Biology subjects

Gaillard, J.

Publications and source records attributed to Gaillard, J..

14 recordsLinked to original sources

bioRxiv2026

Uncoupling microtubule lifetime, stability and post-translational modifications.

Microtubules (MTs) undergo continuous cycles of growth and disassembly. Because the transitions between these states are stochastic, MT age varies widely within a population. As MTs age, they are thought to accumulate post-translational modifications (PTMs) that, directly or indirectly, enhance their stability and thereby extend their lifetime. The rare MTs that withstand prolonged exposure to destabilizing drugs such as nocodazole (NZ) are indeed enriched in PTMs; yet the relationships between MT age, PTMs and stability remain unclear. Using microinjection of labelled tubulin, we measured microtubule network turnover in immortalized mouse embryonic fibroblasts. Half of the network was renewed within 3 minutes and 80% within 10 minutes, while approximately 5% of microtubules persisted for more than 20 minutes. These dynamics were comparable in quiescent and senescent cells, although the fraction of slowly renewing or non-renewing microtubules rose to 20% in senescent cells. Unexpectedly, neither the amount of PTMs (acetylation and detyrosination) nor resistance to NZ increased with MT age, and resistance to NZ was independent of these PTMs. Degrees of acetylation and detyrosination should therefore not be taken as readouts of MT age or stability. Because these PTMs do not accumulate on MTs over time, the chemical modification of polymerized tubulin is likely more reversible and dynamic than previously assumed.

cell biology↗

bioRxiv2026

Direct labeling of microtubule turnover reveals in-lattice repair and stabilization patterns in developing neurons

The microtubule cytoskeleton is the backbone of neuronal morphogenesis, driving the development of the dendrites and axon, and supporting trafficking to distant compartments. How neuronal microtubules are maintained and renewed in the face of this dynamic endeavor and long-term commitment remains unclear. Recently, in-lattice repair has emerged as an alternative mechanism of microtubule renewal that could allow for continuity of the existing network and for the emergence of cell polarity. Here, we directly assessed microtubule turnover using microinjection of labeled tubulin in cultured hippocampal neurons that exhibit well defined stages of development and polarization during the first 24 hours in culture. Sizeable tubulin integration was visible minutes after microinjection, indicating fast turnover of microtubules in developing neurons. Once it appeared, a longer neurite that would become the nascent axon showed decreased turnover, both for its acetylated and non-acetylated populations of microtubules. Combining microinjection with expansion microscopy allowed us to visualize tubulin integration sites along single microtubules, unambiguously demonstrating the existence of in-lattice integration along neurites. In thick neurites, we observed preferential integration within non-acetylated cortical microtubules, but in-lattice integration sites were also visible in the deeper core bundles of acetylated microtubules. Overall, our results link previous observations of microtubule stabilization patterns in developing neurons to their actual turnover. Mapping these patterns of turnover strengthens the notion that microtubules establish an organized network that participates in axon emergence and the establishment of neuronal polarity.

cell biology↗

bioRxiv2025

Cryopreservation of Human Cortical Organoids Using Vitrification.

Cryopreservation at ultra-low temperatures is a valuable tool for preserving cells and tissues used in research. However, few protocols exist for the preservation of brain organoid models. Current methods for preserving human cortical organoids (hCOs) rely on conventional slow cooling approaches with organoids suspended in a medium containing a cocktail of cryoprotectants. In contrast, we have optimized a vitrification technique previously used to cryopreserve human embryos and oocytes for application to hCOs. We have successfully cryopreserved hCOs that were generated by two different protocols. The vitrified organoids demonstrate a growth rate, cytoarchitecture, cell type composition and electrical activity comparable to non-vitrified controls. Our hCO cryopreservation method provides a useful alternative approach for bio-banking and cross-institutional collaboration using cortical organoids as their model system. Highlights- Human pluripotent stem cell (hPSC) derived hCOs can be cryopreserved using vitrification. - Vitrified hCOs develop similarly to non-vitrified controls. - Vitrified hCOs can be stored long-term and shipped to other institutions. MotivationMany methods of cryopreservation have been developed for the maintenance of cell lines, organoid models and tissues. Most organoid models have been successfully preserved using conventional slow-cooling methods; however, human cortical organoids have been difficult to preserve. Our findings demonstrate that the vitrification of cortical organoids preserves their structure, cell type diversity and function upon rewarming and continued culture.

neuroscience↗

bioRxiv2023

Microtubules under mechanical pressure can breach dense actin networks

The crosstalk between actin network and microtubules is key to the establishment of cell polarity. It ensures that the asymmetry of actin architec ture along cell periphery directs the organization of microtubules in cell interior. In particular, the way the two networks are physically inter-twined regulates the spatial organization and the distribution of forces in the microtubule network. While their biochemical crosstalk is getting uncovered, their mechanical crosstalk is still poorly understood. Here we designed an in vitro reconstitution assay to study the physical interaction between dynamic microtubules with various structures made of actin fil aments. We found that microtubules can align and move by their polymerization force along linear bundles of actin filaments. But they cannot enter dense and branched actin meshworks, such as those found in the lamellipodium along cell periphery. However, when microtubules are immobilized, by their crosslinking with actin structures or others means, the force of polymerization builds up pressure in the microtubules that is sufficient to allow them to breach and penetrate these dense actin meshworks. This mechanism may explain the final progression of microtubules up to cell periphery through the denser parts of the actin network.

biophysics↗

bioRxiv2022

Actin architecture steers microtubules in active cytoskeletal composite

Cytoskeletal motility assays use surface-immobilised molecular motors to propel cytoskeletal filaments. These assays have been widely employed to characterise the motor properties and interactions of cytoskeletal elements with themselves or with external factors. Moreover, the motility assays are a promising class of bio-inspired active tools for nanotechnological applications. While effective utilisation of these assays involves 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 motor-propelled microtubules and networks of actin filaments. We demonstrate that the microtubules respond to a network of actin filaments and that this response depends on the networks architecture. Both linear actin filaments and a network of actin branched by the Arp2/3 complex 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 architecture by microtubules, is a conceptually new means of controlling the filament gliding in the motility assay with potential application in the design of active materials and cytoskeletal nano-devices.

biophysics↗

bioRxiv2022

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 network 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 taking place 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 pre-existing green microtubule network 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. We then characterized the size and spatial frequency of these incorporations as a function of injected tubulin concentration and post-injection delay. The saturation of these measurements contradicted the hypothesis of nonspecific adsorption along microtubules and 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 micrometers 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. These results provide evidences that microtubules do self-repair in living cells, and they provide a quantitative characterization of the temporal and spatial dynamics of this process in PtK2 cells.

cell biology↗

bioRxiv2022

The architecture of the actin network can balance the pushing forces produced by growing microtubules.

The position of centrosome, the main microtubule-organizing center (MTOC), is instrumental in the definition of cell polarity. It is defined by the balance of tension and pressure forces in the network of microtubules (MTs). As MTs polymerize against the cell periphery, pressure increases and produces pushing forces on the MTOC. How the mechanical interplay between MTs and the actin network is involved in the regulation of these forces remains poorly understood, in particular because its investigation is technically limited by the structural and biochemical complexity of the cell cytoplasm. Here, in a cell-free assay, we used purified proteins to reconstitute the interaction of an aster of dynamic MTs with actin networks of various compositions and architectures in cell-sized microwells. In the absence of actin filaments, the positioning of the MTOC was highly sensitive to variations in MT length. The presence of a bulk actin filament network limited MTs deformation and displacement, and MTOCs were hold in place. In contrast, the assembly of a dense and branched actin network along the edges of the wells centered the MTOCs by preventing MT slippage and thus maintaining an isotropic balance of pushing forces. In agreement with this, an asymmetric peripheral actin network caused the MTOC to decenter by creating an asymmetry in the pushing forces. Numerical simulations demonstrated that steric hindrance by actin networks, at the tip or along the entire length of MTs, can modulate MTOC positioning, as observed in the experiments. Overall, our results show that actin networks can limit the sensitivity of MTOC positioning to MT length and enforce robust MTOC centering or decentering depending on its architecture.

cell biology↗

bioRxiv2022

Actin-microtubule dynamic composite forms responsive active matter with memory

Active cytoskeletal materials in vitro demonstrate self-organising properties similar to those observed in their counterparts in cells. However, the search to emulate phenomena observed in the living matter has fallen short of producing a cytoskeletal network that would be structurally stable yet possessing adaptive plasticity. Here, we address this challenge by combining cytoskeletal polymers in a composite, where self-assembling microtubules and actin filaments collectively self-organise due to the activity of microtubules-percolating molecular motors. We demonstrate that microtubules spatially organise 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 behaviour in changing mechanochemical environment. We thus establish artificial active actin-microtubule composite as a system demonstrating architectural stability and plasticity.

biophysics↗

bioRxiv2021

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 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 evolution intermediate between prokaryotic FtsZ and eukaryotic microtubule-forming tubulins.

evolutionary biology↗

bioRxiv2020

Bacterial inhibition of CD8+ T-cells mediated cell death promotes neuroinvasion and within-host persistence

Central nervous system infections are amongst the most severe1,2, yet the mechanisms by which pathogens access the brain remain poorly understood. The model microorganism Listeria monocytogenes (Lm) is a major foodborne pathogen that causes neurolisteriosis, one of the deadliest central nervous system infections3,4. While immunosuppression is a well-established host risk factor for neurolisteriosis3,5, little is known regarding the bacterial factors underlying Lm neuroinvasion. We have developed a clinically-relevant experimental model of neurolisteriosis, using hypervirulent neuroinvasive strains6 inoculated in a humanized mouse model of infection7, and we show that the bacterial protein InlB protects infected monocytes from CD8+ T-cells Fas-mediated cell death, in a c-Met/PI3-kinase/FLIP-dependent manner. This blockade of anti-Lm specific cellular immune response lengthens infected monocytes lifespan, favoring Lm transfer from infected monocytes to the brain. The intracellular niche created by InlB-mediated cell-autonomous immunosuppression also promotes Lm fecal shedding, accounting for its selection as a Lm core virulence gene. Here, we have uncovered an unanticipated specific mechanism by which a bacterial pathogen confers to the cells it infects an increased lifespan by rendering them resistant to cell-mediated immunity. This promotes Lm within-host persistence and dissemination to the central nervous system, and transmission.

immunology↗

bioRxiv2019

CLASP mediates microtubule repair by promoting tubulin incorporation into damaged lattices

Microtubule network plays 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 either be induced by mechanical stress or occur spontaneously during polymerization [2-4]. Tubulin incorporation can restore microtubule integrity; moreover, \"islands\" of freshly incorporated GTP-tubulin can inhibit microtubule disassembly and promote rescues [3-7]. Microtubule repair occurs in vitro in the presence of tubulin alone [2-4, 8]. However, in cells, it is likely to be regulated by specific factors, the nature of which is currently unknown. CLASP is an interesting candidate for microtubule repair, because it induces microtubule nucleation, stimulates rescue and suppresses catastrophes by stabilizing incomplete growing plus ends with lagging protofilaments and promoting their conversion into complete ones [9-16]. Here, we used in vitro reconstitution assays combined with laser microsurgery and microfluidics to show that CLASP2 indeed stimulates microtubule lattice repair. CLASP2 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. A single CLASP2 domain, TOG2, which suppresses catastrophes when tethered to microtubules, was sufficient to stimulate microtubule repair, indicating 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.

cell biology↗

bioRxiv2018

Self-repair protects microtubules from their destruction by molecular motors

Microtubules are dynamic polymers that are used for intracellular transport and chromosome segregation during cell division. Their instability stems from the low energy of tubulin dimer interactions, which sets the growing polymer close to its disassembly conditions. Microtubules function in coordination with kinesin and dynein molecular motors, which use ATP hydrolysis to produce mechanical work and move on microtubules. This raises the possibility that the forces 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 the mechanical work of molecular motors can remove tubulin dimers from the lattice and rapidly destroy microtubules. This effect was not observed when free tubulin dimers were present in the assay. Using fluorescently labelled tubulin dimers we 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 kinesin and dynein motors and the renewal of the microtubule lattice.

cell biology↗

bioRxiv2018

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 were correlated with reduced amounts of microtubules. Furthermore, lymphocyte activation resulted in centrosomal-actin disassembly 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. The two assays demonstrated that actin filaments perturb microtubule growth by steric hindrance. Finally, we showed that cell adhesion and spreading leads to lower densities of centrosomal actin thus resulting in higher microtubule growth. Hence we propose a novel mechanism by which the number of centrosomal microtubules is regulated by cell adhesion and actin-network architecture.

cell biology↗

bioRxiv2018

Lattice defects induce microtubule self-renewal

The dynamic instability of microtubules is powered by the addition and removal of tubulin dimers at the ends of the microtubule. Apart from the end, the microtubule shaft is not considered to be dynamic. However recent evidence suggests that free dimers can be incorporated into the shaft of a microtubule damaged by mechanical stress. Here we explored whether dimer exchange was a core property of the microtubule lattice independently of any external constraint. We found that dimers can be removed from and incorporated into the lattice at sites along the microtubule shaft. Furthermore, we showed by experiment and by modeling that rapid dimer renewal requires structural defects in the lattice, which occur in fast growing microtubules. Hence long-lived microtubules have the capacity to self-renew despite their apparent stability and thereby can potentially regulate signaling pathways and structural rearrangements associated with tubulin-dimer exchange at sites along their entire length.

cell biology↗