Investigating glycation-induced changes in the cell nucleus using atomic force microscopy

Details

Supervisors

Dumitru, Andra-Cristina ; Alcaraz Hurtado, Mar

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité spécialisée: biotechnologie

Abstract

Diabetes mellitus (DM) is a chronic metabolic disorder that results in persistently elevated blood glucose levels, which, if left untreated, can lead to severe and often life-threatening complications such as cardiovascular disease, kidney failure, neuropathy, and retinopathy. One of the notable consequences of DM is the increased rigidity of organs and connective tissues, driven by the accumulation of advanced glycation end-products (AGEs). AGEs are formed when sugars and their reactive by-products non-enzymatically modify proteins, leading to cross-linking and improper protein folding and function. This structural alteration in turn reduces tissue elasticity, disrupts normal physiological function, and exacerbates the complications associated with DM. While much is known about extracellular glycation, the impact of glycation on intracellular proteins, particularly nuclear proteins, remains poorly understood. Nuclear proteins play a critical role in mechanotransduction, maintaining nuclear structure, regulating gene expression, and facilitating DNA repair, suggesting that glycation-induced mechanical changes at the nuclear level could have far-reaching pathological consequences. This Master’s thesis focuses on investigating the effects of methylglyoxal (MG), a major glycating agent and by-product of glycolysis, on the mechanical properties of the nucleus, using the HeLa cell line as a model system. After developing and optimizing experimental protocols for the isolation of nuclei and the application of MG treatments, we used Atomic Force Microscopy (AFM) to measure the nanomechanical properties of both isolated nuclei and the nuclear regions of intact cells. Complementary light microscopy techniques were employed to visualize and assess the morphology and integrity of the nuclei under different experimental conditions. The results demonstrated a statistically significant increase in the stiffness of nuclei treated with MG across several experimental models, indicating that glycation directly impairs nuclear mechanics by altering the structural properties of nuclear components. These findings not only highlight the nucleus as a glycation target, but also provide a foundation for understanding how AGE-induced stiffness may contribute to broader cellular dysfunction and tissue-level rigidity observed in DM patients. The study offers new insights into the role of compromised nuclear mechanics in the pathophysiology of DM and other AGE-related conditions. These findings underscore the importance of further research into intracellular glycation, potentially paving the way for new therapies to mitigate glycation-induced cellular damage.