טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentShalev Ezra Yael
SubjectTowards Visualization of 3D Chromosome Reconfiguration in
Live Yeast by Multicolor Point-Spread-Function
Engineering
DepartmentDepartment of Biomedical Engineering
Supervisor Dr. Yoav Shechtman
Full Thesis textFull thesis text - English Version


Abstract

Chromatin has a well-defined spatial structure which is crucial for many cellular functions such as gene regulation, DNA breakage repair and error-free cell division. Those cellular functions are affected by the chromatin conformation and dynamics. DNA spatial organization in fixed cells is determined by various biochemical methods, such as Hi-C, while methods for following chromatin dynamics are limited due to high acquisition time. In this project we aim to visualize dynamics of multiple fluorescently labeled DNA loci, in 3D with high precision on the single live cell level, using a localization microscopy method based on Multicolor Point Spread Function (PSF) engineering.


DNA commonly undergoes double-strand-breaks (DSB) due to different environmental conditions (radiation, reactive oxygen species, chemical, etc.). Fundamentally, a DSB is followed by repair mechanisms such as homologous recombination (HR), to prevent chromatin aberrations which may lead to cancer and cell death. Saccharomyces Cerevisiae is a eukaryotic budding yeast that has two sexual appearances as haploid (MATa and MATα), and each can undergo switching to the opposite type. In this process, namely mating type switching, a specific endonuclease creates a DSB within the MAT locus, which is repaired by gene conversion with the aid of silenced loci, located intrachromosomally on opposite arms. The chromosome undergoes dramatic spatial reconfiguration to allow the relevant locus to be in close proximity to the MAT locus. Since this process is easily controlled in the lab, it serves as a model to study homologous recombination for DSB repair.


Multicolor Point Spread Function (PSF) engineering is a technique in which the image of a point source in an optical microscope is modified, to encode its depth and/or color. This enables simultaneous tracking of different emitters in 3D, by utilizing a spatial light modulator (SLM)-based phase mask in the Fourier plane, modulating the standard PSF of the microscope.


Here, we aim to visualize the well characterized and dynamic mating type switching process by tagging the relevant loci, inducing a DSB, and applying multicolor PSF engineering for simultaneous 3D tracking of multiple emitters. Visualization of this process by multicolor PSF engineering is expected to reduce acquisition time dramatically, relative to previous work, and reveal dynamics that were not detected before, including transient and unstable conformation of the chromosome, which are statistically less frequently observed.