Divisive Structures

Fredrik Hurtig Divisive structures

Two billions years of biofilament evolution

Divisive structures Fredrik Hurtig

Fredrik Hurtig Born in 1988 in Stockholm, Sweden. MSc in Microbiology from Stockholm University. Started his PhD studies at Stockholm University in 2014.

ISBN 978-91-7911-058-1

Department of Molecular Biosciences, The Wenner-Gren Institute

Doctoral Thesis in Molecular Bioscience at Stockholm University, Sweden 2020

Divisive structures Two billions years of biofilament evolution Fredrik Hurtig Academic dissertation for the Degree of Doctor of Philosophy in Molecular Bioscience at Stockholm University to be publicly defended on Friday 12 June 2020 at 10.00 in Sal P216, NPQ-huset, Svante Arrhenius väg 20 A, digitally via Zoom: Zoom Meeting ID: 638-3943-9290, https://stockholmuniversity.zoom.us/j/63839439290

Abstract Our understanding of the functional and regulatory complexity that existed in the eukaryotic progenitor is poor, and investigations have been hindered by our nebulous understanding of where eukaryotes stem from. Recently discovered archaeal lineages with hitherto unseen homology to eukaryotic systems suggest archaea can further our understanding of the eukaryotic cell’s ancestry. However, much of archaeal biology remains largely unexplored. Two eukaryotic systems with archaeal homologues, namely the actin and ESCRT-III protein filament systems, are essential for diverse processes in eukaryotic biology. In this thesis, we show that an archaeal homologue of ESCRT-III divides the cell under proteasomal regulation, a regulatory mechanism central to eukaryotic cell cycle regulation. Additionally, we show how predicted putative profilin and gelsolin homologues regulate the postulated proto-cytoskeleton of Asgard archaea. In investigating the function and regulation of these archaeal systems we demonstrate compelling parallels between archaeal and eukaryotic regulatory strategies which stresses the close evolutionary relationship that exists between these two domains.

Keywords: Evolution, Archaea, Eukaryogenesis, ESCRT-III, Actin, Profilin, Gelsolin, Asgard, TACK, Sulfolobus, Cell division, Cytokinesis, Cytoskeleton, Heimdallarchaeota, Actin-binding protein.

Stockholm 2020 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-180989

ISBN 978-91-7911-058-1 ISBN 978-91-7911-059-8

Department of Molecular Biosciences, The Wenner-Gren Institute

Stockholm University, 106 91 Stockholm

DIVISIVE STRUCTURES

Fredrik Hurtig

Divisive structures

Two billions years of biofilament evolution

Fredrik Hurtig ©Fredrik Hurtig, Stockholm University 2020

ISBN print 978-91-7911-058-1 ISBN PDF 978-91-7911-059-8

Printed in Sweden by Universitetsservice US-AB, Stockholm 2020 “What I cannot create, I do not understand.”

Richard P. Feynman

Thesis abstract

Our understanding of the functional and regulatory complexity that existed in the eukaryotic progenitor is poor, and investigations have been hindered by our nebulous understanding of where eukaryotes stem from. Recently discovered archaeal lineages with hitherto unseen homology to eukaryotic systems suggest archaea can further our understanding of the eukaryotic cell’s ancestry. However, much of archaeal biology remains largely unexplored. Two eukaryotic systems with archaeal homologues, namely the actin and ESCRT-III protein filament systems, are essential for diverse processes in eukaryotic biology. In this thesis, we show that an archaeal homologue of ESCRT-III divides the cell under proteasomal regulation, a regulatory mechanism central to eukaryotic cell cycle regulation. Additionally, we show how predicted putative profilin and gelsolin homologues regulate the postulated proto- cytoskeleton of Asgard archaea. In investigating the function and regulation of these archaeal systems we demonstrate compelling parallels between archaeal and eukaryotic regulatory strategies which stresses the close evolutionary relationship that exists between these two domains.

Keywords Evolution, Archaea, Eukaryogenesis, ESCRT-III, Actin, Profilin, Gelsolin, Asgard, TACK, Sulfolobus, Cell division, Cytokinesis, Cytoskeleton, Heimdallarchaeo- ta, Actin-binding protein

I Populärvetenskaplig Sammanfattning

Alla levande organismer tillhör en av livets tre domäner. Bakterier, Eukaryoter - där djur, växter och svampar ingår bland annat, och Arkéer. Denna tredje grupp upp- täcktes först på 70-talet. På ytan liknar de bakterier - små och encelliga, men när vi gräver djupare upptäcker vi att de faktiskt är närmare besläktade med oss, euka- ryoterna. Vissa av dessa likheter går att hitta i de molekylära maskiner som dessa celler kan producera. Maskinerna är gjorda av proteiner, som ofta kallas för livets byggstenar. Celler använder proteiner för många och varierade funktioner såsom verktyg, motorer och vapen, men också som strukturella komponenter. Vissa av dessa strukturella komponenter hittas både i arkéer och eukaryoter, vilken antyder att de fanns innan de två domänerna delade på sig. Denna avhandling undersöker just två av dessa uråldriga system och försöker dra slutsatser om systemen som troligvis fanns i vår gemensamma förfader. ESCRT-III används i eukaryoter för att manipulera det membran som omsluter alla celler. Hos djur är detta system speciellt viktigt då det också används när en cell ska dela sig till två nya celler. Tidigare studier har visat hur ESCRT-III också existerar hos vissa arkéer där det används just i celldelning. Vi har kunnat visa att ESCRT-III i vissa arkéer fungerar genom att en statisk ring byggs upp där cellen kommer dela sig, bestående av ett protein som kallas CdvB. På detta statiska element byggs se- dan ett kontraktilt element upp, bestående av CdvB1 och CdvB2, två andra delar av ESCRT-III i arkéer. Först när CdvB återvinns av proteasomen, en molekylär avfalls- kvarn, aktiveras det kontraktila elementet bestående av CdvB1 och CdvB2 och delar cellen. Detta har visat sig intressant då reglering via proteasomen är en viktig mek- anism i eukaryoter, vilket tyder på att proteasomal reglering kan vara en uråldrig form av kontroll. Aktin är en del av det så kallade cell-skelettet och det är två proteiner som an- vänds för att reglera detta skelett som vi har studerat. Det första av dessa proteiner, profilin, används för att reglera hur mycket av de subenheter som skelettet består av är tillgängliga, men också hur snabbt dessa subenheter kan re-integreras efter att de använts. En studie från 2017 identifierade ett antal gener i de så kallade

II Asgard-arkéerna som förutspåddes vara profilin-homologer, men den biologiska funktionen av dessa proteiner har dock inte studerats än. Det är just den biologiska funktionen av en av dessa förutspådda profiliner (heimProfilin) som vi har under- sökt. I linje med profilins funktion i eukaryoter visar vi att heimProfilin kan stoppa polymerisering av aktin. Vi kan utöver detta visa på att heimProfilin kan interagera med både PIP2, en viktig signalmolekyl, och polyprolin-sekvenser. I eukaryoter inte- ragerar profilin med en mängd andra regulatoriska proteiner just genom att binda till deras polyprolin-sekvenser. Denna interaktion kan tyda på att profilin-polyprolin mekanismen redan hade utvecklas i eukaryoters och arkéers gemensamma anfader. Likt profilin är gelsolin ett protein som reglerar aktin i cell-skelettet. Till skillnad från profilin binder gelsolin inte till subenheterna av aktin främst, utan till de långa kedjor som aktin bildar. Gelsolins roll är att kapa dessa kedjor och sen binda till den växande änden. Genom denna bindning, kallad ”capping”, förhindras filament från att växa tillbaka. Våra studier har kunnat visa hur ett förutspått gelsolin från As- gard-arkéerna (heimGelsolin) effektivt kan montera ner aktin-kedjor och att denna reaktion är beroende på närvaron av kalciumjoner, något som är känt från eukaryoter. De funktionerna som vi har kunnat påvisa i heimProfilin och heimGelsolin re- presenterar basen för att komplext reglerat cell-skelett i Asgard arkéerna. Sammanlagt har vi demonstrerat spännande regulatoriska paralleller i flera system mellan arkéer och eukaryoter. Studerandet av arkéer med nära relation till eukaryoter är ett lovande mål med potential för lärdom relaterat till både hur systemen regleras och fungerar, men även i jämförelse i jakten på kunskap om vår gemensamma anfader.

III Aims of thesis

Main hypothesis. | Archaea and eukaryotes share a common ancestor whose functional and regulatory complexity can be investigated through comparative approaches of descendant organisms.

To test this hypothesis, this thesis was guided by the following aims: I. To investigate the mechanisms underlying cell cycle regulation in S. acidocaldarius II. To investigate the functional diversity of ESCRT-III homologues that govern cell division in S. acidocaldarius III. To study the regulatory mechanism of putative profilin homologues in the Asgard superphylum IV. To investigate the regulatory mechanism of putative gelsolin homology domain proteins in the Asgard superphylum

IV List of papers

Paper I. | Risa G.T.†, Hurtig F.†, Bray S., Hafner A. E., Harker-Kirschneck L., Faull P., Davis C., Papatziamou D., Mutavchiev D. R., Fan C., Meneguello L., Arashiro Pulschen A., Dey G., Culley S., Kilkenny M., Pellegrini L., de Bruin R. A. M., Henriques R., Snijders A. P., Šarić A., Lindås A.-C., Robinson N., Baum B. The proteasome is required to trigger ESCRT-III-mediated cell division in an archaeon. Science (Manuscript under consideration after second revision) †These authors contributed equally.

Paper II. | Survery S., Hurtig F., Haq S. R., Lindås A.-C., Celestine C. Heimdallarchaeota encodes profilin with eukaryotic-like actin regulation and polyproline binding. Manuscript (Submitted)

Paper III. | Survery S., Hurtig F., Haq S. R., Lindås A.-C., Celestine C. Heimdallarchaeota gelsolin is a multi-domain protein with calcium controlled actin regulation. Manuscript

Related publication (Not included in this thesis)

Paper IV. | Laine R. F., Tosheva K. L., Gustafsson N., Gray R. D. M., Almada P., Albrecht D., Risa G. T., Hurtig F., Lindås A.-C., Baum B., Mercer J., Leterrier C., Pereira P. M., Culley S., Henriques R. NanoJ: a high-perfomance open-source super- resolution microscopy toolbox. Journal of Physics D: Applied Physics, 2019, 52.16: 163001.

Paper V. | Haq S. R., Survery S., Hurtig F., Lindås A.-C., Celestine C. NMR backbone assignment and dynamics of Profilin from Heimdallarchaeota. Scientific Reports (Manuscript submitted)

V Contents

Thesis abstract I Populärvetenskaplig sammanfattning II Aims of thesis IV List of papers V List of abbreviations VII

CHAPTER 1. Archaea & the first eukaryote 2.

1.1 | Archaea, the third domain of life 2. 1.2 | The TACK superphylum 3. 1.3 | The Asgard superphylum 4.

CHAPTER 2. The ESCRT & Cdv machineries 6.

2.1 | The varied functions of ESCRT 6. 2.2 | The ESCRT-III complex 6. 2.3 | The Cdv cell division machinery 8.

CHAPTER 3. The actin family & its keepers 11.

3.1 | Once upon an Actin 11. 3.2 | MreB and Crenactin, the lost prokaryotic relatives 14. 3.3 | Actin-binding proteins - Profilin 16. 3.3 | Actin-binding proteins - Gelsolin 19.

PRESENT INVESTIGATIONS 21.

FUTURE DIRECTIONS 30.

ACKNOWLEDGEMENTS 32.

REFERENCES 34.

VI AAA+ ATPase ATPases associated with diverse cellular activities ABP Actin-binding protein ACC Actin critical concentration ADP Adenosine diphosphate ASP Archaeal signature protein ATP Adenosine triphosphate Ca2+ Calcium ion CCT Chaperonin containing TCP-1 Cdk Cyclin-dependent kinase Cdv Cell division CHMP Charged multivesicular body protein DAPI 4′,6-diamidino-2-phenylindole DNA deoxyribonucleic acid Edu 5-Ethynyl-2’-deoxyuridine EM Electron microscopy ER Endoplasmic reticulum ESCRT Endosomal sorting complexes required for transport ESP Eukaryotic signature protein GH Gelsolin homology domain GTP Guanine triphosphate HGT Horizontal gene transfer HIV Human immunodeficiency virus Hsp70 70 kDa heat-shock protein ILV Intralumenal vesicle

IP3 Inositol trisphosphate ITC Isothermal titration calorimetry LAECA Last archaeo-eukaryotic common ancestor LECA Last eukaryotic common ancestor Mg2+ Magnesium ion MIM MIT interacting motif MIT Microtubule interacting and trafficking MVB Multivesicular body NMR Nuclear magnetic resonance PIP Phosphoinositides

PIP2 Phosphatidylinositol 4,5-bisphosphate RMSD Root mean square distance RNA Ribonucleic acid rRNA Ribosomal ribonucleic acid rRNA Ribosomal RNA STK Thymidine kinase TSG101 Tumor susceptibility gene 101 wH Winged helix Vps Vacuolar protein involved in sorting Vta1 Vesicle Trafficking 1

VII

Preface. Protein-based filament systems exist in most, if not all, living cells. These biological filaments are characterized by their ability to polymerize monomeric subunits into large complexes, drastically expanding the spatial dimensions over which they can exert influence. As a result, protein filaments are involved in major cellular processes and transitions such as motility, morphology, DNA segregation and cell division. Many of these protein filaments have ancient origins, and can be found in all domains of life. Arguably, the most well-studied of these filaments belong to the eukaryotic cytoskeleton which has been studied for over a 100 years, beginning with the discovery of actin. It was only in the past few decades that an analogous prokaryotic cytoskeleton was conceptualized following the discovery of the cell division protein, FtsZ. While functionally analogous to the role of eukaryotic actin during cell division, FtsZ was found to be a distant relative of tubulin, another member of the eukaryotic cytoskeleton. Since those early discoveries, a host of different filamentous proteins have been discovered both in prokaryotes and eukaryotes. These include several homologues of actin, notably MreB and Crenactin, and novel protein filaments involved in every facet of normal cell function. In the wake of these ongoing discoveries a venerable question has re-emerged: How much functional and regulatory complexity existed in the primordial eukaryotic cell? The question itself poses a challenge - how does one study something that by definition doesn’t exist anymore? While an exhaustive investigation into the functional complexity of the first eukaryotic cell is outside the scope of this thesis, this work endeavours to bring us closer to answering these fundamental biological questions. This thesis investigates the regulation and mechanics of two filament systems, ESCRT-III and actin, from the archaeal TACK and Asgard superphyla. These superphyla contain some of the closest known eukaryotic relatives, and by understanding the functional complexity of filament systems in these organisms, we aim to better understand the complexity that was present in the original eukaryotic cell.

- 1 - CHAPTER 1. | Archaea & the first eukaryote

CHAPTER 1.1 | Archaea, the third domain of life. During the late 1970s, all living organism could be divided into two distinct categories: eukaryotes and prokaryotes. The eukaryotes contained large intracellular structures such as a nucleus, mitochondria and chloroplasts while bacteria, the only known prokaryotic group at the time, did not1. These features defined the classification of organisms. However, the prevailing method of classification was inherently flawed as it was based on morphological and phenotypical differences rather than molecular characteristics. As bacteria lack a large nucleus and organelles used in this method of classification, microbes were largely absent from taxonomy during this period2. At this time, much was already known about the molecular mechanisms underpinning cellular function but the use of molecular information in classifying organisms was still in its infancy and mostly based on protein sequences3,4. It was during this time that Carl Woese and George Fox were working on developing a new way to classify organisms. In a seminal 1977 article, Woese proposed that ribosomal RNA (rRNA) sequences could be used as a universal molecular marker for classifying organisms5. Investigating this hypothesis, Woese and Fox created a library of rRNA sequences from eukaryotic and prokaryotic organisms and in comparing them were able to produce a molecular taxonomy which incorporated both prokaryotic and eukaryotic sequences5. It was this rRNA sequence analysis that revealed a grouping that eventually came to be recognized as the third domain of life, Archaea. The sequences that formed this new group were from a scarcely studied group of prokaryotes known as methanogens. These organisms were able to produce methane from carbon dioxide in anaerobic conditions. Their unique metabolism was considered ancient as it aligned with what was known about the conditions on primordial earth. In reference to phenotype, the group was originally termed “Ar- chaebacteria”, stemming from the Greek for “ancient” and “rod”. This term was soon replaced by the term “Archaea” on the recommendation of Woese and others as the original name incorrectly suggested a strong relation to bacteria6. The “an-

- 2 - cient” part of the name was kept to emphasize its perceived relationship with its sister group, the eukaryotes6.

Chapter 1.2 | The TACK superphylum. The TACK superphylum of archaea contains organisms living in some of the most extreme environments ever described. The name TACK refers to the component phyla of this superphylum: the Thaumarchae- ota, Aigararchaeota, Crenarchaeota and Korarchaeota. The Thaumarchaeota consist of mesophilic archaea with various roles in environmental element recycling. Recent years have highlighted the importance of these organisms in the global carbon- and nitrogen-cycle7. Notably, Thaumarchaeota have been estimated to represent 20% of all prokaryotic cells in oceanic environments8. Although initially identified in marine environments, we now know that they are present in terrestrial environments as well9. The Aigarchaeota is one of the least characterized archaeal phyla with most members having been identified in a variety of geothermal habitats. They are only known through 16S rRNA sequencing and remain uncultured to this day. It has been suggested that the group is a deeply rooted branch of Thaumarchaeota rather than a separate phylum10. Similarly, the Korarchaeota remain uncultured and mostly uncharacterized. However, the group is widely thought to be thermophilic and exists in both terrestrial and marine habitats. Inter- estingly, cells observed using electron microscopy (EM) in an enrichment culture revealed ultra-thin and needle-like cells measuring 0.2 µm in width and up to 100 µm in length11. The Crenarchaeota consist mainly of hyperthermophilic and acidophilic organisms. Members of this phylum constitute some of the most well-studied archaea. In particular, members of the genus Sulfolobus are used as model organisms for hy- perthermophiles. The cell cycle of Sulfolobus acidocaldarius has been extensively studied and have an ordered cell cycle consisting of distinct G1 and G2 gap phases separated by rounds of DNA replication reminiscent of many eukaryotes12. The creation of genetic manipulation and synchronization techniques has positioned this organism as a potent model to study archaeal cell cycle regulation13–16. Until recently, the Crenarchaeota represented some of the closest known eukaryotic

- 3 - relatives, making them a powerful organism in understanding eukaryogenesis. Contemporarily, S. acidocaldarius is arguably the closest experimentally tractable living relative of eukaryotes and the Asgard superphylum, while not experimentally tractable, comprise the closest eukaryotic relatives that has been sequenced17.

Chapter 1.3 | The Asgard superphylum. A secondary effect of Woese’s work was highlighting a long-standing question in biology: What was the nature of the first eukaryotic cell5,18? While the origin of the mitochondrion is generally accepted as deriving from alphaproteobacteria19, and the origin of the chloroplast a cyanobacterial ancestor20, the provenance of the host has remained elusive. The incipient taxonomy proposed by Woese’s study propelled archaea to the forefront of potential proto-eukaryotic hosts. A cluster of recent discoveries in the wake of the next- generation sequencing revolution describing a new archaeal superphylum, the Asgard archaea, and might finally provide concrete evidence of the origin and complexity of the eukaryotic cell, and the nature of the last archaeo-eukaryotic common ancestor (LAECA)17,21. The pioneering study was an environmental meta- genomic sequencing investigation by Spang et al in 2015, that identified a novel archaeon with a hitherto unseen similarity to eukaryotes21. The sample was col- lected north of a deep sea hydrothermal vent area known as Loki’s Castle and the candidate phyla was named Lokiarchaeota in reference. Subsequent work described a number of sister groups to Lokiarchaeota: Odinarchaeota, Heimdallar- chaeota and Thorarchaeota. Collectively, these candidate phyla have come to be known as the Asgard superphyla. Analysis of 16S and 23S rRNA, as well as a set of ribosomal proteins conserved between archaea and eukaryotes have been able to robustly show that the Asgard superphyla formed a monophyletic group with high support. More importantly, the eukarya formed a monophyletic group alongside, and potentially nested inside, the Asgard archaea (Fig. 1)17. While originally contro- versial, consensus has moved in support of the Asgard archaea22–24. In addition to rRNA and ribosomal protein homology, comparative genomics indicated that all of the assembled Asgard superphylum genomes include an unusually high number of eukaryotic signature proteins (ESPs), many of which had previously never been

- 4 - detected outside of eukaryotic organisms17. The signature protein terminology was originally used to denote proteins that were wide-spread in archaea (archaeal signature proteins, ASPs), however this terminology was later adapted for eukaryotes as well25,26. Signature proteins are generally defined as being extensively distribut- ed within their domain, while being fully absent from all other domains. The Asgard archaea collectively contain homologues for a wide range of proteins including but not limited to: Actin and tubulin homologues, profilin- and gelsolin-domain proteins (actin-binding proteins which alter the dynamics and structure of the actin filament), a large family of small GTPases (proteins which are involved in various regulatory roles in eukaryotes including membrane remodelling, signal transduc- tion, transport and trafficking), TRAPP domain proteins (in eukaryotes these form a multi-subunit vesicle-anchoring complex involved in trafficking), Sec23/24 (essential components in the COPII complex which transports protein cargo from the ER to the Golgi). Furthermore, the Asgard archaea contain proteins with homology to members of the ESCRT-I, ESCRT-II and ESCRT-III complexes. Interestingly, protein homologues of ESCRT-I and ESCRT-II have previously not been detected outside of eukaryotes. In contrast, ESCRT-III has been identified and characterized in several members of the TACK superphylum of archaea. The cell division system of the hyperthermophilic model organism Sulfolobus acidocaldarius, along with many other archaea, relies on a system homologous to the eukaryotic ESCRT-III complex.

Figure 1. Maximum-likelihood analysis of concatenated large and small rRNA gene sequences showing strong support for a phylogenetic association between eukaryotes and Asgard archaea. Re- printed with permission from Zaremba- Niedzwiedzka et al (2017).

- 5 - CHAPTER 2. | The ESCRT & Cdv machineries

CHAPTER 2.1 | The varied functions of ESCRT. The ESCRT machinery was originally discovered in Saccharomyces cerevisiae in the early 90s27. Genetic screens revealed a number of mutant phenotypes that prevented the sorting of transmembrane proteins into intraluminal vesicles (ILVs) which bud into the lumen of endosomal vesicles28,29. Early studies characterized a set of complexes which came to be known as ESCRT (Endosomal Sorting Complexes Required for Transport). It was discovered that the ESCRT-I, ESCRT-II and ESCRT-0 complexes were able to bind transmembrane proteins with cytosolic ubiquitin modifications and that this was essential for subsequent degradation in lysosomal compartments30–33. While it was clear that ILVs were central to this process, the creation of the ILVs in the first place was still poorly understood. Some years later it was discovered in yeast that purified components of the ESCRT-III complex were enough to catalyse the scission reaction in synthetic liposomes, a reaction functionally analogous to ILV for- mation34. A subsequent study using the same experimental system demonstrated that an ESCRT-I and ESCRT-II supercomplex was able to generate the type of membrane buds required for scission by ESCRT-III35. With this, a general model of ESCRT function was proposed. ESCRT-0, -I and -II recruits and concentrates cargo into small buds which can then be pinched off by ESCRT-III.

CHAPTER 2.2 | The ESCRT-III complex. The ESCRT-III complex is comprised of a number of homologous subunits (7 in S. cerevisiae and 10 in H. sapiens) which are all predicted to share a three-dimensional structure. However, structure determination of the different subunits has proven difficult. Several ESCRT-III domains and subunits have been determined through a variety of methods, however, the structure of the larger complex has remained elusive36–39. Critical to the difficulties in characterizing ESCRT-III is that the subunits do not form a stable protein complex. Rather, the proteins have two states, which exist in equilibrium: 1) as soluble cytoplasmic monomers, the result of a C-terminal auto-inhibitory α-helix36,40,41, or 2) associated with a membrane where they polymerize into an ESCRT-III filament upon release from auto-inhibition via an amphipathic N-terminal α-helix knows as

- 6 - an ANCHR motif, which is inserted into the membrane42,42. The result of this is a highly dynamic protein complex whose study is further complicated by the fact that several of the ESCRT-III proteins appears to act as modulators to the main filament protein. In S. cerevisiae, Vps20 (CHMP6 in H. sapiens) initiates assembly by nucle- ating Snf7 (CHMP4 in H. sapiens), the main filament protein43. Vps24 (CHMP3 in H. sapiens) and Vps2 (CHMP2 in H. sapiens) inhibit polymerization and recruit the associated protein Vps440,44,45. Additionally, there are three ESCRT-III accessory proteins: Vps60, Did2 and Ist1 which are thought to regulate the activity of associated proteins46,47. The C-terminus of ESCRT-III subunits contain a MIT interacting motif (MIM). Vps2, Vps24 and Did2 contain MIM1; Vps20, Snf7, Vps60 contain MIM2; Ist1 contains both48. These motifs are responsible for interactions with the ESCRT-III associated protein Vps4 via its MIT (Microtubule interacting and trafficking) domain. While Vps4 has historically been seen as a dodecamer formed by two closed hexameric rings, recent studies have revealed the active conformation of Vps4 to be an open hexameric ring. Additionally, each Vps4 subunit binds one molecule of the cofactor Vta149–51. The Vps4:Vta1 complex has been shown to disassemble ESCRT-III subunits in an ATP-dependent fashion, returning them to a soluble monomeric state39,48,52. The ESCRT-III complex is integral to a long list of cellular processes including nuclear envelope reformation and repair53,54, multivesicular body (MVB) biogenesis40 and autophagy55,56. Further, a diverse group of viruses including Ebola, HIV and Hepatitis C co-opt the ESCRT-III machinery to facilitate egress from the cell57. Inter- estingly, viral co-option of ESCRT-III appears to have evolved independently several times, reflecting the unique reverse topology of ESCRT-III-mediated membrane deformation57. An excellent review of the many and varied functions of ESCRT-III was recently published by Vietri et al (2019)58. The role ESCRT-III plays in cytokinesis in metazoans is of special relevance to this thesis. During the final stages of cytokinesis, the nascent daughter cells are connected by an intercellular bridge. Microtubules stemming from the mitotic spindle run through the bridge to a medial structure called the midbody. The midbody acts as a platform from which cytokinetic abscission is mediated in an ESCRT-III-dependent manner. ESCRT-III is recruit-

- 7 - ed to the midbody through two parallel processes: the ESCRT-I protein TSG101 enlists the ESCRT-II complex which then recruits CHMP6 (Vps20 in S. cerevisiae) and CHMP4B (Snf7 in S. cerevisiae). Simultaneously, the ESCRT-I associated protein ALIX (Bro1 in S. cerevisiae) nucleate CHMP4B directly through its Bro1 domain59–61. ESCRT-III filaments are established on both sides of the midbody where they un- dergo continuous remodelling in a process dependent on the Vps4:Vta1 complex (Fig. 2)62. How ESCRT-III subunits assemble and carry out their function still remains unclear. The prevailing model proposes that the sequential assembly of a stable ESCRT-III filament, composed mainly of Snf7, recruits Vps2/Vps24. These two late- arriving proteins have been seen forming rigid dome structures in vitro, and this structure is hypothesised to guide the membrane towards fission through interactions with the membrane37. Alternatively the binding of Vps2/Vps24 might induce sharper curvature of the assembled Snf7 filament, inducing fission63. However, neither model has been observed in vivo.

CHAPTER 2.3 | The Cdv cell division machinery. The ESCRT complexes are widely conserved within the eukaryotic domain with the exception of ESCRT-0 which is restricted to fungi and metazoans64. In recent years ESCRT-III orthologues have been described in archaea65,66. Chiefly studied in the Crenarchaeal phylum, the Cdv (Cell division) machinery is homologous to ESCRT-III67. Originally discovered, and

Figure 2. Model showing the dynamic assembly of ESCRT-III filaments, regulated by Vps4 during constriction. Reprinted with permission from Mierzwa et al (2017).

- 8 - extensively studied, in Sulfolobus acidocaldarius, the Cdv system has been implicated in cell division66,67 and membrane vesicle secretion68. The composition of the Cdv system is fairly well conserved within the crenarchaeota, although not ubiquitous, and consists of the cdvA, cdvB and cdvC genes organized in one operon. Initial studies found this operon to be expressed in a single transcriptional wave67, but subsequent studies have argued against this69. Furthermore, between two and four additional cdvB paralogues can be found in crenarchaeal genomes which encode for the core Cdv system. These paralogues are generally known as cdvB1, cdvB2 and cdvB3. CdvA has not been identified outside of the TACK superphylum and is customarily modelled as being the first protein to localize to the site of constriction due to its ability to bind lipid membranes. Asgard archaea generally seem to lack CdvA and instead carry subunits of the ESCRT-I and ESCRT-II complexes, conceiva- bly substituting for CdvA in recruiting CdvB to the membrane. Although many studies into this machinery have been conducted in S. acidocaldarius, others have used related TACK archaea; notably Sulfolobus solfataricus, Sulfolobus islandicus (a recent study proposed these two organisms be reclassified into a new genus called Saccharolobus70) and Metallosphaera sedula. Although closely related, there are indications that there are functional differences in how the Cdv system works in each of these organisms. For clarity, I will first describe what is currently known of this system in S. acidocaldarius before describing the state of the field in related organisms. The membrane of S. acidocaldarius’ is mainly comprised of membrane-spanning tetraether lipids, forming a rigid monolayer71. Experiments using recombinant CdvA revealed a strong interaction with tetraether liposomes purified from S. acidocaldarius. No such interaction has been detected for CdvB, which is the homologue of ESCRT-III. Additionally, CdvA is able to recruit CdvB to membranes through a C- terminal E3B domain which binds an N-terminal winged-helix domain (wH-domain) on CdvB. Interestingly, the recruitment of CdvA and CdvB to liposomes is enough to cause membrane deformation in vitro. Correspondingly, when overexpressed in vivo the CdvB wH-domain causes a three-fold increase of DNA-free cells, indicating a cell division defect69. In line with a role in cell division, immunofluorescent mi-

- 9 - croscopy on fixed cells showed CdvA and CdvB forming band-like structures between segregated chromosomes66,67. CdvA was also seen forming bands in unseg- regated cells, further indicating that CdvA precedes CdvB assembly69. Interestingly, negative staining of recombinant CdvA from M. sedula, a related TACK archaeon, revealed long filaments bound to DNA. Treatment with DNase leads to the dissolu- tion of the CdvA polymers. The authors suggested a predicted medial coiled-coil region in CdvA as a possible binding surface72. Whether or not this interaction has biological significance awaits further investigation. The AAA+ ATPase CdvC shares strong homology to eukaryotic Vps4 on both a sequence (approximately 35% identity versus Saccharomyces cerevisiae) and structural level (S. solfataricus root mean square deviation (RMSD) = 1.62 Å)73. The Vps4 associated protein Vta1 is absent from all known archaea and likely evolved later. Similarly to eukaryotic Vps4, CdvC is thought to form a catalytically active hexameric complex72,73. Overexpression of a catalytically inactive Walker-B mutant form of CdvC in S. solfataricus results in a large number of cells lacking DNA. Furthermore, this overexpression produced a population of giant cells, up to four times in size over wild-type, with intense DAPI staining, indicating elevated DNA content66. CdvC interacts with CdvB through a conserved MIM2 motif on CdvB which can interact with a MIT domain on CdvC66. The CdvB paralogues CdvB1 and CdvB2 lack the wH-domain, meaning they cannot interact with CdvA. They do however contain a truncated MIM2 motif and are able to interact with CdvC with marginal affinity (Kd > 100 µM) compared to CdvB (Kd ≈ 30 µM)66. CdvB3 lacks both the MIM2 and wH-domains completely and subsequently appears unable to interact with CdvC or CdvA. Little is currently known about the biological function of CdvB3. However, data indicates a role in native and virus-induced membrane budding. C-terminal truncations of CdvB, CdvB1 and CdvB2 MIM2 motifs in S. islandicus arrested constricting cells at various stages of cytokinesis which indicates that the truncated MIM2 motif is likely of biological significance. Particularly surprising is that CdvB2ΔC resulted in daughter cells connected by what appeared to be an intercellular bridge with a medial midbody (Fig. 3)74. This discovery poses some fascinating questions concerning the evolutionary origin of these structures. The same study also demonstrated that CdvB1 and

- 10 - Figure 3. Electron micrograph of S. islandicus cells expressing a truncated CdvB2ΔC showing a midbody-like phenotype. Reprinted with permission from Liu et al (2017).

CdvB2 are able to form band-like structures between segregating nucleoids, clearly implicating their involvement in the division machinery of these organisms. A transposon screen of essential genes identified the core Cdv operon along with CdvB2 as essential while cdvB1 and cdvB3 were ostensibly dispensable75. Taken as a whole, these studies have convincingly demonstrated that the Cdv division system and ESCRT-III complex not only have a common origin, but that driving cytokinesis could be the original function of ESCRT-III. As mentioned earlier, the Cdv system is wide-spread among TACK archaea but not ubiquitous. Notably, of the three main crenarchaeal orders (Sulfolobales, Thermoproteales and Desul- furcoccales), Cdv is widespread in the Sulfolobales and Desulfurcoccales. However, no such genes have been identified in Thermoproteales76. Instead this order is hypothesised to use a system based on crenactin, a close homologue of eukaryotic actin77. Additionally, close homologues to both the ESCRT-III complex and actin are widespread within the Asgard archaea17, making the Asgard group a compelling target for studying early eukaryotic cellular complexity.

CHAPTER 3. | The actin family & its keepers

CHAPTER 3.1 | Once upon an Actin. Actin is arguably one of the most well-studied proteins in biology. It is ubiquitous in eukaryotic cells where it is essential for diverse processes such as motility, cell division and muscle contraction. Although all eukaryotic organisms express actin, the number of paralogues can vary significantly. Budding and fission yeast have only one single variant while soybean (Glycine

- 11 - max) has 17 actin paralogues78. Most mammals express six isoforms that are classified as either muscle or non-muscle actins; although non-muscle actins can usually be found in small quantities in muscle tissue as well79,80. The two mammalian non- muscle actin isoforms differ only by four residues but are still both expressed near ubiquitously in non-muscle cells. Furthermore, there appears to be no functional diversity between the two isoforms. Rather, spatial organization in the cell causes emergent functional diversity81–83. Eukaryotic actins are generally highly conserved between organisms. For instance, actin from H. sapiens and S. cerevisiae are >88% identical. Monomeric actin is usually referred to as G-actin (globular actin), while the filamentous form is known as F-actin. The globular moniker is however quite far from the de facto structure of actin. Structural analysis of actin reveals a rather flat molecule consisting of two lobes, divided by a deep central cleft, with each lobe in turn divided into two subdomains (Fig. 4)84–86. Determination of the structure was for a long time frustrated as many of the conditions used in crystallography, e.g. high salt concentration and high protein concentration cause the protein to polymerize. Even today, all structures of eukaryotic actin use proteins, molecules or mutations that hinder G-actin from forming polymers. The stability of the complex is dependent on the binding of an Mg2+-nucleotide complex at the base of the cleft. Loss of this complex quickly leads to denaturation and renaturation is dependent on the ubiquitous cytoplasmic chaperon CCT87,88. Although ATP is hydrolysed to ADP during polymerization of the actin filament, this is, strictly speaking, not a required feature of said polymerization. ADP-actin can polymerize, albeit at a slower rate than ATP-actin89. Additionally, actin also polymerizes in the presence of non-hydrolysable ATP analogues90,90. Importantly, the nature of the bound nucleotide will confer an asymmetry to the elongating filaments, favouring polymerization towards one end over the other91,92. The result of this is an overall directionality of the actin filament, where ATP-actin is added on one end, called the (+)-end or barbed end, and disassociation of ADP-actin monomers on the other end, called the (-)-end or pointed end. The polymerization of actin can principally be divided into three stages: nucleation, elongation and a steady-state called treadmilling. Nucleation is the rate-limiting step of polymerization and relative to all other reac-

- 12 - tions in actin polymerization, the formation of the incipient filament is exceptional- ly inefficient. Polymerization starts with the formation of a nucleus consisting of three to four actin subunits. In vivo, spontaneous nucleation of actin is efficiently restricted, and instead governed by actin-binding proteins93. Once formed, the nucleus is able to rapidly elongate into long actin filaments during the second, elongation phase. The third phase, treadmilling, is characterized by a balance in association and disassociation of subunits between the barbed and pointed ends94. Beyond these dynamics, the actin ultrastructure does not form a single straight filament, but rather a twisted filament (approximately 190° per subunits) which forms a unidirectional staggered double helix together with a second actin filament (Fig. 5)77,95,96. The concentration of the monomeric G-actin pool during steady-state treadmilling is known as the actin critical concentration (ACC)97. Actin will not polymerize if the concentration is below the ACC for any given condition. If the concentration of G-actin is above the ACC the system will reach a steady-state if left unperturbed. This depend- ence on protein concentration regulates actin polymerization in cells along with actin-binding proteins. A large and diverse collection of proteins, known as actin-binding proteins (ABPs), collectively modify the behaviour of both G-

Figure 4. Crystal structure of monomeric rabbit skeletal muscle ATP-actin (PDB ID: 1NWK) showing subdomains and nucleotide binding site. Reprinted with permission from Graceffa and Dominguez (2003).

- 13 - actin and F-actin. These proteins are able to alter every aspect of actin behaviour ranging from nucleation, elongation rate, monomer sequestration, severing and filament branching.

CHAPTER 3.2 | MreB and Crenactin, the lost prokaryotic relatives. Although actin was originally thought to be unique to eukaryotic cell, numerous actins exist in all domains of life. This section will briefly introduce two prokaryotic actins, MreB and crenactin. It should be noted however, that the actin superfamily is surprisingly diverse. A review of the varied roles of different actin superfamily members and their relation to polymerization can be found here98. One of these divergent actin proteins, the non-polymerizing chaperone Hsp70/DnaK, was only classified into the actin superfamily after their crystal structure was determined. This stems from the fact that although Hsp70/DnaK’s tertiary structure is quite similar to actin’s (RMSD = 2.3 Å over 241 pairs), the DNA sequence is not (16.2% identity over 241 pairs)99. This pattern of low sequence similarity but conserved tertiary structure is a recur- ring element when studying prokaryotic homologues. Sequence similarity between E. coli MreB and H. sapiens γ-actin is 23% (pairwise identity); about the same as one would expect when comparing two random sequences. Unlike actin, MreB forms antiparallel in-register double filaments. It is found in most walled bacteria with elongated cell shape morphology and is thought to bind the membrane direct- ly100,101. MreB was previous thought to found long cell-spanning helical filaments which organized cell wall synthesis, but recently this view has been losing support. Recent developments in imaging technique instead supports the hypothesis that MreB forms short fragments which move circumferentially with the cell wall synthesis machinery, locally organising cell wall synthesis102–105. Additionally, MreB has been implicated in chromosome segregation in some bacterial species106, and in cell division for others107,108. More recently, a number of actin and MreB homologues have been discovered in archaea but remain poorly studied. Although some methanogenic archaea contain bona fide MreB homologues, the first archaeal actin homologue to be studied was crenactin, which was discovered in the TACK archaeon Pyrobaculum calidifon-

- 14 - tis. The crenactin gene, cren-1, is encoded in a five-gene operon with four other genes named rkd-1 through -4, which were hypothesised to be novel actin-binding proteins109. Crenactin can form cell-spanning helical structures, which colocalize with the rkd- gene products. Specifically, the crenactin-binding protein arcadin-2 (rkd-2 gene product) is able to sequester crenactin monomers in vitro, functionally homologous to the actin-binding protein thymosin β477. Arcadin-1 (rkd-1 gene product) interacts with crenactin (Kd = 15 µM) but does not appear to interfere with polymerization in vitro. Notably, although the crystal structure was first reported as single helical filaments110,111, subsequent studies using reconstructed cryo-EM images reported a staggered, antiparallel, helical double filament, very similar to the filament ultrastructure formed by eukaryotic F-actin (Fig. 5)77. When the Asgard archaea were discovered, they were found to contain several actin homologues with previously unseen sequence homology to eukaryotic actin. Among these actin homologues were also apparent homologues of putative actin- binding proteins which are ubiquitous within the eukaryotic clade17,21. This thesis touches on two of these, profilin and gelsolin, which are described below.

Figure 5. Crystal structure of H. sapiens actin interaction (top left) and filament configuration (bottom left). Crystal structure of crenactin from P. calidifontis (top right) and filament configuration (bottom right). Reprinted with permission from Wagstaff and Löwe (2019).

- 15 - CHAPTER 3.3 | Actin-binding proteins - Profilin. Profilins are cytosolic proteins expressed in most, if not all, eukaryotic cells and were originally isolated in a complex with actin in the 1970s112. They are relatively small in size, approximately 14- 19 kDa, and although sequence conservation is low across distantly related eukaryotes, conservation of profilin’s tertiary structure remains high113. The tertiary structure of eukaryotic profilin consists of a 7 β-sheet central core flanked by 2 α-helices on each side. Profilin is highly expressed in most mammalian cell types where it acts as a nucleotide exchange factor and a G-actin sequestering protein114. As is common in metazoans, several paralogous genes and protein isoforms exists. There are currently five profilin variants identified in mammalian cells: profilin I, II, III and IV, with profilin II having two splicing isoforms named profilin IIa and IIb. Many cells express several of the profilin variants simultaneously and while the variants display distinct affinities to their various binding partners, the intracellular spatial organization of profilin contributes significantly to function. Interestingly, cyanobacteria encode two actin (ActM) and profilin (PfnM) paralogues. These paralogues share fairly high sequence conservation, with eukaryotic actin and profilin (65% and 57% pairwise identity, respectively). Currently, ActM and PfnM are thought to have integrated into cyanobacterial genomes through horizontal gene transfer (HGT) from a eukaryotic lineage115. Though ActM and PfnM remain poorly studied, it is known that PfnM decorates the ActM filaments in vitro and appears to induce bundling of the filaments. Furthermore, ActM filaments are short (5 to 10 nm) in comparison to its eukaryotic homologues (up to 100 µm in certain cell types)116. Though profilin was originally discovered in association with actin and early functional description focused on profilin’s ability to regulate actin polymerization dynamics, it has further been discovered that profilin is able to interact with several molecules and proteins while simultaneously interacting with actin. It is these characteristics that puts profilin at the centre of a complex regulatory network that controls the polymerization dynamics of one of the most abundant proteins in the eukaryotic cell, actin. Profilin is able to directly bind to the barbed end of both G- actin and F-actin, regulating polymerization dynamics84. In addition, profilin speeds up the ADP to ATP exchange rate of bound actin by up to a 1000-fold117–119. As

- 16 - mentioned earlier (see section 3.1), ATP-actin has a higher affinity for F-actin filaments (KATP-Actin = 0.1 µM) compared to ADP-actin (KADP-Actin = 1.7 µM). In practice, the higher affinity of profilin to ATP-actin in combination with the nucleotide exchange activity of profilin means that the barbed end of an actin filament consists of ATP-actin120. Profilin accelerates the nucleotide exchange rate by inducing a conformational shift in actin’s tertiary structure, opening the central cleft and ex- posing the nucleotide binding site located there121,122. The exchange of ADP- to ATP-actin further strengthens profilin’s affinity for actin123,124. Finally, data indicates that while profilin is able to bind to the barbed end of F-actin, it does so with a significantly lower affinity (KF-actin = 20 µM) compared to G-actin (KG-actin = 0.1 µM)120. Despite the low affinity, profilin’s binding to F-actin’s barbed end is important in competitive regulation with other barbed end-binding proteins125. Profilin’s direct regulation of actin polymerization dynamics is additionally af- fected by two classes of interactions. The first of these is through profilin’s interaction with phosphoinositides. Interaction with a number of different phosphoinositides was originally demonstrated in 1985 when Lassing and Lindberg showed that phosphatidylinositol 4,5-bisphosphate (PIP2) caused profilin to release actin from the actin-profilin complex, leading to actin polymerization126. Subsequent studies showed that a trimeric actin-profilin-PIP2 complex could be formed in a covalently coupled actin-profilin construct, leading to the discovery of a second interaction surface on profilin for phosphoinositides127. This finding was in line with contempo- rary investigations which indicated the presence of two separate interaction sur- 128 faces for phosphoinositides on profilin . Apart from PIP2, profilin is able to bind several other phosphoinositides: PI(4), PI(4,5)P2, PI(3,4,5)P3 and PI(3,4)P2, in order of affinity as determined in vitro129. The biological significance of these interactions is still poorly understood. Our understanding of the second class of molecules which regulate profilin is better, and it relates to an important interaction between profilin and polyproline motifs on a wide range of interaction partners. This discovery was exploited in a lab setting where actin-profilin complexes could quickly and relatively easily be purified using affinity chromatography on polyproline columns130,131. The proline rich

- 17 - stretches interacts with residues in the amino- and carboxyl-terminal helices on profilin. This was elegantly shown by Kaiser and Pollard in 1996 through mutating profilin (W3N and H133S) which lowered the affinity of polyproline for profilin more than a 100-fold132. Today, a wide variety of profilin binding partners containing polyproline motifs has been identified. Generally, these binding partners display polyproline sequences of eight to ten prolines in a row, or an archetypical glycine- pentaproline motif133. The first biological polyproline ligand to be discovered was VASP, vasodilator-stimulation phosphoprotein134, and since then many other polyproline binding interaction partners of profilin have been discovered. Broadly speaking, most of the actin integrated into filaments is complexed with profilin or other ABPs, and it is through profilin’s polyproline-mediated interactions the recruitment of the actin-profilin complex to the barbed end of actin filaments is governed. The nucleotide exchange activity of profilin in conjunction with polyproline- mediated recruitment makes for a much more efficient polymerization system than actin could accomplish in isolation124,135–138. In essence, these polyproline-mediated interactions allow for a spatiotemporal recruitment of ATP-actin, whilst keeping the free G-actin pool below the critical concentration, preventing spontaneous polymerization of actin filaments. Consequently, it is unsurprising that polyproline- mediated interactions appear to be essential for the function of profilin 139. Currently, very little is known about predicted profilin homologues in the Asgard group. A single publication investigating the structure of several Asgard profilins concluded that the homologues have a relatively high RMSD. Profilin I from Lokiar- chaeota is in the range of 2.0 to 2.4 Å versus a variety of eukaryotes140. For comparison, H. sapiens profilin I RMSD is in the range of 1.3 to 2.0 Å for the same set of eukaryotic profilin structures. However, considering that the Asgard group likely diverged from the last archaeo-eukaryotic common ancestor (LAECA) around 2 billion years ago, this is not necessarily unexpected. In support of these proteins being de facto profilin homologues, Akıl et al were able to show that several Asgard profilins can regulate rabbit muscle actin in a functionally analogous way to H. sapiens profilin, though Asgard profilin’s affinity for rabbit actin was unsurprisingly lower relative to H. sapiens profilin140. Additionally, they were able to show that

- 18 - profilin’s interaction with PIP2, but not polyproline, appears to be an ancestral regulatory mechanism. While more research is needed to better understand the function and regulation of Asgard profilins, this line of investigation has given us the first insights into the complexity of cytoskeletal regulation which likely existed before the divergence of eukaryotes from archaea.

CHAPTER 3.4 | Actin-binding proteins - Gelsolin. The actin binding protein gelsolin is another important regulator of F-actin polymerization dynamics. The eukaryotic multi-domain gelsolin family of proteins is involved in diverse regulatory processes including filament annealing, bundling, capping, nucleation and elongation. In addition, gelsolin is one of the most potent filament severing proteins in the eukaryotic cell141–144. Gelsolin proteins generally consist of three to six homologous domains which are thought to have been produced through a series of gene duplication events: two single-domain duplications followed by a whole-gene duplication pro- ducing three and six domain versions of the proteins144–146. In 1986, Kwiatkowski and collaborators were able to show that human gelsolin contains multiple homologous domains, and that sequence comparison to villin indicated a common ances- try147. Today we know that gelsolin is a part of a larger family including gelsolin, villin, severin and fragmin; the common denominator being the inclusion of gelsolin homology (GH) domain repeats. The potent severing activity of gelsolin is dependent on the presence of calcium cations which trigger major conformational changes, allowing it to bind parallel to the actin filament. While the process of binding gelsolin to the filament is rather rapid, the actual severing is slower, possibly a reflection of the conformational changes required for severing to occur148. Interest- ingly, there are indications that the binding of gelsolin confers a kink in the actin filament, indicating a possible mechanical basis for severing149. After gelsolin has severed a filament, it remains attached to the barbed end of the severed filament. This means that the short filaments created by severing are unable to reanneal or elongate further, compounding gelsolin’s effect150. In addition to gelsolin’s severing activity, it is able to cap both monomeric actin and filamentous actin by binding to their respective barbed ends. And while it is

- 19 - able to bind both G-actin and F-actin, the biological role of gelsolin is heavily skewed towards the capping and severing of F-actin - providing complementary regulation as compared to profilin, where interactions with G-actin is a more prom- 143 inent feature . In addition to calcium regulation, gelsolin can interact with PIP2 which efficiently sequesters gelsolin to the membrane, preventing it from interacting with actin151–153. This sequestration can in turn be reversed by ATP, which com- 154 petitively binds to gelsolin, abolishing the PIP2 interaction . The discovery of predicted profilin and gelsolin homologues in archaea provides a potential basis for complex cytoskeletal regulation in archaea, including filament severing, capping, nucleation, nucleotide exchange, sequestration and elongation. In studying actin and ABP from the Asgard group we have the opportunity of infer- ring information about the nascent cytoskeleton and its regulation, which are likely present in LAECA.

- 20 - Present investigations

The overarching aim of this thesis has been to better understand the complex filament systems in eukaryote-adjacent archaea, and by extension to further our understanding of filament systems in LECA and LAECA. The discovery of archaea has had a profound impact on our understanding of where eukaryotic life came from, and which early forms life may have taken. ESCRT-III serves as an example of how understanding archaea can provide insight into the original protein function of cell division machinery proteins. In metazoan cell division, the role of ESCRT-III is a relatively recent discovery and coincided with the discovery of ESCRT-III’s role in archaeal cell division. Current thinking suggests that cell division is one of the original roles of ESCRT-III, and it appears to be an evolutionarily ancient function which later developed into the multi-faceted ESCRT-III complex now present in eukaryotic cells. Understanding the mechanistic and regulatory functions of archaeal systems and correlating those findings with knowledge from the eukaryotic field provides us with a foundation of cellular complexity preceding eukaryogenesis.

Paper I. | The proteasome is required to trigger ESCRT-III-mediated cell division in an archaeon. Author list. | Risa G.T.†, Hurtig F.†, Bray S., Hafner A. E., Harker-Kirschneck L., Faull P., Davis C., Papatziamou D., Mutavchiev D. R., Fan C., Meneguello L., Arashiro Pulschen A., Dey G., Culley S., Kilkenny M., Pellegrini L., de Bruin R. A. M., Henriques R., Snijders A. P., Šarić A., Lindås A.-C., Robinson N., Baum B. Aim. | The aim of this study was to further our understanding of the regulatory and functional mechanisms which govern the ESCRT-III based division system found in many TACK archaea. Previous investigations produced some insight into the relationship and interactions within the Cdv division machinery, however, the function and regulation of the CdvB homologues, CdvB1 and CdvB2 remained elusive. In this study we aimed to describe the functional diversity of the CdvB protein homologues and to better understand how the cell is able to regulate cytokinesis.

- 21 - Results and discussion. | As mentioned elsewhere in this thesis, S. acidocaldarius exhibits an ordered cell cycle consisting of distinct G1 and G2 gap phases separated by rounds of DNA replication and cell division, similar in structure to many eukaryotes. Additionally, several of the key events that drive cell division are also conserved between S. acidocaldarius and eukaryotes. Archaeal protein homologues of Cdv6/Orc/MCM/GINS initiate DNA replication at multiple origins, homologous to eukaryotic DNA replication. However, archaeal homologues of ESCRT-III and Vps4 have been shown to drive cell division. This begs the question, is cell cycle regulation homologues in Sulfolobus and eukaryotes? In eukaryotes, oscillations in the activity of cycling-dependent kinases order the cell cycle. To date, neither cyclins nor cyclin-dependent kinases (Cdk) have been detected outside of eukaryotes. The oscillating nature of this system is achieved through phasic degradation of cyclin by the proteasome. While the cyclin-Cdk system is absent in archaea, close homologues of the eukaryotic core proteasome machinery are wide-spread. This prompted us to investigate whether the S. acidocaldarius proteasome played any role in cell cycle regulation. We began by performing a detailed structural analysis of the S. acidocaldarius 20S proteasome. Exogenous co-expression in E. coli of core α- and N-terminally truncated β-subunits resulted in the formation of an archetypal but catalytically inactive twenty-eight 20S proteasome. In silico homology modelling was then used to predict the structure of the catalytically active β-subunit using structures from Archaeoglobus fulgidus and Saccharomyces cerevisiae as templates. Docking mod- els indicated that the established eukaryotic proteasome inhibitor bortezomib would be effective in S. acidocaldarius. To verify this we exogenously expressed a catalytically active 20S proteasome from S. acidocaldarius in E. coli. Using an established in vitro assay to measure proteasomal degradation, we demonstrated that bortezomib significantly antagonized the catalytic activity of the S. acidocaldarius proteasome. To investigate a potential role for the proteasome in cell cycle control, we syn- chronized S. acidocaldarius cultures and examined cell cycle progression post- synchronization. Strikingly, when bortezomib was added to cultures in G2, cells

- 22 - successfully segregated their chromosomes but failed to divide. To verify that cells were unable to progress through M- into S-phase, we repeated the experiment with a S. acidocaldarius strain overexpressing thymidine kinase (STK). STK incorpo- rates the thymidine analogue 5-Ethynyl-2’-deoxyuridine (Edu) into newly synthe- sised DNA which can then be visualized. While control cells expressing STK pro- gressed through M-phase and into S-phase, incorporating Edu, bortezomib treated cells did not. Taken together, these data show that the proteasome in S. acidocaldarius plays an important role in regulating cell cycle progression during M-phase. Having observed proteasomal dependent mitotic arrest, we next investigated proteins subject to proteasomal regulation that control cell division. To identify such proteins, we compared arrested cultures to G1-phase control cultures by mass spectrometry. While the majority of known cell division proteins remained un- changed relative to the control, the ESCRT-III homologue CdvB was the most en- riched protein in the entire proteome. While inconspicuous at first glance, why would a protein described as a core component to S. acidocaldarius’ cell division machinery need to be degraded for cells to divide? To investigate further, we examined fluorescently labelled asynchronous cell populations with flow cytometry, focusing on known members of the cell division machinery, including CdvB. Interestingly, while CdvB was generally constitutively expressed, cells selectively degraded CdvB just prior to cell division. Further, expression of the CdvB homologues CdvB1 and CdvB2 was maintained at high levels throughout cell division, ending with a symmetrical partitioning of CdvB1 and CdvB2 between daughter cells. Next, we employed super-resolution microscopy to analyse the subcellular organisation of the CdvB system during cell division. Analy- sis revealed that CdvB formed ring-like structures between segregating nucleoids with a diameter equal to that of a cell about to divide. Further, in many cells a co- localized second ring consisting of CdvB1 and CdvB2 was observed. Interestingly, we observed a more infrequent class of cells exclusively expressing a CdvB1 and CdvB2 ring while lacking a CdvB ring. In these cells, ring diameter was more variable and clearly constricting. Furthermore, cytoplasmic CdvB1/CdvB2 signal intensity increased with cell cycle progression. Cells from cultures treated with bortezomib

- 23 - formed CdvB, CdvB1 and CdvB2 rings. However, CdvB ring expression was mutually exclusive from CdvB1/CdvB2 ring expression under bortezomib treatment. Addi- tionally, the diameter of rings in this population equalled the full cell diameter. Based on these data, we propose a model where cells recruit a non-contractile ESCRT-III ring (CdvB). This ring in turn recruits a contractile co-polymer (CdvB1/CdvB2) which stores tension energy in its polymer. This energy is then re- leased by degradation of non-contractile elements in a proteasome dependent fashion. Subsequently, contractile elements rapidly constrict, dividing the cell. To assess whether this model was a plausible physical mechanism we conducted coarse grain molecular dynamics simulations. However, simulations indicated that constriction of the filament was not enough to successfully divide the cell due to steric hindrance at the very end of division. When a gradual disassembly of subunits from the filament was integrated in the model, equivalent to the cytoplasmic signal observed in vivo, simulated cells were able to complete division. In conclusion, this study revealed important parallels between eukaryotes and archaea, implicating the proteasome in a minimal ESCRT-III-based cell division machinery. Strikingly, these findings indicate that the regulatory role of the proteasome preceded the archaeo-eukaryotic split and the cyclin-Cdk system.

- 24 - Paper II. | Heimdallarchaeota encodes profilin with eukaryotic-like actin regulation and polyproline binding. Author list. | Survery S., Hurtig F., Haq S. R., Lindås A.-C., Celestine C. Aim. | The Asgard archaea contain several predicted putative actin regulating proteins previously unseen in any prokaryotic organism, along with de facto actin homologues. One of these regulatory proteins, profilin, is a core component of the regulatory machinery governing the eukaryotic cytoskeleton. The presence of these proteins has broad implications on the potential cytoskeletal complexity of LECA. However, very few studies have investigated the function of these proteins. This investigation focused on determining whether a predicted putative profilin homologue from Heimdallarchaeota LC_3 was functionally analogous to eukaryotic profilin and its interaction with eukaryotic actin and an actin homologue from Heim- dallarchaeota. Results and discussion. | Our investigation focused on a predicted putative profilin from Heimdallarchaeota, a candidate Asgard phylum. Initial observations utilizing NMR spectroscopy initially indicated a canonical profilin fold with the addition of a long disordered N-terminal loop. A previous investigation by Akıl et al had determined the crystal structure for several predicted putative profilin homologues from Loki-, Thor- and Odinarchaeota. The extended N-terminal loop was absent from these structures, as well as from known eukaryotic profilin structures. Upon closer inspection, the structure of heimProfilin differed further. While still containing the canonical 4 α-helices and 7 β-sheets, the orientation and position of several α- helices has shifted significantly. To assess whether the extended N-terminal loop had functional elements we create a truncated form which we called ΔN- heimProfilin (deleting residues 1-23). We then allowed rabbit actin to polymerize in the presence or absence of heimProfilin and ΔN-heimProfilin and observed the filament network using Airyscan super-resolution microscopy. While at high concentrations, heimProfilin was able to inhibit filament formation in a concentration dependent manner, ΔN-heimProfilin had no effect on the filament network at equivalent concentrations. To verify this result we followed rabbit actin polymerization in a pyrene assay. In line with our microscopic observations, heimProfilin

- 25 - inhibited filament formation in a concentration dependent manner while ΔN- heimProfilin was unable to modulate polymerization dynamics. In an effort to investigate how heimProfilin might interact with archaeal actin, we cloned, purified and expressed two actins from Heimdallarchaeota. A full-length version (heimAc- tin) and a truncation variant (ΔC-heimActin) which lacked the last 35 C-terminal amino acids. Electron micrographs of polymerized heimActin revealed long, thin filaments where ΔC-heimActin did not appear to polymerize. Similarly, ATPase activity measurements showed heimActin to be active while ΔC-heimActin had a severely depleted ATPase activity compared to heimActin. This likely stems from the fact that the truncation removed a large section of what would be the barbed- end in eukaryotic actin. Having observed that heimActin could form polymers, we turned to NMR to map the interaction surface on heimProfilin and ΔN-heimProfilin. Interestingly, when comparing similar residues at similar concentrations, ΔN- heimProfilin displayed a stronger chemical shift changes than heimProfilin, indicating that the extended N-terminal loop on heimProfilin might modulate interaction with heimActin. Additionally, ΔN-heimProfilin showed only marginal interaction with ΔC-heimActin. Polyproline interactions are crucial for profilin function in eukaryotic cells. How- ever, a previous study found no interactions between polyproline and profilin from Loki- or Odinarchaeota, implying that this regulatory feature could have appeared after the eukaryotic domain split from the archaeal domain. To investigate this we turned to NMR and isothermal titration calorimetry (ITC). ITC experiments revealed a moderate interaction (KD ≈ 200 µM) between ΔN-heimProfilin and a polyproline motif from eukaryotic VASP (PPPAPPLPAAQ) which parallels interaction affinity seen between VASP polyproline sequences interacting with profilin not bound to actin (KD ≈ 50 - 230 µM depending on sequence). Conversely, heimProfilin displayed very weak interactions with the polyproline peptide. We were also able to map the binding interface using NMR. Lastly, a previous investigation found that a variety of

Asgard profilins could interact with PIP2, an important signalling phospholipid which regulate the function of many actin binding proteins including profilin. Again, we turned to NMR and ITC to investigate and map interactions between ligand and

- 26 - heimProfilin. In line with our earlier findings, we found that ΔN-heimProfilin could interact with PIP2 while no interaction between PIP2 and heimProfilin was observed. Additionally, we were able to map the residues important for the interaction and tested whether heimProfilin and ΔN-heimProfilin was able to interact with

IP3, a second messenger resulting from the hydrolysis of PIP2. Neither heimProfilin nor ΔN-heimProfilin showed any interaction with IP3. In context, these findings indicate that heimProfilin is able to modulate the polymerization of actin and is regulated by PIP2 and polyproline. Additionally we describe a novel regulatory feature in the form of an extended N-terminal loop which abolishes interaction with ligands. These findings bolster the hypothesis that Asgard archaea contain a proto-cytoskeleton under complex regulation previously though relegated to eukaryotic organisms.

- 27 - Paper III. | Heimdallarchaeota gelsolin is a multi-domain protein with calcium controlled actin regulation. Author list. | Survery S., Hurtig F., Haq S. R., Lindås A.-C., Celestine C. Aim. | Investigations into the origin of the first eukaryotic cell have recently been brought into focus with the discovery of the archaeal Asgard superphylum. Com- parative phylogenetics and initial biochemical studies into these organisms indicate a complex cellular organization reminiscent of eukaryotic organisms. These organisms are predicted to encode a wide-ranging collection of genes which until recently were thought to be specific to eukaryotic organisms. This includes putative actins and gelsolin homology-domain proteins. However, no studies to date have investigated the function or structure of predicted Asgard gelsolin proteins. In this study, we describe the domain diversity of Asgard gelsolin homologues and demonstrate that Heimdallarchaeota gelsolin is capable of tight and complex regulation of actin polymerization, equivalent to capping and severing activities seen in eukaryotic gelsolin. In context, this study supports the hypothesis that the Asgard archaea are closely related to eukaryotic organisms and that the formation of a cytoskeleton with complex regulation likely predated eukaryogenesis. Results and discussion. | Amongst the Asgard archaea, another interesting finding was the encoding of apparent putative gelsolin domain proteins. In eukaryotic organisms, gelsolin is capable of a diverse set of functions including severing, capping and nucleation of actin filaments. The protein consists of several domain repeats, usually numbering between 3 and 6 gelsolin homology domains. Heimdallar- chaeota was found to encode a single predicted gelsolin homologue (heimGelsolin). In silico domain modelling indicates that heimGelsolin contains two gelsolin homology domains and a likely third domain of unknown function. When widening our search we discovered broad variability amongst Asgard archaea regarding the number of gelsolin domains present. We also discover a likely WH2 domain that appears to be conserved throughout Loki-, Thor- and Heimdallarchaeota. Next, we wanted to investigate the in vitro activity of heimGelsolin. The control sample showed the formation of a complex network of actin filaments. Upon addition of equimolar concentration of heimGelsolin on pre-polymerized actin filament,

- 28 - little to no effects could be seen. However, when the reaction was supplemented with 1 mM CaCl2 none to very few filaments could be observed. When the reaction was additionally supplemented with 1 mM EGTA along with 1 mM CaCl2, the filament network mirrored the control. These results strongly imply that much like eukaryotic gelsolin, the severing effect of heimGelsolin is tightly regulated by calcium. To verify these results we turned to pyrene assays to measure the effect on polymerization directly. We could observe a concentration dependent severing effect on pre-polymerized actin filaments which was abolished by the addition of 1 mM EGTA. At lower concentrations of CaCl2 severing could be observed but at a lower rate. Taken together, these results demonstrate how heimGelsolin is able to efficiently and rapidly disassemble pre-polymerized actin filaments under tight regulation of calcium ions. Next we wanted to investigate whether heimGelsolin possessed any other regulatory capabilities known from eukaryotic gelsolin. We combined differing concentrations of heimGelsolin with pyrene labelled actin and followed the polymerization. In line with our earlier findings, we found that heimGelsolin was able to inhibit actin polymerization in a concentration dependant fashion and that this reaction is governed by tight calcium regulation. Taken together, our data shows that Heimdallarchaeota encodes functionally homologous gelsolin domain proteins which act in severing and capping capacities under tight calcium regulation. In the context of previous investigations demon- strating the regulatory capacity of various profilin homologues from Asgard archaea, these results further demonstrate the regulatory complexity which exists in the Asgard archaea. In a broader context, it demonstrates that the last eukaryotic common ancestor likely already had complex regulation of cytoskeletal components.

- 29 - Future directions

The discovery of the Archaea has changed how we view where we come from. From the initial reclassifications of methanogenic prokaryotes5,6, to the more recent discoveries of the Asgard group17,21,155, archaea has brought perspective on the evolution of early life. This thesis has focused on understanding the regulatory complexity of protein filament systems in two archaeal groups, the Asgard and TACK superphyla. However, answering questions often leads to further questions. We have proposed an elegant mechanism for division in S. acidocaldarius through which the Cdv system creates a checkpoint just prior to division which is triggered by proteasomal degradation of a target protein. Upon deeper scrutiny the model creates several interesting problems however. CdvB, the proteasomal target, has been shown to interact with CdvA which interacts with lipid membranes in vitro69. Furthermore, a yeast-two-hybrid screen has shown how CdvB is able to interact with CdvB1, which in turn interacts with CdvB2, the two proposed contractile elements in our model66. However, we have been able to show that CdvB is degraded prior to constriction, leaving CdvB1 and CdvB2. Both of these proteins lack the wH-domain responsible for interaction with CdvA69. The question is then, how does CdvB1 and CdvB2 stay attached to the membrane? A possible answer to this question lies in how eukaryotic ESCRT-III proteins attach to the membrane in the absence of a functional homologue of CdvA. Like the eukaryotic N-terminal ANCHR motif, the N-terminus of S. acidocaldarius CdvB1 has a predicted amphipathic α-helix (data not published). Insertion of this motif into the membrane could provide the contractile ring the attachment needed to constrict the cell. A second question concerns how CdvB is removed from the ring. Preliminary data indicates involvement from both Vps4 and the PAN regulatory ATPase (data not published) but a mechanistic explanation is still lacking. Coupled to this question is why CdvB is rapidly degraded, as opposed to being dismantled. As inhibition of the proteasome inhibits cytokinesis, there appears to be a link between degradation of CdvB and cytokinesis, as dismantling of the CdvB ring is not enough.

- 30 - While the Asgard group has garnered large interest due to their apparent similarity to eukaryotes, no organisms from the Asgard group has currently been isolated. The isolation and successful lab cultivation of an Asgard archaeon would greatly widen the experimental approaches available in the study of these organisms. At- tempts to isolate Asgard archaea are currently underway in several laboratories around the world (personal communication) but we will likely have to wait several years for reliable lab cultivation of these organisms. In a one of a kind 12-year study by Imachi et al, electron micrographs of the Lokiarchaeon Prometheoarchaeum syntrophicum were taken from an enrichment culture. The cells observed showed long tendrils extending from the central body of the cell, potentially created by a process analogous to eukaryotic filopodia. This finding does however had important implications to the endosymbiont theory where one model proposes that endosymbiosis occurred in a gradual process of increasing membrane interac- tions156. Very little is currently known about cytoskeletal proteins and their regulators within the Asgard archaea. Current investigations are focusing on fundamental characterization of predicted putative proteins. The revelation that Heimdallar- chaeota profilin potentially can interact with polyproline sequences is likely to lead to a search for polyproline containing proteins. The discovery of such proteins and any potential regulatory role they have in an archaeal actin cytoskeleton will have broad implications on LAECA and the complex structures Asgard archaea potentially contain.

- 31 - Acknowledgements

I would like to start by thanking Ann-Christin and the late Rolf Bernander for the opportunity to do my master’s and PhD project investigating such a fascinating group of organisms. Although it at times has been difficult and sombre, overall this time has been the most developing period of my life, and foundational for how I view science today. I would also like to thank my co-supervisor Roger, your deep knowledge and passion has been inspiring and I hope to match it one day. To our lab-neighbour Claes, for your ever dependable insights into whatever strange question I might have for the day, and for making the moodiest day a little more light- hearted. And to all my colleagues at MBW, thank you for creating an interesting environment that has felt both inclusive and engaging. Mattias, for five years of companionship, for floorball games bordering on fist- fighting, and for showing me that Belgian beer is actually not half bad. Mohea, you showed me the power of sticking to your beliefs. Now that you’re gone, I kind of miss hearing you laughing from the other side of the department. You made me a better scientist and a better person. Kun, you were always a point of calm in what otherwise could be a pretty stormy group. I still have a ton of that amazing green tea left. Sabeen and Raza, we fought an uphill battle, but in the end we pulled through. Gabriel and everyone else in the Buzz group, thank you for reminding me what science should be like. Study till you bleed I lack the words to describe how much meeting you guys has meant to me. I know we’ll always stay in touch, and I’ll forever remember the study beers and late-night philosophy discussions. Einar, for our endless discussions; I wish we’d gotten to know each other earlier. Jutta, for helping me become the person I want to be. Lexi, for helping me understand that person. Daniel, sometimes I think our hikes were the only things that kept me sane through this. Good luck with the potatoes! Franzi, some of my happiest memories are with you, I will always treasure them. Jackie, I couldn’t have asked for a better neighbour, I hope I became “more fun”. Andreas, whether it was tacos or stock crashes, chatting with you always mean a good time. The corridor felt emptier after

- 32 - you defended. Chit Chat, Raquel, Alice and more recently Manno. This little group kept me sane and it was always comforting to know that you guys were always within reach. Jo Gjende, att få försvinna upp i fjällen varje år med er har varit den bästa semestern jag kunde önskat mig. Jag hoppas vi kommer hålla igång tills vi alla är gamla fjällrävar, kbk! Albin, Viktor & Jerker, thank you for all the game nights full of absurdities. Joan, imgur.com/6x4V6dh Lukie, even before we moved to F4 I had come to know you as that guy who is always in the lab with a smile. You might want to diversity your diet a little bit though. Emily, you’re the coolest Scott I know. Fitz, much like Andreas, engaging with you has always meant a good time. Maybe this is why the two of you in the same lunchroom always ended in hysteric laugh- ter. Gabby, thank you for all the climbing sessions and workout chats; I can’t wait to see how the van turns out! And to all of you who I watched pass through these halls before me, thank you for being a part of the best time of my life.

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