La protéine OSBP est un transporteur de lipides qui régule la distribution cellulaire du cholestérol. OSBP comprend un domaine PH, deux séquences « coiled coil », un motif FFAT (deux phénylalanines dans un environement acide), et un domaine de liaison de lipides (ORD) à son extrémité C-terminale. Le domaine PH interagit avec le PI(4)P et la petite protéine G Arf1-GTP au niveau du Golgi, alors que le motif FFAT interagit avec la protéine VAP-A, résidente du réticulum endoplasmique (RE). En liant simultanément tous ces déterminants, OSBP stabilise des sites de contact membranaire entre RE et Golgi, permettant ainsi un contre-échange cholestérol / PI(4)P par l'ORD. OSBP contient également une longue séquence N-terminale d’environ 80 aa, intrinsèquement désordonnée, composée principalement de glycine, proline et d'alanine. Nous démontrons que la présence de ce N-terminus désordonné augmente le rayon de Stoke de OSBP tronquée du domaine ORD, et limite sa densité d’association sur la membrane portant le PI(4)P. La protéine dépourvue du N terminus favorise l'agrégation symétrique des liposomes PI(4)P (mimant la membrane du Golgi) par les deux domaines PH du dimère OSBP, alors que la présence de la séquence désordonnée empêche cette association symétrique. De même, nous observons que la distribution d’OSBP sur la membrane de vésicules unilamellaires géantes (GUV) varie selon la présence ou l'absence du N-terminus. En présence de la séquence désordonnée, la protéine est répartie de manière homogène sur toute la surface du GUV, alors que la protéine sans N-terminal a tendance à s'accumuler à l'interface entre deux GUV de type Golgi. Cette accumulation locale ralentit fortement la mobilité de la protéine à l’interface. Un effet similaire du N-terminal sur la dynamique des protéines est observé lorsque l’association de membranes de type ER et Golgi est assuré par des protéines monomériques (dépourvue du coiled coil) en présence de Vap-A. Les résultats de nos expériences in vitro ont été confirmés en cellules vivantes, où la séquence intrinsèquement désordonnée contrôle le recrutement d’OSBP sur les membranes Golgiennes, sa mobilité et sa dynamique d’activité au cours des cycles de transfert de lipides. La plupart des protéines de la famille d’OSBP contiennent des séquences N-terminales de faible complexité, suggérant un mécanisme général de régulation. / Oxysterol binding protein (OSBP) is a lipid transfer protein that regulates cholesterol distribution in cell membranes. OSBP consists of a pleckstrin homology (PH) domain, two coiled-coils, a “two phenylalanines in acidic tract” (FFAT) motif and a C-terminal lipid binding OSBP-Related Domain (ORD). The PH domain recognizes PI(4)P and small G protein Arf1-GTP at the Golgi, whereas the FFAT motif interacts with the ER-resident protein VAP-A. By binding all these determinants simultaneously, OSBP creates membrane contact sites between ER and Golgi, allowing the counter-transport of cholesterol and PI(4)P by the ORD. OSBP also contains an intrinsically disordered ~80 aa long N-terminal sequence, composed mostly of glycine, proline and alanine. We demonstrate that the presence of disordered N-terminus increases the Stoke’s radius of OSBP truncated proteins and limits their density and saturation level on PI(4)P-containing membrane. The N-terminus also prevents the two PH domains of OSBP dimer to symmetrically tether two PI(4)P-containing (Golgi-like) liposomes, whereas protein lacking the disordered sequence promotes symmetrical liposome aggregation. Similarly, we observe a difference in OSBP membrane distribution on tethered giant unilamellar vesicles (GUVs), based on the presence/absence of N-terminus. Protein with disordered sequence is homogeneously distributed all over the GUV surface, whereas protein without N-terminus tends to accumulate at the interface between two PI(4)P-containing GUVs. This protein accumulation leads to local overcrowding, which is reflected by slow in-plane diffusion. The effect of N-terminus is also manifested in monomeric OSBPderived proteins that tether ER-like and Golgi-like membranes in the presence of VAP-A. Findings from our in vitro experiments are confirmed in living cells, where N-terminus controls the recruitment of OSBP on Golgi membranes, its motility and the on-and-off dynamics during lipid transfer cycles. Most OSBP-related proteins contain low complexity N-terminal sequences, suggesting a general effect.

The coiled-coil motif is present in proteins from all kingdoms of life. Its structure is based on a repeating sequence of 7 amino acids with hydrophobic residues at positions 1 and 4, which folds into an alpha-helix. Two, or more, alpha-helices wind around each other based on hydrophobic interactions forming the coiled-coil. Structural variations include length, deviations from the canonical form based on the heptad repeat, as well as the orientation and number of alpha-helices. They are involved in a wide variety of cellular processes including vesicle tethering and signal transmission along their length. In order to transmit signal, the protein must be able to dynamically rearrange its structure. An outstanding example of a coiled-coil that needs to rearrange its structure to perform its function is the early endosomal tether EEA1, which has been shown to increase its flexibility upon binding to the active form of the small GTPase Rab5. That conformational change generates an entropic collapse that brings the ends of the protein closer to each other. Nevertheless, the recycling from the more flexible state to its original extended conformation was not addressed. Herein, the entropic collapse mechanism was further studied and the full EEA1 cycle between extended and flexible states described. In addition to these studies, other coiled-coil proteins were assessed to determine if they also experience a binding-induced entropic collapse. One of the strategies to investigate the entropic collapse mechanism was to compare the adhesive forces along the two alpha-helices of the EEA1 dimer in its extended and flexible conformations. To this end, an experiment was designed to unwind the dimer using optical tweezers, a force-spectroscopy method that uses a highly focused laser beam to manipulate microscopic objects. Each EEA1 monomer was attached to a distinct DNA piece using a site-specific enzymatic reaction. The DNA pieces were linked to two optically trapped micron-sized beads. And the distance between the optical traps increased to unwind the EEA1. A second strategy to investigate the entropic collapse was to evaluate EEA1 dynamics in solution using dual color fluorescence cross-correlation spectroscopy (dcFCCS). EEA1 C-termini was labeled with two different fluorophores. Fluctuations on fluorescent intensities caused by the dyes crossing a confocal volume were recorded over time. Based on an analysis of these fluctuations, a conformational change in EEA1 from semi-flexible to flexible upon addition of active Rab5 was described. This is in agreement with the previously reported entropic collapse. More importantly, EEA1 was shown to cycle between semi-flexible and flexible states by adding Rab5:GTP and waiting for the GTP to hydrolyse. To determine whether other proteins experience a binding-induced entropic collapse, coiled-coil proteins that share structural and functional similarities with EEA1 were evaluated. Rotary shadowing EM images of the target protein alone and binding with its suspected allosteric effector were compared. It was found that ELKS, a coiled-coil protein involved in vesicle trafficking, undergoes an increase in flexibility upon binding with the active form of Rab6. Thus, hinting that the entropic collapse may indeed be a general mode of action for at least a sub-group of long coiled-coil proteins. Overall, the major contributions of this thesis are to describe the full entropic collapse cycle on EEA1 and to show a second example of a coiled-coil protein experiencing a binding induced flexibility increase.:List of Figures List of Tables List of Equations List of Abbreviations 1 Introduction 1.1 EEA1 as an endosomal tether 2 Materials and Methods 2.1 Materials 2.2 Methods 2.2.1 Sub-cloning 2.2.2 Protein expression and purification 2.2.3 Protein-protein binding assays 2.2.4 Electron microscopy 2.2.5 Analysis of electron microscopy 2.2.6 Generation of DNA handles for protein-DNA conjugates 2.2.7 Adding SortaseA recognition site to EEA1 2.2.8 Protein-DNA conjugation3 2.2.9 Sample preparation for optical tweezers 2.2.10 Dual color labeling of EEA1 2.2.11 Fluorescence cross-correlation spectroscopy 2.2.12 Generation of dsDNA for dcFCCS calibration 2.2.13 RabGTPase nucleotide loading 2.2.14 Liposome preparation 2.2.15 MCBs preparation 3 Unwinding EEA1 coiled-coil domain 3.1 Introduction 3.1.1 Optical tweezers for EEA1 unwinding 3.1.2 SortaseA-catalysed ligation 3.2 Aims 3.3 Results 3.3.1 Optimization of SortaseA-catalysed ligation 3.3.2 Formation of EEA1-DNA handle conjugate 3.3.3 EEA1 unwinding experiments 3.4 Discussion 4 EEA1 entropic collapse is recyclable 4.1 Introduction 4.1.1 Advantages of dcFCCS vs FCS 4.1.2 Requirements for dcFCCS measurements 4.1.3 dcFCCS for end polymer dynamics analysis 4.2 Aims 4.3 Results 4.3.1 System preparation and dcFCCS calibration 4.3.2 Labelling of EEA1 4.3.3 Comparing FCS vs dcFCCS 4.3.4 EEA1 entropic collapse shown by dcFCCS 4.3.5 EEA1 flexibility change is recyclable 4.4 Discussion 5 Entropic collapse as a general mechanism 5.1 Introduction 5.2 Aims 5.3 Results 5.3.1 ELKS increases its flexibility upon binding active Rab6 5.3.2 p115-GM130 complex observed by rotary shadowing EM 5.4 Discussion 6 Conclusions and outlook References

A mecânica celular jaz nas propriedades materiais da membrana plasmática, fundamentalmente uma bicamada fosfolipídica com espessura de dimensões moleculares. Além de forças elásticas, tal material bidimensional também experimenta tensões viscosas devido ao seu comportamento fluido (presumivelmente newtoniano) na direção tangencial. A despeito da notável concordância entre teoria e experimentos biofísicos sobre a geometria de membranas celulares, ainda não se faz presente um método computacional para simulação de sua (real) dinâmica viscosa governada pela lei de Boussinesq-Scriven. Assim sendo, introduzimos uma formulação variacional mista de três campos para escoamentos viscosos de superfícies fechadas curvas. Nela, o fluido circundante é levado em conta considerando-se uma restrição de volume interior, ao passo que uma restrição de área corresponde à inextensibilidade. As incógnitas são a velocidade, o vetor curvatura e a pressão superficial, todas estas interpoladas com elementos finitos lineares contínuos via estabilização baseada na projeção do gradiente de pressão. O método é semi-implícito e requer a solução de apenas um único sistema linear por passo de tempo. Outro ingrediente numérico proposto é uma força que mimetiza a ação de uma pinça óptica, permitindo interação virtual com a membrana, onde a qualidade e o refinamento de malha são mantidos por remalhagem adaptativa automática. Extensivos experimentos numéricos de dinâmica de relaxação são apresentados e comparados com soluções quasi-analíticas. É observada estabilidade temporal condicional com uma restrição de passo de tempo que escala como o quadrado do tamanho de malha. Reportamos a convergência e os limites de estabilidade de nosso método e sua habilidade em predizer corretamente o equilíbrio dinâmico de compridas e finas elongações cilíndricas (tethers) que surgem a partir de pinçamentos membranais. A dependência de forma membranal com respeito a uma velocidade imposta de pinçamento também é discutida, sendo que há um valor limiar de velocidade abaixo do qual um tether não se forma de início. Testes adicionais ilustram a robustez do método e a relevância dos efeitos viscosos membranais quando sob a ação de forças externas. Sem dúvida, ainda há um longo caminho a ser trilhado para o entendimento completo da mecânica celular (há de serem consideradas outras estruturas tais como citoesqueleto, canais iônicos, proteínas transmembranares, etc). O primeiro passo, porém, foi dado: a construção de um esquema numérico variacional capaz de simular a intrigante dinâmica das membranas celulares. / Cell mechanics lies on the material properties of the plasmatic membrane, fundamentally a two-molecule-thick phospholipid bilayer. Other than bending elastic forces, such a two-dimensional interfacial material also experiences viscous stresses due to its (presumably Newtonian) surface fluid tangential behaviour. Despite the remarkable agreement on membrane shapes between theory and biophysical experiments, there is no computational method for simulating its (actual) viscous dynamics governed by the Boussinesq- Scriven law. Accordingly, we introduce a mixed three-field variational formulation for viscous flows of closed curved surfaces. In it, the bulk fluid is taken into account by means of an enclosed-volume constraint, whereas an area constraint stands for the membranes inextensible character. The unknowns are the velocity, vector curvature and surface pressure fields, all of which are interpolated with linear continuous finite elements by means of a pressure-gradient-projection scheme. The method is semi-implicit and it requires the solution of a single linear system per time step. Another proposed ingredient is a numerical force that emulates the action of an optical tweezer, allowing for virtual interaction with the membrane, where mesh quality and refinement are maintained by adaptively remeshing. Extensive relaxation experiments are reported and compared with quasi-analytical solutions. Conditional time stability is observed, with a time step restriction that scales as the square of the mesh size. We discuss both convergence and the stability limits of our method, its ability to correctly predict the dynamical equilibrium of the tether due to tweezing. The dependence of the membrane shape on imposed tweezing velocities is also addressed. Basically, there is a threshold velocity value below which the tethers shape does not arise at first. Further tests illustrate the robustness of the method and show the significance of viscous effects on membranes deformation under external forces. Undoubtedly, there is still a long way to track toward the understanding of celullar mechanics (one still has to account other structures such as cytoskeleton, ion channels, transmembrane proteins, etc). The first step has given, though: the design of a numerically robust variational scheme capable of simulating the engrossing dynamics of fluid cell membranes.

A mecânica celular jaz nas propriedades materiais da membrana plasmática, fundamentalmente uma bicamada fosfolipídica com espessura de dimensões moleculares. Além de forças elásticas, tal material bidimensional também experimenta tensões viscosas devido ao seu comportamento fluido (presumivelmente newtoniano) na direção tangencial. A despeito da notável concordância entre teoria e experimentos biofísicos sobre a geometria de membranas celulares, ainda não se faz presente um método computacional para simulação de sua (real) dinâmica viscosa governada pela lei de Boussinesq-Scriven. Assim sendo, introduzimos uma formulação variacional mista de três campos para escoamentos viscosos de superfícies fechadas curvas. Nela, o fluido circundante é levado em conta considerando-se uma restrição de volume interior, ao passo que uma restrição de área corresponde à inextensibilidade. As incógnitas são a velocidade, o vetor curvatura e a pressão superficial, todas estas interpoladas com elementos finitos lineares contínuos via estabilização baseada na projeção do gradiente de pressão. O método é semi-implícito e requer a solução de apenas um único sistema linear por passo de tempo. Outro ingrediente numérico proposto é uma força que mimetiza a ação de uma pinça óptica, permitindo interação virtual com a membrana, onde a qualidade e o refinamento de malha são mantidos por remalhagem adaptativa automática. Extensivos experimentos numéricos de dinâmica de relaxação são apresentados e comparados com soluções quasi-analíticas. É observada estabilidade temporal condicional com uma restrição de passo de tempo que escala como o quadrado do tamanho de malha. Reportamos a convergência e os limites de estabilidade de nosso método e sua habilidade em predizer corretamente o equilíbrio dinâmico de compridas e finas elongações cilíndricas (tethers) que surgem a partir de pinçamentos membranais. A dependência de forma membranal com respeito a uma velocidade imposta de pinçamento também é discutida, sendo que há um valor limiar de velocidade abaixo do qual um tether não se forma de início. Testes adicionais ilustram a robustez do método e a relevância dos efeitos viscosos membranais quando sob a ação de forças externas. Sem dúvida, ainda há um longo caminho a ser trilhado para o entendimento completo da mecânica celular (há de serem consideradas outras estruturas tais como citoesqueleto, canais iônicos, proteínas transmembranares, etc). O primeiro passo, porém, foi dado: a construção de um esquema numérico variacional capaz de simular a intrigante dinâmica das membranas celulares. / Cell mechanics lies on the material properties of the plasmatic membrane, fundamentally a two-molecule-thick phospholipid bilayer. Other than bending elastic forces, such a two-dimensional interfacial material also experiences viscous stresses due to its (presumably Newtonian) surface fluid tangential behaviour. Despite the remarkable agreement on membrane shapes between theory and biophysical experiments, there is no computational method for simulating its (actual) viscous dynamics governed by the Boussinesq- Scriven law. Accordingly, we introduce a mixed three-field variational formulation for viscous flows of closed curved surfaces. In it, the bulk fluid is taken into account by means of an enclosed-volume constraint, whereas an area constraint stands for the membranes inextensible character. The unknowns are the velocity, vector curvature and surface pressure fields, all of which are interpolated with linear continuous finite elements by means of a pressure-gradient-projection scheme. The method is semi-implicit and it requires the solution of a single linear system per time step. Another proposed ingredient is a numerical force that emulates the action of an optical tweezer, allowing for virtual interaction with the membrane, where mesh quality and refinement are maintained by adaptively remeshing. Extensive relaxation experiments are reported and compared with quasi-analytical solutions. Conditional time stability is observed, with a time step restriction that scales as the square of the mesh size. We discuss both convergence and the stability limits of our method, its ability to correctly predict the dynamical equilibrium of the tether due to tweezing. The dependence of the membrane shape on imposed tweezing velocities is also addressed. Basically, there is a threshold velocity value below which the tethers shape does not arise at first. Further tests illustrate the robustness of the method and show the significance of viscous effects on membranes deformation under external forces. Undoubtedly, there is still a long way to track toward the understanding of celullar mechanics (one still has to account other structures such as cytoskeleton, ion channels, transmembrane proteins, etc). The first step has given, though: the design of a numerically robust variational scheme capable of simulating the engrossing dynamics of fluid cell membranes.