Generation of a microscopy bioimage analysis to determine Cdk2 and Cdk4 activities at the single cell level

• Grant number: 24/16814-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: November 01, 2024
• End date: October 31, 2026
• Field of knowledge: Biological Sciences - Morphology - Cytology and Cell Biology
• Principal Investigator: Alexandre Bruni Cardoso
• Grantee: Lucas de Oliveira Cambuim
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Cyclin-dependent kinases (Cdks) play a fundamental role in regulating the progression of the cell cycle. Imbalances in Cdk activity can lead to excessive growth or tissue function failure. A useful tool for monitoring and quantifying Cdk activity at the single-cell level is the use of fluorescent sensors. These sensors are composed of a fluorescent protein, peptide segments containing phosphorylation sites for Cdk, and nuclear import and export sequences, which allow for the nuclear translocation of the probe according to phosphorylation. In this project, we propose to obtain datasets of images of EpH4 mammary epithelial cells expressing fluorescent sensors for Cdk2 (DHB-Venus) and Cdk4/6 (mCherry-KTR) and to generate an analysis workflow for these images. The images will be obtained through fluorescence microscopy under various conditions, including high and low cell density and treatments with Cdk2 and Cdk4 inhibitors. The computational workflow will be user-friendly and will consist of all analysis steps, from image processing and segmentation to the quantification of the cytoplasm/nucleus ratio of probe intensity at the single-cell level and the plotting of graphs from the obtained data. The expectation is that our datasets and workflow will contribute to understanding Cdk activity in physiological and pathological contexts, such as cancer.