title: Investigation of the evolution of intermediate filament-forming proteins in Eukaryotes
title: Untersuchung der Evolution von Intermediärfilament-bildenden Proteinen in Eukaryoten
titleType: AlternativeTitle
Creators: name: Lometto, Stefano
Publisher: name: Philipps-Universität Marburg
Year: 2024
Subjects: subject: Evolutionsbiologie
subject: Biochemistry
subject: Chemistry &amp; allied sciences
subject: Chemie
subject: Biochemie
subject: Evolutionary Biology
subject: FOS: Biological sciences

description: The remarkable increased complexity of eukaryotes' cytoskeleton represents one of the major 
transitions in life. Inherently intertwined with eukaryogenesis itself, molecular innovations that 
emerged during cytoskeleton evolution are pivotal for understanding Eukaryotes` evolutionary history. 
Despite distant homologs for two major cytoskeletal components, actin, and tubulin, identified in 
prokaryotes, no clear homolog for intermediate filaments has been found. Intermediate filaments 
represent crucial components in the eukaryotic cytoskeleton, being regarded as one of the major 
molecular innovations that evolved in this kingdom. Long evolutionary distances, poor sequence 
conservation, and many other hurdles render tracing the evolution of such proteins a daunting task. 
One possible route to circumvent such limitations is to investigate single-cell eukaryotes, namely 
protists, whose cell biology is expected to entail many overlooked fiber-forming, intermediate
filament-like proteins. 
The filament-forming mitochondrial citrate synthase described in the series of works that prompted 
this study was an ideal candidate for studying the emergence of intermediate filament proteins over a 
known evolutionary interval. The study of such multifunctional proteins is important for several 
reasons. As part of a well-conserved protein family, the mitochondrial citrate synthase (mCS) makes it 
tractable to study transitions over known and relatively short evolutionary intervals in a phylogenetic 
tree. Furthermore, mCS evolution of fiber formation in protists could be the product of convergent 
evolution, giving rise to novel IF structures and potentially elucidating mechanisms for filament 
formation in non-metazoan lineages. Another possible role of fiber formation is linked to metabolism 
regulation. Therefore, studying the evolutionary interval along which such behavior emerged would 
allow us to pinpoint the evolutionary forces acting on enzyme fiber formation and their possible 
recruitment for structural functions. 
Here, we characterized the properties of in vitro fiber formation of the recombinant mitochondrial 
citrate synthase of Tetrahyemna thermophila. We provide orthogonal evidence for this behavior to be 
a buffer-driven artifact caused by a specific chemical moiety that is most likely absent in the native 
biological context. We also show that buffer-induced aggregation is a feature common to eukaryotic 
mitochondrial citrate synthase but absent in bacterial homologs. 
Using an in-vivo approach, we investigated the protein's localization using Strep and GFP-fibre
deficient tagged mutants. We show that the protein colocalizes with mitochondria and does not form 
clear foci during starvation, which indicates the absence of metabolic regulation via filamentation. 
Furthermore, we monitored the protein's localization during conjugation, reporting the absence of 
any extramitochondrial structure. 
Finally, we show how MES-induced ordered fibers emerged in the last common ancestor of 
Hymenostomatida while other species and ancestral proteins form amorphous aggregates. We 
thereby conclude that ordered fibers likely are a product of neutral processes.