Microtubules: 50 Years on from the Discovery of Tubulin

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microtubule dynamics and organization VIEWPOINT emerge from a defined set of proteins Microtubules: 50 years on from through reconstitution experiments. Jonathon Howard. One of the big questions back when I got into the microtubule the discovery of tubulin business, around 1990, was how motor proteins such as kinesin and dynein use Gary Borisy, Rebecca Heald, Jonathon Howard, Carsten Janke, ATP hydrolysis to generate force for Andrea Musacchio and Eva Nogales transport along microtubules (such as axonal transport) or for cell motility (such as Abstract | Next year will be the 50th anniversary of the discovery of tubulin. ciliary or flagellar motion). The interaction To celebrate this discovery, six leaders in the field of microtubule research reflect of kinesin with microtubules was a model on key findings and technological breakthroughs over the past five decades, system, because it was clear that only a discuss implications for therapeutic applications and provide their thoughts on relatively small number of kinesins must what questions need to be addressed in the near future. be capable of moving small vesicles along microtubules. A related question was how microtubule growth and shrinkage could Identifying the main component of Rebecca Heald. In the mid‑1990s, one generate force to move chromosomes microtubules was obviously the pressing question was why microtubules during mitosis. Polymerization and pressing task in the field around 50 years ago. in cells were so much more dynamic than depolymerization forces were very What key questions were being pursued microtubules assembled from purified mysterious: how could you hold on to the when you entered the arena of tubulin. Neuronal microtubule-associated end of a depolymerizing microtubule? How microtubule research? proteins (MAPs) had been identified could a microtubule grow, and new tubulin and studied (in large part because they subunits get in, if its end was pushing up Gary Borisy. Actually, I had no idea in the co‑purified with tubulin isolated from brain against something? What role did the GTP cap early 1960s that I was entering the arena tissue, where it is most abundant), but these have, and how was energy from GTP used to of microtubule research, because it hadn’t proteins all stabilized microtubules, and generate pulling or pushing forces? How did yet been defined as an arena. Although factors that induced the transition from MAPs regulate growth and shrinkage? the term ‘microtubule’ was first coined in growth to shrinkage (catastrophe) were 1963, the structures had not, in fact, been unknown. A related question was how the Carsten Janke. I entered the field of observed in most cells, because the fixation microtubule cytoskeleton transformed microtubule research somewhat through methods used at the time did not preserve from a relatively stable interphase array to a a back door. During my Ph.D. studies, them. It was only after the introduction of highly dynamic bipolar spindle in mitosis. I worked on the role of the MAP tau in glutaraldehyde as a fixative, also in 1963, At the time, the centrosome was thought neurodegeneration, and in my postdoctoral that they began to be observed routinely. to be the sole ‘microtubule-organizing’ work, I characterized new kinetochore The concept of the microtubule as a centre of the cell, determining the site of protein complexes in budding yeast. This ‘ubiquitous’ cytoskeletal structure wasn’t microtubule growth and their polarized was in the late 1990s, and at the time, the put forward until 1965. My entry point orientation. In my opinion, the discovery field had already expanded a lot: different into the field was mitosis: I wanted to get of a large family of kinesin motor proteins, research communities pursued their own a molecular handle on how cells divide. as well as cytoplasmic dynein, spawned interests. There were many parallel advances Working with Ed Taylor at the University key investigations into how cellular factors at the time, such as the biochemical and of Chicago (Illinois, USA), we were trying affect microtubule behaviour. The diverse functional dissection of the kinetochore, to identify and purify the molecule that activities of different motor proteins to the understanding of the role of primary bound to colchicine, because colchicine was induce catastrophe, crosslink and move cilia as the ‘cell antennae’, and advances in known to specifically inhibit mitosis. We microtubules relative to one another the characterization of neuronal transport had no preconceived idea about what the revealed the ability of microtubule arrays mediated by microtubules. Specifically colchicine target would be, but we believed to ‘self-organize’. This process allows regarding microtubule research, I think that identifying the target would teach us the spindle to form in the absence of a highlight of the 1990s and 2000s was something important about mitosis. So, centrosomes — for example, during female the use of highly sophisticated, in vitro the key questions for us at the time were meiosis in many animal species, or when reconstructions of microtubule assemblies how to isolate, purify and characterize the the centrosome is inactivated genetically from recombinant components, and their colchicine-binding protein and then to or by laser ablation. An important ongoing biophysical characterization. This allowed establish its identity1,2. challenge is to fully understand how the definition of minimal functional units

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of microtubule assemblies such as the agents that targeted tubulin and affected to be used on cytoskeletal filaments. This microtubule arrays of the mitotic spindle. dynamic instability were being discovered. was the time when Dick Wade (CNRS) was A second highlight was the amazing advances But what I remember as the most exciting imaging microtubules and proposed the in imaging of microtubule structures development at the time was the publication lattice accommodation theory to explain the in vivo, which were driven by substantial of the structure of actin by Ken Holmes existence of microtubules with different improvements in light microscopy. and colleagues (Max Planck Institute at protofilament numbers. Also using Heidelberg, Germany). It became obvious cryo‑EM, the Mandelkows and Ron Milligan Andrea Musacchio. When I entered the that there was also a pressing need for a (Scripps Research Institute, California, field, approximately 15 years ago, studies on structure of tubulin. Many were trying to USA) visualized the depolymerization of my main research interest, the kinetochore, crystallize it, like actin, in an inhibited form. microtubules by protofilament peeling, had remained traditionally distinct from At the same time, the new technique of producing images that inspired me and studies of microtubules as cytoskeletal cryoelectron microscopy (cryo‑EM) started influenced my scientific career. components. The search-and-capture model offered a conceptual basis for how microtubules that interact with kinetochores The contributors* might become selectively stabilized to Gary Borisy completed his Ph.D. in 1966 at the University of Chicago, Illinois, USA, and was formerly generate kinetochore fibres, but the a member of faculty at the University of Wisconsin and Northwestern University, USA, and President/ mechanism was obscure. The critical gap in Director of the Marine Biological Laboratory, Massachusetts, USA. He is best known for his work on knowledge was in our understanding of how the molecular biology of the cytoskeleton. Currently, he is a Senior Investigator at the Forsyth kinetochores, which are among the largest Institute in Cambridge, Massachusetts, and is exploring the biogeography of microbial communities (if not the largest) microtubule-binding at the micrometre scale through combinatorial labelling, spectral imaging and metagenomics. machines, bind to and stabilize microtubules Gary Borisy’s homepage: http://forsyth.org/person/scientist/gary-borisy to link them to chromosomes. Research Rebecca Heald is a Professor in the Department of Molecular and Cell Biology at the University of therefore focused on the identification of California Berkeley, USA. She pursued her Ph.D. training with Frank McKeon at Harvard University, potential linking proteins and how they are Cambridge, Massachusetts, USA, and carried out postdoctoral work with Eric Karsenti at the harnessed to align the sister chromatids European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany. Her laboratory is to the metaphase plate, and later to part interested in mechanisms of spindle assembly, cell division and size control. them to the opposite spindle poles. The Jonathon Howard has a Ph.D. from the Australian National University in Canberra, Australia, and identification of an array of new kinetochore was Professor of Physiology and Biophysics at the University of Washington in Seattle, USA, before proteins in the early 2000s facilitated the joining the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, Germany, as a identification of such proteins. Most notably, group leader and director. The Howard laboratory recently moved to Yale University, New Haven, teams led by Arshad Desai (University of Connecticut, USA. His research interests include molecular motors and cytoskeletal polymers, especially their roles in mitosis, in ciliary and flagellar motility and in neuronal architecture. California San Diego, USA) and Ted Salmon Jonathon Howard’s homepage: http://medicine.yale.edu/mbb/faculty/jonathon_howard.profile (University of North Carolina, Chapel Hill, USA), with co‑workers that included Carsten Janke studied biochemistry at the University of Leipzig, Germany. After his Ph.D. in Iain Cheeseman (now at the Whitehead neurochemistry, he held several postdoctoral positions in France and Scotland, working on DNA– protein interactions, on kinetochore complexes in budding yeast and, finally, on post-translational Institute in Cambridge, Massachusetts, USA) modifications of tubulin, a subject that became his major research focus. He started his first and Jennifer DeLuca (now at Colorado State independent group in 2005, and his team discovered most of the enzymes involved in University in Fort Collins, USA), identified post-translational polyglutamylation and polyglycylation of tubulin. Today, the team of Carsten in 2006 the NDC80 complex as a crucial Janke is trying to decipher the tubulin code, by employing an interdisciplinary approach from basic component of this physical linkage. Our biochemistry to the study of mouse models. Carsten Janke was elected a European Molecular laboratory has been particularly interested Biology Organization (EMBO) member in 2014. in understanding the structural organization Carsten Janke’s homepage: https://perso.curie.fr/Carsten.Janke/ of these kinetochore components and their Andrea Musacchio is Director of the Department of Mechanistic Cell Biology at the Max Planck interactions with microtubules. Institute of Molecular Physiology in Dortmund, Germany. After his studies at the University of Rome Tor Vergata, Italy, Musacchio carried out doctoral work with Matti Saraste at the European Eva Nogales. Among my more vivid Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, and postdoctoral work with scientific memories when I was a graduate Stephen C. Harrison at the Harvard Medical School in Boston, Massachusetts, USA. Since student working on tubulin self-assembly establishing his research group (initially in Milan, Italy, and now in Dortmund), Musacchio has been at Daresbury Laboratory, Cheshire, investigating the molecular basis of the attachment of chromosomes to spindle microtubules during mitosis, as well as the feedback control mechanisms that regulate this process. UK, are the reports by Eva-Maria and Eckhard Mandelkow (then at the Max Planck Eva Nogales obtained her B.S. in Physics (Autonomous University of Madrid, Spain) and her Ph.D. in Unit for Structural Biology, Hamburg, Biophysics (University of Keele, Staffordshire, UK), working at the Synchrotron Radiation Source, Germany) and Marie-France Carlier (at the UK. As a postdoctoral researcher with Ken Downing at the Lawrence Berkeley National Laboratory (LBNL), California, USA, she described the first atomic structure of tubulin. She is a Molecular and National Centre for Scientific Research Cell Biology Professor at the University of California Berkeley, a Faculty Scientist at LBNL, and a (CNRS), Paris, France) of really fascinating Howard Hughes Medical Institute (HHMI) Investigator. Her laboratory uses cryoelectron microtubule ‘oscillations’, in which a microscopy to study microtubule structure, dynamics and interactions, as well as macromolecular whole population of microtubules was assemblies involved in gene regulation. synchronized to undergo periods of assembly Eva Nogales’ homepage: http://cryoem.berkeley.edu and disassembly in the test tube. It was also *Listed in alphabetical order very interesting that a number of antimitotic

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Glossary

Axonemes Fluorescent speckle microscopy Peptide antibodies Microtubule-based superstructures that run along the lengths A live-imaging technique in which a low number of Antibodies raised against synthetic short peptides of cilia and flagella. They typically contains two central randomly incorporated labelled self-assembling subunits representing an epitope of a protein. If they are generated microtubules surrounded by nine microtubule doublets. generate fluorescence intensity patterns that appear as for a peptide mimicking a specific post-translational distinct puncta, called speckles, that serve as fiduciary modification, the antibodies can be used to determine Density maps markers so that motion and turnover of the polymer can the state of this modification at the native protein. Distribution of mass corresponding to a macromolecule be visualized. obtained by structural biology methods such as X‑ray Search-and-capture model crystallography or cryoelectron microscopy. Kinesin Proposed by Mitchison and Kirschner in 1886, this model A family of small ATPases, distantly related to myosins builds on an intrinsic property of microtubules named Detyrosination–tyrosination cycle and G proteins, that bind specifically to microtubules and dynamic instability. The search-and-capture model A post-translational modification of α-tubulin, consisting undergo conformational changes upon ATP binding, proposed that this property allows the dynamic ends of of the enzymatic removal of the gene-encoded, hydrolysis and product (ADP and inorganic microtubules to explore space randomly, until their capture carboxy-terminal Tyr residue (by enzymes yet unknown) phosphate) release. The conformational changes by defined binding targets (for example, kinetochores) and its enzymatic re‑ligation by tubulin Tyr ligase. can be used to drive directed motion, to generate endowed with the ability to suppress instability, which force and to alter the growth or depolymerization would therefore selectively stabilize on‑target microtubules. Dynamic instability of microtubules. Many aspects of this model have since been confirmed, A property of microtubules discovered in 1984 by including the strong stabilization of microtubules after Tim Mitchison and Mark Kirschner (then at the University Kinetochore kinetochore binding. of California San Francisco, USA), by which individual A large protein assembly of nearly 100 proteins that links microtubules switch stochastically between phases of centromeric DNA to spindle microtubules, thereby Total internal reflection fluorescence microscopy growth and shrinking, powered by GTP hydrolysis that coupling forces generated by microtubule dynamics to (TIRF). An optical technique that exploits the ‘trapping’ of closely follows incorporation of a tubulin dimer into a power chromosome movement during mitosis and meiosis. light within high-refractive-index structures such as optical growing microtubule end. fibres and even ‘fluorescent’ signs such as those sometimes Lattice accommodation theory used to advertise menus in cafes or restaurants, in which Dynein and dynactin A geometrical model that explains how tubulin contacts writing on the glass surface allows the light to leak out. Dynein is a complex, multi-subunit microtubule can be maintained in microtubules with different minus-end-directed motor. Studies in vitro suggest that its protofilaments by skewing of the protofilaments with Tubulin code processive movement on microtubules may require respect to the microtubule axis. Describes the process of generating specialized dynactin, which is a very large multi-subunit assembly, microtubules by directly changing the tubulin building as well as additional adaptor subunits. Optical or magnetic tweezers blocks, either by the incorporation of specific tubulin Optical tweezers, also known as optical traps, are isotypes (a set of gene products of α- and β-tubulins) into GTP cap instruments that use a focused laser beam to the lattice, or by the addition of specific post-translational A GTP–tubulin-rich structure at the growing tip of a generate forces on micrometre-scale objects. Magnetic modifications to tubulin. microtubule, created by the binding of GTP-bound tubulin tweezers are instruments that can generate forces or subunits from solution onto the microtubule end. torque. Both instruments can operate in biologically Vinca alkaloids meaningful force ranges, typically piconewtons, and are A set of natural or semi-synthetic alkaloid agents derived End-binding proteins therefore ideally suited to study the interactions of from vinca plants, which have the capacity to inhibit Proteins that bind with higher affinity to a region at the microtubules with motors and microtubule assembly and are therefore used in dynamic, growing end of a microtubule. microtubule-associated proteins. cancer therapy.

Which technical breakthrough or from the purified protein. This permitted microtubules within a population, as well breakthroughs, in your opinion, have analysis of the mechanisms of assembly as the stochastic transition between these been the most significant in driving and disassembly, and the identification of two states, and enabled precise measurement microtubule research? What questions did proteins that specifically interacted with of these dynamic parameters. This was a particular techniques enable scientists microtubules. A decade before the advent prerequisite for investigating how specific to address? of GFP, the use of fluorescently labelled proteins affected microtubule behaviour, tubulin micro-injected into cells led to the for example, by altering growth rate or the G.B. One breakthrough was demonstrating realization that microtubules were highly frequency of catastrophes. The development that colchicine did, in fact, bind to a dynamic structures, transforming our of fluorescently tagged tubulin was of specific molecule. In retrospect, colchicine concepts of the cytoskeleton. The concept great significance, and techniques such was one of the first examples of how a of dynamic instability gave new insights as fluorescent speckle microscopy and other small-molecule probe could be used to into how cytoplasmic microtubules could approaches that labelled one end of a explore a biological pathway. However, the be rapidly remodelled. The discovery of microtubule or generated traceable fiduciary task of identifying the colchicine target molecular motors and that microtubules marks along its length permitted tracking of with the subunit of microtubules was not serve as tracks for transport of cellular cargo microtubule movements inside cells. This straightforward, as the biochemistry was transformed our understanding of molecular enabled researchers to fully characterize confusing and messy. A breakthrough was in traffic in the cell. dynamic changes in microtubule finding a good source of colchicine-binding organization, for example, in migrating cells activity from which the protein could be R.H. Innovations in microscopy, particularly or dividing cells. To elucidate the structural isolated. This, surprisingly, turned out to be fluorescence and electron microscopy, basis of microtubule dynamic instability, brain tissue — which we now understand have been instrumental in revealing it was essential to obtain high-resolution to be logical, because microtubules are a the basis of microtubule dynamics and images, and the development of cryo-EM principal structural component of neurons. organization. Time-lapse video microscopy and image-analysis tools have been Another breakthrough was the discovery was required to directly visualize the instrumental. These tools are being applied of how to reassemble microtubules in vitro growth and shrinkage of individual to gain detailed insight into the features of

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tubulin that underlie the amazing intrinsic led to the discovery, at the end of the 1970s, E.N. I am biased towards visualization tools! behaviour of microtubules, as well as of multiple α- and β-tubulin genes. Later, Ted Salmon and Clare Waterman (University important information about how specific the development of recombinant protein of North Carolina) pioneering speckle proteins associate with microtubules, production, together with biochemistry microscopy has enabled many to visualize and may even reveal what lurks inside! using Xenopus laevis egg extracts, enabled cellular microtubules, whether they are great progress in the field, as they allowed moving, growing, shrinking or treadmilling. J.H. Single-molecule techniques had a huge us to form microtubule spindles in these But, of course, cryo‑EM remains among impact. Detecting single molecules, especially extracts and study the role of specific my personal favourites. A number of using total internal reflection fluorescence proteins by removing them from or adding laboratories, including those of Milligan, microscopy (TIRF), but also manipulating them into the extracts. The huge success Wade and Linda Amos (MRC Laboratory them with optical traps, really blew open of monoclonal and (especially) peptide of Molecular Biology, Cambridge, UK), the problem of how proteins generate force. antibodies was also one of the driving forces produced the first cryo‑EM structures The experimental obstacle back then was of microtubule research, as these allowed of microtubules decorated with kinesin, how to assay the movement of a motor detection of different tubulin subtypes, such in which the crystal structure of kinesin, protein, such as kinesin, along its filament, as tubulin isotypes and post-translational then just solved by Ron Vale (University the microtubule. In the case of DNA, the modifications, in the 1980s. Later, as I of California San Francisco), could be position of a RNA polymerase, which can be mentioned above, improvements in light docked into the density maps. And then the thought of as another type of motor protein, microscopy and biochemical reconstructions structure of tubulin itself was obtained using could be determined biochemically by of microtubule assemblies in vitro were the electron crystallography of zinc-induced footprinting — that is, sequencing the short key technologies that advanced the field. sheets through the work of Sharon Wolf but unique oligonucleotide that the enzyme Interestingly, when looking back, it seems and myself, under the leadership of protects from proteolysis. This even had that not only have technological advances Ken Downing (Lawrence Berkeley National single-nucleotide resolution! In the case of helped the microtubule field, but research on Laboratory, California). Cryo-EM continues cytoskeletal filaments such as microtubules microtubules has also driven many of these to produce beautiful molecular images of or actin filaments, this approach did not technological advances. We certainly hope complex cellular microtubule structures. work, because the filaments are periodic as that this synergy will continue in the future. Among my favourites are the structures by all the subunits are the same. Single-molecule Daniela Nicastro (Brandeis University and techniques allowed the movement of A.M. The data obtained by in vitro UT Southwestern, USA) of whole axonemes. individual motors to be directly visualized. reconstitution of interactions between Our most detailed visualization of tubulin It was discovered that many cytoskeletal MAPs and microtubule motors with static has come from X‑ray structures, pioneered motor proteins, such as kinesin‑1 (REF. 3), or dynamic microtubules using fluorescence by Marcel Knossow (CNRS), and more were processive, meaning that they could microscopy have superseded the data recently describing the binding of several take many steps along their filament, like obtained by the murky, poorly characterized antimitotic agents by Michel Steinmetz RNA and DNA polymerases. This facilitated assays that had previously been used for (Paul Scherrer Institut, Switzerland). Finally, the detailed study of their mechanisms (using measuring microtubule binding, nucleation I love what the field of single-molecule purified proteins) or their functions (using or bundling. The use of fluorescence biophysics is doing to increase our labelled proteins in living cells). As optics microscopy enabled quantitative analyses of mechanistic understanding of microtubule techniques, labelling strategies, detectors microtubule dynamics. Such experiments motors, using single-molecule fluorescence, and image processing improved, it became can now be complemented by technically Förster resonance energy transfer (FRET) possible to track proteins with nanometre more demanding force measurements or laser tweezers, with contributions from precision4, enabling the progression of with optical or magnetic tweezers. Ron Vale, Steve Block (Stanford University, motors and the growth of microtubules Real-time measurements, often achieving California), Ahmet Yildiz (University of to be studied at the single-molecule and single-molecule sensitivity, are revealing California Berkeley) or Sam Reck-Peterson single-subunit level. The fluorescence the variety of interaction modes that MAPs (Harvard University, Cambridge, techniques, in all of their different ‘flavours’, and motors establish with microtubules, the Massachusetts), to cite a few. could even be used inside living cells, dynamics of their residency on microtubules, allowing biochemical studies — of binding and the consequences of their binding on How successfully has our accumulated kinetics, oligomerization, activity state microtubule stability and growth rates. knowledge of microtubule structure and so on — to be done in situ. Studies on the MAPs XMAP215 and and function been translated to the clinic, end-binding protein 1 (EB1) come to mind in terms of our understanding of how C.J. In the early years of microtubule in this context, but there are many other microtubules drive disease and their research, major advances in electron noteworthy examples. Structural biology importance as possible therapeutic targets? microscopy and biochemistry drove has also had a primary role in microtubule What facets are limiting translation and how the discovery of the microtubule, then the research. For instance, the recent elucidation might they be addressed? discovery of tubulin, and, shortly after, of the structures of dynein and dynactin, the discovery of the detyrosination–tyrosination and of tubulin and microtubules, at atomic G.B. So many extraordinary technological cycle of α-tubulin — which was an amazing or near-atomic resolution are clear advances over the past several decades finding, as it showed that amino acids can landmarks. The revolutionary recent have revolutionized biology in general and be enzymatically incorporated into proteins developments in cryo-EM hold great promise of course had a profound impact on our independently of an RNA template and for future elucidations of the mechanistic understanding of microtubule structure translation. Advances in cloning techniques basis of microtubule function. and function. DNA cloning and sequencing

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enabled the identification of the tubulin proteins. Taxol, the microtubule-stabilizing cancer being a primary example, but the gene family and the enormous variety of diterpenoid isolated from the bark of yew molecular basis for microtubule targeting microtubule-interacting proteins. X‑ray (used in the witches brew), was an essential remains incompletely understood. At the crystallography and cryo-EM have provided reagent for studying microtubules in vitro. end of the last decade, live-cell microscopy structures of tubulin and microtubules at the Before a synthesis was developed, which was began to be exploited to study how mitotic atomic level. Genetically encoded fluorescent an incredible feat, taxol was only available in cells behave when treated with agents proteins such as GFP have enabled analysis miniscule quantities from the US National that interfere with microtubule function of dynamic functions in vivo. The revolution Institutes of Health (NIH). Taxol has been (microtubule poisons) or with the function in imaging has enabled unprecedented very successful in treating ovarian and other of the mitotic checkpoint, which becomes spatial and temporal visualization of cancers. But magic bullets like taxol are activated in the presence of such poisons. cellular components. One striking example rare; I think systems approaches to therapy In this context, a crucial question was of translating this knowledge to the clinic in which multiple, softer tools push the whether some aspects of tumour cells is the use of taxol as an anticancer drug. organism back into a healthy, hopefully stable — for instance, pre-existing aneuploidy — Taxol binds to microtubules and stabilizes state offer a promising avenue in the future. might explain the differential sensitivity them by inhibiting their disassembly, of tumour cells to microtubule poisons thus freezing their dynamics. Taxol has C.J. Microtubules are a prime target of and therefore be exploited for therapy. proven to be a powerful therapeutic for cancer chemotherapy: taxol derivatives The results that have been obtained so far breast and ovarian cancer. However, much and vinca alkaloids are microtubule are complex and have not yet resolved the greater opportunities for translational poisons that account for one-third of all issue in full. Nevertheless, stratification and impact remain. Microtubules are involved chemotherapeutic drugs used. While selection of potential drug targets based in biological processes in virtually every it is extremely successful, this type of on defined cellular behaviours continues cell, tissue and organ in the body, and treatment is very crude, as it affects the to be an appealing therapeutic approach. microtubule disturbances underlie many entire organism and perturbs many cellular Eventually, selected combinations of drugs diseases such as Alzheimer disease and functions, and thus it has considerable might be harnessed to force cancer cells into Parkinson disease, as well as cancer. So, the side effects. Furthermore, some types of particular cell cycle stages — most notably, potential of microtubule-based therapeutics cancers develop resistance to this treatment, mitosis — in which a crucial dependence is great. However, the microtubule system is notably by expressing a specific tubulin (and thus weakness) can be exposed. very complex, and achieving full translational gene, TUBB3, which encodes the otherwise impact will require a deconstruction of that neuron-specific tubulin β3 isotype. E.N. I think that there is still a long way complexity — what today would be called a Microtubules containing this isotype appear to go. We do not yet fully understand systems biology approach. less susceptible to taxol. Considerable efforts microtubule function and dynamics are underway to find new microtubule drugs in healthy cells and organisms, so our R.H. Microtubule-targeting drugs are of that could act more specifically on cancer understanding of their role in disease great value in the clinic, but it seems that we cells, such as antibody–drug conjugates states remains limited. This lack of a do not fully understand how and why they that deliver tubulin drugs specifically into comprehensive understanding stems work. For cancer therapies, microtubule- cancer cells. Another therapeutic tactic from the complexity of the microtubule targeting drugs affect the spindle and cell could be to develop drugs that are specific to cytoskeleton and the many roles that division. In my opinion as a cell biologist, particular forms of tubulin, such as isotypes microtubules have in the cell. The functional translation is limited by our incomplete or post-translationally modified tubulins; complexity is paralleled by the molecular knowledge of the spindle, and basic however, so far no cancer-specific tubulin complexity of tubulin isoforms, their post- research into how the spindle forms and forms have been identified. Of course, there translational modifications and the myriad functions will be instrumental in informing are also drugs that affect other essential of cellular factors that regulate tubulin therapeutic approaches. This requires the microtubule-interacting proteins that are transcription and translation, folding of contributions of researchers working in being actively pursued. the αβ-dimer, and microtubule nucleation a wide variety of systems and organisms, Considering the importance of and assembly, as well as microtubule not just cultured cell-based assays, together microtubules in other clinical conditions, localization and organization. It is clear that with an open-minded and curious attitude tubulin drugs hold great promise many questions remain to be answered, towards the underlying biology. Increasing for the treatment of ciliopathies and and the number of questions may continue the emphasis on ‘translational’ research is neurodegenerative disorders, as at least a to increase the more we learn about these not going to solve the problem of finding subset of these pathologies are linked to fascinating polymers. My work is very effective therapeutic approaches if we failures in microtubule function. Finally, distant from applications in the clinic, but do not understand how the microtubule some very exciting reports were published we know that the effects of some broadly cytoskeleton operates in the context of a cell, recently on the positive effects of taxol and used anti-tubulin drugs for the treatment of tissue, and organism. another tubulin drug, epothilone B, on cancer are still not well defined. For example, axon regeneration. Overall, I think there it is not entirely understood how the J.H. Given the central role that microtubules is a greatly underestimated potential in administration of taxol ultimately results in have in cell division, in cilia and flagella, tubulin drugs. tumour remission, as it is most likely that and in intracellular transport, especially taxol does more than just causing mitotic in neurons, it is not surprising that many A.M. I will limit myself to a few general arrest. More basic as well as translational and genetic diseases have been traced to defects comments. There is clearly a rationale in clinical work is clearly necessary to develop in microtubule motors and other associated targeting microtubules in disease, with improved therapies.

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As the field of microtubule research has leverage evolutionary differences to elucidate since it had always been thought that matured in the five decades since mechanisms that modulate microtubule microtubules only change their composition tubulin was identified as the main component dynamics and organization, thereby by depolymerization and repolymerization, of these cellular structures, which of the setting spindle size and shape? How do not by insertion of tubulin units into the emerging basic principles have you found these mechanisms impact the fidelity of lattice. This discovery reopened the great most unexpected? What question are you chromosome segregation by the spindle? question of how perfectly microtubules are most intrigued about now? Cancer cells are almost always distorted assembled in our cells. in size and morphology, and expression of I am personally driven by my interest G.B. Of particular interest to me are the spindle-associated proteins is frequently in the tubulin code, which turns out to be qualities of cellular organization that go altered. Investigating the molecular basis a very challenging code to crack. As we beyond anything yet engineered by us of spindle variation across species and cell know today, the entire diversity of the humans. Cells are ensembles of molecules types is likely to reveal new principles of cellular tubulin pool, which is generated interacting within boundaries. Some of the microtubule regulation and may identify by the expression of different tubulin genes molecules are organized into supramolecular important factors that contribute to genome (isotypes) and by tubulin post-translational assemblies that have been likened to instability and oncogenesis. modifications, leads in most cases to only molecular machines. But the machine subtle changes in microtubule behaviour, metaphor is rather limiting if we imagine J.H. The discovery I found most unexpected changes that are difficult to measure with machines of the human-made type such was that the kinesin-related protein NCD classical methods. I guess this could be as we see around us. What human-made (of the kinesin‑14 family) moves backwards! another example in which technological machine assembles itself from parts? That is, it moves in the opposite direction advances will be needed to appreciate the What machine is constantly in material flux, to kinesin‑1, the founding member of the full scale of microtubule functions that are tossing out parts and taking in new ones? family. In addition to posing all sorts of regulated by the tubulin code. What machine can repair itself? Yet these questions about the structural basis of motor are all properties of many of the molecular directionality, this discovery presaged the A.M. Structural biologists like myself machines found in cells — including the extraordinary range of activities of kinesins: are usually addressing systems whose microtubule machine. Organization in the some move one way, some the other way, parts self-assemble in conditions of cell depends on a continuity of form, not on and some can move both ways; some can thermodynamic equilibrium. Microtubule individual molecules. It operates far from slide microtubules, while some depolymerize assembly ultimately defies this approach equilibrium and disintegrates if not supplied microtubules and some can even polymerize because it is dissipative, and this allows more with an input of energy. A big challenge for them! In addition, there are many other flexible biochemical behaviours. Using some cell biology is to understand the emergent, proteins, such as XMAP215 and EB1, that tricks, microtubules are of course amenable self-organizing properties of interdependent also regulate the dynamic growth and to structural analysis, but their nature is to molecular systems. It is likely that a successful shrinkage of microtubules and that couple behave dynamically. The description of response to this challenge will require microtubule ends to a large variety of cellular the dynamic instability of microtubules5 multidisciplinary approaches, including a structures — for example, chromosomes, and the development of a theory for how heavy dose of biophysics, systems analysis basal bodies, centrosomes and endoplasmic it may influence cellular morphogenesis and computer modelling. It may be, following reticulum — and figuring out how all this — the search-and-capture model6 of a quote from Richard Feynman, that we will works is a tremendous challenge. Tim Mitchison and Marc Kirschner (both at not be able to fully understand these systems The question that most intrigues me the Harvard Medical School) — has until we can create them. The frontier may now is the length control problem: what sets revolutionized the field. The fundamental well be synthetic cell biology. the size of microtubules and microtubule- concept behind this model is that self- based structures. How do small molecules organization emerges from the dynamic R.H. The most amazing aspect of the like tubulin, motors and MAPs know how behaviour of the constituent parts, which microtubule cytoskeleton to me is that the long a microtubule is? This is a mechanical in turn reflects their ability to operate basic components are conserved across signalling question, and we have ideas away from thermodynamic equilibrium. species, yet a tremendous diversity of about how motor activity can feed back on I suspect that future inspiration at this point microtubule-based structures can form microtubule dynamics to control length. is likely to arrive from the reconstitution in different cell types and organisms. of microtubule-based macromolecular Compare microtubule arrays in a tiny C.J. I’m not certain about some of the structures of adequate complexity, where 2 μm yeast cell to those in a giant 2 mm early discoveries, but the discovery by the simultaneous control of multiple single-cell protist like Stentor! Microtubules Tim Mitchison and Marc Kirschner (at the parameters is possible — something that is have essentially the same structure and time at the University of California San still very difficult to do in non-reconstituted similar associated factors, and carry out Francisco) of the dynamic instability systems. To be able to gain a holistic view some common and some very different of microtubules was certainly a great of self-organization of microtubule-based functions, in these two cell types. How breakthrough. I also think that the discovery structures, we must increase the complexity are vastly different microtubule arrays of enzymatic detyrosination and tyrosination of the reconstituted systems well beyond assembled and remodelled using the same was a great surprise at the time, as it showed current limits. We try to keep these concepts basic cytoskeletal system? My laboratory is that gene-encoded amino acid sequences in mind as we reconstitute larger and larger obsessed with understanding the mitotic can be modified by enzymes. More recently, parts of the kinetochore to understand its spindle, whose architecture and size adapt I found the discovery of microtubule dynamic interactions with microtubules. to fit a wide variety of cell types. Can we self-repair very surprising and exciting, I wish microtubules a good anniversary!

PERSPECTIVES

E.N. How antimitotic agents interfere factors at the structural level. Although Andrea Musacchio is at the Department of Mechanistic with microtubule dynamic instability is we have made some progress, both on Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, something I have been interested in since the understanding of the basic structural Germany; and the Centre for Medical Biotechnology, my Ph.D., and it remains close to my heart. principles of microtubule dynamic Faculty of Biology, University Duisburg-Essen, But I am also fascinated by how cellular instability and on the visualization of Universitätsstrasse, 45141 Essen, Germany. factors regulate dynamic instability, and pairwise interactions between microtubules [email protected] how others factors utilize microtubule and associated factors, it is time to look Eva Nogales is at the Biochemistry, Biophysics and dynamics for their own purposes, such at larger and more complex networks Structural Biology Division, Molecular and Cell Biology as moving with a growing or shrinking of interactions as they occur on the Department, University of California, Berkeley, microtubule end. There has been some microtubule surface. A combination of California 94720-3220, USA; the Howard Hughes Medical Institute, Maryland, USA; beautiful imaging of these molecular biochemical reconstitution approaches and and the Lawrence Berkeley National Laboratory, USA. interactions, as well as single-molecule cellular imaging will certainly be needed. [email protected] force studies, which have provided unique doi:10.1038/nrm.2016.45 Gary Borisy is at the Forsyth Institute, 245 First Street, mechanistic insights — such as the work Published online 22 April 2016 Cambridge, Massachusetts 02142, USA. by Thomas Surrey (at the Francis Crick [email protected] 1. Borisy, G. G. & Taylor, E. W. The mechanism of action of Institute, London, UK) on end-binding colchicine. Binding of colchincine‑3H to cellular protein. proteins, by Joe Howard (Yale University, Rebecca Heald is at the Division of Cell and J. Cell Biol. 34, 525–533 (1967). Developmental Biology and Molecular and Cell Biology 2. Borisy, G. G. & Taylor, E. W. The mechanism of action of colchicine. Colchicine binding to sea urchin eggs and the New Haven, Connecticut, USA) on the Department, University of California, Berkeley, mitotic apparatus. J. Cell Biol. 34, 535–548 (1967). microtubule polymerase XMAP215, California 94720, USA. 3. Howard, J., Hudspeth, A. J. & Vale, R. D. Movement of or by Stefan Westermann (Institute of [email protected] microtubules by single kinesin molecules. Nature 342, Molecular Pathology, Vienna, Austria) and 154–158 (1989). Jonathon Howard is at the Department of Molecular 4. Svoboda, K., Schmidt, C. F., Schnapp, B. J. & Block, S. M. Chip Asbury (University of Washington, Biophysics and Biochemistry, Yale University, Direct observation of kinesin stepping by optical trapping interferometry. Nature 365, 721–727 (1993). USA) on kinetochores, to cite some of New Haven, Connecticut 06510, USA. 5. Mitchison, T. & Kirschner, M. Dynamic instability of my own favourites — that have been real [email protected] microtubule growth. Nature 312, 237–242 (1984). 6. Kirschner, M. & Mitchison, T. Beyond self-assembly: eye-openers for me. The complexity of Carsten Janke is at the Institut Curie, PSL Research from microtubules to morphogenesis. Cell 45, these molecular activities intrigues me University, CNRS UMR3348, Centre Universitaire, 329–342 (1986). and motivates me to study the interaction Bâtiment 110, F-91405 Orsay, France. Competing interests statement of microtubules with these regulatory [email protected] The authors declare no competing interests.