Single Microtubule Research Articles

The Cryo-EM Structure and Activity of Kinesin-5 from Plasmodium falciparum: Mechanistic Lessons from a Parasite Kinesin

Kinesin superfamily members use ATP to drive mechanical output on the microtubule cytoskeleton. This mechanochemical behaviour has been specialised in the different kinesin families, and also between species. It has also been hypothesised that such differences could permit kinesin family, and even species specific inhibition by small-molecules. This triggered our structural and biochemical studies on the kinesin-5 family member from the malaria causing parasite – Plasmodium falciparum Kinesin-5 (PfK5). From this data we aimed to gain insight into the key aspects of kinesin mechanochemistry, and how this motor function may be adapted between species. In addition, we explored whether these adaptions can be exploited for the development of species specific inhibition of kinesin-5s - potentially opening the door to new anti-malarial therapeutics. To do this we employed single particle cryo-electron microscopy to visualize PfK5 in nucleotide-free and ATP-like stages of its mechanochemical cycle bound to microtubules. We used new techniques in 3D image processing and molecular modelling to obtain a near-atomic representation of PfK5's mechanochemical cycle. In conjunction with studies on PfK5's steady state ATP activity, and single molecule microtubule binding using total internal reflection microscopy, we have described PfK5 molecular properties. We find that PfK5 exhibits nucleotide dependent conformational changes and biochemical behaviour that are characteristic of a kinesin-5 family member. Conversely, we also observe evidence of some atypical biochemical properties which are explained by several unexpected conformations seen in our 3D reconstructions. Strikingly, a Plasmodium species specific insertion radically alters the chemical environment of the kinesin drug binding site, offering the possibility of PfK5-specific inhibition. Our results broaden our understanding of how evolution adapts the core function of eukaryotic kinesin mechanochemistry, and how we may utilise these differences in therapeutic development.

Complex nearly immotile behaviour of enzymatically driven cargos.

We report a minimal microtubule-based motile system displaying signatures of unconventional diffusion. The system consists of a single model cargo driven by an ensemble of N340K NCD motors along a single microtubule. Despite the absence of cytosolic or cytoskeleton complexity, the system shows complex behavior, characterized by sub-diffusive motion for short time lag scales and linear mean squared displacement dependence for longer time lags. The latter is also shown to have non-Gaussian character and cannot be ascribed to a canonical diffusion process. We use single particle tracking and analysis at varying temperatures and motor concentrations to identify the origin of these behaviors as enzymatic activity of mutant NCD. Our results show that signatures of non-Gaussian diffusivities can arise as a result of an active process and suggest that some immotility of cargos observed in cells may reflect the ensemble workings of mechanochemical enzymes and need not always reflect the properties of the cytoskeletal network or the cytosol.

Collective dynamics of microtubule-based 3D active fluids from single microtubules.

Self-organization of kinesin-driven, microtubule-based 3D active fluids relies on the collective dynamics of single microtubules. However, the connection between macroscopic fluid flows and microscopic motion of microtubules remains unclear. In this work, the motion of single microtubules was characterized by means of 2D gliding assays and compared with the flows of 3D active fluids. While the scales of the two systems differ by ∼1000×, both were driven by processive, non-processive or an equal mixture of both molecular motor proteins. To search for the dynamic correlation between both systems, the motor activities were tuned by varying temperature and ATP concentration, and the changes in both systems were compared. Motor processivity played an important role in active fluid flows but only when the fluids were nearly motionless; otherwise, flows were dominated by hydrodynamic resistance controlled by sample size. Furthermore, while the motors' thermal reaction led active fluids to flow faster with increasing temperature, such temperature dependence could be reversed by introducing temperature-varying depletants, emphasizing the potential role of the depletant in designing an active fluid's temperature response. The temperature response of active fluids was nearly immediate (⪅10 s). Such a characteristic enables active fluids to be controlled with a temperature switch. Overall, this work not only clarifies the role of temperature in active fluid activity, but also sheds light on the underlying principles of the relationship between the collective dynamics of active fluids and the dynamics of their constituent single microtubules.

Transport of microtubules according to the number and spacing of kinesin motors on gold nano-pillars.

Motor proteins function in in vivo ensembles to achieve cargo transport, flagellum motion, and mitotic cell division. Although the cooperativity of multiple motors is indispensable for physiological function, reconstituting the arrangement of motors in vitro is challenging, so detailed analysis of the functions of motor ensembles has not yet been achieved. Here, we developed an assay platform to study the motility of microtubules driven by a defined number of kinesin motors spaced in a definite manner. Gold (Au) nano-pillar arrays were fabricated on a silicon/silicon dioxide (Si/SiO2) substrate with spacings of 100 nm to 500 nm. The thiol-polyethylene glycol (PEG)-biotin self-assembled monolayer (SAM) and silane-PEG-CH3 SAM were then selectively formed on the pillars and SiO2 surface, respectively. This allowed for both immobilization of kinesin molecules on Au nano-pillars in a precise manner and repulsion of kinesins from the SiO2 surface. Using arrayed kinesin motors, we report that motor number and spacing do not influence the motility of microtubules driven by kinesin-1 motors. This assay platform is applicable to all kinds of biotinylated motors, allows the study of the effects of motor number and spacing, and is expected to reveal novel behaviors of motor proteins.

CCDC102B functions in centrosome linker assembly and centrosome cohesion.

The proteinaceous centrosome linker is an important structure that allows the centrosome to function as a single microtubule-organizing center (MTOC) in interphase cells. However, the assembly mechanism of the centrosome linker components remains largely unknown. In this study, we identify CCDC102B as a new centrosome linker protein that is required for maintaining centrosome cohesion. CCDC102B is recruited to the centrosome by C-Nap1 (also known as CEP250) and interacts with the centrosome linker components rootletin and LRRC45. CCDC102B decorates and facilitates the formation of rootletin filaments. Furthermore, CCDC102B is phosphorylated by Nek2A (an isoform encoded by NEK2) and is disassociated from the centrosome at the onset of mitosis. Together, our findings reveal a molecular role for CCDC102B in centrosome cohesion and centrosome linker assembly.This article has an associated First Person interview with the first authors of the paper.

Label-free high-speed wide-field imaging of single microtubules using interference reflection microscopy.

The cytoskeleton gives a cell its shape and plays a major role in its movement and division. It's also helps organise the content of cells and is the base for intracellular transport. Important components of the cytoskeleton are microtubules, which are hollow cylindrical beams (25 nm in diameter) that assemble from protein building blocks called tubulin. Deficiencies in microtubules are related to many diseases including cancer and Alzheimer. Given their important role, microtubules are heavily investigated in many laboratories. One way to study microtubules is to isolate them from cells and image them using light microscopy. Over the years a number of imaging techniques have been used. These techniques have a number of drawbacks which are addressed by ongoing efforts which this work is a part of. Here, we present a method based on light interference that produce high quality images of microtubules. The technique is cheap and easy to implement making it accessible to a wide base of researchers.

Dual-spindle formation in zygotes keeps parental genomes apart in early mammalian embryos.

At the beginning of mammalian life, the genetic material from each parent meets when the fertilized egg divides. It was previously thought that a single microtubule spindle is responsible for spatially combining the two genomes and then segregating them to create the two-cell embryo. We used light-sheet microscopy to show that two bipolar spindles form in the zygote and then independently congress the maternal and paternal genomes. These two spindles aligned their poles before anaphase but kept the parental genomes apart during the first cleavage. This spindle assembly mechanism provides a potential rationale for erroneous divisions into more than two blastomeric nuclei observed in mammalian zygotes and reveals the mechanism behind the observation that parental genomes occupy separate nuclear compartments in the two-cell embryo.

The Central Stalk Determines the Motility of Mitotic Kinesin-14 Homodimers

Mitotic kinesin-14 homodimers that contain an N-terminal nonmotor microtubule-binding tail contribute to spindle organization by preferentially crosslinking two different spindle microtubules rather than interacting with a single microtubule to generate processive motility. However, the mechanism underlying such selective motility behavior remains poorly understood. Here, we show that when a flexible polypeptide linker is inserted into the coiled-coil central stalk, two homodimeric mitotic kinesin-14s of distinct motility-the processive plus-end-directed KlpA from Aspergillus nidulans [1] and the nonprocessive minus-end-directed Ncd from Drosophila melanogaster [2]-both switch to become processive minus-end-directed motors. Our results demonstrate that the polypeptide linker introduces greater conformational flexibility into the central stalk. Importantly, we find that the linker insertion significantly weakens the ability of Ncd to preferentially localize between and interact with two microtubules. Collectively, our results reveal that besides the canonical role of enabling dimerization, the central stalk also functions as a mechanical component to determine the motility of homodimeric mitotic kinesin-14 motors. We suggest that the central stalk is an evolutionary design that primes these kinesin-14 motors for nontransport roles within the mitotic spindle.

Molecular force spectroscopy of kinetochore-microtubule attachment in silico: Mechanical signatures of an unusual catch bond and collective effects.

Measurement of the lifetime of attachments formed by a single microtubule (MT) with a single kinetochore (kt) in vitro under force-clamp conditions had earlier revealed a catch-bond-like behavior. In the past, the physical origin of this apparently counterintuitive phenomenon was traced to the nature of the force dependence of the (de)polymerization kinetics of the MTs. Here, first the same model MT-kt attachment is subjected to external tension that increases linearly with time until rupture occurs. In our force-ramp experiments in silico, the model displays the well known "mechanical signatures" of a catch bond probed by molecular force spectroscopy. Exploiting this evidence, we have further strengthened the analogy between MT-kt attachments and common ligand-receptor bonds in spite of the crucial differences in their underlying physical mechanisms. We then extend the formalism to model the stochastic kinetics of an attachment formed by a bundle of multiple parallel microtubules with a single kt considering the effect of rebinding under force-clamp and force-ramp conditions. From numerical studies of the model we predict the trends of variation of the mean lifetime and mean rupture force with the increasing number of MTs in the bundle. Both the mean lifetime and the mean rupture force display nontrivial nonlinear dependence on the maximum number of MTs that can attach simultaneously to the same kt.

Complete microtubule\u2013kinetochore occupancy favours the segregation of merotelic attachments

Kinetochores are multi-protein complexes that power chromosome movements by tracking microtubules plus-ends in the mitotic spindle. Human kinetochores bind up to 20 microtubules, even though single microtubules can generate sufficient force to move chromosomes. Here, we show that high microtubule occupancy at kinetochores ensures robust chromosome segregation by providing a strong mechanical force that favours segregation of merotelic attachments during anaphase. Using low doses of the microtubules-targeting agent BAL27862 we reduce microtubule occupancy and observe that spindle morphology is unaffected and bi-oriented kinetochores can still oscillate with normal intra-kinetochore distances. Inter-kinetochore stretching is, however, dramatically reduced. The reduction in microtubule occupancy and inter-kinetochore stretching does not delay satisfaction of the spindle assembly checkpoint or induce microtubule detachment via Aurora-B kinase, which was so far thought to release microtubules from kinetochores under low stretching. Rather, partial microtubule occupancy slows down anaphase A and increases incidences of lagging chromosomes due to merotelically attached kinetochores.

LED-based interference-reflection microscopy combined with optical tweezers for quantitative three-dimensional microtubule imaging.

Optical tweezers combined with various microscopy techniques are a versatile tool for single-molecule force spectroscopy. However, some combinations may compromise measurements. Here, we combined optical tweezers with total-internal-reflection-fluorescence (TIRF) and interference-reflection microscopy (IRM). Using a light-emitting diode (LED) for IRM illumination, we show that single microtubules can be imaged with high contrast. Furthermore, we converted the IRM interference pattern of an upward bent microtubule to its three-dimensional (3D) profile calibrated against the optical tweezers and evanescent TIRF field. In general, LED-based IRM is a powerful method for high-contrast 3D microscopy.

The preprophase band-associated kinesin-14 OsKCH2 is a processive minus-end-directed microtubule motor

In animals and fungi, cytoplasmic dynein is a processive minus-end-directed motor that plays dominant roles in various intracellular processes. In contrast, land plants lack cytoplasmic dynein but contain many minus-end-directed kinesin-14s. No plant kinesin-14 is known to produce processive motility as a homodimer. OsKCH2 is a plant-specific kinesin-14 with an N-terminal actin-binding domain and a central motor domain flanked by two predicted coiled-coils (CC1 and CC2). Here, we show that OsKCH2 specifically decorates preprophase band microtubules in vivo and transports actin filaments along microtubules in vitro. Importantly, OsKCH2 exhibits processive minus-end-directed motility on single microtubules as individual homodimers. We find that CC1, but not CC2, forms the coiled-coil to enable OsKCH2 dimerization. Instead, our results reveal that removing CC2 renders OsKCH2 a nonprocessive motor. Collectively, these results show that land plants have evolved unconventional kinesin-14 homodimers with inherent minus-end-directed processivity that may function to compensate for the loss of cytoplasmic dynein.

Bidirectional motility of kinesin-5 motor proteins: structural determinants, cumulative functions and physiological roles.

Mitotic kinesin-5 bipolar motor proteins perform essential functions in mitotic spindle dynamics by crosslinking and sliding antiparallel microtubules (MTs) apart within the mitotic spindle. Two recent studies have indicated that single molecules of Cin8, the Saccharomyces cerevisiae kinesin-5 homolog, are minus end-directed when moving on single MTs, yet switch directionality under certain experimental conditions (Gerson-Gurwitz et al., EMBO J 30:4942-4954, 2011; Roostalu et al., Science 332:94-99, 2011). This finding was unexpected since the Cin8 catalytic motor domain is located at the N-terminus of the protein, and such kinesins have been previously thought to be exclusively plus end-directed. In addition, the essential intracellular functions of kinesin-5 motors in separating spindle poles during mitosis can only be accomplished by plus end-directed motility during antiparallel sliding of the spindle MTs. Thus, the mechanism and possible physiological role of the minus end-directed motility of kinesin-5 motors remain unclear. Experimental and theoretical studies from several laboratories in recent years have identified additional kinesin-5 motors that are bidirectional, revealed structural determinants that regulate directionality, examined the possible mechanisms involved and have proposed physiological roles for the minus end-directed motility of kinesin-5 motors. Here, we summarize our current understanding of the remarkable ability of certain kinesin-5 motors to switch directionality when moving along MTs.

Geometry Matters for Cargos Navigating 3D Microtubule Intersections

Author(s): Bovyn, Matthew Jacob | Advisor(s): Allard, Jun F; Gross, Steven P | Abstract: Eukaryotic cells transport cargos along microtubules to control their distribution within the cell and deliver them to distant locations. While we understand how molecular motors can transport cargos along individual microtubules, the cell's microtubules are usually arranged in a complex 3D network. While traversing this network, cargos need to navigate intersections where microtubules cross at a wide variety of separation distances and angles. To gain insight into how cargos navigate these intersections, we have used a recently established 3D construction technique based on holographic optical trapping to build single 3D microtubule intersections in vitro with relevant nanoscale precision. We then used these fully suspended microtubule structures to perform motility assays on kinesin-1 coated cargos. We find that some intersection geometries influence cargos to pass along their current microtubule, while other geometries influence them to switch to the intersecting one. To understand how, we use a 3D Brownian dynamics simulation of cargo transport to investigate the mechanisms which give rise to the observed switching probabilities. Using these stochastic simulations, we find that switching probability is often determined by a competition between a stronger motor team on the primary microtubule and the intersecting microtubule sterically hindering that team's progress. This understanding of the basic mechanisms of switching at single intersections in 3D helps lay a foundation for understanding how the cell may regulate switching to control how cargos navigate the MT network and ultimately their spatial organization.

Label Free High Speed Wide Field Imaging of Single Microtubules using Interference Reflection Microscopy

When studying microtubules, label free imaging of single microtubules is necessary when the quantities of purified tubulin is too low for efficient fluorescent labeling. Commonly used techniques for observing unlabeled microtubules, such as video enhanced differential interference (VE-DIC), dark-field (DF) and more recently laser-based interferometric scattering (iSCAT) microscopy, suffer from a number of drawbacks. For example, contrast dependence on microtubule orientation (DIC), high sensitivity to impurities and to misalignments (DF), and complexity and limited field of view (iSCAT). In addition, all of these techniques require costly optical components such as DIC prisms, dark-field condensers, and lasers and laser scanners. Here we show wide-field high-speed imaging of single microtubules using interference reflection microscopy (IRM) that does not suffer from any of the aforementioned drawbacks. The technique only requires the incorporation of a 50/50 mirror in a fluorescence microscope. We optimized the microscope settings to achieve highest signal-to-noise ratio. We also compared IRM to DIC and fluorescence imaging. Finally, we demonstrated the strength of the technique by high speed (100 fps) imaging and tracking of dynamic microtubules. In conclusion, the image quality of IRM is comparable to aforementioned techniques and, with minimal microscope modification, can be used to study the dynamics of unlabeled microtubules.

Label-free Imaging and Bending Analysis of Microtubules by ROCS Microscopy and Optical Trapping

Mechanical manipulation of single cytoskeleton filaments and their monitoring over long times is difficult because of fluorescence bleaching or phototoxic protein degradation. The integration of label-free microscopy techniques, capable of imaging freely diffusing, weak scatterers such as microtubules (MTs) in real-time, and independent of their orientation, with optical trapping and tracking systems, would allow many new applications. Here, we show that rotating-coherent-scattering microscopy (ROCS) in dark-field mode can also provide strong contrast for structures far from the coverslip such as arrangements of isolated MTs and networks. We could acquire thousands of images over up to 30 min without loss in image contrast or visible photodamage. We further demonstrate the combination of ROCS imaging with fast and nanometer-precise 3D interferometric back-focal-plane tracking of multiple beads in time-shared optical traps using acoustooptic deflectors to specifically construct and microrheologically probe small microtubule networks with well-defined geometries. Thereby, we explore the frequency-dependent elastic response of single microtubule filaments between 0.5 Hz and 5 kHz, which allows for investigating their viscoelastic response up to the fourth-order bending mode. Our spectral analysis reveals constant filament stiffness at low frequencies and frequency-dependent stiffening following a power law ∼ωp with a length-dependent exponent p(L). We find further evidence for the dependence of the MT persistence length on the contour length L, which is still controversially debated. We could also demonstrate slower stiffening at high frequencies for longer filaments, which we believe is determined by the molecular architecture of the MT. Our results shed new light on the nanomechanics of this essential, multifunctional cytoskeletal element and pose new questions about the adaptability of the cytoskeleton.

The Microtubule-Associated Protein Tau Mediates the Organization of Microtubules and Their Dynamic Exploration of Actin-Rich Lamellipodia and Filopodia of Cortical Growth Cones

Proper organization and dynamics of the actin and microtubule (MT) cytoskeleton are essential for growth cone behaviors during axon growth and guidance. The MT-associated protein tau is known to mediate actin/MT interactions in cell-free systems but the role of tau in regulating cytoskeletal dynamics in living neurons is unknown. We used cultures of cortical neurons from postnatal day (P)0-P2 golden Syrian hamsters (Mesocricetus auratus) of either sex to study the role of tau in the organization and dynamics of the axonal growth cone cytoskeleton. Here, using super resolution microscopy of fixed growth cones, we found that tau colocalizes with MTs and actin filaments and is also located at the interface between actin filament bundles and dynamic MTs in filopodia, suggesting that tau links these two cytoskeletons. Live cell imaging in concert with shRNA tau knockdown revealed that reducing tau expression disrupts MT bundling in the growth cone central domain, misdirects trajectories of MTs in the transition region and prevents single dynamic MTs from extending into growth cone filopodia along actin filament bundles. Rescue experiments with human tau expression restored MT bundling, MT penetration into the growth cone periphery and close MT apposition to actin filaments in filopodia. Importantly, we found that tau knockdown reduced axon outgrowth and growth cone turning in Wnt5a gradients, likely due to disorganized MTs that failed to extend into the peripheral domain and enter filopodia. These results suggest an important role for tau in regulating cytoskeletal organization and dynamics during growth cone behaviors.SIGNIFICANCE STATEMENT Growth cones are the motile tips of growing axons whose guidance behaviors require interaction of the dynamic actin and microtubule cytoskeleton. Tau is a microtubule-associated protein that stabilizes microtubules in neurons and in cell-free systems regulates actin-microtubule interaction. Here, using super resolution microscopy, live-cell imaging, and tau knockdown, we show for the first time in living axonal growth cones that tau is important for microtubule bundling and microtubule exploration of the actin-rich growth cone periphery. Importantly tau knockdown reduced axon outgrowth and growth cone turning, due to disorganized microtubules that fail to enter filopodia and co-align with actin filaments. Understanding normal tau functions will be important for identifying mechanisms of tau in neurodegenerative diseases such as Alzheimer's.

Targeted Activation of Molecular Transportation by Visible Light.

Regulated transportation of nanoscale objects with a high degree of spatiotemporal precision is a prerequisite for the development of targeted molecular delivery. In vitro integration of the kinesin-microtubule motor system with synthetic molecules offers opportunities to develop controllable molecular shuttles for lab-on-a-chip applications. We attempted a combination of the kinesin-microtubule motor system with push-pull type azobenzene tethered inhibitory peptides (azo-peptides) through which reversible, spatiotemporal control over the kinesin motor activity was achieved locally by a single, visible wavelength. The fast thermal relaxation of the cis-isomers of azo-peptides offered us quick and complete resetting of the trans-state in the dark, circumventing the requirement of two distinct wavelengths for two-way switching of kinesin-driven microtubule motility. Herein, we report the manipulation of selected, single microtubule movement while keeping other microtubules at complete rest. The photoresponsive inhibitors discussed herein would help in realizing complex bionanodevices.

Computational vibration and buckling analysis of microtubule bundles based on nonlocal strain gradient theory

A nonlocal strain gradient model is proposed to study buckling and vibrational responses of microtubules in axons with attention to different size effect parameters based on finite element method. Supporting effects of surrounding cytoplasm and MAP Tau proteins are considered. Microtubules are modeled as elastically connected improved Timoshenko nano-beams resting on a two-parameter Pasternak foundation. Differential equations are discretized using Galerkin method. Finally, two eigenvalue problems are solved to achieve critical buckling loads and frequencies of single and doubled-microtubule systems. The nonlocal strain gradient model is employed in order to show both hardening and softening effects of structural stiffness, based on relative magnitudes of nanoscale parameters. Influence of size effects, including nonlocal nanoscale parameter, gradient coefficient and surface effects, are examined for various boundary conditions and some benchmark results are reported. It is observed that these effects are more prominent at higher modes. Based on presented numerical results, MAP Tau proteins strengthen doubled microtubule systems to bear 11.7% more buckling load than a single microtubule. Furthermore, vibration frequencies of microtubules depend on their physical surrounding condition in cell; such as cell matrix and membrane, and since microtubules could be employed as biosensors, this property may be used in order to detect malignant tumors, based on vibrational damping.

Calcium-axonemal microtubuli interactions underlie mechanism(s) of primary cilia morphological changes.

We have used cell culture of astrocytes aligned within microchannels to investigate calcium effects on primary cilia morphology. In the absence of calcium and in the presence of flow of media (10μL.s-1) the majority (90%) of primary cilia showed reversible bending with an average curvature of 2.1±0.9×10-4nm-1. When 1.0mM calcium was present, 90% of cilia underwent bending. Forty percent of these cilia demonstrated strong irreversible bending, resulting in a final average curvature of 3.9±1×10-4nm-1, while 50% of cilia underwent bending similar to that observed during calcium-free flow. The average length of cilia was shifted toward shorter values (3.67±0.34μm) when exposed to excess calcium (1.0mM), compared to media devoid of calcium (3.96±0.26μm). The number of primary cilia that became curved after calcium application was reduced when the cell culture was pre-incubated with 15μM of the microtubule stabilizer, taxol, for 60min prior to calcium application. Calcium caused single microtubules to curve at a concentration ≈1.0mM in vitro, but at higher concentration (≈1.5mM) multiple microtubule curving occurred. Additionally, calcium causes microtubule-associated protein-2 conformational changes and its dislocation from the microtubule wall at the location of microtubule curvature. A very small amount of calcium, that is 1.45×1011 times lower than the maximal capacity of TRPPs calcium channels, may cause gross morphological changes (curving) of primary cilia, while global cytosol calcium levels are expected to remain unchanged. These findings reflect the non-linear manner in which primary cilia may respond to calcium signaling, which in turn may influence the course of development of ciliopathies and cancer.