Biology subjects

Guichet, A.

Publications and source records attributed to Guichet, A..

9 recordsLinked to original sources

bioRxiv2026

The Spectraplakin Short Stop (Shot) Organizes an Acentrosomal Microtubule Network in Early Oogenesis, Essential for Nuclear Positioning.

Nuclear positioning relies on the coordinated organization of cytoskeletal networks to generate and balance intracellular forces. In the Drosophila oocyte, the nucleus undergoes a centering phase during mid-oogenesis before migrating asymmetrically to the cortex in a microtubule (MT)-dependent manner, a process essential for dorso-ventral axis specification. However, the origin and organization of the MT network responsible for nuclear positioning remain to be fully understood. Here, we identify a previously uncharacterized non-centrosomal Microtubule-Organizing Center (ncMTOC) at the posterior cortex of the oocyte. This ncMTOC is defined by the polarized accumulation of the spectraplakin Short Stop (Shot) together with the MT minus- end-binding protein Patronin prior to nuclear migration. Loss of Shot or Patronin results in posterior displacement of the nucleus, demonstrating that this cortical MT array generates pushing forces that maintain the nucleus at the oocyte center before directional migration. Within this ncMTOC, Shot plays a key role in recruiting both the MT severing enzyme Katanin and the MT polymerase of the XMAP215 family protein Mini-spindle (Msps), ensuring the formation of MTs that emanate from the vicinity of the posterior plasma membrane towards the oocyte cytoplasm. In addition, this ncMTOCs is also associated with {gamma}-TuRC which function independently of centrosomes. Together, these activities establish a dynamic posterior acentrosomal MT network essential for nuclear centering during early oogenesis.

cell biology↗

bioRxiv2025

Girdin controls the pace of 3D tracheal cell intercalation by coupling adherens junctions to the actin cytoskeleton in Drosophila

Morphogenesis is orchestrated through coordinated cell movements, including cell intercalation, which drives extensive changes in cell shape and position. This process requires precise regulation of interactions between Adherens Junctions (AJs) and the cortical actin network to generate the necessary mechanical forces. Although the Myosin molecular motor II plays a central role in generating morphogenetic forces, it is dispensable for processes such as dorsal branch (DB) morphogenesis in the Drosophila tracheal system, where coordinated cell migration and three-dimensional intercalation rely on alternative, yet poorly characterised mechanisms of force generation. Here, we show that Vinculin and Ajuba LIM protein, two key regulators of mechano-transduction and cell adhesion, do not localise to AJs of DBs, while the cytoskeletal adaptor Girdin does. We demonstrate that Girdins function relies on AJ components -Catenin and E-Cadherin and is specifically required in tracheal cells to ensure the proper pace of cell intercalation. In addition, Girdin contributes to the actin network enrichment at AJs. Its role as an integral AJ component is further elucidated through the development of a novel genetic tool enabling in vivo cell-specific actin depolymerisation. Summary StatementDrosophila Girdin contributes to the pace of tracheal cell intercalation by linking adherens junctions to actin. A genetic tool for actin depolymerisation reveals Girdin as a key adherens junction component.

developmental biology↗

bioRxiv2025

The fastest movements and the most extreme deformations of the cell nucleus are driven by microtrubules in the model fungus Podospora anserina

A growing number of studies shows that nuclear movement, positioning, and the mechanical constraints applied to the nucleus play important roles in cellular physiology and gene regulation. This process, known as nuclear mechanotransduction, is an emerging field of research, and its deregulation can lead to severe human pathologies. In this study, we examine the behavior of female and male nuclei during sexual reproduction in the model fungus Podospora anserina. After plasmogamy, the two types of nuclei do not immediately undergo karyogamy. Instead, once inside the trichogyne (the female-specific hyphae extending from the ascogonium) the male nuclei migrate over distances of up to several hundred micrometers from the trichogyne tip, where plasmogamy occurs, to the ascogonium at the center of the protoperithecium. Our study highlights the contrasting behaviors of the two nuclear types: female nuclei remain immobile and spherical, while male nuclei move rapidly and exhibit striking deformations and stretching. Trichogynes are branched and compartmentalized, consisting of articles separated by septa that communicate through the septal pores. We show that during their migration across these compartments, male nuclei undergo the most extreme movements, deformations, and speeds reported to date for nuclei in motion. We also investigated the role of the actin and microtubule (MT) cytoskeletons in male nuclei behavior. Both cytoskeletal networks are highly dynamic and abundant in trichogynes, particularly at septa. We demonstrate that while actin depolymerization does not impair male nuclei migration, MT disruption causes a complete arrest of both nuclear movement and stretching, indicating a critical role for MTs. Finally, we reveal that the MT network in trichogynes is highly polarized, offering new insights into how male nuclei navigate within branched trichogynes.

cell biology↗

bioRxiv2022

Multifaceted modes of γ-tubulin complex recruitment and microtubule nucleation at mitotic centrosomes

Microtubule nucleation is mediated by {gamma}-tubulin ring complexes ({gamma}-TuRCs). In most eukaryotes, a GCP4/5/4/6 "core" complex promotes {gamma}-tubulin small complex ({gamma}-TuSC) association to generate cytosolic {gamma}-TuRCs. Unlike {gamma}-TuSCs, however, this core complex is non-essential in various species and absent from budding yeasts. In Drosophila, Spindle defective-2 (Spd-2) and Centrosomin (Cnn) redundantly recruit {gamma}-tubulin complexes to mitotic centrosomes. Here we show that Spd-2 recruits {gamma}-TuRCs formed via the GCP4/5/4/6 core, but that Cnn can recruit {gamma}-TuSCs directly via its well-conserved CM1 domain, similar to its homologues in budding yeast. When centrosomes fail to recruit {gamma}-tubulin complexes, they still nucleate microtubules via the TOG domain protein Mini-spindles (Msps), but these microtubules have different dynamic properties. Our data therefore help explain the dispensability of the GCP4/5/4/6 core and highlight the robustness of centrosomes as microtubule organising centres. They also suggest that the dynamic properties of microtubules are influenced by how they were nucleated.

cell biology↗

bioRxiv2022

Kinesin-1 promotes centrosome clustering and nuclear migration in the Drosophila oocyte

Accurate positioning of the nucleus is essential. Microtubules and their associated motors are important players in this process. Although nuclear migration in Drosophila oocytes is controlled by microtubule, a role for microtubule-associated molecular motors in nuclear positioning has yet to be reported. We characterize novel landmarks that allow a precise description of the pre-migratory stages. Using these newly defined stages, we report that, prior to migration, the nucleus moves from the oocyte anterior side toward the center and concomitantly the centrosomes cluster at the posterior of the nucleus. In absence of Kinesin-1, centrosome clustering is impaired and the nucleus fails to position and migrate properly. The maintenance of a high level of Polo-kinase at centrosomes prevents centrosome clustering and impairs nuclear positioning. In absence of Kinesin-1, SPD2 an essential component of the pericentriolar material is increased at the centrosomes, suggesting that Kinesin-1 associated defects result from a failure to reduce centrosome activity. Consistently, depleting centrosomes rescues the nuclear migration defects induced by Kinesin-1 inactivation. Our results suggest that Kinesin-1 controls nuclear migration in the oocyte by modulating centrosome activity. Summary statementIn this study, we identified a crucial role of Kinesin-1 in centrosome clustering required for nuclear positioning and migration in the Drosophila oocyte.

developmental biology↗

bioRxiv2021

Progenitor cell integration into a barrier epithelium during adult organ turnover

Barrier epithelial organs face the constant challenge of sealing the interior body from the external environment while simultaneously replacing the cells that contact this environment. These replacement cells--the progeny of basal stem cells--are born without apical, barrier-forming structures such as a protective, lumen-facing membrane and occluding junctions. How stem cell progeny acquire these structures to become part of the barrier is unknown. Here we use Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM), Correlative Light-Electron Microscopy (CLEM), and volumetric imaging of live and fixed organs to investigate progenitor integration in the intestinal epithelium of adult Drosophila. We find that stem cell daughters gestate their future lumenal-apical membrane beneath a transient, basal niche formed by an umbrella-shaped occluding junction that shelters the growing cell and adheres it to mature neighbor cells. The umbrella junction both targets formation of a deep, microvilli-lined, apical invagination and closes it off from the contents of the gut lumen. When the growing cell is sufficiently mature, the umbrella junction retracts to expose this Pre-Assembled Apical Compartment (PAAC) to the gut lumen, thus incorporating the new cell into the intestinal barrier. When we block umbrella junctions, stem cell daughters grow and attempt to differentiate but fail to integrate; when we block cell growth, no umbrella junctions form and daughters arrest in early differentiation. Thus, stem cell progeny build new barrier structures in the shelter of a transient niche, where they are protected from lumenal insults until they are prepared to withstand them. By coordinating this dynamic junctional niche with progenitor cell differentiation, a physiologically active epithelial organ incorporates new cells while upholding integrity of its barrier.

developmental biology↗

bioRxiv2020

Phospho-regulated auto-inhibition of Cnn controls microtubule nucleation during cell division

{gamma}-tubulin ring complexes ({gamma}-TuRCs) nucleate microtubules. They are recruited to centrosomes in dividing cells via binding to N-terminal CM1 domains within {gamma}-TuRC-tethering proteins, including Drosophila Cnn. Binding promotes microtubule nucleation and is restricted to centrosomes in dividing cells, but the mechanism regulating binding remains unknown. Here we identify an extreme N-terminal "CM1 auto-inhibition" (CAI) domain found specifically within the centrosomal isoform of Cnn (Cnn-C) that inhibits {gamma}-TuRC binding. Robust binding occurs after removal of the CAI domain or with the addition of phospho-mimetic mutations, suggesting that phosphorylation helps relieve inhibition. We show that regulation of Cnn binding to {gamma}-TuRCs is isoform-specific and that mis-regulation of binding can result in ectopic cytosolic microtubules and major defects during cell division. We also find that human CDK5RAP2 is auto-inhibited from binding {gamma}-TuRCs, suggesting conservation across species. Overall, our results shed light on how and why CM1 domain binding to {gamma}-TuRCs is regulated. Summary (for the online JCB table of contents and alerts)We show that auto-inhibition regulates the binding between microtubule nucleating complexes and proteins that tether them to sites of microtubule nucleation. Failure to properly regulate this binding can lead to ectopic cytosolic microtubule nucleation and major defects during cell division.

cell biology↗

bioRxiv2019

The Somatic Golgi nucleates microtubules that are directed by Kinesin-2 to maintain microtubule polarity within neurons

Neurons contain polarised microtubule arrays essential for neuronal function. How microtubule nucleation and polarity are regulated within neurons remains unclear. We show that {gamma}-tubulin localises asymmetrically to the somatic Golgi within Drosophila neurons. Microtubules originate from the Golgi with an initial growth preference towards the axon. Their growing plus ends also turn towards and into the axon, adding to the plus-end-out microtubule pool. Any plus ends that reach a dendrite, however, do not readily enter, maintaining minus-end-out polarity. Both turning towards the axon and exclusion from dendrites depend on Kinesin-2, a plus-end-associated motor that guides growing plus ends along adjacent microtubules. We propose that Kinesin-2 engages with a polarised microtubule network within the soma to guide growing microtubules towards the axon; while at dendrite entry sites engagement with microtubules of opposite polarity generates a backward stalling force that prevents entry into dendrites and thus maintains minus-end-out polarity within proximal dendrites.

cell biology↗

bioRxiv2018

The Kinesin-1 binding domain of Ensconsin/MAP7 promotes Kinesin-1 activation in vivo

Centrosome separation in Drosophila larval neuroblasts and asymmetric transport of embryonic determinants in oocytes are both microtubule-dependent processes that require Kinesin-1 activation by Ensconsin/microtubule-associated protein 7 (MAP7). However, the molecular mechanism used by Ensconsin to activate Kinesin-1 remains elusive. Ensconsin/ MAP7 contains an N-terminal microtubule-binding domain (MBD) and a C-terminal Kinesin-binding domain (KBD). Using rescue experiments in live flies, we show that KBD expression alone is sufficient to fully rescue Ensconsin-dependent centrosome separation defects, but not the fast oocyte streaming and the localization patterns of Staufen and Gurken proteins. Interestingly, we show here for the first time that KBD binds and stimulates Kinesin-1 binding to Mts in vivo and in vitro. We propose that the KBD/Kinesin-1 motor represents a minimal activation module that stimulates Kinesin-1 binding to Mts. Addition of the MBD, present in the full length Ensconsin allows this activation to occur directly on the Mt. Our data also suggest that in a very large cell with a complex microtubule network, but not in smaller cells, this dual activation by Ensconsin is essential for optimal Kinesin-1 targeting to the microtubule cytoskeleton.

cell biology↗