Regulation of microtubule sliding and its effect on mitotic spindle length in human retinal cells RPE-1

doctoral thesis

• Title (english): Regulacija klizanja mikrotubula te utjecaj na duljinu diobenog vretena u stanicama retina čovjeka RPE-1
• Author: Ivana Ponjavić
• Mentor: Iva Marija Tolić (mentor)
• Committee member: Andreja Ambriović Ristov (predsjednik povjerenstva)
• Committee member: Inga Urlić (član povjerenstva)
• Committee member: Marin Barišić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2023-09-22, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology
• Universal decimal classification (UDC): 576 - Cellular and subcellular biology. Cytology

Abstract

Poleward flux of the microtubules in the mitotic spindle is a complex process driven by multiple
motor proteins driving sliding of overlapping microtubules (MTs). However, coordination of
the sliding in the midzone with the MT depolymerization at the pole remains unknown. Here I
show, by using speckle microscopy to measure flux of individual MTs, that after kinesin-5
(EG5) inhibition while the spindle is collapsing, kinetochore fiber (k-fiber) movement away
from the midplane is not impaired, however, bridging fiber movement from the midplaine is
30% slower when compared to the untreated metaphase spindles suggesting that sliding of
overlapping MTs is impaired. At the same time poleward flux is similar as in untreated
metaphase spindles. After dynein inhibition with Ciliobrevin D, the spindle retains its length
but the bridging fiber overlap length is reduced and its connection with k-fibers is perturbed.
Furthermore, k-fiber flux is significantly reduced, suggesting that dynein acts as a crosslinker
of bridging and k-fibers. When EG5 is inhibited together with kinesin family member 4A
(KIF4A), bridging fiber and k-fiber movement away from the midplane is significantly reduced,
indicating that these two motors act together to slide overlapping MTs. Taken together, our
results demonstrate that EG5 alone has a role in the sliding of antiparallel MTs and that in
metaphase EG5 and KIF4A in the overlaps of the bridging fibers generate sliding forces which
are transmitted to the associated k-fibers through dynein. We propose that the spindle consists
of two mechanically distinct parts, the midzone part which remains functional during collapse,
and the pole-proximal part of the spindle which is shortened during spindle collapse.

Abstract (english)

Tok mikrotubula prema polu u mitotskom diobenom vretenu složen je proces kojeg pokreću
višestruki motorni proteini koji pokreću klizanje preklapajućih mikrotubula. Međutim,
koordinacija klizanja u središnjoj zoni vretena s depolimerizacijom mikrotubula na polu ostaje
nepoznata. Ovdje pokazujem, korištenjem speckle mikroskopije za mjerenje toka pojedinačnih
mikrotubula, da nakon inhibicije kinezina-5 (EG5) motornog proteina dok vreteno kolabira,
kretanje kinetohornih vlakana (k-vlakana) od središnje ravnine nije poremećeno, međutim,
kretanje premoščujućih vlakana je 30% sporije u usporedbi s netretiranim metafaznim
vretenima što sugerira da je klizanje preklapajućih mikrotubula usporeno. U isto vrijeme tok
mikrotubula prema polu sličan je kao i u netretiranim metafaznim vretenima. Nakon inhibicije
dineina Ciliobrevinom D, vreteno zadržava svoju duljinu, ali se duljina preklapanja premosnog
vlakna smanjuje i njegova veza s k-vlaknima je poremećena. Nadalje, protok k-vlakana
značajno je smanjen, što sugerira da dinein djeluje kao poprečni povezivač premošćujućih i kvlakana. Kada je EG5 inhibiran zajedno s kinezinom-4A (KIF4A), kretanje premosnih vlakana
i k-vlakana od središnje ravnine je značajno smanjeno, što ukazuje da ova dva motora djeluju
zajedno kako bi klizali mikrotubule koji se preklapaju. Zajedno, moji rezultati pokazuju da EG5
sam ima ulogu u klizanju antiparalelnih mikrotubula te da u metafazi EG5 i KIF4A u
preklapanjima premosnih vlakana stvaraju sile klizanja koje se prenose na pridružena k-vlakna
preko dineina. Predlažem da se vreteno sastoji od dva mehanički različita dijela, dijela srednje
zone koji ostaje funkcionalan tijekom kolapsa i polu-proksimalnog dijela vretena koji se
skraćuje tijekom kolapsa vretena.

• Keywords: microscopy; mitosis; poleward flux; motor proteins; bridging fibers
• Keywords (english): mikroskopija; mitoza; motorni proteini; diobeno vreteno; tok mikrotubula
• Language: english
• URN:NBN: urn:nbn:hr:217:600314
• Promotion: 2023
• Study programme: Title: Doctoral study; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti u području prirodnih znanosti (doktor/doktorica znanosti u području prirodnih znanosti)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-11-15 13:08:19