Identification of evolutionary conserved mechanisms promoting rapid actin dynamics

Cells are the minimal compartments of life that constantly respond to cues coming from both inside and outside of their environment. In response, cells migrate, transport different molecules and undergo sudden shape changes to meet their environmental demands. These processes are regulated by the actin cytoskeleton, a ubiquitous component of eukaryotic organisms that dynamically cycles between monomeric and polymeric form at the sites of cellular rearrangements. Polymerization of actin against plasma membranes and organelles provides force to the constant remodeling of cells. Consequently, the actin cytoskeleton is critical for many fundamental cellular processes, including cell division, cell migration, cellular morphogenesis, endo- and exocytosis. Actin molecules bind ATP and polymerize to polar filaments with structurally two different ends. Actin filaments grow from the ‘plus’ (+) ends by the incorporation of ATP-actin monomers, after which they convert through ATP-hydrolysis to ADP-state that populates the ‘minus’ (-) end of the filaments. ADP-state renders actin molecules to a state of depolymerization, and thus actin filaments slowly shrink from the (-) ends in steady-state conditions. This is a fundamental property of actin filaments that drives their directional growth in the force production. Thus, cells require a constant supply of actin monomers from depolymerization of ‘old’ filaments and conversion of these actin monomers from ADP- to ATP-state. Several proteins, including evolutionary conserved ADF/cofilin and cyclase-associated protein (CAP), are involved in the regulation of these steps in cells, but the exact molecular mechanisms have remained elusive. In this work, I studied the molecular principles of how actin filaments are dynamically converted between monomeric and filamentous forms. CAP is a multidomain protein and earlier shown to be critical for actin dynamics in various model organisms. However, the molecular mechanisms by which CAP regulates actin dynamics have remained unknown. In the first and second parts of my thesis, I utilized X-ray crystallography, mutagenesis and biochemical assays to reveal how CAP interacts with both actin monomers and filaments. I show that the N-terminal domains of CAP are responsible for binding to the (-) ends of actin filaments and catalysis of rapid filament depolymerization in synergy with ADF/cofilin. The C-terminal domains of CAP scavenge the depolymerized ADP-actin molecules from ADF/cofilin and catalyze their conversion to ATP-state. These data establish CAP as a recycling machine for actin molecules and explain the earlier in vivo observations why different domains of CAP are vital for rapid actin dynamics in cells. In the fourth part of my thesis, I show how rapid actin dynamics are achieved in an evolutionary distant organism, pathological Leishmania parasite that contains only a very simple actin-regulatory machinery. By utilizing the molecular tools developed in the third study, combined with cryo-electron microscopy, mutagenesis and biochemical assays, I show that Leishmania actin filaments are inherently dynamic and susceptible to disassembly by ADF/cofilin, and reveal the atomic mechanisms behind these attributes. These data help to understand the mechanisms that regulate the actin cytoskeleton evolved in eukaryotic organisms and could provide a molecular rationale for development of inhibitory compounds against Leishmania parasite actin in future. Together, this work provides a detailed molecular level understanding of new mechanism by which the dynamics of actin cytoskeleton are regulated in eukaryotic organisms. This work also provides insight into the evolution of the actin cytoskeleton and its regulatory mechanisms. Finally, the findings presented here help understanding the basic mechanisms of biological processes, and also provide molecular level understanding of the mechanisms of human diseases.Solut ovat elämän perusyksiköitä, jotka reagoivat ympäristössä tapahtuviin muutoksiin solun ulkoisten tai sisäisten viestien sanelemana. Näiden seurauksena solut voivat liikkua ympäristössään, muuttaa muotoaan ja vaihtaa molekyylejä ympäristönsä kanssa. Nämä solun perustoiminnot ovat riippuvaisia solujen aktiinitukirangasta. Tämä pääasiassa aktiini-nimisestä proteiinista koostuva verkosto on jokaisen aitotumallisen solun erityisrakenne, joka vaihtelee aktiivisesti monomeerisen ja säikeisen muodon välillä. Aktiinin säikeistyminen tuottaa voimaa solukalvojen ja soluelinten muodonmuutoksille sekä uudelleenjärjestelylle. Tästä syystä aktiinitukiranka on tärkeä useissa erilaisissa solutoiminnoissa, solujakautumisesta solujen liikkumiseen ja muodonmuutoksiin, sekä ekso- ja endosytoosiin. Aktiinimolekyylit sitovat ATP:tä ja säikeistyvät kaksinapaisiksi rakenteiksi. Tasapainotilassa aktiinisäikeet kasvavat liittämällä ATP-tilassa olevia aktiinimonomeereja ’plus’ (+) päähän, minkä jälkeen ne muuntuvat ATP-hydrolyysireaktion kautta ADP-tilaan ja purkautuvat hitaasti säikeen ’miinus’ (-) päästä. Tämä aktiinisäikeiden perusominaisuus kasvaa (+) päistä ja pienentyä (-) päistä on tärkeää kohdennetussa voiman tuotossa. Solut tarvitsevatkin jatkuvasti ATP-tilassa olevia aktiinimonomeerejä kierrättämällä niitä ’vanhoista’ aktiinisäikeistä ADP/ATP-nukleotidin vaihtoreaktiossa. Näitä aktiinin kierrätysvaiheita säätelevät soluissa useat proteiinit, mukaan lukien ADF/kofiliini ja syklaasiin-assosioituva proteiini. Kyseisten säätelytekijöiden molekulaariset perustat aktiinin kierrätykselle ovat kuitenkin huonosti ymmärrettyjä. Tässä tutkielmassa tutkin molekylaarisia mekanismeja, joilla aktiinisäikeitä säädellään aktiivisesti säikeisen ja monomeerisen tilan välillä. Syklaasiin-assosioituva proteiini on useista domeeneista koostuva proteiini, joka on aiemmin havaittu tärkeäksi aktiinin säätelytekijäksi eri malliorganismeissa. Tärkeydestään huolimatta tämän proteiinin molekylaarinen perusta aktiinitukirangan säätelyssä on huonosti ymmärretty. Tutkielmani ensimmäisessä ja toisessa osatyössä selvitin syklaasiin-assosioituvan proteiinin ja aktiinin välisiä vuorovaikutuksia hyödyntäen röntgensädekristallografiaa ja biokemiallisia aktiivisuuskokeita soluista eristetyillä proteiineilla. Tämän tutkielman tulokset osoittavat molekyylitason mekanismin, joilla syklaasiin-assosioituvan proteiinin aminopäässä oleva domeeni vuorovaikuttaa aktiinisäikeiden (-) pään aktiinimolekyyleihin niiden purkautumista nopeuttavalla tavalla, mikä on riippuvaista ADF/kofiliinin ja aktiinin välisistä vuorovaikutuksista. Lisäksi nämä tulokset osoittavat molekyylitasolla miten syklaasiin-assosioituvan proteiinin karboksipäässä oleva domeeni vastaanottaa (-) päästä purettuja ADP-tilassa olevia aktiinimolekyylejä, ja katalysoi niiden ADP/ATP-nukleotidivaihtoreaktiota. Nämä tulokset havainnollistavat molekyylitasolla miksi syklaasiin-assosioituva proteiini on elintärkeä tekijä aktiinitukirangan säätelyssä aitotumallisissa soluissa. Tutkielmani neljännen osatyön tulokset osoittavat kuinka aktiinitukirankaa säädellään Leishmania-loisen soluissa. Hyödyntäen apunani kolmannessa osatyössä kehitettyjä työkaluja, kryoelektronimikroskopiaa ja biokemiallisia aktiviisuuskokeita, osoitimme että Leishmania-parasiitin aktiinisäikeet purkautuvat luonnostaan hyvin nopeasti ja ovat erityisesti alttiita ADF/kofiliinin säätelylle. Pystyimme myös selvittämään näiden erityisominaisuuksien molekylaarisen perustan. Työni tulokset lisäävät ymmärrystä aktiinitukirangan evoluutiosta ja voivat auttaa tulevaisuudessa kehittämään Leishmania-loisen aktiinitukirangan toimintaa estäviä yhdisteitä. Tämä väitöskirjatyö havainnollistaa uusia molekyylitason mekanismeja, joilla aktiinisäikeitä säädellään aitotumallisissa soluissa. Lisäksi tämä työ lisää ymmärrystä aktiinitukirangan muutoksista evoluutiossa sekä sen säätelystä aitotumallisissa soluissa. Nämä havainnot ovat tärkeitä solun perusmekanismien ymmärtämisessä, ja voivat siten auttaa myös ymmärtämään eri sairauksien perusmekanismeja

Mechanism of Borrelia immune evasion by FhbA-related proteins

Author summaryRelapsing fever and Lyme Disease are infectious diseases caused by borrelia bacteria. Relapsing fever occurs sporadically worldwide, whereas distribution of Lyme Disease is restricted to the Northern Hemisphere. Both infections are transmitted to humans by blood eating ticks or lice. These infections are often difficult to diagnose due to nonspecific symptoms. To be able to cause infection, borrelia must circumvent the human immune responses. Here we describe a mechanism, how borrelia bacteria protect themselves in the human host by utilizing host proteins. By using X-ray crystallography, we solved the structure of an outer membrane protein FhbA from a relapsing fever causing borreliae, Borrelia hermsii, in complex with human complement regulator factor H. FhbA has a unique alpha-helical fold that has not been reported earlier. The structure of the complex revealed how FhbA binds factor H in a very specific manner. Factor H bound to FhbA on the surface of borrelia protects bacteria from the complement system and lysis. Based on the structure, we performed structure-guided sequence database analysis, which suggests that similar proteins are present in all relapsing fever causing borrelia and possibly in some Lyme disease agents. Immune evasion facilitates survival of Borrelia, leading to infections like relapsing fever and Lyme disease. Important mechanism for complement evasion is acquisition of the main host complement inhibitor, factor H (FH). By determining the 2.2 angstrom crystal structure of Factor H binding protein A (FhbA) from Borrelia hermsii in complex with FH domains 19-20, combined with extensive mutagenesis, we identified the structural mechanism by which B. hermsii utilizes FhbA in immune evasion. Moreover, structure-guided sequence database analysis identified a new family of FhbA-related immune evasion molecules from Lyme disease and relapsing fever Borrelia. Conserved FH-binding mechanism within the FhbA-family was verified by analysis of a novel FH-binding protein from B. duttonii. By sequence analysis, we were able to group FH-binding proteins of Borrelia into four distinct phyletic types and identified novel putative FH-binding proteins. The conserved FH-binding mechanism of the FhbA-related proteins could aid in developing new approaches to inhibit virulence and complement resistance in Borrelia.Peer reviewe

Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

Coordinated polymerization of actin filaments provides force for cell migration, morphogenesis and endocytosis. Capping protein (CP) is a central regulator of actin dynamics in all eukaryotes. It binds to actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. However, in cells, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism remains unclear. Here, we report that the conserved cytoskeletal regulator twinfilin is responsible for CP’s rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association between CP and cellular F-actin arrays, as well as to its retrograde movement throughout leading-edge lamellipodia. These were accompanied by diminished F-actin turnover rates. In vitro single-filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while enabling subsequent filament depolymerization. These results uncover a bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells.Peer reviewe

Regulation of INF2-mediated actin polymerization through site-specific lysine acetylation of actin itself

INF2 is a formin protein that accelerates actin polymerization. A common mechanism for formin regulation is autoinhibition, through interaction between the N-terminal diaphanous inhibitory domain (DID) and C-terminal diaphanous autoregulatory domain (DAD). We recently showed that INF2 uses a variant of this mechanism that we term "facilitated autoinhibition," whereby a complex consisting of cyclase-associated protein (CAP) bound to lysine-acetylated actin (KAc-actin) is required for INF2 inhibition, in a manner requiring INF2-DID. Deacetylation of actin in the CAP/KAc-actin complex activates INF2. Here we use lysine-to-glutamine mutations as acetylmimetics to map the relevant lysines on actin for INF2 regulation, focusing on K50, K61, and K328. Biochemically, K50Q- and K61Q-actin, when bound to CAP2, inhibit full-length INF2 but not INF2 lacking DID. When not bound to CAP, these mutant actins polymerize similarly to WT-actin in the presence or absence of INF2, suggesting that the effect of the mutation is directly on INF2 regulation. In U2OS cells, K50Q- and K61Q-actin inhibit INF2-mediated actin polymerization when expressed at low levels. Direct-binding studies show that the CAP WH2 domain binds INF2-DID with submicromolar affinity but has weak affinity for actin monomers, while INF2-DAD binds CAP/K50Q-actin 5-fold better than CAP/WT-actin. Actin in complex with full-length CAP2 is predominately ATP-bound. These interactions suggest an inhibition model whereby CAP/KAc-actin serves as a bridge between INF2 DID and DAD. In U2OS cells, INF2 is 90-fold and 5-fold less abundant than CAP1 and CAP2, respectively, suggesting that there is sufficient CAP for full INF2 inhibition.Peer reviewe

Structural basis of rapid actin dynamics in the evolutionarily divergent Leishmania parasite

The authors report here the structure-function analysis of highly divergent actin from Leishmania parasite. The study reveals remarkably rapid dynamics of parasite actin as well as the underlying molecular basis, thus providing insight into evolution of the actin cytoskeleton. Actin polymerization generates forces for cellular processes throughout the eukaryotic kingdom, but our understanding of the 'ancient' actin turnover machineries is limited. We show that, despite > 1 billion years of evolution, pathogenic Leishmania major parasite and mammalian actins share the same overall fold and co-polymerize with each other. Interestingly, Leishmania harbors a simple actin-regulatory machinery that lacks cofilin 'cofactors', which accelerate filament disassembly in higher eukaryotes. By applying single-filament biochemistry we discovered that, compared to mammalian proteins, Leishmania actin filaments depolymerize more rapidly from both ends, and are severed > 100-fold more efficiently by cofilin. Our high-resolution cryo-EM structures of Leishmania ADP-, ADP-Pi- and cofilin-actin filaments identify specific features at actin subunit interfaces and cofilin-actin interactions that explain the unusually rapid dynamics of parasite actin filaments. Our findings reveal how divergent parasites achieve rapid actin dynamics using a remarkably simple set of actin-binding proteins, and elucidate evolution of the actin cytoskeleton.Peer reviewe

Structural basis underlying specific biochemical activities of non-muscle tropomyosin isoforms

Publisher Copyright: © 2022 The Author(s)The actin cytoskeleton is critical for cell migration, morphogenesis, endocytosis, organelle dynamics, and cytokinesis. To support diverse cellular processes, actin filaments form a variety of structures with specific architectures and dynamic properties. Key proteins specifying actin filaments are tropomyosins. Non-muscle cells express several functionally non-redundant tropomyosin isoforms, which differentially control the interactions of other proteins, including myosins and ADF/cofilin, with actin filaments. However, the underlying molecular mechanisms have remained elusive. By determining the cryogenic electron microscopy structures of actin filaments decorated by two functionally distinct non-muscle tropomyosin isoforms, Tpm1.6 and Tpm3.2, we reveal that actin filament conformation remains unaffected upon binding. However, Tpm1.6 and Tpm3.2 follow different paths along the actin filament major groove, providing an explanation for their incapability to co-polymerize on actin filaments. We also elucidate the molecular basis underlying specific roles of Tpm1.6 and Tpm3.2 in myosin II activation and protecting actin filaments from ADF/cofilin-catalyzed severing.Peer reviewe

Tropomyosin Isoforms Specify Functionally Distinct Actin Filament Populations In Vitro

Actin filaments assemble into a variety of networks to provide force for diverse cellular processes [1]. Tropomyosins are coiled-coil dimers that form head-to-tail polymers along actin filaments and regulate interactions of other proteins, including actin-de polymerizing factor (ADF)/cofilins and myosins, with actin [2-5]. In mammals, >40 tropomyosin isoforms can be generated through alternative splicing from four tropomyosin genes. Different isoforms display non-redundant functions and partially non-overlapping localization patterns, for example within the stress fiber network [6, 7]. Based on cell biological studies, it was thus proposed that tropomyosin isoforms may specify the functional properties of different actin filament populations [2]. To test this hypothesis, we analyzed the properties of actin filaments decorated by stress-fiber-associated tropomyosins (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2). These proteins bound F-actin with high affinity and competed with a-actinin for actin filament binding. Importantly, total internal reflection fluorescence (TIRF) microscopy of fluorescently tagged proteins revealed that most tropomyosin isoforms cannot co-polymerize with each other on actin filaments. These isoforms also bind actin with different dynamics, which correlate with their effects on actin-binding proteins. The long isoforms Tpm1.6 and Tpm1.7 displayed stable interactions with actin filaments and protected filaments from ADF/cofilin-mediated disassembly, but did not activate non-muscle myosin Ila (NMIIa). In contrast, the short isoforms Tpm3.1, Tpm3.2, and Tpm4.2 displayed rapid dynamics on actin filaments and stimulated the ATPase activity of NMIla, but did not efficiently protect filaments from ADF/cofilin. Together, these data provide experimental evidence that tropomyosin isoforms segregate to different actin filaments and specify functional properties of distinct actin filament populations.Peer reviewe

Mechanism of synergistic actin filament pointed end depolymerization by cyclase-associated protein and cofilin

The ability of cells to generate forces through actin filament turnover was an early adaptation in evolution. While much is known about how actin filaments grow, mechanisms of their disassembly are incompletely understood. The best-characterized actin disassembly factors are the cofilin family proteins, which increase cytoskeletal dynamics by severing actin filaments. However, the mechanism by which severed actin filaments are recycled back to monomeric form has remained enigmatic. We report that cyclase-associated-protein (CAP) works in synergy with cofilin to accelerate actin filament depolymerization by nearly 100-fold. Structural work uncovers the molecular mechanism by which CAP interacts with actin filament pointed end to destabilize the interface between terminal actin subunits, and subsequently recycles the newly-depolymerized actin monomer for the next round of filament assembly. These findings establish CAP as a molecular machine promoting rapid actin filament depolymerization and monomer recycling, and explain why CAP is critical for actin-dependent processes in all eukaryotes.Peer reviewe

Leishmania profilin interacts with actin through an unusual structural mechanism to control cytoskeletal dynamics in parasites

Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids

Large-scale long-term passive-acoustic monitoring reveals spatio-temporal activity patterns of boreal bats

The distribution ranges and spatio-temporal patterns in the occurrence and activity of boreal bats are yet largely unknown due to their cryptic lifestyle and lack of suitable and efficient study methods. We approached the issue by establishing a permanent passive-acoustic sampling setup spanning the area of Finland to gain an understanding on how latitude affects bat species composition and activity patterns in northern Europe. The recorded bat calls were semi-automatically identified for three target taxa; Myotis spp., Eptesicus nilssonii or Pipistrellus nathusii and the seasonal activity patterns were modeled for each taxa across the seven sampling years (2015-2021). We found an increase in activity since 2015 for E. nilssonii and Myotis spp. For E. nilssonii and Myotis spp. we found significant latitude -dependent seasonal activity patterns, where seasonal variation in patterns appeared stronger in the north. Over the years, activity of P. nathusii increased during activity peak in June and late season but decreased in mid season. We found the passive-acoustic monitoring network to be an effective and cost-efficient method for gathering bat activity data to analyze spatio-temporal patterns. Long-term data on the composition and dynamics of bat communities facilitates better estimates of abundances and population trend directions for conservation purposes and predicting the effects of climate change