Microtubules under mechanical pressure can breach dense actin networks.

The crosstalk between the actin network and microtubules is essential for cell polarity. It orchestrates microtubule organization within the cell, driven by the asymmetry of actin architecture along the cell periphery. The physical intertwining of these networks regulates spatial organization and force distribution in the microtubule network. Although their biochemical interactions are becoming clearer, the mechanical aspects remain less understood. To explore this mechanical interplay, we developed an in vitro reconstitution assay to investigate how dynamic microtubules interact with various actin filament structures. Our findings revealed that microtubules can align and move along linear actin filament bundles through polymerization force. However, they are unable to pass through when encountering dense branched actin meshworks, similar to those present in the lamellipodium along the periphery of the cell. Interestingly, immobilizing microtubules through crosslinking with actin or other means allow the buildup of pressure, enabling them to breach these dense actin barriers. This mechanism offers insights into microtubule progression towards the cell periphery, with them overcoming obstacles within the denser parts of the actin network and ultimately contributing to cell polarity establishment.

Microtubules self-repair in living cells.

Microtubule self-repair has been studied both in vitro and in vivo as an underlying mechanism of microtubule stability. The turnover of tubulin dimers along the microtubule has challenged the pre-existing dogma that only growing ends are dynamic. However, although there is clear evidence of tubulin incorporation into the shaft of polymerized microtubules in vitro, the possibility of such events occurring in living cells remains uncertain. In this study, we investigated this possibility by microinjecting purified tubulin dimers labeled with a red fluorophore into the cytoplasm of cells expressing GFP-tubulin. We observed the appearance of red dots along the pre-existing green microtubule within minutes. We found that the fluorescence intensities of these red dots were inversely correlated with the green signal, suggesting that the red dimers were incorporated into the microtubules and replaced the pre-existing green dimers. Lateral distance from the microtubule center was similar to that in incorporation sites and in growing ends. The saturation of the size and spatial frequency of incorporations as a function of injected tubulin concentration and post-injection delay suggested that the injected dimers incorporated into a finite number of damaged sites. By our low estimate, within a few minutes of the injections, free dimers incorporated into major repair sites every 70 µm of microtubules. Finally, we mapped the location of these sites in micropatterned cells and found that they were more concentrated in regions where the actin filament network was less dense and where microtubules exhibited greater lateral fluctuations.

Actin Architecture Steers Microtubules in Active Cytoskeletal Composite.

Motility assays use surface-immobilized molecular motors to propel cytoskeletal filaments. They have been widely used to characterize motor properties and their impact on cytoskeletal self-organization. Moreover, the motility assays are a promising class of bioinspired active tools for nanotechnological applications. While these assays involve controlling the filament direction and speed, either as a sensory readout or a functional feature, designing a subtle control embedded in the assay is an ongoing challenge. Here, we investigate the interaction between gliding microtubules and networks of actin filaments. We demonstrate that the microtubule's behavior depends on the actin architecture. Both unbranched and branched actin decelerate microtubule gliding; however, an unbranched actin network provides additional guidance and effectively steers the microtubules. This effect, which resembles the recognition of cortical actin by microtubules, is a conceptually new means of controlling the filament gliding with potential application in the design of active materials and cytoskeletal nanodevices.

Actin network architecture can ensure robust centering or sensitive decentering of the centrosome.

The orientation of cell polarity depends on the position of the centrosome, the main microtubule-organizing center (MTOC). Microtubules (MTs) transmit pushing forces to the MTOC as they grow against the cell periphery. How the actin network regulates these forces remains unclear. Here, in a cell-free assay, we used purified proteins to reconstitute the interaction of a microtubule aster with actin networks of various architectures in cell-sized microwells. In the absence of actin filaments, MTOC positioning was highly sensitive to variations in microtubule length. The presence of a bulk actin network limited microtubule displacement, and MTOCs were held in place. In contrast, the assembly of a branched actin network along the well edges centered the MTOCs by maintaining an isotropic balance of pushing forces. An anisotropic peripheral actin network caused the MTOC to decenter by focusing the pushing forces. Overall, our results show that actin networks can limit the sensitivity of MTOC positioning to microtubule length and enforce robust MTOC centering or decentering depending on the isotropy of its architecture.

Actin-microtubule dynamic composite forms responsive active matter with memory.

Active cytoskeletal materials in vitro demonstrate self-organizing properties similar to those observed in their counterparts in cells. However, the search to emulate phenomena observed in living matter has fallen short of producing a cytoskeletal network that would be structurally stable yet possess adaptive plasticity. Here, we address this challenge by combining cytoskeletal polymers in a composite where self-assembling microtubules and actin filaments collectively self-organize due to the activity of microtubule-percolating molecular motors. We demonstrate that microtubules spatially organize actin filaments that in turn guide microtubules. The two networks align in an ordered fashion using this feedback loop. In this composite, actin filaments can act as structural memory and, depending on the concentration of the components, microtubules either write this memory or get guided by it. The system is sensitive to external stimuli, suggesting possible autoregulatory behavior in changing mechanochemical environments. We thus establish an artificial active actin-microtubule composite as a system demonstrating architectural stability and plasticity.

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.

Reconstituting the Interaction Between Purified Nuclei and Microtubule Network.

The nucleus is the stiffest organelle in the cell. Several morphogenetic processes depend on its deformation such as cell migration, cell differentiation, or senescence. Recent studies have revealed various mechanisms involved in the regulation of nucleus stiffness and deformation. The implication of chromatin swelling, lamin density, actin filament, and microtubule network revealed that nucleus shape is the outcome of a fine balance between various sources of external forces and numerous means of internal resistance. In adherent cells, the actin network is the dominant player in external force production, whereas in nonadherent cells microtubules seem to take over. It is therefore important to set up reconstitution assays in order to decipher the exact contribution of each player in this mechanical balance. In this method, we describe a nucleus purification protocol that is suitable for nonadherent cells. We also show that purified nuclei can interact with microtubules and that nuclei purified from distinct cell types get differentially wrapped into the array of microtubules. A combination with a microtubule gliding assay offers the possibility to counterbalance the binding to the nucleus membrane by active motor-based forces pulling on microtubules. So this protocol allows an in-depth study of microtubule-nucleus interactions in vitro.

Structure and dynamics of Odinarchaeota tubulin and the implications for eukaryotic microtubule evolution.

Tubulins are critical for the internal organization of eukaryotic cells, and understanding their emergence is an important question in eukaryogenesis. Asgard archaea are the closest known prokaryotic relatives to eukaryotes. Here, we elucidated the apo and nucleotide-bound x-ray structures of an Asgard tubulin from hydrothermal living Odinarchaeota (OdinTubulin). The guanosine 5'-triphosphate (GTP)-bound structure resembles a microtubule protofilament, with GTP bound between subunits, coordinating the "+" end subunit through a network of water molecules and unexpectedly by two cations. A water molecule is located suitable for GTP hydrolysis. Time course crystallography and electron microscopy revealed conformational changes on GTP hydrolysis. OdinTubulin forms tubules at high temperatures, with short curved protofilaments coiling around the tubule circumference, more similar to FtsZ, rather than running parallel to its length, as in microtubules. Thus, OdinTubulin represents an evolutionary stage intermediate between prokaryotic FtsZ and eukaryotic microtubule-forming tubulins.

Self-repair protects microtubules from destruction by molecular motors.

Microtubule instability stems from the low energy of tubulin dimer interactions, which sets the growing polymer close to its disassembly conditions. Molecular motors use ATP hydrolysis to produce mechanical work and move on microtubules. This raises the possibility that the mechanical work produced by walking motors can break dimer interactions and trigger microtubule disassembly. We tested this hypothesis by studying the interplay between microtubules and moving molecular motors in vitro. Our results show that molecular motors can remove tubulin dimers from the lattice and rapidly destroy microtubules. We also found that dimer removal by motors was compensated for by the insertion of free tubulin dimers into the microtubule lattice. This self-repair mechanism allows microtubules to survive the damage induced by molecular motors as they move along their tracks. Our study reveals the existence of coupling between the motion of molecular motors and the renewal of the microtubule lattice.

A new perspective on microtubule dynamics: destruction by molecular motors and self-repair.

Microtubules are dynamic polymers, permanently assembling and disassembling, that serve as tracks for intra-cellular transport by molecular motors. We recently found that the low energy of tubulin dimer interactions allows for spontaneous loss of tubulin dimers from the microtubule lattice [1]. This raised the possibility that the mechanical work produced by molecular motors as they move on microtubules can break dimer interactions and trigger microtubule disassembly. In a recent study, we tested this hypothesis by studying the interplay between microtubules and moving molecular motors in vitro [2]. Our results show that molecular motors can remove tubulin dimers from the lattice and rapidly destroy microtubules. We also found that dimer removal by motors was compensated by the insertion of free tubulin dimers into the microtubule lattice. This self-repair mechanism allows microtubules to survive the damage induced by molecular motors as they move along their tracks. Our study reveals the existence of coupling between the motion of molecular motors and the renewal of the microtubule lattice.

Les microtubules sont des polymères dynamiques, s'assemblant et se désassemblant en permanence, qui servent de pistes pour le transport intracellulaire par des moteurs moléculaires. Nous avons récemment découvert que la faible énergie des interactions entre les dimères de tubuline permet la perte spontanée des dimères de tubuline le long d'un microtubule [1]. Le travail mécanique produit par les moteurs moléculaires lorsqu'ils se déplacent sur les microtubules pourrait donc rompre ces faibles interactions entre dimères et déclencher le désassemblage des microtubules. Dans une étude récente, nous avons testé cette hypothèse en étudiant l'interaction entre les microtubules et les moteurs moléculaires en mouvement in vitro [2]. Nos résultats montrent que les moteurs moléculaires peuvent retirer les dimères de tubuline du réseau et détruire rapidement les microtubules. Nous avons également constaté que l'élimination des dimères par les moteurs était compensée par l'insertion de dimères de tubuline libres dans le réseau de microtubules. Ce mécanisme d'autoréparation permet aux microtubules de survivre aux dommages induits par les moteurs moléculaires lors de leurs déplacements. Notre étude révèle donc l'existence d'un couplage entre le mouvement des moteurs moléculaires et le renouvellement du réseau de microtubules.

CLASP Mediates Microtubule Repair by Restricting Lattice Damage and Regulating Tubulin Incorporation.

Microtubules play a key role in cell division, motility, and intracellular trafficking. Microtubule lattices are generally regarded as stable structures that undergo turnover through dynamic instability of their ends [1]. However, recent evidence suggests that microtubules also exchange tubulin dimers at the sites of lattice defects, which can be induced by mechanical stress, severing enzymes, or occur spontaneously during polymerization [2-6]. Tubulin incorporation can restore microtubule integrity; moreover, "islands" of freshly incorporated GTP-tubulin can inhibit microtubule disassembly and promote rescues [3, 4, 6-8]. Microtubule repair occurs in vitro in the presence of tubulin alone [2-6, 9]. However, in cells, it is likely to be regulated by specific factors, the nature of which is currently unknown. CLASPs are interesting candidates for microtubule repair because they induce microtubule nucleation, stimulate rescue, and suppress catastrophes by stabilizing incomplete growing plus ends with lagging protofilaments and promoting their conversion into complete ones [10-17]. Here, we used in vitro reconstitution assays combined with laser microsurgery and microfluidics to show that CLASP2α indeed stimulates microtubule lattice repair. CLASP2α promoted tubulin incorporation into damaged lattice sites, thereby restoring microtubule integrity. Furthermore, it induced the formation of complete tubes from partial protofilament assemblies and inhibited microtubule softening caused by hydrodynamic-flow-induced bending. The catastrophe-suppressing domain of CLASP2α, TOG2, combined with a microtubule-tethering region, was sufficient to stimulate microtubule repair, suggesting that catastrophe suppression and lattice repair are mechanistically similar. Our results suggest that the cellular machinery controlling microtubule nucleation and growth can also help to maintain microtubule integrity.

Lattice defects induce microtubule self-renewal.

Microtubules are dynamic polymers, which grow and shrink by addition and removal of tubulin dimers at their extremities. Within the microtubule shaft, dimers adopt a densely packed and highly ordered crystal-like lattice structure, which is generally not considered to be dynamic. Here we report that thermal forces are sufficient to remodel the microtubule shaft, despite its apparent stability. Our combined experimental data and numerical simulations on lattice dynamics and structure suggest that dimers can spontaneously leave and be incorporated into the lattice at structural defects. We propose a model mechanism, where the lattice dynamics is initiated via a passive breathing mechanism at dislocations, which are frequent in rapidly growing microtubules. These results show that we may need to extend the concept of dissipative dynamics, previously established for microtubule extremities, to the entire shaft, instead of considering it as a passive material.

Actin filaments regulate microtubule growth at the centrosome.

The centrosome is the main microtubule-organizing centre. It also organizes a local network of actin filaments. However, the precise function of the actin network at the centrosome is not well understood. Here, we show that increasing densities of actin filaments at the centrosome of lymphocytes are correlated with reduced amounts of microtubules. Furthermore, lymphocyte activation resulted in disassembly of centrosomal actin and an increase in microtubule number. To further investigate the direct crosstalk between actin and microtubules at the centrosome, we performed in vitro reconstitution assays based on (i) purified centrosomes and (ii) on the co-micropatterning of microtubule seeds and actin filaments. These two assays demonstrated that actin filaments constitute a physical barrier blocking elongation of nascent microtubules. Finally, we showed that cell adhesion and cell spreading lead to lower densities of centrosomal actin, thus resulting in higher microtubule growth. We therefore propose a novel mechanism, by which the number of centrosomal microtubules is regulated by cell adhesion and actin-network architecture.

Microtubules acquire resistance from mechanical breakage through intralumenal acetylation.

Eukaryotic cells rely on long-lived microtubules for intracellular transport and as compression-bearing elements. We considered that long-lived microtubules are acetylated inside their lumen and that microtubule acetylation may modify microtubule mechanics. Here, we found that tubulin acetylation is required for the mechanical stabilization of long-lived microtubules in cells. Depletion of the tubulin acetyltransferase TAT1 led to a significant increase in the frequency of microtubule breakage. Nocodazole-resistant microtubules lost upon removal of acetylation were largely restored by either pharmacological or physical removal of compressive forces. In in vitro reconstitution experiments, acetylation was sufficient to protect microtubules from mechanical breakage. Thus, acetylation increases mechanical resilience to ensure the persistence of long-lived microtubules.

Self-repair promotes microtubule rescue.

The dynamic instability of microtubules is characterized by slow growth phases stochastically interrupted by rapid depolymerizations called catastrophes. Rescue events can arrest the depolymerization and restore microtubule elongation. However, the origin of these rescue events remains 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.

Actin nucleation at the centrosome controls lymphocyte polarity.

Cell polarity is required for the functional specialization of many cell types including lymphocytes. A hallmark of cell polarity is the reorientation of the centrosome that allows repositioning of organelles and vesicles in an asymmetric fashion. The mechanisms underlying centrosome polarization are not fully understood. Here we found that in resting lymphocytes, centrosome-associated Arp2/3 locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. Therefore, F-actin nucleation at the centrosome--regulated by the availability of the Arp2/3 complex--determines its capacity to polarize in response to external stimuli.

The centrosome is an actin-organizing centre.

Microtubules and actin filaments are the two main cytoskeleton networks supporting intracellular architecture and cell polarity. The centrosome nucleates and anchors microtubules and is therefore considered to be the main microtubule-organizing centre. However, recurring, yet unexplained, observations have pointed towards a connection between the centrosome and actin filaments. Here we have used isolated centrosomes to demonstrate that the centrosome can directly promote actin-filament assembly. A cloud of centrosome-associated actin filaments could be identified in living cells as well. Actin-filament nucleation at the centrosome was mediated by the nucleation-promoting factor WASH in combination with the Arp2/3 complex. Pericentriolar material 1 (PCM1) seemed to modulate the centrosomal actin network by regulating Arp2/3 complex and WASH recruitment to the centrosome. Hence, our results reveal an additional facet of the centrosome as an intracellular organizer and provide mechanistic insights into how the centrosome can function as an actin-filament-organizing centre.

Microtubules self-repair in response to mechanical stress.

Microtubules--which define the shape of axons, cilia and flagella, and provide tracks for intracellular transport--can be highly bent by intracellular forces, and microtubule structure and stiffness are thought to be affected by physical constraints. Yet how microtubules tolerate the vast forces exerted on them remains unknown. Here, by using a microfluidic device, we show that microtubule stiffness decreases incrementally with each cycle of bending and release. Similar to other cases of material fatigue, the concentration of mechanical stresses on pre-existing defects in the microtubule lattice is responsible for the generation of more extensive damage, which further decreases microtubule stiffness. Strikingly, damaged microtubules were able to incorporate new tubulin dimers into their lattice and recover their initial stiffness. Our findings demonstrate that microtubules are ductile materials with self-healing properties, that their dynamics does not exclusively occur at their ends, and that their lattice plasticity enables the microtubules' adaptation to mechanical stresses.

Microtubule-sliding activity of a kinesin-8 promotes spindle assembly and spindle-length control.

Molecular motors play critical roles in the formation of mitotic spindles, either through controlling the stability of individual microtubules, or by crosslinking and sliding microtubule arrays. Kinesin-8 motors are best known for their regulatory roles in controlling microtubule dynamics. They contain microtubule-destabilizing activities, and restrict spindle length in a wide variety of cell types and organisms. Here, we report an antiparallel microtubule-sliding activity of the budding yeast kinesin-8, Kip3. The in vivo importance of this sliding activity was established through the identification of complementary Kip3 mutants that separate the sliding activity and microtubule-destabilizing activity. In conjunction with Cin8, a kinesin-5 family member, the sliding activity of Kip3 promotes bipolar spindle assembly and the maintenance of genome stability. We propose a slide-disassemble model where the sliding and destabilizing activity of Kip3 balance during pre-anaphase. This facilitates normal spindle assembly. However, the destabilizing activity of Kip3 dominates in late anaphase, inhibiting spindle elongation and ultimately promoting spindle disassembly.

Quantification of MAP and molecular motor activities on geometrically controlled microtubule networks.

The spatial organization of the microtubule (MT) network directs cell polarity and mitosis. It is finely regulated by hundreds of different types of microtubule-associated proteins and molecular motors whose specific functions are difficult to investigate directly in cells. Here, we have investigated their functions using geometrically controlled MT networks in vitro in cell-free assay. This was achieved by developing a new method to spatially define MT nucleation using MT microseeds adsorbed on a micropatterned glass substrate. This method could be used to control MT growth and the induction of complex MT networks. We selected the interaction of two radial arrays of dynamic and polarized MTs to analyze the formation of the central antiparallel MT bundle. We investigated the effects of the MT cross-linker anaphase spindle elongation 1 (Ase1) and the kinesin motor Klp2, which are known to regulate MT organization in the spindle midzone. We thus identified the respective roles of each protein and revealed their synergy on the establishment of stable antiparallel MT bundles by quantifying MT interactions over hundreds of comparable MT networks.