Free Microtubules Research Articles

CSPP1 stabilizes microtubules by capping both plus and minus ends.

Although the dynamic instability of microtubules (MTs) is fundamental to many cellular functions, quiescent MTs with unattached free distal ends are commonly present and play important roles in various events to power cellular dynamics. However, how these free MT tips are stabilized remains poorly understood. Here, we report that centrosome and spindle pole protein 1 (CSPP1) caps and stabilizes both plus and minus ends of static MTs. Real-time imaging of laser-ablated MTs in live cells showed deposition of CSPP1 at the newly generated MT ends, whose dynamic instability was concomitantly suppressed. Consistently, MT ends in CSPP1-overexpressing cells were hyper-stabilized, while those in CSPP1-depleted cells were much more dynamic. This CSPP1-elicited stabilization of MTs was demonstrated to be achieved by suppressing intrinsic MT catastrophe and restricting polymerization. Importantly, CSPP1-bound MTs were resistant to mitotic centromere-associated kinesin-mediated depolymerization. These findings delineate a previously uncharacterized CSPP1 activity that integrates MT end capping to orchestrate quiescent MTs.

WDR47 protects neuronal microtubule minus ends from katanin-mediated severing.

Axons and dendrites are long extensions of neurons that contain arrays of noncentrosomal microtubules. Calmodulin-regulated spectrin-associated proteins (CAMSAPs) bind to and stabilize free microtubule minus ends and are critical for proper neuronal development and function. Previous studies have shown that the microtubule-severing ATPase katanin interacts with CAMSAPs and limits the length of CAMSAP-decorated microtubule stretches. However, how CAMSAP and microtubule minus end dynamics are regulated in neurons is poorly understood. Here, we show that the neuron-enriched protein WDR47 interacts with CAMSAPs and is critical for axon and dendrite development. We find that WDR47 accumulates at CAMSAP2-decorated microtubules, is essential for maintaining CAMSAP2 stretches, and protects minus ends from katanin-mediated severing. We propose a model where WDR47 protects CAMSAP2 at microtubule minus ends from katanin activity to ensure proper stabilization of the neuronal microtubule network.

Kinesin-14 motors drive a right-handed helical motion of antiparallel microtubules around each other

Within the mitotic spindle, kinesin motors cross-link and slide overlapping microtubules. Some of these motors exhibit off-axis power strokes, but their impact on motility and force generation in microtubule overlaps has not been investigated. Here, we develop and utilize a three-dimensional in vitro motility assay to explore kinesin-14, Ncd, driven sliding of cross-linked microtubules. We observe that free microtubules, sliding on suspended microtubules, not only rotate around their own axis but also move around the suspended microtubules with right-handed helical trajectories. Importantly, the associated torque is large enough to cause microtubule twisting and coiling. Further, our technique allows us to measure the in situ spatial extension of the motors between cross-linked microtubules to be about 20 nm. We argue that the capability of microtubule-crosslinking kinesins to cause helical motion of overlapping microtubules around each other allows for flexible filament organization, roadblock circumvention and torque generation in the mitotic spindle.

Microtubule Minus-End Binding Protein CAMSAP2 and Kinesin-14 Motor KIFC3 Control Dendritic Microtubule Organization

SummaryNeuronal dendrites are characterized by an anti-parallel microtubule organization. The mixed oriented microtubules promote dendrite development and facilitate polarized cargo trafficking; however, the mechanism that regulates dendritic microtubule organization is still unclear. Here, we found that the kinesin-14 motor KIFC3 is important for organizing dendritic microtubules and to control dendrite development. The kinesin-14 motor proteins (Drosophila melanogaster Ncd, Saccharomyces cerevisiae Kar3, Saccharomyces pombe Pkl1, and Xenopus laevis XCTK2) are characterized by a C-terminal motor domain and are well described to organize the spindle microtubule during mitosis using an additional microtubule binding site in the N terminus [1, 2, 3, 4]. In mammals, there are three kinesin-14 members, KIFC1, KIFC2, and KIFC3. It was recently shown that KIFC1 is important for organizing axonal microtubules in neurons, a process that depends on the two microtubule-interacting domains [5]. Unlike KIFC1, KIFC2 and KIFC3 lack the N-terminal microtubule binding domain and only have one microtubule-interacting domain, the motor domain [6, 7]. Thus, in order to regulate microtubule-microtubule crosslinking or sliding, KIFC2 and KIFC3 need to interact with additional microtubule binding proteins to connect two microtubules. We found that KIFC3 has a dendrite-specific distribution and interacts with microtubule minus-end binding protein CAMSAP2. Depletion of KIFC3 or CAMSAP2 results in increased microtubule dynamics during dendritic development. We propose a model in which CAMSAP2 anchors KIFC3 at microtubule minus ends and immobilizes microtubule arrays in dendrites.

Effect of viscoelastic medium on wave propagation along protein microtubules

An Orthotropic Kelvin-like model is developed here to study wave dispersion relation along microtubules when they are embedded in viscoelastic material. Owing anisotropicity of elastic shell like microtubules, an orthotropic elastic shell model is derived while the surrounding environment of microtubules is modeled as Kelvin like material. Symmetrical and asymmetrical waves are studied in embedded microtubules. We compared the wave velocities for embedded and free microtubules as well as the comparison of wave velocities for isotropic and orthotropic microtubules are also given. Longitudinal, Torsional and Radial wave velocities are obtained, noticing that torsional and radial wave velocities are lower in embedded microtubules as compared to longitudinal wave velocities in embedded microtubules. The radial wave frequency is considerably low because in cylindrical microtubules, pressure is exerted from the surroundings. The decrease in wave velocities is due to strong mechanical coupling of microtubules with surrounding medium and this decrement is more obvious when the wave length is long.

Coming into Focus: Mechanisms of Microtubule Minus-End Organization.

Microtubule organization has a crucial role in regulating cell architecture. The geometry of microtubule arrays strongly depends on the distribution of sites responsible for microtubule nucleation and minus-end attachment. In cycling animal cells, the centrosome often represents a dominant microtubule-organizing center (MTOC). However, even in cells with a radial microtubule system, many microtubules are not anchored at the centrosome, but are instead linked to the Golgi apparatus or other structures. Non-centrosomal microtubules predominate in many types of differentiated cell and in mitotic spindles. In this review, we discuss recent advances in understanding how the organization of centrosomal and non-centrosomal microtubule networks is controlled by proteins involved in microtubule nucleation and specific factors that recognize free microtubule minus ends and regulate their localization and dynamics.

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 non-centrosomal microtubule networks, which are essential for cell division, polarization and differentiation. Here, we show that the C-terminal CKK domain of CAMSAPs is widely present among eukaryotes and autonomously recognizes microtubule minus ends. Through a combination of structural approaches, we uncover how mammalian CKK binds between two tubulin dimers at the inter-protofilament interface on the outer microtubule surface. In vitro reconstitution assays combined with high resolution fluorescence microscopy and cryo-electron tomography suggest 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 inter-protofilament interface at this site form 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.

Centrosome nucleates numerous ephemeral microtubules and only few of them participate in the radial array.

It is generally accepted that long microtubules (MTs) grow from the centrosome with their minus ends anchored there and plus ends directed towards cell membrane. However, recent findings show this scheme to be an oversimplification. To further analyze the relationship between the centrosome and the MT array we undertook a detailed study on the MTs growing from the centrosome after microinjection of Cy3 labeled tubulin and transfection of cells with EB1-GFP. To evaluate MTs around the centrosome two approaches were used: path photobleaching across the centrosome area (Komarova et al., ) and sequential image subtraction analysis (Vorobjev et al., ). We show that about 50% of MTs had been nucleated at the centrosome are short-living: their mean length was 1.8 ± 0.8 μm and their life span - 7 ± 2 s. MTs initiated from the centrosome also rarely reach cell margin, since their elongation was limited and growth after shortening (rescue) was rare. After initial growth all MTs associated with the centrosome converted to pause or shortening. After pause MTs associated with the centrosome mainly depolymerized via the plus end shortening. Stability of the minus ends of cytoplasmic MTs was the same as for centrosomal ones. We conclude that in fibroblasts (1) the default behavior of free MTs in the cell interior is biased dynamic instability (i.e., random walk of the plus ends with significant positive drift); (2) MTs born at the centrosome show "dynamic instability" type behavior with no boundary; and (3) that the extended radial array is formed predominantly by MTs not associated with the centrosome.

The Ran-GTP Gradient Spatially Regulates XCTK2 in the Spindle

Ran is a small GTP binding protein that was originally identified as a regulator of nucleocytoplasmic transport [1] and subsequently found to be important for spindle formation [2-5]. In mitosis, a gradient of Ran-GTP emanates from chromatin and diminishes toward spindle poles [6]. Ran-GTP promotes spindle self-organization through the release of importin-bound spindle assembly factors (SAFs), which stimulate microtubule (MT) nucleation and organization and regulate MT dynamics [7-9]. Although many SAFs are non-motile MT-associated proteins, such as NuMA, TPX2, and HURP [7, 10-12], Ran also controls motor proteins, including Kid and HSET/XCTK2 [13, 14]. The Kinesin-14 XCKT2 is important for spindle assembly and pole organization [15-20], and Ran-GTP is proposed to promote XCKT2 MT crosslinking activity by releasing importin α/β from a bipartite nuclear localization signal (NLS) located in the tail domain [14]. Here, we show that the Ran-GTP gradient spatially regulates XCTK2 within the spindle. A flattened Ran-GTP gradient blocked the ability of excess XCTK2 to stimulate bipolar spindle assembly and resulted in XCTK2-mediated bundling of free MTs. These effects required the XCTK2 tail, which promoted the motility of XCTK2 within the spindle independent of the Ran-GTP gradient. In addition, the turnover kinetics of XCTK2 were spatially controlled: they were faster near the poles relative to the chromatin, but not with a mutant XCTK2 that cannot bind to importin α/β. Our results support a model in which the Ran-GTP gradient spatially coordinates motor localization with motility to ensure efficient spindle formation.

Microtubule minus-end binding protein CAMSAP2 controls axon specification and dendrite development.

In neurons, most microtubules are not associated with a central microtubule-organizing center (MTOC), and therefore, both the minus and plus-ends of these non-centrosomal microtubules are found throughout the cell. Microtubule plus-ends are well established as dynamic regulatory sites in numerous processes, but the role of microtubule minus-ends has remained poorly understood. Using live-cell imaging, high-resolution microscopy, and laser-based microsurgery techniques, we show that the CAMSAP/Nezha/Patronin family protein CAMSAP2 specifically localizes to non-centrosomal microtubule minus-ends and is required for proper microtubule organization in neurons. CAMSAP2 stabilizes non-centrosomal microtubules and is required for neuronal polarity, axon specification, and dendritic branch formation in vitro and in vivo. Furthermore, we found that non-centrosomal microtubules in dendrites are largely generated by γ-Tubulin-dependent nucleation. We propose a two-step model in which γ-Tubulin initiates the formation of non-centrosomal microtubules and CAMSAP2 stabilizes the free microtubule minus-ends in order to control neuronal polarity and development.

CAMSAPs Add to the Growing Microtubule Minus-End Story

Free microtubule minus ends, found in many differentiated cells, contribute to polarized motility. Work from Jiang etal. (2014) in this issue of Developmental Cell shows how mammalian CAMSAP proteins stabilize minus ends, providing a key piece to the puzzle of how these minus ends are formed and stabilized.

KIFC3 promotes mitotic progression and integrity of the central spindle in cytokinesis

Kinesin-14 motor proteins play a variety of roles during metaphase and anaphase. However, it is not known whether members of this family of motors also participate in the dramatic changes in mitotic spindle organization during the transition from telophase to cytokinesis. We have identified the minus-end-directed motor, KIFC3, as an important contributor to central bridge morphology at this stage. KIFC3’s unique motor-dependent localization at the central bridge allows it to congress microtubules, promoting efficient progress through cytokinesis. Conversely, when KIFC3 function is perturbed, abscission is delayed, and the central bridge is both widened and extended. Examination of KIFC3 on growing microtubules in interphase indicates that it caps microtubules released from the centrosome, both in the region of the centrosome and in the cell periphery. In line with other kinesin-14 family members, KIFC3 may guide free microtubules to their destination at the bridge and/or may slide and crosslink central bridge microtubules in order to stage the cells for abscission.

Motors or dynamics: What really moves chromosomes?

Motors or dynamics: What really moves chromosomes?

A Physical Model Reveals the Mechanochemistry Responsible for Dynein's Processive Motion

The molecular motor dynein is associated with various cellular activities, such as directed transport along microtubules and the rhythmic beating of the axoneme. Because of the size and complexity of the protein, a detailed understanding of the mechanochemistry that drives dynein's processive motion is lacking. To overcome this deficiency, we developed the first (to our knowledge) computational model for two-headed dynein that couples conformational changes of the motor's subunits to the biochemical steps involved in ATP hydrolysis. Analysis of the model provides what we believe are several novel insights into how the protein functions: 1), structural constraints limit the motion of the free microtubule binding domain to one dimension, increasing the efficiency with which this domain finds a binding site; 2), in addition to the power stroke of the bound head, recovery of the free head to a pre-power-stroke conformation is required for this head to reach a forward binding site; 3), the order in which the power stroke and recovery transitions occur affects the probability of back-stepping; and 4), the existence of multiple equilibria in the motor's bending energy provides a mechanism for processive back-stepping. To the best of our knowledge, our computational model provides the first complete mechanochemical description of the motor protein dynein, and the findings presented here should motivate new experimental investigations to test its predictions.

Buckling of embedded microtubules in elastic medium

Motivated by the application of Winkler-like models for the buckling analysis of embedded carbon nanotubes, an orthotropic Winkler-like model is developed to study the buckling behavior of embedded cytoskeletal microtubules within the cytoplasm. Experimental observations of the buckling of embedded cytoskeletal microtubules reveal that embedded microtubules bear a large compressive force as compared with free microtubules. The present theoretical model predicts that embedded microtubules in an elastic medium bear large compressive forces than free microtubules. The estimated critical pressure is in good agreement with the experimental values of the pressure-induced buckling of microtubules. Moreover, due to the mechanical coupling of microtubules with the surrounding elastic medium, the critical buckling force is increased considerably, which well explains the theory that the mechanical coupling of microtubules with an elastic medium increases compressive forces that microtubules can sustain. The model presented in the paper is a good approximation for the buckling analysis of embedded microtubules.

Microtubule and katanin-dependent dynamics of microtubule nucleation complexes in the acentrosomal Arabidopsis cortical array

Microtubule nucleation in interphase plant cells primarily occurs through branching from pre-existing microtubules at dispersed sites in the cell cortex. The minus ends of new microtubules are often released from the sites of nucleation, and the free microtubules are then transported to new locations by polymer treadmilling. These nucleation-and-release events are characteristic features of plant arrays in interphase cells, but little is known about the spatiotemporal control of these events by nucleating protein complexes. We visualized the dynamics of two fluorescently-tagged γ-tubulin complex proteins, GCP2 and GCP3, in Arabidopsis thaliana. These probes labelled motile complexes in the cytosol that transiently stabilized at fixed locations in the cell cortex. Recruitment of labelled complexes occurred preferentially along existing cortical microtubules, from which new microtubule was synthesized in a branching manner, or in parallel to the existing microtubule. Complexes localized to microtubules were approximately 10-fold more likely to display nucleation than were complexes recruited to other locations. Nucleating complexes remained stable until daughter microtubules were either completely depolymerized from their plus ends or released by katanin-dependent severing activity. These observations suggest that the nucleation complexes are primarily activated on association with microtubule lattices, and that nucleation complex stability depends on association with daughter microtubules and is regulated in part by katanin activity.

Patronin Regulates the Microtubule Network by Protecting Microtubule Minus Ends

Tubulin assembles into microtubule polymers that have distinct plus and minus ends. Most microtubule plus ends in living cells are dynamic; the transitions between growth and shrinkage are regulated by assembly-promoting and destabilizing proteins. In contrast, minus ends are generally not dynamic, suggesting their stabilization by some unknown protein. Here, we have identified Patronin (also known as ssp4) as a protein that stabilizes microtubule minus ends in Drosophila S2 cells. In the absence of Patronin, minus ends lose subunits through the actions of the Kinesin-13 microtubule depolymerase, leading to a sparse interphase microtubule array and short, disorganized mitotic spindles. In vitro, the selective binding of purified Patronin to microtubule minus ends is sufficient to protect them against Kinesin-13-induced depolymerization. We propose that Patronin caps and stabilizes microtubule minus ends, an activity that serves a critical role in the organization of the microtubule cytoskeleton.

Mechanistic Analysis of Kar3Cik1 for Mitotic Function

Kar3Cik1 is a S. cerevisae Kinesin-14 motor protein that functions to shorten cytoplasmic microtubule (MT) during yeast mating for nuclear fusion, yet crosslinks interpolar MTs (ipMTs) during anaphase. The Kar3 head contains both an ATP and MT binding site, yet the Cik1 head lacks an ATP catalytic site. Presteady-state and steady state experiments were conducted to define the mechanochemical pathway by which Kar3Cik1 stabilizes anti-parallel ipMTs for its mitotic function. To initiate the cycle, we used a high ADP strategy to promote MT-binding by the Cik1 head at 4.9 ∈1/4M-1s-1. The initial association is then followed by a 4-5 s-1 conformational change to induce Kar3 head binding to the MT with rapid ADP release from the active site at 109 s-1. MantATP binding to the nucleotide free MT•Kar3Cik1 is fast at 2.1 ∈1/4M-1s-1 with koff = 16.6 s-1. Pulse-chase methodology further reveals that MgATP binding to MT•Kar3Cik1 follows a two step process, formation of a collision complex followed by a 64 s-1 isomerization step. ATP hydrolysis occurs at 26 s-1 followed by motor detachment from microtubule at 11.5 s-1. The rate-limiting step for steady-state ATP turnover at 5 s-1 is hypothesized to be the conformational change leading to Kar3 head binding to MT. These initial results suggest a model in which Kar3Cik1 interacts with the MT through an alternating cycle of Cik1 binding followed by Kar3 binding. Because Cik1 does not have a nucleotide binding site, we propose that head-head communication is mediated by a strain-dependent mechanism. Supported by NIH GM54141.

MCAK (Kinesin-13) has an Unconventional ATP Hydrolysis Cycle Adapted for Microtubule Depolymerization

Unlike members of the kinesin-1 subfamily, the microtubule-depolymerising kinesin-13, MCAK, has no translocation activity. Rather it diffuses on the microtubule lattice to accelerate targeting to both ends, where it carries out ATP-dependent catalytic depolymerisation. The ATP hydrolysis cycle of MCAK has been largely overlooked. However, it may hold the key to the strikingly different behavior of kinesin-13 proteins compared to the conventional translocating kinesins that move directionally on the lattice. We have elucidated the ATP hydrolysis cycle of MCAK in solution and in the presence of both free tubulin dimers and microtubules. In contrast to most other kinesins and also myosins, for which product release is rate-limiting, ATP cleavage limits the hydrolysis cycle of MCAK in solution. Therefore MCAK meets the microtubule from solution in the ATP-containing state which binds tightly. Lattice-stimulated ATP cleavage drives MCAK into a weakly-bound nucleotide state, which diffuses on the lattice to target the microtubule end. An end-specific feature of the microtubule acts as a nucleotide exchange factor, promoting exchange of ADP for ATP by increasing the rate constant for ADP dissociation by more than 20-fold over the equivalent process in solution. Nucleotide exchange triggers tight binding of ATP-MCAK at the microtubule end, deforming the bound tubulin dimer causing lattice destabilization, leading to depolymerization. Tubulin-stimulated ATP hydrolysis is required to allow dissociation of tubulin-MCAK complexes released from the MT end, thereby allowing catalytic depolymerization. The altered ATP hydrolysis cycle of MCAK, relative to kinesin-1, tailors its affinity for tubulin to produce the characteristic weakly-bound diffusive interaction with the microtubule lattice and the strong microtubule end-dependent binding that promotes depolymerization.

Interactions between Subunits in Heterodimeric Ncd Molecules

The nonprocessive minus-end-directed kinesin-14 Ncd is involved in the organization of the microtubule (MT) network during mitosis. Only one of the two motor domains is involved in the interaction with the MT. The other head is tethered to the bound one. Here we prepared, purified, and characterized mutated Ncd molecules carrying point mutations in one of the heads, thus producing heterodimeric motors. The mutations tested included substitutions in Switch I and II: R552A, E585A, and E585D; the decoupling mutant N600K; and a deletion in the motor domain in one of the subunits resulting in a single-headed molecule (NcN). These proteins were isolated by two sequential affinity chromatography steps, followed by measurements of their affinities to MT, enzymatic properties, and the velocity of the microtubule gliding test in vitro. A striking observation is a low affinity of the single-headed NcN for MT both without nucleotides and in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate, implying that the tethered head has a profound effect on the structure of the Ncd-MT complex. Mutated homodimers had no MT-activated ATPase and no motility, whereas NcN had motility comparable with that of the wild type Ncd. Although the heterodimers had one fully active and one inactive head, the ATPase and motility of Ncd heterodimers varied dramatically, clearly demonstrating that interactions between motor domains exist in Ncd. We also show that the bulk property of dimeric proteins that interact with the filament with only one of its heads depends also on the distribution of the filament-interacting subunits.