Development of CRISPR-Cas9 tools for the study of mitochondrial structure using expansion microscopy

• Grant number: 23/12636-9
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2024
• End date: December 31, 2024
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Sandra Martha Gomes Dias
• Grantee: Mell Kimberly Damini
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Campinas , SP, Brazil
• Associated research grant: 21/05726-6 - Metabolism in the microenvironment and the role of metabolic exchanges in tumor progression, AP.TEM

Abstract

Cellular biology has experienced a revolution in the past decade, largely driven by technological advances in microscopy techniques. The ability to visualize cellular structures in unprecedented detail has provided profound insights into the complexity and dynamics of the living cell. With the recent advances in expansion microscopy, especially techniques like proExM (Protein-retention Expansion Microscopy) and ONE (one-step nanoscale expansion), it is now possible to view molecular details with unmatched clarity. These emerging techniques have the potential to revolutionize our understanding of cellular structures, allowing us to delve deeper into the intricate networks of molecular interactions. The research group has expertise in understanding mitochondrial morphology with the filament formation of the distinct isoforms of the enzyme glutaminase - which plays a significant role in tumor metabolism -, already observed in situ and modulating mitochondrial morphology. The formation of these filaments inside the mitochondria is reported in literature as being crucial in modulating tumor progression and interfering with cognitive processes in patients with mutations capable of modulating this phenomenon. In this context, the project aims to explore the frontier of expansion microscopy to elucidate mitochondrial morphology through the oligomerization of the enzyme glutaminase, using endogenous expression systems with genome modification by CRISPR for the knock-in of fluorescent proteins and to evaluate in situ the ability of glutaminases to form filaments and modulate mitochondrial morphology. Through these advanced techniques, the goal is to achieve unprecedented resolution of cell ultrastructure, offering insights into the dynamics and organization of metabolic enzymes and fostering groundbreaking structural discoveries.