University of Zagreb. Faculty of Science. Department of Biology.
Abstract
Glavna struktura koja upravlja procesom je diobeno vreteno, a jedna od klasa mikrotubula u toj strukturi su kinetohorna vlakna koja vežu kromosome putem kinetohora na centromeri. Trenutne spoznaje govore da dva sestrinska kinetohorna vlakna nisu u međusobnoj izravnoj interakciji. U ovom radu pažnja je usmjerena na opisivanje nove klase mikrotubula u vretenu a nazvana je premošćujući mikrotubuli. Ta nova klasa tvori vlakna antiparalelnih mikrotubula i lateralno povezuje sestrinska kinetohorna vlakna te sudjeluje u mehanici diobenog vretena. U provedenom istraživanju korišten je konfokalni mikroskop za snimanje živih stanica HeLa te je korištena laserska mikrodisekcija vanjskog kinetohornog vlakna, da bi se pokazala prisutnost premošćujućih mikrotubula te ispitala njihova uloga u distribuciji sila u vretenu. Ove su rezultate potvrdili eksperimenti sa stanicama sa smanjenom razinom proteina PRC1 koji je sastavni dio metafaznog vretena. Procesi u regiji između sestrinskih kromosoma analizirani su u stanicama s obilježenim proteinom EB3, uključenim u rast mikrotubula na + kraju. U oba eksperimenta potvrđena je prisutnost kompresivne sile i sile tenzije u metafaznom diobenom vretenu.Main structure that orchestrates mitosis is the mitotic spindle, and one of the microtubule classes comprised within are the kinetochore fibers, which bind to chromosomes via kinetochores on the centromere. It is currently believed that there is no direct interaction between two sister kinetochore fibers. The focus of this research is to describe a novel class of microtubules in the spindle, which was named bridging microtubules. This new class forms antiparallel bundles, laterally connects sister kinetochore fibers and contributes to spindle mechanics. In conducted research, confocal microscope was used for live-cell imaging of HeLa, and the laser microsurgery was applied on outermost kinetochore fiber in order to confirm bridging microtubule presence and to test their function in force distribution in the spindle. By using cells with decreased level of PRC1 protein, protein involved in metaphase spindle mechanics, we showed that bridging bundle contributes to force balance in the spindle. Microtubule dynamics in the region between sister kinetochores was also analyzed in cells with labeled EB3 protein involved in the growth of microtubule 's + end. Laser microsurgery confirmed compressive and tensile force in the metaphase spindle in both experiments
