Optogenetic method for rapid and reversible removal of proteins from the mitotic spindle

Abstract

Na početku mitoze mikrotubuli i pripadajući proteini formiraju diobeno vreteno, strukturu koja omogućuje točnu raspodjelu kromosoma između dvije stanice kćeri. U ovom radu razvijena je optogenetička metoda koja omogućuje brzu i reverzibilnu translokaciju proteina iz diobenog vretena na mitohondrije korištenjem plavog svjetla. Metoda je primijenjena za proučavanje proteina TACC3 i PRC1 koji sudjeluju u formiranju paralelnih, odnosno antiparalelnih svežnjeva mikrotubula. Translokacija oba proteina događala se na vremenskim skalama od nekoliko minuta. Mijenjanjem koncentracije proteina PRC1 na diobenom vretenu bilo je moguće inducirati raspadanje i ponovno formiranje antiparalelnih svežnjeva mikrotubula što je rezultiralo promjenom oblika diobenog vretena. Dakle, ova metoda predstavlja novi moćni alat za proučavanje proteina diobenog vretena kontrolom njihove lokalizacije u pojedinim fazama mitoze.At the onset of mitosis microtubules and microtubule associated proteins form a spindle, which is responsible for proper segregation of chromosomes between two daughter cells. In this thesis, I have developed optogenetic method for rapid and reversible translocation of proteins from the mitotic spindle to mitochondria using blue light. This method was used for studying TACC3 and PRC1, proteins which are required for formation of parallel and antiparallel microtubule bundles, respectively. Translocation of both proteins occurred at time scales of several minutes. By changing concentration of PRC1 in the mitotic spindle, I was able to induce disassembly and formation of antiparallel microtubule bundles, which resulted in changes in the spindle shape. Thus, this method represents powerful new tool for studying spindle proteins by controlling their localization at specific stages of mitosis