Optimization and application of Trim-Away for studying a liquid-like spindle domain in mammalian oocytes
von Chun So
Datum der mündl. Prüfung:2019-08-19
Erschienen:2019-10-01
Betreuer:Dr. Melina Schuh
Gutachter:Dr. Melina Schuh
Gutachter:Prof. Dr. Stefan Jakobs
Gutachter:Prof. Dr. Jörg Enderlein
Gutachter:Prof. Dr. Rüdiger Behr
Gutachter:Dr. Roland Dosch
Gutachter:Prof. Dr. Markus Zweckstetter
Dateien
Name:Final thesis (no CV)_20190925.pdf
Size:9.76Mb
Format:PDF
Zusammenfassung
Englisch
In this thesis, I reported a membraneless structure that permeated a major region of the female meiotic spindle and formed droplet-like protrusions around the spindle poles. Proteins within this previously unknown structure were highly dynamic and could redistribute rapidly throughout the entire spindle region. I found that this unusual structure behaves similar to a liquid and forms by phase separation and hence, termed it the liquid-like meiotic spindle domain (LISD). Interestingly, the LISD was not only present in the spindle of mouse oocytes, but also cow, pig and sheep oocytes and is thus widely conserved among mammals. Similar structures were not observed in wildtype or centrosome-depleted somatic cells, suggesting that the LISD is likely exclusive to the specialized spindle in oocytes. To identify the key proteins driving LISD assembly, I further optimized Trim-Away, the technique for acute depletion of endogenous proteins in mammalian cells. By combining in vitro and in vivo assays, I identified Aurora A kinase and two LISD proteins, transforming acidic coiled-coil-containing protein 3 (TACC3) and clathrin heavy chain 17 (CHC17), as the proteins essential for LISD assembly. Disruption of the LISD via different means released microtubule regulatory factors that reside within this domain into the cytosol and caused severe spindle defects. Spindles were half of their original size and failed to segregate chromosomes properly. Microtubule growth rates were reduced, and their overall turnover was increased. Both kinetochore fibers as well as interpolar microtubules were strongly depleted. A TACC3 mutant that loses its ability to phase-separate failed to rescue the substantial loss of microtubules in TACC3-depleted oocytes. Together, these data suggest that the LISD is necessary for the efficient assembly of stable acentrosomal spindle in mammalian oocytes.
Keywords: LISD; Liquid-like meiotic spindle domain; Trim-Away; Mammalian oocytes; Meiotic spindle; Meiosis; Phase separation; Microtubule