequently, stochastic motor attachment and detachment events can lead to directional reversals without the need for regulators. Intracellular transport along microtubules involves runs, pauses and directional reversals. Here, D’Souza et al . mimic these dynamics in vitro using a minimal system of Dynein-Dynactin-BICD2 and Kinesin-3 on vesicles without the need for regulators.

Mammalian Hedgehog (Hh) signal transduction requires a primary cilium, a microtubule-based organelle, and the Gli–Sufu complexes that mediate Hh signalling, which are enriched at cilia tips. Kif7, a kinesin-4 family protein, is a conserved regulator of the Hh signalling pathway and a human ciliopathy protein. Here we show that Kif7 localizes to the cilium tip, the site of microtubule plus ends, where it limits cilium length and controls cilium structure. Purified recombinant Kif7 binds the plus ends of growing microtubules in vitro , where it reduces the rate of microtubule growth and increases the frequency of microtubule catastrophe. Kif7 is not required for normal intraflagellar transport or for trafficking of Hh pathway proteins into cilia. Instead, a central function of Kif7 in the mammalian Hh pathway is to control cilium architecture and to create a single cilium tip compartment, where Gli–Sufu activity can be correctly regulated. Anderson and colleagues report that the kinesin-4 family member Kif7 binds to microtubule plus ends at cilium tips to regulate their length and structure, and to ensure the fidelity of Hedgehog signalling.

Key Points More than 650 members of the kinesin superfamily have been discovered in eukaryotic organisms. They are categorized into 14 subfamilies on the basis of sequence homology and classified as mitotic kinesins, which are involved in cell division, and non-mitotic kinesins, which are principally involved in intracellular transport. The mitotic spindle is a validated target in cancer chemotherapy, and several agents that target tubulin and that interfere with microtubule dynamics are in clinical use. This success has triggered the search for additional mitotic spindle targets, including mitotic kinases (such as cyclin-dependent kinases, Aurora kinase A, Aurora kinase B and Polo-like kinase 1) and specific mitotic kinesins. EG5 and centromere-associated protein E (CENPE) are two mitotic kinesins that have received much of this attention. Specific inhibitors have been developed and are currently in multiple Phase I or Phase II clinical trials. Other kinesins are also considered potential therapeutic targets and candidate inhibitors have been described. Several non-mitotic kinesins are also involved in tumorigenesis and in the development of resistance to anticancer agents. Several kinesins have multiple functions in mitosis or in intracellular transport. Therefore, careful validation is needed to identify any important side effects and to clarify whether they are viable cancer drug targets. Kinesins — a family of molecular motors that travel unidirectionally along microtubule tracks — have emerged as potential targets for cancer drug development. As discussed in this Review, several compounds that inhibit mitotic kinesins have entered clinical trials and others are being developed, raising the possibility that the range of kinesin-based drug targets may expand in the future. Kinesins are a family of molecular motors that travel unidirectionally along microtubule tracks to fulfil their many roles in intracellular transport or cell division. Over the past few years kinesins that are involved in mitosis have emerged as potential targets for cancer drug development. Several compounds that inhibit two mitotic kinesins (EG5 (also known as KIF11) and centromere-associated protein E (CENPE)) have entered Phase I and II clinical trials either as monotherapies or in combination with other drugs. Additional mitotic kinesins are currently being validated as drug targets, raising the possibility that the range of kinesin-based drug targets may expand in the future.