Microtubule Tips Research Articles

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.

Cross-linkers at growing microtubule ends generate forces that drive actin transport.

SignificanceComplex cellular processes such as cell migration require coordinated remodeling of both the actin and the microtubule cytoskeleton. The two networks for instance exert forces on each other via active motor proteins. Here we show that, surprisingly, coupling via passive cross-linkers can also result in force generation. We specifically study the transport of actin filaments by growing microtubule ends. We show by cell-free reconstitution experiments, computer simulations, and theoretical modeling that this transport is driven by the affinity of the cross-linker for the chemically distinct microtubule tip region. Our work predicts that growing microtubules could potentially rapidly relocate newly nucleated actin filaments to the leading edge of the cell and thus boost migration.

Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate dynamic instability

Kinesin-14 molecular motors represent an essential class of proteins that bind microtubules and walk toward their minus-ends. Previous studies have described important roles for Kinesin-14 motors at microtubule minus-ends, but their role in regulating plus-end dynamics remains controversial. Kinesin-14 motors have been shown to bind the EB family of microtubule plus-end binding proteins, suggesting that these minus-end-directed motors could interact with growing microtubule plus-ends. In this work, we explored the role of minus-end-directed Kinesin-14 motor forces in controlling plus-end microtubule dynamics. In cells, a Kinesin-14 mutant with reduced affinity to EB proteins led to increased microtubule lengths. Cell-free biophysical microscopy assays were performed using Kinesin-14 motors and an EB family marker of growing microtubule plus-ends, Mal3, which revealed that when Kinesin-14 motors bound to Mal3 at growing microtubule plus-ends, the motors subsequently walked toward the minus-end, and Mal3 was pulled away from the growing microtubule tip. Strikingly, these interactions resulted in an approximately twofold decrease in the expected postinteraction microtubule lifetime. Furthermore, generic minus-end-directed tension forces, generated by tethering growing plus-ends to the coverslip using λ-DNA, led to an approximately sevenfold decrease in the expected postinteraction microtubule growth length. In contrast, the inhibition of Kinesin-14 minus-end-directed motility led to extended tip interactions and to an increase in the expected postinteraction microtubule lifetime, indicating that plus-ends were stabilized by nonmotile Kinesin-14 motors. Together, we find that Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate microtubule lengths in cells.

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

Microtubules (MTs), mesoscopic cellular filaments, grow primarily by the addition of GTP-bound tubulin dimers at their dynamic flaring plus-end tips. MTs can perform mechanical work by cycling between phases of growth and shortening, driven by both GTP hydrolysis in tubulin subunits and dynamics of individual protofilaments (PFs) at the plus-end tips. The mechanochemical basis of energy conversion and transduction by MTs has remained elusive and controversial, despite extensive work. Moreover, recent experimental findings have created the paradox that the plus-end MT tips look very similar during both growing and shrinking phases, thereby challenging the traditional textbook picture.

A liquid +TIP-network drives microtubule dynamics through tubulin condensation

Tubulin dimers assemble into a dynamic microtubule network throughout the cell. Microtubule dynamics and network organization must be precisely tuned for the microtubule cytoskeleton to regulate a dazzling array of dynamic cell behaviors. Since tubulin concentration determines microtubule growth, we studied here a novel regulatory mechanism of microtubule dynamics: local tubulin condensation. We discovered that two microtubule tip-binding proteins, CLIP-170 and EB3, undergo phase separation and form a liquid-like network at the growing microtubule tip in cells.

Magnesium increases the chemo-mechanical energy released by curling protofilaments during microtubule disassembly

Disassembling microtubules provide forces that drive chromosomal movement in dividing cells. This motion can occur independently of conventional motor activity, and instead relies on the tenacious coupling of kinetochores to dynamic microtubule tips. During microtubule disassembly, kinetochores maintain load-bearing tip attachments even as thousands of tubulin subunits are lost from the tip per second. Despite recent advances in our structural and functional understanding of kinetochores and microtubules, it remains unknown how these molecular machines sustain such persistent attachments.

Spastin accumulates on shrinking microtubule tips by slowing them down

The microtubule-severing enzyme spastin is an important regulator of microtubule dynamics and its mutation is implicated in hereditary spastic paraplegia. In addition to severing microtubules, spastin also slows their depolymerization and promotes their transition from shrinkage to growth (rescue). This activity, in combination with severing, can lead to the amplification of microtubule number and mass. The effects of spastin on microtubule dynamics may be enhanced by its observed accumulation on the tips of shrinking microtubules, but the mechanism of tip accumulation is unclear.

Microtubule assembly and disassembly dynamics model: Exploring dynamic instability and identifying features of Microtubules’ Growth, Catastrophe, Shortening, and Rescue

Microtubules (MTs), a cellular structure element, exhibit dynamic instability and can switch stochastically from growth to shortening; but the factors that trigger these processes at the molecular level are not understood. We developed a 3D Microtubule Assembly and Disassembly DYnamics (MADDY) model, based upon a bead-per-monomer representation of the αβ-tubulin dimers forming an MT lattice, stabilized by the lateral and longitudinal interactions between tubulin subunits. The model was parameterized against the experimental rates of MT growth and shortening, and pushing forces on the Dam1 protein complex due to protofilaments splaying out. Using the MADDY model, we carried out GPU-accelerated Langevin simulations to access dynamic instability behavior. By applying Machine Learning techniques, we identified the MT characteristics that distinguish simultaneously all four kinetic states: growth, catastrophe, shortening, and rescue. At the cellular 25 μM tubulin concentration, the most important quantities are the MT length L, average longitudinal curvature κlong, MT tip width w, total energy of longitudinal interactions in MT lattice Ulong, and the energies of longitudinal and lateral interactions required to complete MT to full cylinder Ulongadd and Ulatadd. At high 250 μM tubulin concentration, the most important characteristics are L, κlong, number of hydrolyzed αβ-tubulin dimers nhyd and number of lateral interactions per helical pitch nlat in MT lattice, energy of lateral interactions in MT lattice Ulat, and energy of longitudinal interactions in MT tip ulong. These results allow greater insights into what brings about kinetic state stability and the transitions between states involved in MT dynamic instability behavior.

Catching the Conformational Wave: Measuring the Working Strokes of Protofilaments as They Curl Outward from Disassembling Microtubule Tips.

Optical traps have enabled foundational studies of how mechanoenzymes such as kinesins and dynein motors walk along microtubules, how myosins move along F-actin, and how nucleic acid enzymes move along DNA or RNA. Often the filamentous substrates serve merely as passive tracks for mechanoenzymes but microtubules and F-actin are themselves dynamic protein polymers, capable of generating movement and force independently of conventional motors. Microtubule-driven forces are particularly important during mitosis, when they align duplicated chromosomes at the metaphase plate and then pull them apart during anaphase. These vital movements depend on specialized protein assemblies called kinetochores that couple the chromosomes to the tips of dynamic microtubule filaments, thereby allowing filament shortening to produce pulling forces. Although great strides have been made toward understanding the structures and functions of many kinetochore subcomplexes, the biophysical basis for their coupling to microtubule tips remains unclear. During tip disassembly, strain energy is released when straight protofilaments in the microtubule lattice curl outward, creating a conformational wave that propagates down the microtubule. A popular viewpoint is that the protofilaments as they curl outward hook elements of the kinetochore and tug on them, transferring some of their curvature strain energy to the kinetochore. As a first step toward testing this idea, we recently developed a laser trap assay to directly measure the working strokes generated by curling protofilaments. Our "wave" assay is based on an earlier pioneering study, with improvements that allow measurement of curl-driven movements as functions of force and quantification of their conformational strain energy. In this chapter, we provide a detailed protocol for our assay and describe briefly our instrument setup and data analysis methods.

Visualization and Quantification of Microtubule Self-Repair.

Since its discovery, several decades ago, microtubule dynamic instability has been the subject of countless studies that demonstrate its impact on cellular behavior in health and disease. Recent studies reveal a new dimension of microtubule dynamics. Microtubules are not only dynamic at their tips but also exhibit loss and incorporation of tubulin subunits along their lattice far from the tips. Although this phenomenon has been observed to occur under various conditions in vitro as well as in cells, many questions remain regarding the regulation of lattice dynamics and their contribution to overall microtubule network organization and function. Compared to microtubule tip dynamics, the dynamics of tubulin incorporation along the lattice are more challenging to investigate as they are hidden in classical experimental setups, which is likely the reason they were overlooked for a long time. In this chapter, we present a strategy to visualize and quantify the incorporation of tubulin subunits into the microtubule lattice in vitro. The proposed method does not require specialized equipment and can thus be carried out readily in most research laboratories.

Lattice defects induced by microtubule-stabilizing agents exert a long-range effect on microtubule growth by promoting catastrophes

Microtubules are dynamic cytoskeletal polymers that spontaneously switch between phases of growth and shrinkage. The probability of transitioning from growth to shrinkage, termed catastrophe, increases with microtubule age, but the underlying mechanisms are poorly understood. Here, we set out to test whether microtubule lattice defects formed during polymerization can affect growth at the plus end. To generate microtubules with lattice defects, we used microtubule-stabilizing agents that promote formation of polymers with different protofilament numbers. By employing different agents during nucleation of stable microtubule seeds and the subsequent polymerization phase, we could reproducibly induce switches in protofilament number and induce stable lattice defects. Such drug-induced defects led to frequent catastrophes, which were not observed when microtubules were grown in the same conditions but without a protofilament number mismatch. Microtubule severing at the site of the defect was sufficient to suppress catastrophes. We conclude that structural defects within the microtubule lattice can exert effects that can propagate over long distances and affect the dynamic state of the microtubule end.

Overexpression of the microtubule-binding protein CLIP-170 induces a +TIP network superstructure consistent with a biomolecular condensate

Proper regulation of microtubule (MT) dynamics is critical for cellular processes including cell division and intracellular transport. Plus-end tracking proteins (+TIPs) dynamically track growing MTs and play a key role in MT regulation. +TIPs participate in a complex web of intra- and inter- molecular interactions known as the +TIP network. Hypotheses addressing the purpose of +TIP:+TIP interactions include relieving +TIP autoinhibition and localizing MT regulators to growing MT ends. In addition, we have proposed that the web of +TIP:+TIP interactions has a physical purpose: creating a dynamic scaffold that constrains the structural fluctuations of the fragile MT tip and thus acts as a polymerization chaperone. Here we examine the possibility that this proposed scaffold is a biomolecular condensate (i.e., liquid droplet). Many animal +TIP network proteins are multivalent and have intrinsically disordered regions, features commonly found in biomolecular condensates. Moreover, previous studies have shown that overexpression of the +TIP CLIP-170 induces large “patch” structures containing CLIP-170 and other +TIPs; we hypothesized that these structures might be biomolecular condensates. To test this hypothesis, we used video microscopy, immunofluorescence staining, and Fluorescence Recovery After Photobleaching (FRAP). Our data show that the CLIP-170-induced patches have hallmarks indicative of a biomolecular condensate, one that contains +TIP proteins and excludes other known condensate markers. Moreover, bioinformatic studies demonstrate that the presence of intrinsically disordered regions is conserved in key +TIPs, implying that these regions are functionally significant. Together, these results indicate that the CLIP-170 induced patches in cells are phase-separated liquid condensates and raise the possibility that the endogenous +TIP network might form a liquid droplet at MT ends or other +TIP locations.

Moving the ER tip by tip.

Endoplasmic reticulum (ER) and microtubule (MT) interactions have been observed in different cell types. However, how these interactions are regulated remains unknown. In this issue of Developmental Cell, Nourbakhsh etal. show that an ER-localized kinase, TAOK2, catalyzes the dynamic tethering of the ER tip to the MT tip.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons.

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

On the Relationship Between EB-3 Profiles and Microtubules Growth in Cultured Cells.

Microtubules are dynamic structures undergoing rapid growth and shrinkage in living cells and in vitro. The growth of microtubules in vitro was analyzed with subpixel precision (Maurer et al., Current Biology, 2014, 24 (4), 372–384); however, to what extent these results could be applied for microtubules growing in vivo remains largely unknown. Particularly, the question is whether microtubule growth velocity in cells could be sufficiently approximated by a Gaussian distribution or its variability requires a more sophisticated description? Addressing this question, we used time-lapse microscopy and mathematical modeling, and we analyzed EB-3 comets forming on microtubules of cultured cells with subpixel precision. Parameters of comets (shape, form, and velocity) were used as topological characteristics of 3D voxel objects. Using regression analysis, we determined the real positions of the microtubule tips in time-lapse sequences. By exponential decay fitting of the restored comet intensity profile, we found that in vivo EB-3 rapidly exchanges on growing microtubule ends with a decoration time ∼ 2 s. We next developed the model showing that the best correlation between comet length and microtubule end growth velocity is at time intervals close to the decoration time. In the cells, EB comet length positively correlates with microtubule growth velocity in preceding time intervals, while demonstrating no correlation in subsequent time intervals. Correlation between comet length and instantaneous growth velocity of microtubules remains under nocodazole treatment when mean values of both parameters decrease. Our data show that the growth of microtubules in living cells is well-approximated by a constant velocity with large stochastic fluctuations.

Optimizing microtubule arrangements for rapid cargo capture

Cellular functions such as autophagy, cell signaling, and vesicular trafficking involve the retrograde transport of motor-driven cargo along microtubules. Typically, newly formed cargo engages in slow undirected movement from its point of origin before attaching to a microtubule. In some cell types, cargo destined for delivery to the perinuclear region relies on capture at dynein-enriched loading zones located near microtubule plus ends. Such systems include extended cell regions of neurites and fungal hyphae, where the efficiency of the initial diffusive loading process depends on the axial distribution of microtubule plus ends relative to the initial cargo position. We use analytic mean first-passage time calculations and numerical simulations to model diffusive capture processes in tubular cells, exploring how the spatial arrangement of microtubule plus ends affects the efficiency of retrograde cargo transport. Our model delineates the key features of optimal microtubule arrangements that minimize mean cargo capture times. Namely, we show that configurations with a single microtubule plus end abutting the distal tip and broadly distributed other plus ends allow for efficient capture in a variety of different scenarios for retrograde transport. Live-cell imaging of microtubule plus ends in Aspergillus nidulans hyphae indicates that their distributions exhibit these optimal qualitative features. Our results highlight important coupling effects between the distribution of microtubule tips and retrograde cargo transport, providing guiding principles for the spatial arrangement of microtubules within tubular cell regions.

Regulation of microtubule dynamics, mechanics and function through the growing tip.

Microtubule dynamics and their control are essential for the normal function and division of all eukaryotic cells. This plethora of functions is, in large part, supported by dynamic microtubule tips, which can bind to various intracellular targets, generate mechanical forces and couple with actin microfilaments. Here, we review progress in the understanding of microtubule assembly and dynamics, focusing on new information about the structure of microtubule tips. First, we discuss evidence for the widely accepted GTP cap model of microtubule dynamics. Next, we address microtubule dynamic instability in the context of structural information about assembly intermediates at microtubule tips. Three currently discussed models of microtubule assembly and dynamics are reviewed. These are considered in the context of established facts and recent data, which suggest that some long-held views must be re-evaluated. Finally, we review structural observations about the tips of microtubules in cells and describe their implications for understanding the mechanisms of microtubule regulation by associated proteins, by mechanical forces and by microtubule-targeting drugs, prominently including cancer chemotherapeutics.

Structure and regulation of the microtubule plus-end tracking protein Kar9

In many eukaryotes, coordination of chromosome segregation with cell cleavage relies on the patterned interaction of specific microtubules with actin filaments through dedicated microtubule plus-end tracking proteins (+TIPs). However, how these+TIPs are spatially controlled is unclear. The yeast+TIP Kar9 drives one of the spindle aster microtubules along actin cables to align the mitotic spindle with the axis of cell division. Here, we report the crystal structure of Kar9's folded domain, revealing spectrin repeats reminiscent of the+TIPs MACF/ACF7/Shot and PRC1/Ase1. Point mutations abrogating spectrin-repeat-mediated dimerization of Kar9 reduced and randomized Kar9 distribution to microtubule tips, and impaired spindle positioning. Six Cdk1 sites surround the Kar9 dimerization interface. Their phosphomimetic substitution inhibited Kar9 dimerization, displaced Kar9 from microtubules, and affected its interaction with the myosin motor Myo2. Our results provide molecular-level understanding on how diverse cell types may regulate and pattern microtubule-actin interactions to orchestrate their divisions.

A CLK1-KKT2 Signaling Pathway Regulating Kinetochore Assembly in Trypanosoma brucei.

ABSTRACTDuring mitosis, eukaryotic cells must duplicate and separate their chromosomes in a precise and timely manner. The apparatus responsible for this is the kinetochore, which is a large protein structure that links chromosomal DNA and spindle microtubules to facilitate chromosome alignment and segregation. The proteins that comprise the kinetochore in the protozoan parasite Trypanosoma brucei are divergent from yeast and mammals and comprise an inner kinetochore complex composed of 24 distinct proteins (KKT1 to KKT23, KKT25) that include four protein kinases, CLK1 (KKT10), CLK2 (KKT19), KKT2, and KKT3. We recently reported the identification of a specific trypanocidal inhibitor of T. brucei CLK1, an amidobenzimidazole, AB1. We now show that chemical inhibition of CLK1 with AB1 impairs inner kinetochore recruitment and compromises cell cycle progression, leading to cell death. Here, we show that KKT2 is a substrate for CLK1 and identify phosphorylation of S508 by CLK1 to be essential for KKT2 function and for kinetochore assembly. Additionally, KKT2 protein kinase activity is required for parasite proliferation but not for assembly of the inner kinetochore complex. We also show that chemical inhibition of the aurora kinase AUK1 does not affect CLK1 phosphorylation of KKT2, indicating that AUK1 and CLK1 are in separate regulatory pathways. We propose that CLK1 is part of a divergent signaling cascade that controls kinetochore function via phosphorylation of the inner kinetochore protein kinase KKT2.

Two modes of PRC1-mediated mechanical resistance to kinesin-driven microtubule network disruption

The proper organization of the microtubule-based spindle during cell division requires the collective activity of many different proteins. These include non-motor microtubule-associated proteins (MAPs), whose functions include crosslinking microtubules to regulate filament sliding rates and assemble microtubule arrays. One such protein is PRC1, an essential MAP that has been shown to preferentially crosslink overlapping antiparallel microtubules at the spindle midzone. PRC1 has been proposed to act as a molecular brake, but insight into the mechanism of how PRC1 molecules function cooperatively to resist motor-driven microtubule sliding and to allow for the formation of stable midzone overlaps remains unclear. Here, we employ a modified microtubule gliding assay to rupture PRC1-mediated microtubule pairs using surface-bound kinesins. We discovered that PRC1 crosslinks always reduce bundled filament sliding velocities relative to single-microtubule gliding rates and do so via two distinct emergent modes of mechanical resistance to motor-driven sliding. We term these behaviors braking and coasting, where braking events exhibit substantially slowed microtubule sliding compared to coasting events. Strikingly, braking behavior requires the formation of two distinct high-density clusters of PRC1 molecules near microtubule tips. Our results suggest a cooperative mechanism for PRC1 accumulation when under mechanical load that leads to a unique state of enhanced resistance to filament sliding and provides insight into collective protein ensemble behavior in regulating the mechanics of spindle assembly.