טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKatzir Ofer
SubjectA Novel Sufficiency Test to Define Cell-Cell
Fusion Proteins
DepartmentDepartment of Biology
Supervisor Professor Benjamin Podbilewicz
Full Thesis textFull thesis text - English Version


Abstract

Cell-cell fusion is the process when two cells or more fuse their plasma membranes. Their cytoplasm unites to generate a hybrid cell with multiple nuclei. Cell-cell fusion is critical for many developmental processes such as placenta formation, muscle development and bone maintenance. Cell fusion has also been implicated in neuronal function and stem-cell reprogramming. However, the fusion mechanisms are poorly understood. Furthermore, life itself begins in a fusion event. Despite the significance of the cell-cell fusion process in fertilization little is known about this process. We still do not know which proteins mediate membrane fusion between the egg and the sperm in any organism.    
I studied the process of gametes fusion and my goal was to identify the proteins that mediate it. I investigated organisms from different kingdoms and phyla while Caenorhabditis elegans (C. elegans) served as my fusion model organism. C. elegans’ EFF-1 was shown to be essential and sufficient for fusion in worms and heterologous cells. Both cells need to express it in order to fuse, thus, it acts in a homotypic way. Based on eff-1 mutant hypodermis fusion pattern I developed an assay to identify new cell-cell fusion proteins. I checked if a specific or combination of proteins could substitute EFF-1 and be sufficient for cells fusion. The strategy I used was to induce over-expression of genes encoding candidate fusion proteins and screen them for fusion events in wild-type and eff-1(-) backgrounds and gave a fusion score. This score was based on how many embryos showed fusion event/s after the candidate/s induction. I obtained this quantitative score using an embryonic co-transformation marker that indicated which embryos bore the candidate proteins.   
Overall this research provided an optimized protocol to identify new cell-cell fusion proteins that can substitute EFF-1 in C. elegans embryos and showed negative results for eight candidate fusion proteins genes using the sufficiency test. The negative results suggest that all eight proteins are not sufficient for fusion. However, preliminary results suggest that the inducible HAP2-YFP fusogen candidate co-localizes in the basolateral membranes and mediate ectopic fusion. I observed cytoplasmic content mixing between two adjacent cells that normally do not fuse.  There is the possibility that one of the eight candidates is a true fusogen but it acts in a heterotypic way and in my assay it lacked its receptor. If I could find such a pair, expressing them together in my assay will probably result in cells fusion.