Revising the actin disassembly machinery : The role of GMF and twinfilin in turnover of dendritic actin arrays

Polymerization of actin filaments against cellular membranes and contraction of actomyosin fibers generate pushing and pulling forces for cell migration, endocytosis, cell division, and maintenance of cell morphology, as well as for intracellular motility and morphogenesis of organelles. Thus, the actin cytoskeleton is a fundamental cellular component in development, immune responses, and in several other aspects of physiology. Moreover, the actin cytoskeleton is hijacked by viruses and pathogens during the infection process. Owing to their central role in above-mentioned cellular processes, actin and actin-binding proteins have been in the limelight of cancer research. Actin is a globular protein, which can polymerize into filaments and depolymerize back to monomers. Dozens of actin binding proteins regulate actin dynamics in cells. Whereas regulation of actin filament nucleation and filament elongation are relatively well understood, the disassembly is far more enigmatic topic. ADF/cofilin is the key actin disassembly factor. It belongs to a family of six actin depolymerizing homology (ADF-H) domain proteins, which all interact with actin or actin-related proteins. However, apart from ADF/cofilin, biochemical and cellular functions of the members of this protein family have remained elusive. In this work, I studied the cellular and biochemical roles of two ADF-H domain proteins, glia maturation factor (GMF) and twinfilin. I show that they both promote the disassembly of dendritic actin networks in cells, but by distinct mechanisms. GMF, which binds actin-related proteins (Arp) in the Arp2/3 complex, debranches dendritic actin networks in vitro. The data presented here show that GMF regulates the dynamics of lamellipodial, dendritic actin network in Drosophila cells and promotes collective border cell migration in vivo. Moreover, Drosophila GMF display a strong genetic interaction in cells and in vivo with another actin-regulatory protein, actin-interacting protein 1 (Aip1), indicating that they facilitate actin disassembly in a synergistic manner. Twinfilin interacts with actin monomers and actin filament barbed ends to inhibit actin polymerization. Moreover, it binds heterodimeric Capping Protein (CP) and membrane phosphatidylinositol phosphates (PIPs), which inhibit the actin-binding function of twinfilin. However, the molecular mechanism of this interaction has remained unknown. Thus, in the second part of the thesis I utilized a combination of mutagenesis and biochemistry, supplemented with molecular dynamics simulations, to reveal how PIPs inhibit twinfilin. Interestingly, twinfilin interacts with PIPs with a two-step mechanism. First, the CP-interaction motif in the carboxy-terminal (C-terminal) tail of twinfilin anchors the protein to plasma membrane. Subsequently, the actin-binding interface interacts with lipids, leading to inhibition of both the CP- and actin-binding activities of twinfilin. Cellular functions of twinfilin have remained elusive despite extensive studies in past decades. In the third part of the thesis, I generated mouse twinfilin knockout cell lines and showed that twinfilin regulates both actin and CP turnover in lamellipodia. Surprisingly, twinfilin promotes CP dynamics in cells and in vitro by uncapping filament barbed ends, thus providing an explanation why the localization of CP in cells is restricted to the very distal edge of lamellipodia. Moreover, biochemical experiments demonstrated that twinfilin itself does not accelerate filament disassembly after uncapping, but instead allows filaments to disassemble after removal of CP from actin filament barbed ends. These findings explain the diminished actin filament disassembly rates in lamellipodia of twinfilin-deficient cells. Together, the work presented here highlights the important roles of twinfilin and GMF in regulation of lamellipodial actin networks. Their distinct roles in actin disassembly show that actin turnover in dendritic arrays is maintained by several functionally different proteins which, in concert, facilitate the turnover of branched actin filament networks in cells.Aktiinisäikeiden solun kalvorakenteita vasten tuottama työntövoima sekä aktiini- ja myosiinikimppujen synnyttämä kimppujen supistumisvoima ylläpitävät muun muassa solujen liikkumista, kalvoliikennettä, jakautumista sekä solujen muodon ja rakenteen säilymistä. Näin ollen solujen aktiinitukiranka on välttämätön muun muassa yksilönkehityksessä ja immuunipuolustusjärjestelmässä. Useat virukset ja taudinaiheuttajat hyödyntävät aktiinikoneistoa päästäkseen soluun sisälle. Lisäksi aktiinitukirangan merkitys edellä mainituissa solubiologisissa tapahtumissa on tuonut aktiinin ja aktiinitukirankaa säätelevät proteiinit syöpätutkimuksen valokeilaan. Aktiini on pallomainen proteiini, joka kykenee pidentymään pitkiksi säikeiksi ja purkautumaan yksittäisiksi monomeereiksi. Aktiinisäikeiden pidentyminen ja sen säätelijät tunnetaan varsin hyvin. Sen sijaan purkautumiseen osallistuvat proteiinit ja niiden rooli on huonommin tiedossa. Useat tutkimukset viime vuosikymmeninä ovat osoittaneet, että ADF/kofiliinilla on keskeinen rooli aktiinisäikeiden purkautumisen säätelyssä. ADF/kofiliini kuuluu kuuden proteiinin muodostamaan proteiiniperheeseen, jotka kaikki sitoutuvat joko aktiiniin tai aktiinin kaltaisiin proteiineihin. Toisin kuin ADF/kofiliinin, muiden tämän perheen jäsenten biokemialliset ja solubiologiset toiminnot ovat huonosti ymmärrettyjä. Tässä tutkielmassa tutkin kahden tämän perheen proteiinin, GMF:n ja twinfiliinin, solubiologisia ja biokemiallisia toimintoja. Näytän, että ne molemmat osallistuvat haaroittuneiden aktiinisäieverkostojen purkautumiseen omilla hyvin erilaisilla tavoilla. GMF, joka sitoutuu aktiinin kaltaiseen proteiiniin (Arp) Arp2/3-kompleksissa, purkaa aktiinisäieverkostojen haaroja. Tämän tutkielman tulokset osoittavat, että GMF säätelee solun levyjalan aktiinisäikeiden kierrätystä ja on tärkeässä roolissa rajasolujen liikkumisessa banaanikärpäsen munakammion kehittymisen aikana. Lisäksi GMF:n ja toisen aktiinia säätelevän proteiinin, Aip1:n, geeniluennan samanaikainen hiljentäminen johti aktiinisäikeiden kertymiseen sekä viljellyissä soluissa että kärpäsen munakammioissa. Aiemmin on osoitettu, että twinfiliini sitoutuu sekä yksittäisiin aktiinimonomeereihin että aktiinisäikeiden nopeasti kasvaviin pluspäihin, estäen näin säikeiden pidentymistä. Tämän lisäksi twinfiliini sitoutuu aktiinisäikeiden pluspäihin sitoutuvaan CP-tulppaproteiiniin ja solukalvon PIP-lipideihin. PIP-lipidit estävät twinfiliinin sitoutumisen aktiiniin, mutta tämän säätelyn tarkka mekanismi on ollut tuntematon. Tässä työssä käytimme puhdistettuja proteiineja biokemiallisissa kokeissa sekä hyödynsimme tietokonemallinnusta selvittääksemme, miten twinfiliini sitoutuu PIP-lipideihin. Tuloksemme osoittavat, että twinfiliini sitoutuu lipideihin kaksiosaisella mekanismilla. Aluksi twinfiliinin häntä ankkuroi proteiinin solukalvoon, minkä jälkeen loppu proteiini aktiininsitoumisalueineen sitoutuu kalvoon. Näin ollen twinfiliinin kyky sitoa aktiinia ja CP:ia estyvät sen sitoutuessa solukalvon PIP-lipideihin. Twinfiliinin tarkka rooli aktiinisäikeiden säätelyssä soluissa on jäänyt toistaiseksi epäselväksi. Tässä tutkielmassa käytin hiiren soluja, joista olin estänyt twinfiliinin geenin ilmentymisen mutaatiolla, ja vertasin näitä soluja villityyppisiin soluihin. Näin osoitan, että twinfiliini säätelee sekä aktiinisäikeiden että CP:n dynamiikkaa solujen levyjaloissa. Twinfiliini poistaa CP:n aktiinisäikeiden plus-päistä ja siten edistää aktiinisäikeiden purkautumista soluissa. Tämä havainto selittää sen, miksi CP paikantuu solujen levyjalassa aivan solukalvon lähelle ja sen, miksi CP:n dynamiikka on huomattavasti nopeampaa soluissa kuin mitä sen biokemialliset ominaisuudet ennustavat. Tulokset selittävät myös sen, miksi aktiinisäikeiden lyhentyminen on hitaampaa soluissa, joista twinfiliinin ilmentyminen on estetty mutaatiolla. Tämä väitöskirjatyö osoittaa, että twinfiliinillä ja GMF:llä on tärkeä rooli solujen aktiiniverkostojen säätelyssä. Niiden hyvin erilaiset roolit aktiinisäikeiden purkautumisen säätelyssä osoittavat, että aktiinisäikeiden kierrätystä ylläpitää soluissa useat proteiinit yhteistyössä toistensa kanssa

Field biomass as global energy source

Current (1997–2006) and future (2050) global field biomass bioenergy potential was estimated based on FAO (2009) production statistics and estimations of climate change impacts on agriculture according to emission scenario B1 of IPCC. The annual energy potential of raw biomass obtained from crop residues and bioenergy crops cultivated in fields set aside from food production is at present 122–133 EJ, 86–93 EJ or 47–50 EJ, when a vegetarian, moderate or affluent diet is followed, respectively. In 2050, with changes in climate and increases in population, field bioenergy production potential could be 101–110 EJ, 57–61 EJ and 44–47 EJ, following equivalent diets. Of the potential field bioenergy production, 39–42 EJ now and 38–41 EJ in 2050 would derive from crop residues. The residue potential depends, however, on local climate, and may be considerably lower than the technically harvestable potential, when soil quality and sustainable development are considered. Arable land could be used for bioenergy crops, particularly in Australia, South and Central America and the USA. If crop production technology was improved in areas where environmental conditions allow more efficient food production, such as the former Soviet Union, large areas in Europe could also produce bioenergy in set aside fields. The realistic potential and sustainability of field bioenergy production are discussed

Uncertainty in multispectral lidar signals caused by incidence angle effects

Multispectral terrestrial laser scanning (TLS) is an emerging technology. Several manufacturers already offer commercial dual or three wavelength airborne laser scanners, while multispectral TLS is still carried out mainly with research instruments. Many of these research efforts have focused on the study of vegetation. The aim of this paper is to study the uncertainty of the measurement of spectral indices of vegetation with multispectral lidar. Using two spectral indices as examples, we find that the uncertainty is due to systematic errors caused by the wavelength dependency of laser incidence angle effects. This finding is empirical, and the error cannot be removed by modelling or instrument modification. The discovery and study of these effects has been enabled by hyperspectral and multispectral TLS, and it has become a subject of active research within the past few years. We summarize the most recent studies on multi-wavelength incidence angle effects and present new results on the effect of specular reflection from the leaf surface, and the surface structure, which have been suggested to play a key role. We also discuss the consequences to the measurement of spectral indices with multispectral TLS, and a possible correction scheme using a synthetic laser footprint.publishedVersionPeer reviewe

Urban Aerosol Particle Size Characterization in Eastern Mediterranean Conditions

Characterization of urban particle number size distribution (PNSD) has been rarely reported/performed in the Middle East. Therefore, we aimed at characterizing the PNSD (0.01–10 µm) in Amman as an example for an urban Middle Eastern environment. The daily mean submicron particle number concentration (PNSub) was 6.5 × 103–7.7 × 104 cm−3 and the monthly mean coarse mode particle number concentration (PNCoarse) was 0.9–3.8 cm−3 and both had distinguished seasonal variation. The PNSub also had a clear diurnal and weekly cycle with higher concentrations on workdays (Sunday–Thursday; over 3.3 × 104 cm−3) than on weekends (below 2.7 × 104 cm−3). The PNSub constitute of 93% ultrafine fraction (diameter < 100 nm). The mean particle number size distributions was characterized with four well-separated submicron modes (Dpg,I, Ni): nucleation (22 nm, 9.4 × 103 cm−3), Aitken (62 nm, 3.9 × 103 cm−3), accumulation (225 nm, 158 cm−3), and coarse (2.23 µm, 1.2 cm−3) in addition to a mode with small geometric mean diameter (GMD) that represented the early stage of new particle formation (NPF) events. The wind speed and temperature had major impacts on the concentrations. The PNCoarse had a U-shape with respect to wind speed and PNSub decreased with wind speed. The effect of temperature and relative humidity was complex and require further investigations

Increase in perennial forage yields driven by climate change, at Apukka Research Station, Rovaniemi, 1980-2017

The official variety trials at Rovaniemi, Finland (66.58°N, 26.01°E) in 1980–2017 show a substantial increase in dry matter yields (DMY) of timothy (Phleum pratense), meadow fescue (Festuca pratensis) and tall fescue (Festuca arundinacea), coinciding with a 156 °Cd increase in the average growing season Tsum and a 461 °Cd decrease in the average winter frost sum for the same period. The annual DMY of timothy was 3128, 4668, 8385 and 9352 kg ha-1 in the periods (P) 1980–1989 (P1), 1990–1999 (P2), 2000–2009 (P3), and 2010–2017 (P4). The first cut yielded 1792, 2166, 4008 and 4473, and the second cut 1337, 2503, 4378 and 4879 kg ha-1, respectively. Yields of meadow fescue followed a similar pattern. The first cut was about ten days and the second cut about one week earlier on P4 than on P1. Shorter snow cover period, milder winters, higher live ground cover of timothy in spring, and higher temperature sum during the growing season were most likely responsible for the yield increase. The results indicate a strong impact of climate change on DMY of perennial forage crops in the north

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