Microtubule Rescue Research Articles

Abl2 repairs microtubules and phase separates with tubulin to promote microtubule nucleation

Abl family kinases are evolutionarily conserved regulators of cell migration and morphogenesis. Genetic experiments in Drosophila suggest that Abl family kinases interact functionally with microtubules to regulate axon guidance and neuronal morphogenesis. Vertebrate Abl2 binds to microtubules and promotes their plus-end elongation, both invitro and in cells, but the molecular mechanisms by which Abl2 regulates microtubule (MT) dynamics are unclear. We report here that Abl2 regulates MT assembly via condensation and direct interactions with both the MT lattice and tubulin dimers. We find that Abl2 promotes MT nucleation, which is further facilitated by the ability of the Abl2 C-terminal half to undergo liquid-liquid phase separation (LLPS) and form co-condensates with tubulin. Abl2 binds to regions adjacent to MT damage, facilitates MT repair via fresh tubulin recruitment, and increases MT rescue frequency and lifetime. Cryo-EM analyses strongly support a model in which Abl2 engages tubulin C-terminal tails along an extended MT lattice conformation at damage sites to facilitate repair via fresh tubulin recruitment. Abl2Δ688-790, which closely mimics a naturally occurring splice isoform, retains binding to the MT lattice but does not bind tubulin, promote MT nucleation, or increase rescue frequency. In COS-7 cells, MT reassembly after nocodazole treatment is greatly slowed in Abl2 knockout COS-7 cells compared with wild-type cells, and these defects are rescued by re-expression of Abl2, but not Abl2Δ688-790. We propose that Abl2 locally concentrates tubulin to promote MT nucleation and recruits it to defects in the MT lattice to enable repair and rescue.

Microtubule rescue control by drugs and MAPs examined with in vitro pedestal assay

Microtubules are essential cytoskeletal polymers, which exhibit stochastic transitions between assembly and disassembly, known as catastrophes and rescues. Understanding of catastrophes, rescues, and their control by drugs and microtubule associated proteins (MAPs) has been informed by in vitro reconstitutions of microtubule dynamics. In such experiments microtubules are typically observed on a flat surface of the coverslip. In contrast, we have recently proposed a modified setup in which microtubules assemble from stabilized seeds, overhanging from microfabricated pedestals, so that their dynamic extensions are fully isolated from contact with the coverslip. This assay allows to eliminate potential artifacts, which may substantially affect the frequency of microtubule rescues in vitro. Here we use the pedestal assay to study the sensitivity of microtubules to paclitaxel, one of the best-known inhibitors of microtubule dynamics. By comparing observations in the conventional and the pedestal assays, we find that microtubule dynamics are substantially more sensitive to paclitaxel when the polymers can contact the coverslip. We interpret this as a consequence of the coverslip-induced microtubule assembly perturbation, leading to formation of lattice with defects, and thereby enhancing the efficiency of paclitaxel binding to microtubules in the conventional assay. To test this idea, we use vinblastine, another small-molecule inhibitor, which had been previously shown to cause microtubule growth perturbations. We find that in the pedestal assay vinblastine sensitizes microtubules to paclitaxel to the level, observed in the conventional assay. Interestingly, a minimal fragment of MAP called CLASP2, a previously characterized rescue factor, has a strong effect on microtubule rescues, regardless of the type of assay. Overall, our study underscores the role of microtubule damage in promoting rescues and highlights the utility of the in vitro pedestal assay to study microtubule dynamics modulation by tubulin inhibitors and MAPs.

Abl2 mediates microtubule nucleation and repair via tubulin co-condensation.

Abl family kinases are evolutionarily conserved regulators of cell migration and morphogenesis. Genetic experiments in Drosophila suggest that Abl family kinases interact functionally with microtubules to regulate axon guidance and neuronal morphogenesis. Vertebrate Abl2 binds to microtubules and promotes their plus-end elongation both in vitro and in cells, but the molecular mechanisms by which Abl2 regulates microtubule (MT) dynamics were unclear. We report here that Abl2 regulates MT assembly via condensation and direct interactions with both the MT lattice and tubulin dimers. We discovered that Abl2 promotes MT nucleation, which is further facilitated by the ability of the Abl2 C-terminal half to undergo phase separation and form co-condensates with tubulin. A naturally occurring tubulin binding-deficient Abl2 splice isoform fails to promote nucleation. Abl2 binds to regions adjacent to MT damage and facilitates their repair via fresh tubulin recruitment and increases MT rescue frequency and lifetime. MT recovery after nocodazole treatment is greatly slowed in Abl2 knockout COS-7 cells compared to wild type cells. Our results lead us to propose a model in which Abl2 locally concentrates tubulin and recruits it to MT tips or to defects in the MT lattice to promote MT repair, rescue, and nucleation.

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

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

Theory of tip structure–dependent microtubule catastrophes and damage-induced microtubule rescues

Microtubules are essential cytoskeletal polymers that exhibit stochastic switches between tubulin assembly and disassembly. Here, we examine possible mechanisms for these switches, called catastrophes and rescues. We formulate a four-state Monte Carlo model, explicitly considering two biochemical and two conformational states of tubulin, based on a recently conceived view of microtubule assembly with flared ends. The model predicts that high activation energy barriers for lateral tubulin interactions can cause lagging of curled protofilaments, leading to a ragged appearance of the growing tip. Changes in the extent of tip raggedness explain some important but poorly understood features of microtubule catastrophe: weak dependence on tubulin concentration and an increase in its probability over time, known as aging. The model predicts a vanishingly rare frequency of spontaneous rescue unless patches of guanosine triphosphate tubulin are artificially embedded into microtubule lattice. To test our model, we used invitro reconstitution, designed to minimize artifacts induced by microtubule interaction with nearby surfaces. Microtubules were assembled from seeds overhanging from microfabricated pedestals and thus well separated from the coverslip. This geometry reduced the rescue frequency and the incorporation of tubulins into the microtubule shaft compared with the conventional assay, producing data consistent with the model. Moreover, the rescue positions of microtubules nucleated from coverslip-immobilized seeds displayed a nonexponential distribution, confirming that coverslips can affect microtubule dynamics. Overall, our study establishes a unified theory accounting for microtubule assembly with flared ends, a tip structure-dependent catastrophe frequency, and a microtubule rescue frequency dependent on lattice damage and repair.

Microtubule rescue at midzone edges promotes overlap stability and prevents spindle collapse during anaphase B

During anaphase B, molecular motors slide interpolar microtubules to elongate the mitotic spindle, contributing to the separation of chromosomes. However, sliding of antiparallel microtubules reduces their overlap, which may lead to spindle breakage, unless microtubules grow to compensate sliding. How sliding and growth are coordinated is still poorly understood. In this study, we have used the fission yeast S. pombe to measure microtubule dynamics during anaphase B. We report that the coordination of microtubule growth and sliding relies on promoting rescues at the midzone edges. This makes microtubules stable from pole to midzone, while their distal parts including the plus ends alternate between assembly and disassembly. Consequently, the midzone keeps a constant length throughout anaphase, enabling sustained sliding without the need for a precise regulation of microtubule growth speed. Additionally, we found that in S. pombe, which undergoes closed mitosis, microtubule growth speed decreases when the nuclear membrane wraps around the spindle midzone.

Vimentin intermediate filaments stabilize dynamic microtubules by direct interactions

The cytoskeleton determines cell mechanics and lies at the heart of important cellular functions. Growing evidence suggests that the manifold tasks of the cytoskeleton rely on the interactions between its filamentous components—actin filaments, intermediate filaments, and microtubules. However, the nature of these interactions and their impact on cytoskeletal dynamics are largely unknown. Here, we show in a reconstituted in vitro system that vimentin intermediate filaments stabilize microtubules against depolymerization and support microtubule rescue. To understand these stabilizing effects, we directly measure the interaction forces between individual microtubules and vimentin filaments. Combined with numerical simulations, our observations provide detailed insight into the physical nature of the interactions and how they affect microtubule dynamics. Thus, we describe an additional, direct mechanism by which cells establish the fundamental cross talk of cytoskeletal components alongside linker proteins. Moreover, we suggest a strategy to estimate the binding energy of tubulin dimers within the microtubule lattice.

Stress-induced phosphorylation of CLIP-170 by JNK promotes microtubule rescue.

The stress-induced c-Jun N-terminal kinase (JNK) controls microtubule dynamics by enhancing both microtubule growth and rescues. Here, we show that upon cell stress, JNK directly phosphorylates the microtubule rescue factor CLIP-170 in its microtubule-binding domain to increase its rescue-promoting activity. Phosphomimetic versions of CLIP-170 enhance its ability to promote rescue events in vitro and in cells. Furthermore, while phosphomimetic mutations do not alter CLIP-170's capability to form comets at growing microtubule ends, both phosphomimetic mutations and JNK activation increase the occurrence of CLIP-170 remnants on the microtubule lattice at the rear of comets. As the CLIP-170 remnants, which are potential sites of microtubule rescue, display a shorter lifetime when CLIP-170 is phosphorylated, we propose that instead of acting at the time of rescue occurrence, CLIP-170 would rather contribute in preparing the microtubule lattice for future rescues at these predetermined sites.

Effects of Severing Enzymes on the Length Distribution and Total Mass of Microtubules

Microtubules are cytoskeletal polymers of αβ-tubulin whose dynamics is highly regulated as eukaryotic cells undergo cell division, migration and tissue development. Microtubule severing enzymes, spastin, katanin and fidgetin use ATP-hydrolysis to fragment microtubules into shorter filaments. Paradoxically, in vivo experiments have shown that severases can increase total microtubule number and mass inside the cells, suggesting that severases possess a nucleation-like activity. We have resolved this paradox by reconstituting a dynamic microtubule severing system in vitro and discovered that Drosophila spastin has two additional properties in addition to severing: it increases microtubule rescue and slows down shrinkage. Under the right circumstances these effects lead to an exponential increase in the number and mass of microtubules. In this abstract, we address the question of how the exponential rate constant and the length distribution depend on severing and microtubule dynamics. A mathematical model reveals that the amplification rate depends on the balance of creating new microtubules through severing and the loss of existing microtubule through shrinkage. The mean length depends on the ratio of the growth rate to the severing rate and can be well-approximated by a simplified no-catastrophe model, which we have solved analytically. Unexpectedly, dynamic instability has only a small impact on microtubule length. On the other hand, dynamic instability can influence the amplification rate. Thus differential regulation of the dynamic parameters can separately modulate different aspects of the microtubule network. Our results establish the quantitative basis for how severing and dynamics conjunctly constitute a versatile mechanism to regulate microtubule length and mass in the complex cellular environment.

Structures of TOG1 and TOG2 from the human microtubule dynamics regulator CLASP1.

Tubulin-binding TOG domains are found arrayed in a number of proteins that regulate microtubule dynamics. While much is known about the structure and function of TOG domains from the XMAP215 microtubule polymerase family, less in known about the TOG domain array found in animal CLASP family members. The animal CLASP TOG array promotes microtubule pause, potentiates rescue, and limits catastrophe. How structurally distinct the TOG domains of animal CLASP are from one another, from XMAP215 family TOG domains, and whether a specific order of structurally distinct TOG domains in the TOG array is conserved across animal CLASP family members is poorly understood. We present the x-ray crystal structures of Homo sapiens (H.s.) CLASP1 TOG1 and TOG2. The structures of H.s. CLASP1 TOG1 and TOG2 are distinct from each other and from the previously determined structure of Mus musculus (M.m.) CLASP2 TOG3. Comparative analyses of CLASP family TOG domain structures determined to date across species and paralogs supports a conserved CLASP TOG array paradigm in which structurally distinct TOG domains are arrayed in a specific order. H.s. CLASP1 TOG1 bears structural similarity to the free-tubulin binding TOG domains of the XMAP215 family but lacks many of the key tubulin-binding determinants found in XMAP215 family TOG domains. This aligns with studies that report that animal CLASP family TOG1 domains cannot bind free tubulin or microtubules. In contrast, animal CLASP family TOG2 and TOG3 domains have reported microtubule-binding activity but are structurally distinct from the free-tubulin binding TOG domains of the XMAP215 family. H.s. CLASP1 TOG2 has a convex architecture, predicted to engage a hyper-curved tubulin state that may underlie its ability to limit microtubule catastrophe and promote rescue. M.m. CLASP2 TOG3 has unique structural elements in the C-terminal half of its α-solenoid domain that our modeling studies implicate in binding to laterally-associated tubulin subunits in the microtubule lattice in a mode similar to, yet distinct from those predicted for the XMAP215 family TOG4 domain. The potential ability of the animal CLASP family TOG3 domain to engage lateral tubulin subunits may underlie the microtubule rescue activity ascribed to the domain. These findings highlight the structural diversity of TOG domains within the CLASP family TOG array and provide a molecular foundation for understanding CLASP-dependent effects on microtubule dynamics.

Severing Enzymes Amplify Microtubule Arrays through Lattice GTP-Tubulin Incorporation

Spastin and katanin sever and destabilize microtubules. Their mutation causes debilitating diseases. Paradoxically, despite their destructive activity they increase microtubule mass in spindles, plant cortical arrays and neurons. Combining electron and single-molecule TIRF microscopy we show that the elemental step in microtubule-severing is the generation of nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers. These damage sites are repaired spontaneously by GTP-tubulin incorporation. As a result, the microtubule shaft is rejuvenated with GTP-tubulin islands that stabilize it against depolymerization and newly severed ends emerge with a high-density of GTP-tubulin that protects against depolymerization. Consequently, spastin and katanin increase microtubule rescue frequency without affecting growth rates and catastrophe. The stabilization of the newly severed plus-ends and the higher rescue frequency synergize to amplify microtubule number and mass. Thus, severing enzymes regulate microtubule architecture and dynamics by promoting GTP-tubulin incorporation within the microtubule shaft.

A unified model for microtubule rescue.

How microtubules transition from depolymerization to polymerization, known as rescue, is poorly understood. Here we examine two models for rescue: 1) an “end-driven” model in which the depolymerizing end stochastically switches to a stable state; and 2) a “lattice-driven” model in which rescue sites are integrated into the microtubule before depolymerization. We test these models using a combination of computational simulations and in vitro experiments with purified tubulin. Our findings support the “lattice-driven” model by identifying repeated rescue sites in microtubules. In addition, we discover an important role for divalent cations in determining the frequency and location of rescue sites. We use “wash-in” experiments to show that divalent cations inhibit rescue during depolymerization, but not during polymerization. We propose a unified model in which rescues are driven by embedded rescue sites in microtubules, but the activity of these sites is influenced by changes in the depolymerizing ends.

Strengthening Microtubules by Cuts that Heal

Microtubule-severing enzymes, which can remove tubulin dimers from microtubule lattices, participate in cytoskeletal remodeling in various contexts. A recent study showed that partially damaged microtubule shafts and new microtubule ends generated by these enzymes can incorporate GTP-tubulin and serve as sites of microtubule rescue and re-growth, explaining how severing enzymes can amplify microtubule arrays.

Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation.

Spastin and katanin sever and destabilize microtubules. Paradoxically, despite their destructive activity they increase microtubule mass in vivo. We combined single-molecule total internal reflection fluorescence microscopy and electron microscopy to show that the elemental step in microtubule severing is the generation of nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers. These damage sites are repaired spontaneously by guanosine triphosphate (GTP)-tubulin incorporation, which rejuvenates and stabilizes the microtubule shaft. Consequently, spastin and katanin increase microtubule rescue rates. Furthermore, newly severed ends emerge with a high density of GTP-tubulin that protects them against depolymerization. The stabilization of the newly severed plus ends and the higher rescue frequency synergize to amplify microtubule number and mass. Thus, severing enzymes regulate microtubule architecture and dynamics by promoting GTP-tubulin incorporation within the microtubule shaft.

An isolated CLASP TOG domain suppresses microtubule catastrophe and promotes rescue.

Microtubules are heavily regulated dynamic polymers of αβ-tubulin that are required for proper chromosome segregation and organization of the cytoplasm. Polymerases in the XMAP215 family use arrayed TOG domains to promote faster microtubule elongation. Regulatory factors in the cytoplasmic linker associated protein (CLASP) family that reduce catastrophe and/or increase rescue also contain arrayed TOGs, but how CLASP TOGs contribute to activity is poorly understood. Here, using Saccharomyces cerevisiae Stu1 as a model CLASP, we report structural, biochemical, and reconstitution studies that clarify functional properties of CLASP TOGs. The two TOGs in Stu1 have very different tubulin-binding properties: TOG2 binds to both unpolymerized and polymerized tubulin, and TOG1 binds very weakly to either. The structure of Stu1-TOG2 reveals a CLASP-specific residue that likely confers distinctive tubulin-binding properties. The isolated TOG2 domain strongly suppresses microtubule catastrophe and increases microtubule rescue in vitro, contradicting the expectation that regulatory activity requires an array of TOGs. Single point mutations on the tubulin-binding surface of TOG2 ablate its anti-catastrophe and rescue activity in vitro, and Stu1 function in cells. Revealing that an isolated CLASP TOG can regulate polymerization dynamics without being part of an array provides insight into the mechanism of CLASPs and diversifies the understanding of TOG function.

A Novel Plasma Membrane-Anchored Protein Regulates Xylem Cell-Wall Deposition through Microtubule-Dependent Lateral Inhibition of Rho GTPase Domains

Spatial control of cell-wall deposition is essential for determining plant cell shape [1]. Rho-type GTPases, together with the cortical cytoskeleton, play central roles in regulating cell-wall patterning [2]. In metaxylem vessel cells, which are the major components of xylem tissues, active ROP11 Rho GTPases form oval plasma membrane domains that locally disrupt cortical microtubules, thereby directing the formation of oval pits in secondary cell walls [3-5]. However, the regulatory mechanism that determines the planar shape of active Rho of Plants (ROP) domains is still unknown. Here we show that IQD13 associates with cortical microtubules and the plasma membrane to laterally restrict the localization of ROP GTPase domains, thereby directing the formation of oval secondary cell-wall pits. Loss and overexpression of IQD13 led to the formation of abnormally round and narrow secondary cell-wall pits,respectively. Ectopically expressed IQD13 increased the presence of parallel cortical microtubules by promoting microtubule rescue. A reconstructive approach revealed that IQD13 confines the area of active ROP domains within the lattice of the cortical microtubules, causing narrow ROP domains to form. This activity required the interaction of IQD13 with the plasma membrane. These findings suggest that IQD13 positively regulates microtubule dynamics as well as their linkage to the plasma membrane, which synergistically confines the area of active ROP domains, leading to the formation of oval secondary cell-wall pits. Thisfinding sheds light on the role of microtubule-plasma membrane linkage as a lateral fence thatdetermines the planar shape of Rho GTPase domains.

Localized Mechanical Stress Promotes Microtubule Rescue

Microtubule dynamics rely on the properties of tubulin and are regulated by microtubule-associated proteins. GTP-tubulin assembles into hollow polymers, which can depolymerize upon GTP hydrolysis. Depolymerizing microtubules may stop shrinking and resume growth. Such rescues are regulated by microtubule-associated proteins like CLIP-170 and the CLASPs [1, 2]. Microtubule domains prone to rescues contain discrete regions (previously termed "GTP islands") that retain a GTP-tubulin-like conformation in the main body of the microtubule [3]. However, the exact nature of these domains and the mechanisms controlling their occurrence and distribution are largely unknown. Here we show that collisions between growing microtubules and mechanical obstacles (including other microtubules) invitro result in the higher abundance of GTP-like islands in stressed microtubule regions. Furthermore, these islands were found to be efficiently generated by both lateral contacts and mechanical constraints applied to the main body of the microtubules. They were also particularly prominent where shifts in the number of protofilaments occur in the microtubule lattice. GTP-like islands and rescues frequently co-occurred at microtubule intersections invitro and in living cells, both in crossing and in crossed microtubules. We also observed that CLIP-170 recognizes GTP-like islands invivo and is retained at microtubule crossings. Therefore, we propose that rescues occur via a two-stage mechanism: (1) lattice defects determine potential rescue-promoting islands in the microtubule structure, and (2) CLIP-170 detects these islands to stimulate microtubule rescue. Our results reveal the interplay between rescue-promoting factors and microtubule architecture and organization to control microtubule dynamics.

Self-repair promotes microtubule rescue

SummaryThe dynamic instability of microtubules is characterised by slow growth phases stochastically interrupted by rapid depolymerisations called catastrophes. Rescue events can arrest the depolymerisation and restore microtubule elongation. However the origin of these rescue events remain unexplained. Here we show that microtubule lattice self-repair, in structurally damaged sites, is responsible for the rescue of microtubule growth. Tubulin photo-conversion in cells revealed that free tubulin dimers can incorporate along the shafts of microtubules, especially in regions where microtubules cross each other, form bundles or become bent due to mechanical constraints. These incorporation sites appeared to act as effective rescue sites ensuring microtubule rejuvenation. By securing damaged microtubule growth, the self-repair process supports a mechanosensitive growth by specifically promoting microtubule assembly in regions where they are subjected to physical constraints.

Separase Promotes Microtubule Polymerization by Activating CENP-E-Related Kinesin Kin7

Microtubules play an essential role in breaking cellular symmetry. We have previously shown that separase associates with microtubules and regulates microtubule-dependent establishment of cell polarity in Arabidopsis. However, separase lacks microtubule-binding activity, raising questions about mechanisms underlying this phenomenon. Here we report that the N-terminal non-catalytic domain of separase binds to the C-terminal tail domain of three homologs of the centromeric protein CENP-E Kinesin 7 (Kin7). Conformational changes of Kin7 induced upon binding to separase facilitate recruitment of Kin7/separase complex (KISC) onto microtubules. KISC operates independently of proteolytic activity of separase in promoting microtubule rescue and pauses, as well as in suppressing catastrophes. Genetic complementation experiments in conditional separase mutant rsw4 background demonstrate the importance of KISC for the establishment of cell polarity and for plant development. Our study establishes a mechanism governing microtubule dynamics via the separase-dependent activation of CENP-E-related kinesins.

The linear and rotational motions of the fission yeast nucleus are governed by the stochastic dynamics of spatially distributed microtubules

Dynamic nuclei are involved in a wide variety of fundamental biological processes including cell migration, cell division and fertilization. Here, we develop a mathematical model, in combination with live-cell imaging at high temporal resolution, to quantitatively elucidate how the linear and rotational motions of the nucleus are governed by the stochastic dynamics of the microtubule cytoskeleton. Our simulation and experimental results demonstrate that microtubule rescue and catastrophe frequencies are the decisive factors in regulating the nuclear movement. Lower rescue and catastrophe frequencies can lead to significantly larger angular and translational oscillations of the nucleus. In addition, our model also suggests that the stochastic dynamics of individual spatially distributed microtubules works collectively as a restoring force to maintain nuclear centering and hence ensures symmetric cell division, in excellent agreement with direct experimental observations.