Thierry Lorca

Microtubules are dynamic, conserved cytoskeletal filaments that are essential for all eukaryotic cells. Microtubule dynamic properties are primarily characterized by measuring how fast they grow and shrink (growth and shrinkage rates) and how often they switch between assembly and disassembly (catastrophe and rescue frequencies). These four parameters measured for individual filaments can inform on the behavior of the entire microtubule network at the cell level. By comparing microtubule dynamics in Caenorhabditis elegans one-cell embryos using different genetically-encoded fluorescent probes, we observed an unexpected high variability in these parameters. Microtubule dynamics parameters were consistently higher in C. elegans strains expressing a fluorescently labelled microtubule end-binding protein than in strains relying on tubulin labelling, with microtubule growth rates differing by nearly a factor of two between the two conditions. This discrepancy was not limited to C. elegans , as we observed a similar effect in embryos of the tunicate Phallusia mammillata . Despite this, spindle size and assembly timing were only mildly affected. However, embryos expressing labelled end-binding protein exhibited higher frequency of mitotic defects and perturbed embryonic development upon exposure to various stresses such as elevated temperature or a compromised spindle assembly checkpoint. Thus, our work reveals both the remarkable robustness of mitotic spindle assembly in response to extreme microtubule dynamics plasticity, and the requirement for strict control of microtubule dynamics across successive early embryonic divisions. Our findings should also serve as a cautionary note when using tagged end-binding proteins to measure microtubule dynamics.

Meiotic progression relies on maintaining a precise balance between cyclin B/CDK1 activity and the phosphatase PP2A-B55. The latter is negatively regulated by the Greatwall kinase (Gwl). In maturing Xenopus oocytes, we show that the loss of Gwl and the subsequent hyperactivation of PP2A-B55 prevents phosphorylation of key proteins involved in spindle formation, chromosome condensation and spindle migration as well as the phosphorylation of Wee1/Myt1 and the APC/C complex. As a consequence, in these oocytes bipolar spindles cannot be formed and migrate to the cortex and chromosomes are partially decondensed preventing meiotic I progression. The APC/C remains inactive impairing cyclin B3 degradation and ultimately preventing Erp1 accumulation. Finally, the c-Mos-MAPK-Rsk1/2 pathway fails to activate due to the absence of c-Mos as a consequence of its improper degradation. Overall, our findings reveal a crucial role of Gwl in the coordination and progression of meiotic divisions.

The evolutionarily conserved, intrinsically disordered protein Bora is critical for initiating the activation of mitotic kinases. Once phosphorylated at Ser112 by Cyclin A-Cdk1 kinase, phospho-Bora activates unphosphorylated Aurora A kinase (AURKA), directing it towards Polo-like kinase 1 (Plk1), thus promoting Cyclin B-Cdk1 activation and mitotic entry. Here, by combining structural modeling and in vitro assays, we provide evidence that Bora wraps around the N-terminal lobe of AURKA to position its phospho-Ser112 near AURKA’s T-loop, mimicking T-loop phosphorylation. Additionally, Bora transiently interacts with the αC helix of the Plk1 kinase domain through a conserved motif, guiding AURKA activity towards the Plk1 T-loop, which is otherwise impervious to phosphorylation by AURKA. We highlight the importance of this motif for Bora function in vitro and during mitotic entry in Xenopus laevis egg extracts. Our results reveal critical molecular details of mitotic kinase activation, which could lead to the development of pathway-specific inhibitors.

Cell adhesion tightly controls cell proliferation and homeostasis in stratified epithelia by mechanisms that remain unclear. Focal adhesion kinase (FAK) transduces cell adhesion signals, is frequently deregulated in epithelial cancer, and it has been associated with proliferation and resistance to treatments. The mechanisms by which FAK controls the epithelial cell cycle are still intriguing. We previously unraveled a mitosis‐differentiation checkpoint that is the limiting factor in the keratinocyte cell cycle. To investigate whether FAK plays a role in this checkpoint, we inactivated the protein in normal human oral keratinocytes by specific shRNAs or by the specific inhibitor defactinib. Inactivation of FAK very rapidly and strikingly blocked entry into mitosis and triggered a differentiation response. This response was independent of DNA damage. Tumor suppressor P53 was induced shortly after inhibition of FAK, while mitotic Cyclin B was not translocated into the nucleus. Human epidermal N‐TERT cells that were synchronized in prometaphase failed to execute mitosis. Concomitant inhibition of FAK‐downstream Rho‐associated kinase (Rock) rescued mitotic progression. The results unveil a rapid Rock‐dependent mitosis switch upon inactivation of FAK, inducing terminal differentiation, pointing at a mitotic automatic mechanism of epithelia to suppress suprabasal proliferation of precancerous cells.

SUMMARY: Meiotic progression relies on maintaining a precise balance between cyclin B/CDK1 activity and the phosphatase PP2A-B55. The latter is negatively regulated by the Greatwall kinase (Gwl). In Xenopus oocytes, we show that the loss of Gwl and the subsequent hyperactivation of PP2A-B55 severely disrupt the transition from meiosis I to meiosis II. This disruption prevents phosphorylation of both Wee1/Myt1 and the APC/C complex, thereby blocking APC/C activation. As a consequence, APC/C remains inactive during the MI–MII transition, which impairs cyclin degradation and the partial CDK1 inactivation that is normally required at this stage. Additionally, the mos-MAPK-Rsk1/2 pathway fails to activate due to insufficient Mos accumulation, thereby preventing metaphase II arrest. Finally, the lack of APC/C activation during meiosis I inhibits the degradation of its inhibitor Erp1, revealing a critical feedback loop between APC/C and Erp1. Overall, our findings reveal that Gwl is a key coordinator of both meiotic divisions, acting through dual regulation of APC/C and the Mos/MAPK/Rsk1/2 pathways by modulating PP2A-B55 activity.

The evolutionarily conserved, intrinsically disordered protein Bora is critical for initiating the activation of mitotic kinases. Once phosphorylated at Ser112 by Cyclin A-Cdk1 kinase, phospho-(p)Bora activates unphosphorylated Aurora A kinase (AURKA), directing it towards Polo-like kinase 1 (Plk1), thus promoting Cyclin B-Cdk1 activation and mitotic entry. Here, by combining structural modelling and in vitro assays, we provide evidence that Bora wraps around the N-terminal lobe of AURKA to position its phospho-Ser112 near AURKA’s T-loop, mimicking T-loop phosphorylation. Additionally, Bora transiently interacts with the Alpha C helix of the Plk1 kinase domain through a conserved motif, guiding AURKA activity towards the Plk1 T-loop, which is otherwise impervious to phosphorylation by AURKA. We highlight the importance of this motif for Bora function in vitro and during mitotic entry in Xenopus egg extracts. Our results reveal critical molecular details of mitotic kinase activation, which could lead to the development of pathway-specific inhibitors. Highlights: MITOKINAC: a versatile approach that reconstitutes Bora and AURKA-dependent Plk1 T-loop phosphorylation in E. coli . AlphaFold 3 structural modeling of the phospho-Bora – AURKA complex tested by MITOKINAC using 39 Bora variants. Phospho-Bora wraps around the N-terminal lobe of AURKA to position its essential phospho-Serine 112 in the activation loop of AURKA. pBora delivers the AURKA activity to the Plk1 T-loop, which is otherwise impervious to AURKA phosphorylation.

MAST-like, or Greatwall (Gwl), an atypical protein kinase related to the evolutionarily conserved MAST kinase family, is crucial for cell cycle control during mitotic entry. Mechanistically, Greatwall is activated by Cyclin B-Cdk1 phosphorylation of a 550 amino acids-long insertion in its atypical activation segment. Subsequently, Gwl phosphorylates Endosulfine and Arpp19 to convert them into inhibitors of PP2A-B55 phosphatase, thereby preventing early dephosphorylation of M-phase targets of Cyclin B-Cdk1. Here, searching for an elusive Gwl-like activity in C. elegans , we show that the single worm MAST kinase, KIN-4, fulfills this function in worms and can functionally replace Greatwall in the heterologous Xenopus system. Compared to Greatwall, the short activation segment of KIN-4 lacks a phosphorylation site, and KIN-4 is active even when produced in E. coli . We also show that a balance between Cyclin B-Cdk1 and PP2A-B55 activity, regulated by KIN-4, is essential to ensure asynchronous cell divisions in the early worm embryo. These findings resolve a long-standing puzzle related to the supposed absence of a Greatwall pathway in C. elegans , and highlight a novel aspect of PP2A-B55 regulation by MAST kinases.

Entry into mitosis has been classically attributed to the activation of a cyclin B/Cdk1 amplification loop via a partial pool of this kinase becoming active at the end of G2 phase. However, how this initial pool is activated is still unknown. Here we discovered a new role of the recently identified PP2A-B55 inhibitor FAM122A in triggering mitotic entry. Accordingly, depletion of the orthologue of FAM122A in C. elegans prevents entry into mitosis in germline stem cells. Moreover, data from Xenopus egg extracts strongly suggest that FAM122A-dependent inhibition of PP2A-B55 could be the initial event promoting mitotic entry. Inhibition of this phosphatase allows subsequent phosphorylation of early mitotic substrates by cyclin A/Cdk, resulting in full cyclin B/Cdk1 and Greatwall (Gwl) kinase activation. Subsequent to Greatwall activation, Arpp19/ENSA become phosphorylated and now compete with FAM122A, promoting its dissociation from PP2A-B55 and taking over its phosphatase inhibition role until the end of mitosis.

Entry into mitosis has been classically attributed to the activation of cyclin B/cdk1 amplification loop by a partial pool of this kinase that becomes active at the end of G2. However, how this pool is activated is still unknown. Here we discovered a new role of the recently identified PP2A-B55 inhibitor FAM122A in triggering mitotic entry. Accordingly, the depletion of the orthologue of FAM122A in C. elegans , prevents entry into mitosis in germline stem cells. Moreover, our data in Xenopus egg extract strongly supports that FAM122A-dependent inhibition of PP2A-B55 could be the initial event promoting mitotic entry. The inhibition of this phosphatase allows the subsequent phosphorylation of first mitotic substrates by cyclin A/cdk resulting in cyclin B/cdk1 and Greatwall (Gwl) activation. However, interestingly, from Gwl activation, Arpp19/ENSA become phosphorylated and compete with FAM122A promoting its dissociation from PP2A-B55 and taking over its inhibition until the end of mitosis.

PP2A is a major serine/threonine phosphatase class involved in the regulation of cell signaling through the removal of protein phosphorylation. This class of phosphatases is comprised of different heterotrimeric complexes displaying distinct substrate specificities. The present review will focus on one specific heterocomplex, the phosphatase PP2A-B55. Herein, we will report the direct substrates of this phosphatase identified to date, and its impact on different cell signaling cascades. We will additionally describe its negative regulation by its inhibitors Arpp19 and ENSA and their upstream kinase Greatwall. Finally, we will describe the essential molecular features defining PP2A-B55 substrate specificity that confer the correct temporal pattern of substrate dephosphorylation. The main objective of this review is to provide the reader with a unique source compiling all the knowledge of this particular holoenzyme that has evolved as a key enzyme for cell homeostasis and cancer development.