Periodic Reporting for period 1 - MechanoATR (ATR-mediated mechanotrasduction at the nuclear envelope)
Reporting period: 2021-12-01 to 2023-11-30
Cells can sense and respond to mechanical forces, translating these cues into coordinated gene expression changes that impact processes such as cell differentiation, tissue renewal, and homeostasis. ATR kinase is essential for maintaining genome integrity by stabilizing replication forks and coordinating DNA repair with cell cycle progression. While the role of ATR in mediating the DNA damage response is well-established, recent findings suggest that ATR also senses mechanical forces and translocates to the nuclear envelope (NE), where it plays a role in nuclear mechanoadaptation. Understanding how ATR mediates nuclear responses to mechanical forces can lead to improved treatments for conditions where NE instability and genome integrity are compromised. This includes not only laminopathies but also cancer, aging-related diseases, and other genetic disorders. Our study aimed to elucidate the mechanism by which ATR responds to mechanical forces and the downstream effects of this response on nuclear integrity. Using advanced molecular biology techniques, cutting-edge imaging modalities, and the vertebrate model Xenopus laevis, we will investigate the downstream consequences of NE-ATR translocation, focusing on nuclear actin dynamics. Our findings reveal a novel mechanotransduction pathway where ATR, upon sensing mechanical forces, translocates to the NE and phosphorylates RASSF1A. This phosphorylation event regulates nuclear actin . This ATR/RASSF1A pathway represents a crucial mechanism by which cells maintain genome integrity and adapt to mechanical stress, with potential implications for understanding diseases characterized by genome instability and impaired mechanotransduction.
The investigation commenced by elucidating the specific type of mechanical stimulation necessary for the association of ATR (Ataxia Telangiectasia and Rad3-related protein) with NE, achieved through monitoring the localization of endogenous ATR during cyclic mechanical stretch. Pharmacologic modulators targeting the cytoskeleton were then employed to discern the roles of cytoskeletal and nuclear tension in initiating ATR translocation to the NE in response to mechanical stimuli. Intriguingly, inhibition of actin polymerization, Rho-associated kinase (ROCK), or actinomyosin contractility failed to impede NE association of ATR. However, the abrogation of LINC (linker of nucleoskeleton and cytoskeleton) utilizing a dominant-negative nesprin (DN-KASH) complex effectively inhibited NE-ATR recruitment upon mechanical stretch.
The translocation of ATR to the NE in response to mechanical stress instigates the phosphorylation of RASSF1A (Ras association domain family 1 isoform A). Combining hyperosmolarity and mechanical stretching with either an ATR inhibitor or siRNA against ATR, the study demonstrated that inhibiting the ATR phosphorylation site of RASSF1A blocked its recruitment to the NE in response to mechanical stimulation. Crucially, inactivation of the ATR/RASSF1A axis led to a reduction in cells exhibiting nuclear actin filaments, underscoring the pivotal role of this axis in modulating nuclear actin dynamics, which is essential for various cellular processes, including transcription, DNA repair, and chromatin organization.
These findings will be presented at the 5th International meeting in Laminopathies, scheduled for May 2025 in Paris. The presentation promises to illuminate novel insights into the molecular mechanisms underlying laminopathies, offering potential avenues for therapeutic interventions targeting the ATR/RASSF1A axis and its associated pathways.
The translocation of ATR to the NE in response to mechanical stress instigates the phosphorylation of RASSF1A (Ras association domain family 1 isoform A). Combining hyperosmolarity and mechanical stretching with either an ATR inhibitor or siRNA against ATR, the study demonstrated that inhibiting the ATR phosphorylation site of RASSF1A blocked its recruitment to the NE in response to mechanical stimulation. Crucially, inactivation of the ATR/RASSF1A axis led to a reduction in cells exhibiting nuclear actin filaments, underscoring the pivotal role of this axis in modulating nuclear actin dynamics, which is essential for various cellular processes, including transcription, DNA repair, and chromatin organization.
These findings will be presented at the 5th International meeting in Laminopathies, scheduled for May 2025 in Paris. The presentation promises to illuminate novel insights into the molecular mechanisms underlying laminopathies, offering potential avenues for therapeutic interventions targeting the ATR/RASSF1A axis and its associated pathways.
The project represents a significant advancement beyond the current state of the art by uncovering a previously unknown mechanotransduction pathway operating at the nuclear envelope (NE). By elucidating the roles of ATR, RASSF1A, and nuclear actin, the project provided a detailed understanding of NE mechanotransduction, shedding light on fundamental processes governing cellular responses to mechanical cues. Insights gained from the project may lead to the identification of novel therapeutic targets for diseases associated with NE instability, such as laminopathies. By targeting components of the ATR/RASSF1A/nuclear actin axis, it may be possible to develop interventions aimed at restoring NE integrity and alleviating the symptoms of these debilitating disorders. Moreover, defects in ATR pathways are linked to various cancers, and understanding its role in protecting the NE provides new insights into tumor suppression and cancer progression.