The centrosome - diverse functions in fertilization and development across species.

The centrosome is a non-membrane-bound organelle that is conserved across most animal cells and serves various functions throughout the cell cycle. In dividing cells, the centrosome is known as the spindle pole and nucleates a robust microtubule spindle to separate genetic material equally into two daughter cells. In non-dividing cells, the mother centriole, a substructure of the centrosome, matures into a basal body and nucleates cilia, which acts as a signal-transducing antenna. The functions of centrosomes and their substructures are important for embryonic development and have been studied extensively using in vitro mammalian cell culture or in vivo using invertebrate models. However, there are considerable differences in the composition and functions of centrosomes during different aspects of vertebrate development, and these are less studied. In this Review, we discuss the roles played by centrosomes, highlighting conserved and divergent features across species, particularly during fertilization and embryonic development.

Microtubules under mechanical pressure can breach dense actin networks.

The crosstalk between the actin network and microtubules is essential for cell polarity. It orchestrates microtubule organization within the cell, driven by the asymmetry of actin architecture along the cell periphery. The physical intertwining of these networks regulates spatial organization and force distribution in the microtubule network. Although their biochemical interactions are becoming clearer, the mechanical aspects remain less understood. To explore this mechanical interplay, we developed an in vitro reconstitution assay to investigate how dynamic microtubules interact with various actin filament structures. Our findings revealed that microtubules can align and move along linear actin filament bundles through polymerization force. However, they are unable to pass through when encountering dense branched actin meshworks, similar to those present in the lamellipodium along the periphery of the cell. Interestingly, immobilizing microtubules through crosslinking with actin or other means allow the buildup of pressure, enabling them to breach these dense actin barriers. This mechanism offers insights into microtubule progression towards the cell periphery, with them overcoming obstacles within the denser parts of the actin network and ultimately contributing to cell polarity establishment.

Cryo-EM of α-tubulin isotype-containing microtubules revealed a contracted structure of α4A/ß2A microtubules.

Microtubules are hollow α/ß-tubulin heterodimeric polymers that play critical roles in cells. In vertebrates, both α- and ß-tubulins have multiple isotypes encoded by different genes, which are intrinsic factors in regulating microtubule functions. However, the structures of microtubules composed of different tubulin isotypes, especially α-tubulin isotypes, remain largely unknown. Here, we purify recombinant tubulin heterodimers composed of different mouse α-tubulin isotypes, including α1A, α1C and α4A, with the ß-tubulin isotype ß2A. We further assemble and determine the cryo-electron microscopy (cryo-EM) structures of α1A/ß2A, α1C/ß2A, and α4A/ß2A microtubules. Our structural analysis demonstrates that α4A/ß2A microtubules exhibit longitudinal contraction between tubulin interdimers compared with α1A/ß2A and α1C/ß2A microtubules. Collectively, our findings reveal that α-tubulin isotype composition can tune microtubule structures, and also provide evidence for the "tubulin code" hypothesis.

Length limitation of astral microtubules orients cell divisions in murine intestinal crypts.

Planar spindle orientation is critical for epithelial tissue organization and is generally instructed by the long cell-shape axis or cortical polarity domains. We introduced mouse intestinal organoids in order to study spindle orientation in a monolayered mammalian epithelium. Although spindles were planar, mitotic cells remained elongated along the apico-basal (A-B) axis, and polarity complexes were segregated to basal poles, so that spindles oriented in an unconventional manner, orthogonal to both polarity and geometric cues. Using high-resolution 3D imaging, simulations, and cell-shape and cytoskeleton manipulations, we show that planar divisions resulted from a length limitation in astral microtubules (MTs) which precludes them from interacting with basal polarity, and orient spindles from the local geometry of apical domains. Accordingly, lengthening MTs affected spindle planarity, cell positioning, and crypt arrangement. We conclude that MT length regulation may serve as a key mechanism for spindles to sense local cell shapes and tissue forces to preserve mammalian epithelial architecture.

Kinesin-1 promotes centrosome clustering and nuclear migration in the Drosophila oocyte.

Microtubules and their associated motors are important players in nucleus positioning. Although nuclear migration in Drosophila oocytes is controlled by microtubules, a precise role for microtubule-associated molecular motors in nuclear migration has yet to be reported. We characterize novel landmarks that allow a precise description of the pre-migratory stages. Using these newly defined stages, we report that, before migration, the nucleus moves from the oocyte anterior side toward the center and concomitantly the centrosomes cluster at the posterior of the nucleus. In the absence of Kinesin-1, centrosome clustering is impaired and the nucleus fails to position and migrate properly. The maintenance of a high level of Polo-kinase at centrosomes prevents centrosome clustering and impairs nuclear positioning. In the absence of Kinesin-1, SPD-2, an essential component of the pericentriolar material, is increased at the centrosomes, suggesting that Kinesin-1-associated defects result from a failure to reduce centrosome activity. Consistently, depleting centrosomes rescues the nuclear migration defects induced by Kinesin-1 inactivation. Our results suggest that Kinesin-1 controls nuclear migration in the oocyte by modulating centrosome activity.

A model for generating differences in microtubules between axonal branches depending on the distance from terminals.

In the remodeling of axonal arbor, the growth and retraction of branches are differentially regulated within a single axon. Although cell-autonomously generated differences in microtubule (MT) turnover are thought to be involved in selective branch regulation, the cellular system whereby neurons generate differences of MTs between axonal branches has not been clarified. Because MT turnover tends to be slower in longer branches compared with neighboring shorter branches, feedback regulation depending on branch length is thought to be involved. In the present study, we generated a model of MT lifetime in axonal terminal branches by adapting a length-dependent model in which parameters for MT dynamics were constant in the arbor. The model predicted that differences in MT lifetime between neighboring branches could be generated depending on the distance from terminals. In addition, the following points were predicted. Firstly, destabilization of MTs throughout the arbor decreased the differences in MT lifetime between branches. Secondly, differences of MT lifetime existed even before MTs entered the branch point. In axonal MTs in primary neurons, treatment with a low concentration of nocodazole significantly decreased the differences of detyrosination (deTyr) and tyrosination (Tyr) of tubulins, indicators of MT turnover. Expansion microscopy of the axonal shaft before the branch point revealed differences in deTyr/Tyr modification on MTs. Our model recapitulates the differences in MT turnover between branches and provides a feedback mechanism for MT regulation that depends on the axonal arbor geometry.

The effect of motor-induced shaft dynamics on microtubule stability and length.

Control of microtubule abundance, stability, and length is crucial to regulate intracellular transport as well as cell polarity and division. How microtubule stability depends on tubulin addition or removal at the dynamic ends is well studied. However, microtubule rescue, the event when a microtubule switches from shrinking to growing, occurs at tubulin exchange sites along the shaft. Molecular motors have recently been shown to promote such exchanges. Using a stochastic theoretical description, we study how microtubule stability and length depend on motor-induced tubulin exchange and thus rescue. Our theoretical description matches our in vitro experiments on microtubule dynamics in the presence of kinesin-1 molecular motors. Although the overall dynamics of a population of microtubules can be captured by an effective rescue rate, by assigning rescue to exchange sites, we reveal that the dynamics of individual microtubules within the population differ dramatically. Furthermore, we study in detail a transition from bounded to unbounded microtubule growth. Our results provide novel insights into how molecular motors imprint information of microtubule stability on the microtubule network.

Modeling and mechanical perturbations reveal how spatially regulated anchorage gives rise to spatially distinct mechanics across the mammalian spindle.

During cell division, the spindle generates force to move chromosomes. In mammals, microtubule bundles called kinetochore-fibers (k-fibers) attach to and segregate chromosomes. To do so, k-fibers must be robustly anchored to the dynamic spindle. We previously developed microneedle manipulation to mechanically challenge k-fiber anchorage, and observed spatially distinct response features revealing the presence of heterogeneous anchorage (Suresh et al., 2020). How anchorage is precisely spatially regulated, and what forces are necessary and sufficient to recapitulate the k-fiber's response to force remain unclear. Here, we develop a coarse-grained k-fiber model and combine with manipulation experiments to infer underlying anchorage using shape analysis. By systematically testing different anchorage schemes, we find that forces solely at k-fiber ends are sufficient to recapitulate unmanipulated k-fiber shapes, but not manipulated ones for which lateral anchorage over a 3 µm length scale near chromosomes is also essential. Such anchorage robustly preserves k-fiber orientation near chromosomes while allowing pivoting around poles. Anchorage over a shorter length scale cannot robustly restrict pivoting near chromosomes, while anchorage throughout the spindle obstructs pivoting at poles. Together, this work reveals how spatially regulated anchorage gives rise to spatially distinct mechanics in the mammalian spindle, which we propose are key for function.

Unrestrained growth of correctly oriented microtubules instructs axonal microtubule orientation.

In many eukaryotic cells, directed molecular transport occurs along microtubules. Within neuronal axons, transport over vast distances particularly relies on uniformly oriented microtubules, whose plus-ends point towards the distal axon tip (anterogradely polymerizing, or plus-end-out). However, axonal microtubules initially have mixed orientations, and how they orient during development is not yet fully understood. Using live imaging of primary Drosophila melanogaster neurons, we found that, in the distal part of the axon, catastrophe rates of plus-end-out microtubules were significantly reduced compared to those of minus-end-out microtubules. Physical modelling revealed that plus-end-out microtubules should therefore exhibit persistent long-term growth, while growth of minus-end-out microtubules should be limited, leading to a bias in overall axonal microtubule orientation. Using chemical and physical perturbations of microtubule growth and genetic perturbations of the anti -catastrophe factor p150, which was enriched in the distal axon tip, we confirmed that the enhanced growth of plus-end-out microtubules is critical for achieving uniform microtubule orientation. Computer simulations of axon development integrating the enhanced plus-end-out microtubule growth identified here with previously suggested mechanisms, that is, dynein-based microtubule sliding and augmin-mediated templating, correctly predicted the long-term evolution of axonal microtubule orientation as found in our experiments. Our study thus leads to a holistic explanation of how axonal microtubules orient uniformly, a prerequisite for efficient long-range transport essential for neuronal functioning.

For humans to be able to wiggle their toes, messages need to travel from the brain to the foot, a distance well over a meter in many adults. This is made possible by neurons, the cells that form the nervous system, which transmit electrical signals along long extensions called 'axons'. Axons can only transmit signals if all the required molecules, which are produced in a part of the neuron known as the cell body, are ferried to the ends of the axons. This ferrying around of molecules is carried out by long, filamentous molecules called microtubules, which act as a directed carrier system, shuttling molecules along the axon, either towards or away from the cell body. Microtubules can be thought of as asymmetrical rods. One end ­ known as the plus end ­ is dynamic and can undergo growth or shrinkage, while the other end ­ called the minus end ­ is stable. For transport along the axon to happen efficiently, microtubules in the neuron need to be oriented with their plus end pointing towards the ends of the axon. Microtubules in growing neurons develop this orientation, but how that is achieved is not fully understood. To understand the basis of this cellular phenomenon, Jakobs, Zemel and Franze examined the behaviour of microtubules in developing neurons from fruit fly larvae. A fluorescent protein, which emits light when the microtubules are growing, helped the researchers visualise the plus end of microtubules, the microtubule orientation, and their growth in developing axons. This experiment showed that microtubules that had their plus end pointing towards the axon end shrank more slowly than those with the opposite orientation, leading them to grow longer. This resulted in a higher proportion of the correctly-oriented microtubules in the axon. Treating the neurons with Nocodazole, a chemical that disrupts microtubule growth, or with sodium chloride, which changes the osmotic pressure, caused the microtubules that were oriented with their plus end towards the axon to grow less, and disrupted the uniform orientation of the microtubules in the axon. The next step was to determine whether specific axonal proteins such as p150 ­ a protein that is enriched at the tip of the axon and decreases microtubule shrinkage rates ­ are involved in this process. Reducing the levels of p150 in fruit flies using molecular and genetic methods resulted in microtubules with their plus end pointing towards the axon tip shrinking faster, reducing the proportion of microtubules with this orientation in the axon. This role of proteins enriched in the axonal tip, along with previously discovered mechanisms, explains how microtubules align unidirectionally in axons. These findings open new avenues of research into neurodegenerative diseases like Alzheimer's and Parkinson's, which might manifest due to a breakdown of transport along microtubules in neurons.

Cross-linker design determines microtubule network organization by opposing motors.

During cell division, cross-linking motors determine the architecture of the spindle, a dynamic microtubule network that segregates the chromosomes in eukaryotes. It is unclear how motors with opposite directionality coordinate to drive both contractile and extensile behaviors in the spindle. Particularly, the impact of different cross-linker designs on network self-organization is not understood, limiting our understanding of self-organizing structures in cells but also our ability to engineer new active materials. Here, we use experiment and theory to examine active microtubule networks driven by mixtures of motors with opposite directionality and different cross-linker design. We find that although the kinesin-14 HSET causes network contraction when dominant, it can also assist the opposing kinesin-5 KIF11 to generate extensile networks. This bifunctionality results from HSET's asymmetric design, distinct from symmetric KIF11. These findings expand the set of rules underlying patterning of active microtubule assemblies and allow a better understanding of motor cooperation in the spindle.

Extracellular stiffness induces contractile dysfunction in adult cardiomyocytes via cell-autonomous and microtubule-dependent mechanisms.

The mechanical environment of the myocardium has a potent effect on cardiomyocyte form and function, yet an understanding of the cardiomyocyte responses to extracellular stiffening remains incomplete. We therefore employed a cell culture substrate with tunable stiffness to define the cardiomyocyte responses to clinically relevant stiffness increments in the absence of cell-cell interactions. When cultured on substrates magnetically actuated to mimic the stiffness of diseased myocardium, isolated rat adult cardiomyocytes exhibited a time-dependent reduction of sarcomere shortening, characterized by slowed contraction and relaxation velocity, and alterations of the calcium transient. Cardiomyocytes cultured on stiff substrates developed increases in viscoelasticity and microtubule detyrosination in association with early increases in the α-tubulin detyrosinating enzyme vasohibin-2 (Vash2). We found that knockdown of Vash2 was sufficient to preserve contractile performance as well as calcium transient properties in the presence of extracellular substrate stiffening. Orthogonal prevention of detyrosination by overexpression of tubulin tyrosine ligase (TTL) was also able to preserve contractility and calcium homeostasis. These data demonstrate that a pathologic increment of extracellular stiffness induces early, cell-autonomous remodeling of adult cardiomyocytes that is dependent on detyrosination of α-tubulin.

Molecular Link between DNA Damage Response and Microtubule Dynamics.

Microtubules are major components of the cytoskeleton that play important roles in cellular processes such as intracellular transport and cell division. In recent years, it has become evident that microtubule networks play a role in genome maintenance during interphase. In this review, we highlight recent advances in understanding the role of microtubule dynamics in DNA damage response and repair. We first describe how DNA damage checkpoints regulate microtubule organization and stability. We then highlight how microtubule networks are involved in the nuclear remodeling following DNA damage, which leads to changes in chromosome organization. Lastly, we discuss how microtubule dynamics participate in the mobility of damaged DNA and promote consequent DNA repair. Together, the literature indicates the importance of microtubule dynamics in genome organization and stability during interphase.

Beyond the centrosome: The mystery of microtubule organising centres across mammalian preimplantation embryos.

Mammalian preimplantation embryogenesis depends on the spatio-temporal dynamics of the microtubule cytoskeleton to enable exceptionally fast changes in cell number, function, architecture, and fate. Microtubule organising centres (MTOCs), which coordinate the remodelling of microtubules, are therefore of fundamental significance during the first days of a new life. Despite its indispensable role during early mammalian embryogenesis, the origin of microtubule growth remains poorly understood. In this review, we summarise the most recent discoveries on microtubule organisation and function during early human embryogenesis and compare these to innovative studies conducted in alternative mammalian models. We emphasise the differences and analogies of centriole inheritance and their role during the first cleavage. Furthermore, we highlight the significance of non-centrosomal MTOCs for embryo viability and discuss the potential of novel in vitro models and light-inducible approaches towards unravelling microtubule formation in research and assisted reproductive technologies.

Tools for studying the cytoskeleton during plant cell division.

The plant cytoskeleton regulates fundamental biological processes, including cell division. How to experimentally perturb the cytoskeleton is a key question if one wants to understand the role of both actin filaments (AFs) and microtubules (MTs) in a given biological process. While a myriad of mutants are available, knock-out in cytoskeleton regulators, when nonlethal, often produce little or no phenotypic perturbation because such regulators are often part of a large family, leading to functional redundancy. In this review, alternative techniques to modify the plant cytoskeleton during plant cell division are outlined. The different pharmacological and genetic approaches already developed in cell culture, transient assays, or in whole organisms are presented. Perspectives on the use of optogenetics to perturb the plant cytoskeleton are also discussed.

The microtubule cytoskeleton in cardiac mechanics and heart failure.

The microtubule network of cardiac muscle cells has unique architectural and biophysical features to accommodate the demands of the working heart. Advances in live-cell imaging and in deciphering the 'tubulin code' have shone new light on this cytoskeletal network and its role in heart failure. Microtubule-based transport orchestrates the growth and maintenance of the contractile apparatus through spatiotemporal control of translation, while also organizing the specialized membrane systems required for excitation-contraction coupling. To withstand the high mechanical loads of the working heart, microtubules are post-translationally modified and physically reinforced. In response to stress to the myocardium, the microtubule network remodels, typically through densification, post-translational modification and stabilization. Under these conditions, physically reinforced microtubules resist the motion of the cardiomyocyte and increase myocardial stiffness. Accordingly, modified microtubules have emerged as a therapeutic target for reducing stiffness in heart failure. In this Review, we discuss the latest evidence on the contribution of microtubules to cardiac mechanics, the drivers of microtubule network remodelling in cardiac pathologies and the therapeutic potential of targeting cardiac microtubules in acquired heart diseases.

Microtubule polarity is instructive for many aspects of neuronal polarity.

Many neurons in bilaterian animals are polarized with functionally distinct axons and dendrites. Microtubule polarity, microtubule stability, and the axon initial segment (AIS) have all been shown to influence polarized transport in neurons. Each of these cytoskeletal cues could act independently to control axon and dendrite identity, or there could be a hierarchy in which one acts upstream of the others. Here we test the hypothesis that microtubule polarity acts as a master regulator of neuronal polarity by using a Drosophila genetic background in which some dendrites have normal minus-end-out microtubule polarity and others have the axonal plus-end-out polarity. In these mosaic dendrite arbors, we found that ribosomes, which are more abundant in dendrites than axons, were reduced in plus-end-out dendrites, while an axonal cargo was increased. In addition, we determined that microtubule stability was different in plus-end-out and minus-end-out dendrites, with plus-end-out ones having more stable microtubules like axons. Similarly, we found that ectopic diffusion barriers, like those at the AIS, formed at the base of dendrites with plus-end-out regions. Thus, changes in microtubule polarity were sufficient to rearrange other cytoskeletal features associated with neuronal polarization. However, overall neuron shape was maintained with only subtle changes in branching in mosaic arbors. We conclude that microtubule polarity can act upstream of many aspects of intracellular neuronal polarization, but shape is relatively resilient to changes in microtubule polarity in vivo.

Bending-torsional elasticity and energetics of the plus-end microtubule tip.

SignificanceThe mechanochemical basis of microtubule growth, which is essential for the normal function and division of eukaryotic cells, has remained elusive and controversial, despite extensive work. In particular, recent findings have created the paradox that the microtubule plus-end tips look very similar during both growing and shrinking phases, thereby challenging the traditional textbook picture. Our large-scale atomistic simulations resolve this paradox and explain microtubule growth and shrinkage dynamics as a process governed by energy barriers between protofilament conformations, the heights of which are in turn fine-tuned by different nucleotide states, thus implementing an information-driven Brownian ratchet.

Swap and stop - Kinetochores play error correction with microtubules: Mechanisms of kinetochore-microtubule error correction: Mechanisms of kinetochore-microtubule error correction.

Correct chromosome segregation in mitosis relies on chromosome biorientation, in which sister kinetochores attach to microtubules from opposite spindle poles prior to segregation. To establish biorientation, aberrant kinetochore-microtubule interactions must be resolved through the error correction process. During error correction, kinetochore-microtubule interactions are exchanged (swapped) if aberrant, but the exchange must stop when biorientation is established. In this article, we discuss recent findings in budding yeast, which have revealed fundamental molecular mechanisms promoting this "swap and stop" process for error correction. Where relevant, we also compare the findings in budding yeast with mechanisms in higher eukaryotes. Evidence suggests that Aurora B kinase differentially regulates kinetochore attachments to the microtubule end and its lateral side and switches relative strength of the two kinetochore-microtubule attachment modes, which drives the exchange of kinetochore-microtubule interactions to resolve aberrant interactions. However, Aurora B kinase, recruited to centromeres and inner kinetochores, cannot reach its targets at kinetochore-microtubule interface when tension causes kinetochore stretching, which stops the kinetochore-microtubule exchange once biorientation is established.

Linking path and filament persistence lengths of microtubules gliding over kinesin.

Microtubules and kinesin motor proteins are involved in intracellular transports in living cells. Such intracellular material transport systems can be reconstructed for utilisation in synthetic environments, and they are called molecular shuttles driven by kinesin motors. The performance of the molecular shuttles depends on the nature of their trajectories, which can be characterized by the path persistence length of microtubules. It has been theoretically predicted that the path persistence length should be equal to the filament persistence length of the microtubules, where the filament persistence length is a measure of microtubule flexural stiffness. However, previous experiments have shown that there is a significant discrepancy between the path and filament persistence lengths. Here, we showed how this discrepancy arises by using computer simulation. By simulating molecular shuttle movements under external forces, the discrepancy between the path and filament persistence lengths was reproduced as observed in experiments. Our close investigations of molecular shuttle movements revealed that the part of the microtubules bent due to the external force was extended more than it was assumed in the theory. By considering the extended length, we could elucidate the discrepancy. The insights obtained here are expected to lead to better control of molecular shuttle movements.

Atomic force microscopy reveals distinct protofilament-scale structural dynamics in depolymerizing microtubule arrays.

The dynamic reorganization of microtubule-based cellular structures, such as the spindle and the axoneme, fundamentally depends on the dynamics of individual polymers within multimicrotubule arrays. A major class of enzymes implicated in both the complete demolition and fine size control of microtubule-based arrays are depolymerizing kinesins. How different depolymerases differently remodel microtubule arrays is poorly understood. A major technical challenge in addressing this question is that existing optical or electron-microscopy methods lack the spatial-temporal resolution to observe the dynamics of individual microtubules within larger arrays. Here, we use atomic force microscopy (AFM) to image depolymerizing arrays at single-microtubule and protofilament resolution. We discover previously unseen modes of microtubule array destabilization by conserved depolymerases. We find that the kinesin-13 MCAK mediates asynchronous protofilament depolymerization and lattice-defect propagation, whereas the kinesin-8 Kip3p promotes synchronous protofilament depolymerization. Unexpectedly, MCAK can depolymerize the highly stable axonemal doublets, but Kip3p cannot. We propose that distinct protofilament-level activities underlie the functional dichotomy of depolymerases, resulting in either large-scale destabilization or length regulation of microtubule arrays. Our work establishes AFM as a powerful strategy to visualize microtubule dynamics within arrays and reveals how nanometer-scale substrate specificity leads to differential remodeling of micron-scale cytoskeletal structures.