Visualising the cytoskeletal machinery in neuronal growth cones using cryo-electron tomography.

Neurons extend axons to form the complex circuitry of the mature brain. This depends on the coordinated response and continuous remodelling of the microtubule and F-actin networks in the axonal growth cone. Growth cone architecture remains poorly understood at nanoscales. We therefore investigated mouse hippocampal neuron growth cones using cryo-electron tomography to directly visualise their three-dimensional subcellular architecture with molecular detail. Our data showed that the hexagonal arrays of actin bundles that form filopodia penetrate and terminate deep within the growth cone interior. We directly observed the modulation of these and other growth cone actin bundles by alteration of individual F-actin helical structures. Microtubules with blunt, slightly flared or gently curved ends predominated in the growth cone, frequently contained lumenal particles and exhibited lattice defects. Investigation of the effect of absence of doublecortin, a neurodevelopmental cytoskeleton regulator, on growth cone cytoskeleton showed no major anomalies in overall growth cone organisation or in F-actin subpopulations. However, our data suggested that microtubules sustained more structural defects, highlighting the importance of microtubule integrity during growth cone migration.

Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition.

Plasmodium parasites cause malaria and are responsible annually for hundreds of thousands of deaths. Kinesins are a superfamily of microtubule-dependent ATPases that play important roles in the parasite replicative machinery, which is a potential target for antiparasite drugs. Kinesin-5, a molecular motor that cross-links microtubules, is an established antimitotic target in other disease contexts, but its mechanism in Plasmodium falciparum is unclear. Here, we characterized P. falciparum kinesin-5 (PfK5) using cryo-EM to determine the motor's nucleotide-dependent microtubule-bound structure and introduced 3D classification of individual motors into our microtubule image processing pipeline to maximize our structural insights. Despite sequence divergence in PfK5, the motor exhibits classical kinesin mechanochemistry, including ATP-induced subdomain rearrangement and cover neck bundle formation, consistent with its plus-ended directed motility. We also observed that an insertion in loop5 of the PfK5 motor domain creates a different environment in the well-characterized human kinesin-5 drug-binding site. Our data reveal the possibility for selective inhibition of PfK5 and can be used to inform future exploration of Plasmodium kinesins as antiparasite targets.

A microtubule RELION-based pipeline for cryo-EM image processing.

Microtubules are polar filaments built from αß-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and ß-tubulin. The determination of the αß-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins.

Structure and Dynamics of Single-isoform Recombinant Neuronal Human Tubulin.

Microtubules are polymers that cycle stochastically between polymerization and depolymerization, i.e. they exhibit "dynamic instability." This behavior is crucial for cell division, motility, and differentiation. Although studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure, and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogeneous isotypically pure engineered tubulin. Here, we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/ßIII is a purely neuronal tubulin isoform. The 4.2-Å structure of post-translationally unmodified human α1A/ßIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but it is distinguished by subtle differences at polymerization interfaces, which are hot spots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogeneous microtubules undergo dynamic instability, but they polymerize slower and have fewer catastrophes. Interestingly, we find that epitaxial growth of α1A/ßIII microtubules from heterogeneous brain seeds is inefficient but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogeneous α1A/ßIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well defined tubulin species and is a first and necessary step toward uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics.

Mapping the Processivity Determinants of the Kinesin-3 Motor Domain.

Kinesin superfamily members play important roles in many diverse cellular processes, including cell motility, cell division, intracellular transport, and regulation of the microtubule cytoskeleton. How the properties of the family-defining motor domain of distinct kinesins are tailored to their different cellular roles remains largely unknown. Here, we employed molecular-dynamics simulations coupled with energetic calculations to infer the family-specific interactions of kinesin-1 and kinesin-3 motor domains with microtubules in different nucleotide states. We then used experimental mutagenesis and single-molecule motility assays to further assess the predicted residue-wise determinants of distinct kinesin-microtubule binding properties. Collectively, our results identify residues in the L8, L11, and α6 regions that contribute to family-specific microtubule interactions and whose mutation affects motor-microtubule complex stability and processive motility (the ability of an individual motor to take multiple steps along its microtubule filament). In particular, substitutions of prominent kinesin-3 residues with those found in kinesin-1, namely, R167S/H171D, K266D, and R346M, were found to decrease kinesin-3 processivity 10-fold and thus approach kinesin-1 levels.

MAPping out distribution routes for kinesin couriers.

In the crowded environment of eukaryotic cells, diffusion is an inefficient distribution mechanism for cellular components. Long-distance active transport is required and is performed by molecular motors including kinesins. Furthermore, in highly polarised, compartmentalised and plastic cells such as neurons, regulatory mechanisms are required to ensure appropriate spatio-temporal delivery of neuronal components. The kinesin machinery has diversified into a large number of kinesin motor proteins as well as adaptor proteins that are associated with subsets of cargo. However, many mechanisms contribute to the correct delivery of these cargos to their target domains. One mechanism is through motor recognition of sub-domain-specific microtubule (MT) tracks, sign-posted by different tubulin isoforms, tubulin post-translational modifications, tubulin GTPase activity and MT-associated proteins (MAPs). With neurons as a model system, a critical review of these regulatory mechanisms is presented here, with a particular focus on the emerging contribution of compartmentalised MAPs. Overall, we conclude that - especially for axonal cargo - alterations to the MT track can influence transport, although in vivo, it is likely that multiple track-based effects act synergistically to ensure accurate cargo distribution.

Structure Determination of Microtubules and Pili: Past, Present, and Future Directions.

Historically proteins that form highly polymeric and filamentous assemblies have been notoriously difficult to study using high resolution structural techniques. This has been due to several factors that include structural heterogeneity, their large molecular mass, and available yields. However, over the past decade we are now seeing a major shift towards atomic resolution insight and the study of more complex heterogenous samples and in situ/ex vivo examination of multi-subunit complexes. Although supported by developments in solid state nuclear magnetic resonance spectroscopy (ssNMR) and computational approaches, this has primarily been due to advances in cryogenic electron microscopy (cryo-EM). The study of eukaryotic microtubules and bacterial pili are good examples, and in this review, we will give an overview of the technical innovations that have enabled this transition and highlight the advancements that have been made for these two systems. Looking to the future we will also describe systems that remain difficult to study and where further technical breakthroughs are required.

Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions.

Kinesin-binding protein (KBP) is an important selective inhibitor of specific kinesin family members and its genetic disruption causes Goldberg-Shprintzen syndrome. Cryo-electron microscopy (cryo-EM) has recently been used to reveal the structure of KBP alone (72 kDa) and in complex with the motor domain of the mitotic kinesin-12 KIF15 (110 kDa). KBP is an α-solenoid, tetratricopeptide-repeat protein that interacts with the microtubule-binding region of the kinesin motor domain and blocks microtubule attachment. Numerous challenges arose relating to the behavior of KBP and KBP-kinesin complexes during cryo-EM sample preparation. These included the partial denaturation of KBP by air-water interfaces, protein aggregation resulting from carbon interaction and preferential orientation. Sample preparation with a graphene oxide substrate enabled the eventual structure determination. Here, experiences with preparing these samples are detailed, bringing attention to some of the challenges and opportunities that are likely to arise from protein-surface interactions.

The mechanism of kinesin inhibition by kinesin-binding protein.

Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin-binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment, and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP-kinesin motor domain complex. KBP is a tetratricopeptide repeat-containing, right-handed α-solenoid that sequesters the kinesin motor domain's tubulin-binding surface, structurally distorting the motor domain and sterically blocking its MT attachment. KBP uses its α-solenoid concave face and edge loops to bind the kinesin motor domain, and selected structure-guided mutations disrupt KBP inhibition of kinesin transport in cells. The KBP-interacting motor domain surface contains motifs exclusively conserved in KBP-interacting kinesins, suggesting a basis for kinesin selectivity.

Author Correction: A structural model for microtubule minus-end recognition and protection by CAMSAP proteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression.

EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo-electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α- and ß-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser144 and Ser146 in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression. In addition, constitutive activation of NEK6 or NEK7 reduced the association of EML4 with interphase microtubules. Together, these data support a model in which NEK6- and NEK7-dependent phosphorylation promotes the dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression.

Structural determinants of microtubule minus end preference in CAMSAP CKK domains.

CAMSAP/Patronins regulate microtubule minus-end dynamics. Their end specificity is mediated by their CKK domains, which we proposed recognise specific tubulin conformations found at minus ends. To critically test this idea, we compared the human CAMSAP1 CKK domain (HsCKK) with a CKK domain from Naegleria gruberi (NgCKK), which lacks minus-end specificity. Here we report near-atomic cryo-electron microscopy structures of HsCKK- and NgCKK-microtubule complexes, which show that these CKK domains share the same protein fold, bind at the intradimer interprotofilament tubulin junction, but exhibit different footprints on microtubules. NMR experiments show that both HsCKK and NgCKK are remarkably rigid. However, whereas NgCKK binding does not alter the microtubule architecture, HsCKK remodels its microtubule interaction site and changes the underlying polymer structure because the tubulin lattice conformation is not optimal for its binding. Thus, in contrast to many MAPs, the HsCKK domain can differentiate subtly specific tubulin conformations to enable microtubule minus-end recognition.

Microtubule architecture in vitro and in cells revealed by cryo-electron tomography.

The microtubule cytoskeleton is involved in many vital cellular processes. Microtubules act as tracks for molecular motors, and their polymerization and depolymerization can be harnessed to generate force. The structures of microtubules provide key information about the mechanisms by which their cellular roles are accomplished and the physiological context in which these roles are performed. Cryo-electron microscopy allows the visualization of in vitro-polymerized microtubules and has provided important insights into their overall morphology and the influence of a range of factors on their structure and dynamics. Cryo-electron tomography can be used to determine the unique three-dimensional structure of individual microtubules and their ends. Here, a previous cryo-electron tomography study of in vitro-polymerized GMPCPP-stabilized microtubules is revisited, the findings are compared with new tomograms of dynamic in vitro and cellular microtubules, and the information that can be extracted from such data is highlighted. The analysis shows the surprising structural heterogeneity of in vitro-polymerized microtubules. Lattice defects can be observed both in vitro and in cells. The shared ultrastructural properties in these different populations emphasize the relevance of three-dimensional structures of in vitro microtubules for understanding microtubule cellular functions.

Tubulin isoform composition tunes microtubule dynamics.

Microtubules polymerize and depolymerize stochastically, a behavior essential for cell division, motility, and differentiation. While many studies advanced our understanding of how microtubule-associated proteins tune microtubule dynamics in trans, we have yet to understand how tubulin genetic diversity regulates microtubule functions. The majority of in vitro dynamics studies are performed with tubulin purified from brain tissue. This preparation is not representative of tubulin found in many cell types. Here we report the 4.2-Å cryo-electron microscopy (cryo-EM) structure and in vitro dynamics parameters of α1B/ßI+ßIVb microtubules assembled from tubulin purified from a human embryonic kidney cell line with isoform composition characteristic of fibroblasts and many immortalized cell lines. We find that these microtubules grow faster and transition to depolymerization less frequently compared with brain microtubules. Cryo-EM reveals that the dynamic ends of α1B/ßI+ßIVb microtubules are less tapered and that these tubulin heterodimers display lower curvatures. Interestingly, analysis of EB1 distributions at dynamic ends suggests no differences in GTP cap sizes. Last, we show that the addition of recombinant α1A/ßIII tubulin, a neuronal isotype overexpressed in many tumors, proportionally tunes the dynamics of α1B/ßI+ßIVb microtubules. Our study is an important step toward understanding how tubulin isoform composition tunes microtubule dynamics.

The divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry.

MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% identity) and size (~40% larger) compared to other kinesins. Using cryo-electron microscopy and biophysical assays, we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle, and show that many facets of its mechanism are distinct from other kinesins. While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state, it does not fully dock along the motor domain. Furthermore, the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins, and enhanced by kinesin-6-specific sequences. The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface. Our results emphasize the role of family-specific insertions in modulating kinesin motor function.

A structural model for microtubule minus-end recognition and protection by CAMSAP proteins.

CAMSAP and Patronin family members regulate microtubule minus-end stability and localization and thus organize noncentrosomal microtubule networks, which are essential for cell division, polarization and differentiation. Here, we found that the CAMSAP C-terminal CKK domain is widely present among eukaryotes and autonomously recognizes microtubule minus ends. Through a combination of structural approaches, we uncovered how mammalian CKK binds between two tubulin dimers at the interprotofilament interface on the outer microtubule surface. In vitro reconstitution assays combined with high-resolution fluorescence microscopy and cryo-electron tomography suggested that CKK preferentially associates with the transition zone between curved protofilaments and the regular microtubule lattice. We propose that minus-end-specific features of the interprotofilament interface at this site serve as the basis for CKK's minus-end preference. The steric clash between microtubule-bound CKK and kinesin motors explains how CKK protects microtubule minus ends against kinesin-13-induced depolymerization and thus controls the stability of free microtubule minus ends.

Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins.

Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles--including their nucleotide-free states--at ∼ 7 Šresolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin-microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface.