The mechanisms of microtubule associated proteins in chromosome segregation

doctoral thesis

• Title (croatian): Mehanizmi djelovanja proteina povezanih s mikrotubulima u segregaciji kromosoma
• Author: Kruno Vukušić
• Mentor: Iva Marija Tolić (mentor)
• Committee member: Juraj Simunić (predsjednik povjerenstva)
• Committee member: Maja Matulić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2020-05-15, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology
• Universal decimal classification (UDC): 57 - Biological sciences. Physical anthropology. Biodiversity

Abstract

Successful cell division requires chromosomes to be separated on opposite sides of the cell. This
is achieved by depolymerisation of kinetochore fibers and spindle elongation. Forces required for
spindle elongation in human cells are linked to sliding of antiparallel microtubules and sliding
capacity has been demonstrated in vitro for multiple motor proteins, but the molecular mechanism
of sliding during anaphase in the spindles of human cells remains unknown. Here I show, by
using combined depletion and inactivation assays to explore redundancy between multiple
targets together with CRISPR technology, that PRC1-dependent motor KIF4A/kinesin-4, together
with EG5/kinesin-5 motor is essential for spindle elongation in human cells. Depletion of KIF4A,
or its partner PRC1, together with EG5 inhibition, completely arrests cell division in early
anaphase while depletion of other PRC1-interacting partners had minor effect on anaphase
progression. On the other hand, decreased stability of midzone microtubules measured by
photoactivation assay or their structural changes studied by expansion microscopy cannot explain
the observed anaphase arrest. Conclusively, my work demonstrates that two independent sliding
modules power sliding mechanism that drives anaphase spindle elongation in human cells thus
resolving longstanding question in the field and bridging the gap between extensive in vitro work
on sliding motors and their concrete actions within the cell.

Abstract (croatian)

Uspješna stanična dioba rezultat je točnog razdvajanja genetičkog materijala tijekom anafaze u
dvije stanice kćeri. Iako su sile koje su potrebne kako bi se kromosomi razdvojili u ljudskim
stanicama povezane s klizanjem premošćujućih antiparalelnih mikrotubula i poznati su mnogi
motorni proteini koji mogu klizati antiparalelne microtubule u sustavima in vitro, molekularni
mehanizam klizanja mikrotubula u živim stanicama tijekom anafaze još je uvijek nepoznat.
Koristeći se kombiniranim utišavanjem i inhibicijom mnogih proteina zajedno s CRISPR
tehnologijom u svrhu otkrivanja mogućega nadomjesnog mehanizma u anafazi ljudskih stanica, u
ovom radu pokazao sam kako PRC1-ovisni motor KIF4A/kinezin-4 djeluje zajedno s motornim
proteinom EG5/kinezin-5 te kako su oba ključna za produljivanje diobenog vretena u ljudskim
stanicama. Eksperimenti fotoaktivacije tubulina kombinirani s ekspanzijskom mikroskopijom
pokazuju kako perturbacija oba proteina značajno utječe na klizanje mikrotubula, dok njihova
smanjena stabilnost ne može objasniti opisano zaustavljanje anafaze. Zaključno, elongacija
diobenog vretena u anafazi ljudskih stanica rezultat je klizanja premošćujućih mikrotubula koje
pokreću dva motorna proteina.

• Keywords: mitosis; anaphase; motor proteins; spindle elongation; bridging fibers
• Keywords (english): mitoza; anafaza; motorni proteini; premošćujući mikrotubuli; diobeno vreteno
• Language: english
• URN:NBN: urn:nbn:hr:217:732521
• Promotion: 2021
• Study programme: Title: Biology; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija (doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-01-29 12:53:43