Computational Modeling of 3D Actin Organization through Polymerization, Turnover, Crosslinking, and Motor Pulling

The cellular actin cytoskeleton is an intricate system of actin filaments that supports cell morphology and is crucial for numerous cell functions including cell growth and cell division. Among the most important actin cytoskeleton structures are actin cables and cytokinetic ring, which are bundles of actin filaments. The actin cables span the cell and serve as tracks for vesicle transport while the actin cytokinetic ring forms in the middle and constricts to divide the cell. The focus of my work is to gain a quantitative understanding of how such cables and ring are formed, what are the essential components, how overexpression and underexpression of certain proteins will affect the structure and dynamics. I built a 3D computational model that starts out from the basic physical and mechanical properties of actin filaments and accounts for known interactions with other proteins, to reproduce the experimental observations of the actin cytoskeleton in different cell systems and further make testable predictions for cell mutants.First, I modeled individual actin filament as a semiflexible worm like chain. I coarse grained the filamentous actin segment using a bead-spring model with spring, bending and thermal forces. This model represents of the actin filament\u27s spatial and dynamical properties. I tested that the model reproduces the correct persistence length, relaxation dynamics and equipartition of energy.Second, to obtain a quantitative understanding of these actin structures and dynamics in fission yeast, I extended the individual actin filament model and added actin-interacting factors. Polymerization out of formin cortical sites, bundling by cross-linkers, pulling by type V myosin, and severing by cofilin, are simulated as growth, cross-linking, pulling and turnover of the semiflexible polymers. With the above mechanisms my quantitative model generated actin cable structures and dynamics similar to those observed in live cell experiments. The simulations reproduced the particular actin cable structures in myoVΔ cells and predicted the effect of increased myosin V pulling. I found that increasing cross-linking parameters generated thicker actin cables and led to anti-parallel and parallel phases with straight or curved cables. I further analyzed the cable number, curvature and loop occurrences of experimental images of cells overexpressing crosslinkers and cell treated with drugs that depolymerize actin patches, provided by our collaborator Damien Laporte. Our predictions are in quantitative agreement with the experiments. Furthermore, the model predicts that clustering of formins at cell tips promotes actin cable formation.Third, I adapted the actin cable model to budding yeast, another well-studied model organism. Budding yeast differs from fission yeast in that it has a more complex geometry and different types of interacting proteins. I refined the previous fission yeast actin cable model by considering a more accurate model of orientation-dependent crosslinking by fimbrin and a more accurate aging mechanism for turnover. It also included polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosins stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which I suggest as a model for the assembly of actin filaments to the contractile ring.Finally, my colleague Dr. Tamara Bidone and I further extended the model to simulate the actin contractile ring. We showed that the ring formation region in parameter space lies close to regions leading to clumps, meshworks or double rings, and stars/cables, which are consistent with prior experiments with mutations that alter the morphology of the condensing network. We also quantified tension along actin filaments and forces on nodes during ring assembly and showed that the mechanisms describing ring assembly can also drive ring constriction once the ring is formed.In summary, this work provides a numerical way to study the morphology and dynamics of the actin cytoskeleton in model cell organisms. Combining simulated, analytical and experimental results, the proposed model with minimal set of interactions successfully reproduced experimental observations and made predictions for further studies

UNCOVERING FUNDAMENTAL MECHANISMS OF ACTOMYOSIN CONTRACTILITY USING ANALYTICAL THEORY AND COMPUTER SIMULATION

Actomyosin contractility is a ubiquitous force-generating function of almost all eukaryotic organisms. While more understanding of its dynamic non-equilibrium be- havior has been uncovered in recent years, little is known regarding its self-emergent structures and phase transitions that are observed in vivo. With this in mind, this thesis aims to develop a state-of-the-art computational model for the simulation of actomyosin assemblies, containing detailed cytosolic reaction-diffusion processes such as actin filament treadmilling, cross-linker (un)binding, and molecular motor walking. This is explicitly coupled with novel mechanical potentials for semi-flexible actin filaments. Then, using this simulation framework combined with other ana- lytical approaches, we propose a novel mechanism of contractility in a fundamental actomyosin structural element, derived from a thermodynamic free energy gradi- ent favoring overlapped actin filament states when passive cross-linkers are present. With this spontaneous cross-linking, transient motors such as non-muscle myosin II can generate robust network contractility in a collective myosin II-cross-linker ratcheting mechanism. Finally, we map the phases of contractile behavior of disor- dered actomyosin using this theory, showing explicitly the cross-linking, motor and boundary conditions required for geometric collapse or tension generation in a net- work comprised of those elements. In this theory, we move away from the sarcomeric contractility mechanism typically reconciled in disordered non-muscle structures. It is our hope that this study adds theoretical knowledge as well as computational tools to study the diverse contractile assemblies found in non-muscle actomyosin networks

Dynamics of Competing Structures and Actin Binding Proteins

Actin proteins polymerize into many different filamentous structures within individual cells. These actin structures coexist, each playing a significant role in the function of cells. The biophysical basis of this competition however remains an area in need of further investigation. In fission yeast actin patches (nucleated by the Arp2/3 protein complex) and actin cables (polymerized by formin proteins) coexist and regulate endocytosis and cell tip growth, respectively. The available quantitative data and the existence of only two distinct actin structures offer the possibility of using fission yeast as model system to develop quantitative mathematical models to study the interdependence of actin cytoskeleton structures in cells. Recent experimental studies have shown that actin patches and actin cables compete for the same pool of monomeric actin under the regulation of many proteins such as profilin, fimbrin, cofilin, and tropomyosin. To quantify this competition, we developed a mathematical model using a set of differential equations. The model incorporates the most important regulatory factors revealed by prior experiments while using a minimal set of parameter values. In the model actin can be distributed in three pools: patches, cables and cytoplasm. The Arp2/3 complex contributes to patch nucleation and is consumed in patches. Fimbrin and cofilin incorporate in patches and cables and regulate patch and cable lifetime. Profilin binds to actin monomers in the cytoplasm and regulates the elongation rate of actin filaments in cables.The model captured the main qualitative and quantitative trends in several prior experimental studies, such as the observed increase in ectopic actin filament bundles upon treatment with the drug CK-666 that disassembles actin patches. It can also capture the change in actin patches and actin cables upon underexpression/overexpression of actin , in combination with CK-666, as well as the increase in actin patch number in cofilin and formin mutants. The model can also describe the change in patch number in experiments of profilin overexpression. The model provides predictions that can be tested in future experiments and illustrates the degree of complexity of mutual dependencies among actin cytoskeletal structures.The development of actin networks of different structure and morphology depends on many proteins that regulate the dynamics of actin filaments, such as their length, lifetime and binding interactions. In particular, several actin filament side-binding proteins can sever, stabilize or bundle actin filaments. In this study we focused on three of these proteins, namely tropomyosin, cofilin and fimbrin, which are found in many actin cytoskeletal structures. Recent in vitro studies have shown that their actin side-binding dynamics are affected in the presence of each other. In order to study the kinetics and organization of these competing binders along actin filaments, we use stochastic simulations. In the model the actin filament is represented as two independent lattices, representing the two protofilaments of the actin filament double helix. For simplicity, we neglected the mutual dependence between the bound proteins of one protofilament to the other. In accordance with prior in vitro experiments, in our model the binding of a protein to one or more (for the case of tropomyosin) lattice units excludes the binding of proteins of different or same type to these lattice units. Taking into account their actin binding cooperativity properties, we parametrized the model by fitting prior experimental data and using parameters from previous models. The model reveals the range of concentrations where one protein dominates against the other from the start of the simulation until equilibrium but also areas of concentrations where there is a shift of the dominating protein between early times and equilibrium We find concentration ranges where initially tropomyosin occupies a large portion of the lattice but then either cofilin or fimbrin dominate the equilibrium state. In these cases, we find that while initially cofilin or fimbrin bind in smaller numbers than tropomyosin, they create boundaries that don’t allow for long stable tropomyosin chains, so in time tropomyosin is being removed by the lattice. Simulations of actin polymerization that includes tropomyosin, cofilin and fimbrin showed that fimbrin inhibits the elongation of tropomyosin chains on early times, allowing the binding of cofilin on sites where the actin filament hasn’t released Pi

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

Syklaasiin assosioituva proteiini (CAP) säätelee aktiini-tukirangan dynamiikkaa solujen liikkumisen ja morfogeneesin yhteydessä.

The highly dynamic remodeling of the actin cytoskeleton is responsible for most motile and morphogenetic processes in all eukaryotic cells. In order to generate appropriate spatial and temporal movements, the actin dynamics must be under tight control of an array of actin binding proteins (ABPs). Many proteins have been shown to play a specific role in actin filament growth or disassembly of older filaments. Very little is known about the proteins affecting recycling i.e. the step where newly depolymerized actin monomers are funneled into new rounds of filament assembly. A central protein family involved in the regulation of actin turnover is cyclase-associated proteins (CAP, called Srv2 in budding yeast). This 50-60 kDa protein was first identified from yeast as a suppressor of an activated RAS-allele and a factor associated with adenylyl cyclase. The CAP proteins harbor N-terminal coiled-coil (cc) domain, originally identified as a site for adenylyl cyclase binding. In the N-terminal half is also a 14-3-3 like domain, which is followed by central proline-rich domains and the WH2 domain. In the C-terminal end locates the highly conserved ADP-G-actin binding domain. In this study, we identified two previously suggested but poorly characterized interaction partners for Srv2/CAP: profilin and ADF/cofilin. Profilins are small proteins (12-16 kDa) that bind ATP-actin monomers and promote the nucleotide exchange of actin. The profilin-ATP-actin complex can be directly targeted to the growth of the filament barbed ends capped by Ena/VASP or formins. ADF/cofilins are also small (13-19 kDa) and highly conserved actin binding proteins. They depolymerize ADP-actin monomers from filament pointed ends and remain bound to ADP-actin strongly inhibiting nucleotide exchange. We revealed that the ADP-actin-cofilin complex is able to directly interact with the 14-3-3 like domain at the N-terminal region of Srv2/CAP. The C-terminal high affinity ADP-actin binding site of Srv2/CAP competes with cofilin for an actin monomer. Cofilin can thus be released from Srv2/CAP for the subsequent round of depolymerization. We also revealed that profilin interacts with the first proline-rich region of Srv2/CAP and that the binding occurs simultaneously with ADP-actin binding to C-terminal domain of Srv2/CAP. Both profilin and Srv2/CAP can promote nucleotide exchange of actin monomer. Because profilin has much higher affinity to ATP-actin than Srv2/CAP, the ATP-actin-profilin complex is released for filament polymerization. While a disruption of cofilin binding in yeast Srv2/CAP produces a severe phenotype comparable to Srv2/CAP deletion, an impairment of profilin binding from Srv2/CAP results in much milder phenotype. This suggests that the interaction with cofilin is essential for the function of Srv2/CAP, whereas profilin can also promote its function without direct interaction with Srv2/CAP. We also show that two CAP isoforms with specific expression patterns are present in mice. CAP1 is the major isoform in most tissues, while CAP2 is predominantly expressed in muscles. Deletion of CAP1 from non-muscle cells results in severe actin phenotype accompanied with mislocalization of cofilin to cytoplasmic aggregates. Together these studies suggest that Srv2/CAP recycles actin monomers from cofilin to profilin and thus it plays a central role in actin dynamics in both yeast and mammalian cells.Kaikista aitotumallisista soluista löytyvä solutukiranka koostuu erilaisista säikeisistä proteiineista, jotka täyttävät solun sisäisen tilan ylläpitäen solun muotoa ja auttaen solua kestämään mekaanista rasitusta. Eräs keskeinen solutukirangan proteiini on nimeltään aktiini. Aktiinilla muodostaa säikeitä, jotka samanaikaisesti kasvavat niin sanotuista plus- ja hajoavat vastakkaisista miinus-päistä. Liikkumattomassa hiivasolussa aktiini-säikeillä on tärkeä tehtävä endosytoosissa, kalvorakkuloiden kuljetuksessa solun sisällä sekä solunjakautumisessa. Eläinsoluissa aktiini osallistuu näiden toimintojen lisäksi solun liikkumiseen. Solun työntyessä eteenpäin kasvavat aktiini-säikeiden plus-päät painavat solukalvoa eteenpäin. Säädelläkseen aktiinitukirangan muutoksia, solulla on laaja kirjo aktiinia sitovia proteiineja. Nämä proteiinit aikaansaavat mm. aktiini-säikeiden rakentumista, hajoamista, haaroittumista ja kiinnittymistä toisiinsa. Näitä ilmiöitä on tutkittu paljon, mutta siitä, miten aktiini-monomeerit saadaan ohjattua kasvaviin aktiini-säikeiden päihin, tiedetään hyvin vähän. Yksi keskeinen aktiinin kierrätykseen osallistuva proteiini on nimeltään syklaasiin assosioituva proteiini, CAP (cyclase-associated protein). Hiivan vastaavasta proteiinista käytetään nimeä Srv2/CAP. CAP proteiinit painavat noin 50-60 kilodaltonia ja koostuvat useista toiminnallisista domeenista. Proteiinin amino-terminaalisessa päässä sijaitsee cc(coiled-coil)-domeeni, jota seuraa 14-3-3-domeeni. Tämän jälkeen proteiinissa on kaksi proliini-rikasta aluetta sekä WH2-domeeni. Karboksi-terminaalisessa domeenissa sijaitsee aiemmin karakterisoitu konservoitunut ADP-aktiinin sitomisalue. Tässä väitöskirjatyössä tutkittiin kahden tunnetun aktiinia sitovan proteiinin, profiliinin ja ADF/kofiliinin, sitoutumista Srv2/CAP:iin, sekä niiden yhteistyötä aktiini-tukirangan dynamiikassa. Profiliinit ovat pieniä (12-15 kDa) ATP-aktiinia sitovia proteiineja, joilla on tärkeä tehtävä aktiiniin sitoutuneen nukleotidin, ADP:n, vaihtamisessa ATP-muotoon. ADF/kofiliinit ovat myös pieniä (13-19 kDa), konservoituneita proteiineja, jotka sitoutuvat voimakkaasti ADP-aktiiniin ja hajottavat aktiinisäikeitä. Tutkimuksessa saimme selville Srv2/CAP:in sitoutuvan suoraan hiivan kofiliiniin, mutta vain, jos tähän on sitoutuneena myös ADP-aktiini. Tuloksemme viittaavat siihen, että tämän jälkeen Srv2/CAP:n karboksi-terminaalinen aktiinia sitoa alue kilpailee ADP-aktiinin kofiliinilta. Koska Srv2/CAP sitoutuu hyvin heikosti pelkkään kofiliiniin, tämä todennäköisesti irtoaa kompleksista ja on taas vapaana toistamaan omaa tehtäväänsä. Totesimme myös profiliinin sitoutuvan Srv2/CAP:in ensimmäiseen proliini-rikkaaseen alueeseen samanaikaisesti kun ADP-aktiini on sitoutuneena Srv2/CAP:iin. Aktiinin nukleotidi vaihdetaan ATP:ksi, joko profiliinin tai Srv2/CAP:n toimesta. Profiliini sitoutuu voimakkaasti ATP-aktiiniin ja irtoaa Srv2/CAP:stä. Vapautuvia Profiliini-ATP-aktiini komplekseja käytetään solussa kontrolloituun aktiini-säikeiden kasvuun. Kofiliinin sitomisen poistaminen Srv2/CAP:sta aiheuttaa hiivassa vakavan fenotyypin, johon liittyy ongelmia mm. solun kasvussa ja aktiinirakenteiden muodostumisessa. Tästä voi päätellä kofiliinin sitomisen olevan hyvin keskeinen ominaisuus Srv2/CAP:in toiminnalle. Tutkimuksissamme hiiren CAP proteiineilla, CAP1:llä ja CAP2:lla, havaitsimme CAP2:n ilmentymisen rajoittuvan lihassoluihin, kun taas CAP1 ilmentyi kaikissa muissa solutyypeissä. CAP1:in poistaminen hiiren melanoomasoluista aiheutti kofiliinin kasaantumisen aktiinin kanssa epänormaaleihin kerääntymiin. Nämä tutkimukset osoittavat CAP:in olevan keskeinen komponentti kofiliinin ja aktiinin kierrätyksessä niin hiiva- kuin eläinsoluissakin

Dynamics of epithelial gap closure using microfabrication and micromechanical approaches

Les cellules peuvent migrer sous différentes conditions qui dépendent de l environnement biochimique ou mécanique. Connaître les mécanismes de la migration, les protéines impliquées et leur régulation est essentiel pour comprendre les processus de morphogénèse ou certaines situations pathologiques. Dans ce contexte, la migration collective des cellules est un processus clé qui intervient pendant le développement ainsi que dans la vie adulte. Elle joue un rôle très important pour la formation et l entretien des couches épithéliales, notamment au cours du développement embryonnaire et pendant la cicatrisation des trous épithéliaux résultant, par exemple, d une blessure. Lorsque l épithélium présente une discontinuité, des mécanismes actifs qui impliquent une migration coordonnée des cellules sont nécessaires pour préserver l intégrité des tissus. Dans ce travail, nous avons étudié les mécanismes impliqués dans la fermeture des trous dans un épithélium. Pour des blessures de faible taille, le mode de fermeture dit de purse string est souvent évoqué, impliquant la contraction d un anneau contractile d acto-myosine qui ferme la blessure. Pour des blessures de tailles plus importantes, il est courant d observer un mécanisme différent conduisant { la migration active des cellules du bord qui couvrent la surface libre .Pour étudier ces aspects de manière quantitative et reproductible, nous avons développé une nouvelle méthode basée sur des techniques de microfabrication et de lithographie dite molle qui permet de faire une étude quantitative de la fermeture des trous épithéliaux. Nous avons fabriqué des substrats de micropiliers de diamètre et de forme variés dans les quels les cellules sont libres de pousser entre les microstructures. Lorsqu elles sont parvenues à confluence, on retire le substrat qui laisse apparaître des trous contrôlés.De cette manière, nous avons observé que les cellules épithéliales forment des lamellipodes pour la fermeture de ces trous. Le mécanisme de fermeture dépend de la taille des trous et nous avons pu observer différents régimes en fonction de diamètre des piliers. Les trous petits (de la taille d une seule cellule) sont fermés par un mécanisme passif alors que la fermeture de trous plus larges nécessite un mécanisme actif de migration conduisant à la formation de lamellipodes et à des modes de migration collective. Par la suite, nous nous sommes intéressés à l aspect mécanique de la fermeture des trous épithéliaux. Pour cela, nous avons utilisé un système d ablation laser pour rompre quelques cellules dans une monocouche épithéliale. Nous avons alors mesuré les forces de traction que les cellules exercent au substrat et leur évolution temporelle et spatiale. Nous avons pu mettre en évidence différents modes de traction: au début, les cellules exercent des forces de traction importantes sur leur substrat pour laisser place à des contraintes mécaniques qui sont davantage issues d un processus collectif au travers de la formation d un câble multicellulaire qui les relie les cellules de bord entre elles. En conclusion, ce travail nous a permis d obtenir des informations sur les mécanismes dynamiques de fermeture des tissus épithéliaux qui sont évidemment impliqués dans la cicatrisation des blessures mais aussi dans certains problèmes de malformations congénitales lors l embryogenèse.Most cells migrate under the appropriate conditions or stimuli; understanding the mechanisms of migration, the players involved, and their regulation, is pivotal to tackle the pathological situations where migration becomes an undesired effect. While largely overshadowed by the study of single cell migration, collective cell migration is a very relevant process that takes place during development as well as in adult life. Collective migration is very relevant for the formation and maintenance of epithelial layers: extensive migratory processes occur during the shape of the embryo, as well as during the healing of a skin incision in the adult. When openings or discontinuities appear in the epithelia, it is crucial that the appropriate mechanisms are activated.In the present work we attempt at deciphering what are the mechanisms involved in gap closure. Until now, most of the literature concerning the subject has reported contradictory results, mainly arising from the complexity of the process and the lack of systematic analysis. We have designed a novel approach to address epithelial gap closure under well-defined and controlled conditions. By using our gap patterning method, we have observed that epithelial cells extend lamellipodia when exposed to a newly available space. Interestingly, we found that the closure of such gap depends on the size: small gaps are closed by a passive physical mechanism, while large gaps are closed through a Rac-dependent cell crawling mechanism, in a collective migration-like manner. 11Abstract (English)Most cells migrate under the appropriate conditions or stimuli; understanding the mechanisms of migration, the players involved, and their regulation, is pivotal to tackle the pathological situations where migration becomes an undesired effect. While largely overshadowed by the study of single cell migration, collective cell migration is a very relevant process that takes place during development as well as in adult life. Collective migration is very relevant for the formation and maintenance of epithelial layers: extensive migratory processes occur during the shape of the embryo, as well as during the healing of a skin incision in the adult. When openings or discontinuities appear in the epithelia, it is crucial that the appropriate mechanisms are activated.In the present work we attempt at deciphering what are the mechanisms involved in gap closure. Until now, most of the literature concerning the subject has reported contradictory results, mainly arising from the complexity of the process and the lack of systematic analysis. We have designed a novel approach to address epithelial gap closure under well-defined and controlled conditions. By using our gap patterning method, we have observed that epithelial cells extend lamellipodia when exposed to a newly available space. Interestingly, we found that the closure of such gap depends on the size: small gaps are closed by a passive physical mechanism, while large gaps are closed through a Rac-dependent cell crawling mechanism, in a collective migration-like manner. Next, we also addressed the mechanical component of epithelial gap closure. In this study, we took advantage of a laser-ablation system to disrupt some cells within an epithelial monolayer, and study how the remaining cells sealed that gap. By measuring the traction forces that cells exert on the substrate along the closure, we observed that cells first pulled on the substrate to propel themselves. By the last steps of closure, there is a transition in the direction of the force, so that cells are pulled to the center of the gap due to the assembly of a supracellular actin cable. Altogether, this work provides valuable knowledge on the current understanding of the mechanisms accounting for epithelial gap closure. We believe that a better comprehension of these mechanisms can help to shed light in clinically relevant situations where epithelial gap closure is impaired.PARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF