Amyotrophic Lateral Sclerosis (ALS) is a rare and fatal neurodegenerative disorder resulting in the loss of motor neurons from the spinal cord and frontal cortex. The patterns of neurodegeneration, affected regions, age of onset, and time course of disease progression are all highly variable between and within variants of the disease. Familial ALS (fALS), inherited versions of ALS due to genetic changes, accounts for between 5-20% of all ALS cases, while the rest are sporadic, with either no causative mutation identified or no familial history of ALS. Recently, the discovery of C9orf72 hexanucleotide repeat expansions have been identified as one of the most common causes of familial ALS, with some patients presenting with dual phenotypes of ALS and frontotemporal dementia, leading to new hypotheses about the nature of neurodegenerative diseases. Despite the continued discovery of new ALS causative genes, little is known about the pathogenesis of the disease. While almost all variants include the presence of intracellular protein inclusions, the site of the protein plaques and involved proteins varies between genetic and phenotypic variants of this disease. Due to the lack of clear pathogenic mechanisms, several hypotheses have been developed to explain the process of neurodegeneration. Autophagy, the process of self-eating, leading to destruction of damaged or excess proteins and organelles, has been implicated as being altered in ALS. Multiple variants have demonstrated altered mitochondrial morphology and cellular energetic dynamics, which could explain previous observations that implicate the process of apoptosis in cellular death. Many of the involved proteins in ALS have functional roles for intracellular, nucleocytoplasmic, and axonal transport of various proteins or RNA. These three competing hypotheses are currently the most prominent hypotheses in the pathogenesis of ALS, and have largely been considered as separate and competing in past research. Is there a chance that the true pathogenesis leading to neuronal destruction via apoptosis involve all three hypotheses? Altered protein and RNA transport dynamics could lead to changes in cellular stress responses or overload autophagy pathways, leading to exacerbated cellular stress responses, leading to alterations in mitochondrial morphology and eventually cell death via apoptosis.

Neste trabalho apresentamos um modelo teórico que busca descrever aspectos do movimento bidirecional apresentado por objetos intracelulares (vesículas, organelas, vírus etc, aos quais iremos nos referir simplesmente como (\"vesículas\"), observado, sobretudo em experimentos in vivo. Este movimento nao-difusivo e caracterizado por inversões rápidas em sua direção e é capaz de gerar gradientes de concentração do objeto transportado. Os fenômenos de transporte intracelular são sabidamente mediados por proteínas motoras (como as kinesinas e dinenas) cujo movimento unidirecional sobre _lamentos protéicos e bem caracterizado (kinesinas se movem em direção a extremidade mais enquanto as dinenas se movem em direção a extremidade-menos dos microtúbulos) e é normalmente entendido através de modelos estocásticos que descrevem o comportamento de uma partícula browniana na presença de um potencial assimétrico que varia no tempo (ver Astumian [26], Adjari e Prost [22], Magnasco [23]). Mais recentemente, surgiram na literatura trabalhos que tentam descrever o movimento de partículas motoras interagentes, uma vez que se percebeu que efeitos coletivos que surgem nestas situações podem ser relevantes para os fenômenos de transporte sobre microtúbulos. Uma abordagem para a descrição do comportamento destes sistemas de partículas motoras interagentes é aquela baseada nos modelos para os sistemas difusivos dirigidos\". Em particular, a versão contínua dos modelos do tipo totally asymmetric exclusion processes\" (TASEP) e asymmetric exclusion processes\" (ASEP) tem sido utilizada para o estudo do comportamento da densidade de motores sobre os microtúbulos, através da analise de soluções estacionarias da equação de Burgers correspondente (Parmeggiani et al. [33]). Até agora, entretanto, não existem na literatura tentativas de abordar, com estes modelos, o transporte bidirecional de vesículas mediado por estes motores interagentes. A idéia que apresentamos aqui é associar este estranho tipo de movimento ao movimento de ondas de choque presentes nas soluções transientes da equa_c~ao de Burgers para algumas condições iniciais. Deste modo, as vesículas acompanhando (\"surfando\") os choques fariam o papel de suas correspondentes microscópicas partículas de segunda classe\", introduzidas h_a um bom tempo na literatura [36], [37], [38] para o estudo da dinâmica microscópica dos choques que estão presentes também na versão discreta dos modelos TASEP e ASEP. Neste sentido, é natural que as condições iniciais consideradas, que seriam perturbações no estado estacionário das partículas, possam ser causadas, no sistema real, pela própria interação com a vesícula. É o caso, portanto, de se propor que a geometria deste objeto tenha um papel importante na determinação da direcional de seu próprio movimento no meio intracelular. Esta parece ser, por exemplo, uma alternativa interessante para explicar aspectos do movimento de vírus no interior das células. / In this work we present a theoretical model to describe aspects of the bidirectional movement performed by intracellular structures (vesicles, organelles, viruses etc, to which we refer here simply as \"vesicles\"), observed essentially at in vivo experiments. This nondifusive movement is characterized by rapid inversions in direction and is capable of creating concentration gradients of the transported cargo. The phenomenon of intracellular transport is known to be mediated by motor proteins (such as kinesins and dyneins) whose own unidirectional motion along protein laments is well characterized (kinesins moves to the plus-end direction while dyneins moves to the minus-end direction of the microtubules) and is usually modeled by a stochastic dynamics describing the behavior of a Brownian particle in the presence of a time dependent asymmetrical potential held (see Astumian [26], Adjari and Prost [22], Magnasco [23]). More recently, it appeared in the literature works attempting to describe the movement of interacting motor proteins, since it was realized that collective e_ects emerging from this situation may be relevant to the transport phenomena along microtubules. An approach to describe the behavior of such interacting motor particles is based on existing models for \\driven di_usive systems\". In particular, the continuum versions of the totally asymmetric exclusion processes\" (TASEP) or the asymmetric exclusion processes\" (ASEP) have been used to study the behavior of motors density along microtubules by analyzing the steady state solutions to the corresponding Burgers equation (Parmeggiani et al. [33]). Up to now, however, there are no attempts in the literature to approach in this context the questions related to the bidirecionality of vesicles transported by these interacting motors. The idea we present here is to associate this odd movement to the movement of shock waves presented by the transient solutions of Burgers equation for certain initial conditions. Accordingly, the vesicles accompanying (sur_ng) the shocks fronts would play the role of their microscopic analogous \\particles of second class\" introduced long ago in the literature [36], [37], [38] to study the kinetics of the shocks that are also present in the discrete versions of the TASEP and ASEP. In this regard, it is natural to think that the considered initial conditions, namely perturbations to the motor density with respect to a steady state, can be created in the real systems simply by the interaction with the vesicle. It might then be the case also to propose that the geometry of the vesicle plays an important role to direct its own movement within intracellular environment. This seems to be, for example, an attractive alternative for explaining aspects of virus movement inside the cell.

Intracellular cargo transport involves the movement of critical cellular components (e.g. vesicles, organelles, mRNA, chromosomes) along cytoskeletal tracks by tiny molecular motors. Myosin Va motors have been demonstrated to play a vital role in the transport of cargos destined for the cell membrane by navigating their cargos through the three-dimensional actin networks of the cell. Transport of cargo through these networks presents many challenges, including directional and physical obstacles which teams of myosin Va-bound to a single cargo must overcome. Specifically, myosin Va motors are presented with numerous actin-actin intersections and dense networks of filaments which can act as a physical barrier to transport. Due to the complexities of studying myosin Va cargo transport in cells, much effort has been focused on the in vitro observation and analysis of myosin Va transport along single actin filaments or simple actin cytoskeletal models. However, these model systems often rely on non-physiological cargos (e.g. beads, quantum dots) and two-dimensional arrangements of actin attached to glass surfaces. Interestingly, a disconnect exists between the transport of cargo on these simple model systems and studies of myosin Va transport on suspended 3D actin arrangements or cellular networks which show longer run lengths, increased velocities, and straighter, more directed trajectories. One solution to this discrepancy is that the cell may use the fluidity of the cargo surface, the recruitment of myosin Va motor teams, and the 3D geometry of the actin, to finely tune the transport of intracellular cargo depending on cellular need. To understand how myosin Va motors transport their cargo through 3D networks of actin, we investigated myosin Va motor ensembles transporting fluorescent 350 nm lipid-bilayer cargo through arrangements of suspended 3D actin filaments. This was accomplished using single molecule fluorescent imaging, three-dimensional super resolution Stochastic Optical Reconstruction Microscopy (STORM), optical tweezers, and in silico modeling. We found that when moving along 3D actin filaments, myosin motors could be recruited from across the fluid lipid cargo’s surface to the filaments which enabled dynamic teams to be formed and explore the full actin filaments binding landscape. When navigating 3D actin-actin intersections these teams capable of maneuvering their cargo through the intersection in a way that encouraged the vesicles to continue straight rather than switch filaments and turn at the intersection. We hypothesized that this finding may be the source of the relatively straight directed runs by myosin Va-bound cargo observed in living cells. To test this, we designed 3D actin networks where the vesicles interacted with 2-6 actin filaments simultaneously. Actin forms polarized filaments, which, in cells, generally have their plus-ends facing the exterior of the cell; the same direction in which myosin Va walks. We found that to maintain straight directed trajectories and not become stationary within the network, vesicles needed to move along filaments with a bias in their polarity. This allows for cargo-bound motors to align their motion along the polarized networks and produced productive motion despite physical and directional obstacles. Together this work demonstrates the physical properties of the cargo, the geometric arrangement of the actin, and the mechanical properties of the motor are all critical aspects of a robust myosin Va transport system.

Hook-Proteine spielen eine wichtige Rolle im intrazellulären Transport. Sie binden n-terminal Mikrotubuli, haben eine coiled-coil-Domäne und binden c-terminal Organellen. Da aus humanen Hirnschnitten eine Kolokalisation mit den bei Alzheimerscher Erkrankung auftretenden neurofibrillären Tangles bekannt war, erscheint eine weitere Untersuchung der Expression und Lokalisation von Hook-Proteinen im zentralen Nervensystem interessant. Hierzu wurde die Expression der humanen Hook-Proteine mittels real-time RT-PCR quantifiziert und die Lokalisation der Hook-Proteine in verschiedenen transgenen Mausmodellen der Tauopathie mittels Immunhistochemie dargestellt. Außerdem wurde die cDNA der Hook-Proteine mittels PCR auf alternatives Spleißen untersucht.

The number of motor proteins attached to cellular cargos is widely believed to influence intracellular transport processes and may play a role in transport regulation. However, to date, investigating the biophysics of multiple-motor dynamics has been challenging since the number of motors responsible for cargo motion is not easily characterized. This work examines the transport properties of structurally-defined motor complexes containing two kinesin-1 motors, from both an experimental and theoretical perspective. Motor complexes were synthesized using DNA as a molecular scaffold and engineered DNA-conjugated protein polymers as linkers to couple motors to scaffolds. After anchoring the motor complexes to a bead their dynamic properties were measured using an automated optical trapping instrument that could be used to perform both static (increasing load) and force-feedback (constant load) optical trapping experiments. Data from these experiments is compared to predictions from a microscopic transition rate model of multiple kinesin dynamics. Together, these studies uncovered that multiple kinesins typically cannot cooperate since the microtubule-bound configuration of a motor complex often prevents both kinesins from sharing cargo loads. Furthermore, multiple-motor behaviors are influenced by the fact that motor complexes display hysteretic force-velocity behaviors when applied loads change rapidly in time. Overall, such behaviors suggest the number of kinesins on a cargo will not be a key determinant of intracellular transport processes, and in turn, will not contribute appreciably to mechanisms that regulate cargo motion. However, this work also provides evidence that processive microtubule motors that are less efficient than kinesin (e.g., dynein) will cooperate productively, produce greater responses to motor number, and may therefore act as a regulator of cargo transport.

The protein Hedgehog (Hh) is a highly conserved, secreted ligand (and morphogen) capable of patterning many different tissues during development. Recently, Sonic Hedgehog (SHH) a human homolog of Drosophila Hh was found to be a causative agent in certain cancers. While several drugs are being developed to combat the binding of SHH to its receptor Patched or the Patched-target Smoothened, very little is known about how SHH is secreted from the producing cell, another site for therapeutic targeting. We report here the characterization of a novel proteolytic event and genetic pathway that controls Hh intracellular sorting and axon transport using the Drosophila eye imaginal disc as our model system. In fly larval photoreceptor neurons the developmental signal Hh is guided to the apical (retina) and basal (growth cone, GC) ends where secretion of the morphogen is an inductive factor in photoreceptor differentiation and establishment of eye/brain neural connections. The Hh secreted from the basal side induces lamina development while Hh secreted at the retina induces ommatidial development. Hedgehog processing consists of autocleavage from its 46 kDa form (HhU) to become a lipid-modified N-terminal signaling molecule (HhN; 19kDa) and a C-terminal molecule (HhC24; 24 kDa). Following autocleavage, a fraction of the C-terminal auto-cleavage product then undergoes a second cleavage event leading to 16 kDa (HhC16) and 9 kDa products. Nothing is known about the significance of the C-terminal “2nd cleavage” other than its occurrence in both fly and human tissue. In an effort to identify regulators of Hh sorting, we discovered that the HhC “2nd cleavage” is a determining factor in the sorting of the HhN signaling domain. That is, if a cell induces more cleavage (more HhC16) we observe more HhN in the apical domain. Likewise, if a cell inhibits 2nd cleavage (less HhC16) we see more basal HhN. Creation of a “2nd cleavage mutant” shows that this process has developmental significance. Further, biochemical characterization of the 2nd cleavage suggests it occurs in the ER after autocleavage and that HhC24 can exit the cell in a Golgi independent manner (via lipid droplets) while HhC16 remains intracellular. The ER exit of HhC24 appears to be controlled by a conserved PP2A (Mts) /PKB (Akt) kinase pathway which potentially regulates the size and number of lipid droplets produced. These findings are an important first step in understanding the intracellular sorting and transport of Hh and highlight new targets for the treatment of SHH-related cancers. The discovery of divergent modes of Hh secretion and the “2nd cleavage” open novel avenues for Hh research by offering an alternative, and very direct, line of attack in the treatment of Hh-related cancer.

This thesis uses methods and models from non-equilibrium statistical physics to describe intracellular processes. Bidirectional microtubule-based transport within axons is modeled as a quasi-one-dimensional stochastic lattice gas with two particle species moving in opposite directions under mutual exclusion interaction. Generically occurring clusters of particles in current models for intracellular transport can be dissolved by additionally considering the dynamics of the transport lattice, i.e., the microtubule. An idealized model for the lattice dynamics is used to create a phase transition toward a homogenous state with efficient transport in both directions. In the thermodynamic limit, a steady state property of the dynamic lattice limits the maximal size of clusters. Lane formation mechanisms which are due to specific particle-particle interactions turn out to be very sensitive to the model assumptions. Furthermore, even if some particle-particle interaction is considered, taking the lattice dynamics into account almost always improves transport. Thus the lattice dynamics seems to be the key aspect in understanding how nature regulates intracellular traffic. The last part introduces a model for the dynamics of a microtubule which is limited in its growth by the cell boundary. The action of a rescue-enhancing protein which is added to the growing tip of a microtubule and then slowly dissociates leads to interesting aging effects which should be experimentally observable.

Neste trabalho apresentamos um modelo teórico que busca descrever aspectos do movimento bidirecional apresentado por objetos intracelulares (vesículas, organelas, vírus etc, aos quais iremos nos referir simplesmente como (\"vesículas\"), observado, sobretudo em experimentos in vivo. Este movimento nao-difusivo e caracterizado por inversões rápidas em sua direção e é capaz de gerar gradientes de concentração do objeto transportado. Os fenômenos de transporte intracelular são sabidamente mediados por proteínas motoras (como as kinesinas e dinenas) cujo movimento unidirecional sobre _lamentos protéicos e bem caracterizado (kinesinas se movem em direção a extremidade mais enquanto as dinenas se movem em direção a extremidade-menos dos microtúbulos) e é normalmente entendido através de modelos estocásticos que descrevem o comportamento de uma partícula browniana na presença de um potencial assimétrico que varia no tempo (ver Astumian [26], Adjari e Prost [22], Magnasco [23]). Mais recentemente, surgiram na literatura trabalhos que tentam descrever o movimento de partículas motoras interagentes, uma vez que se percebeu que efeitos coletivos que surgem nestas situações podem ser relevantes para os fenômenos de transporte sobre microtúbulos. Uma abordagem para a descrição do comportamento destes sistemas de partículas motoras interagentes é aquela baseada nos modelos para os sistemas difusivos dirigidos\". Em particular, a versão contínua dos modelos do tipo totally asymmetric exclusion processes\" (TASEP) e asymmetric exclusion processes\" (ASEP) tem sido utilizada para o estudo do comportamento da densidade de motores sobre os microtúbulos, através da analise de soluções estacionarias da equação de Burgers correspondente (Parmeggiani et al. [33]). Até agora, entretanto, não existem na literatura tentativas de abordar, com estes modelos, o transporte bidirecional de vesículas mediado por estes motores interagentes. A idéia que apresentamos aqui é associar este estranho tipo de movimento ao movimento de ondas de choque presentes nas soluções transientes da equa_c~ao de Burgers para algumas condições iniciais. Deste modo, as vesículas acompanhando (\"surfando\") os choques fariam o papel de suas correspondentes microscópicas partículas de segunda classe\", introduzidas h_a um bom tempo na literatura [36], [37], [38] para o estudo da dinâmica microscópica dos choques que estão presentes também na versão discreta dos modelos TASEP e ASEP. Neste sentido, é natural que as condições iniciais consideradas, que seriam perturbações no estado estacionário das partículas, possam ser causadas, no sistema real, pela própria interação com a vesícula. É o caso, portanto, de se propor que a geometria deste objeto tenha um papel importante na determinação da direcional de seu próprio movimento no meio intracelular. Esta parece ser, por exemplo, uma alternativa interessante para explicar aspectos do movimento de vírus no interior das células. / In this work we present a theoretical model to describe aspects of the bidirectional movement performed by intracellular structures (vesicles, organelles, viruses etc, to which we refer here simply as \"vesicles\"), observed essentially at in vivo experiments. This nondifusive movement is characterized by rapid inversions in direction and is capable of creating concentration gradients of the transported cargo. The phenomenon of intracellular transport is known to be mediated by motor proteins (such as kinesins and dyneins) whose own unidirectional motion along protein laments is well characterized (kinesins moves to the plus-end direction while dyneins moves to the minus-end direction of the microtubules) and is usually modeled by a stochastic dynamics describing the behavior of a Brownian particle in the presence of a time dependent asymmetrical potential held (see Astumian [26], Adjari and Prost [22], Magnasco [23]). More recently, it appeared in the literature works attempting to describe the movement of interacting motor proteins, since it was realized that collective e_ects emerging from this situation may be relevant to the transport phenomena along microtubules. An approach to describe the behavior of such interacting motor particles is based on existing models for \\driven di_usive systems\". In particular, the continuum versions of the totally asymmetric exclusion processes\" (TASEP) or the asymmetric exclusion processes\" (ASEP) have been used to study the behavior of motors density along microtubules by analyzing the steady state solutions to the corresponding Burgers equation (Parmeggiani et al. [33]). Up to now, however, there are no attempts in the literature to approach in this context the questions related to the bidirecionality of vesicles transported by these interacting motors. The idea we present here is to associate this odd movement to the movement of shock waves presented by the transient solutions of Burgers equation for certain initial conditions. Accordingly, the vesicles accompanying (sur_ng) the shocks fronts would play the role of their microscopic analogous \\particles of second class\" introduced long ago in the literature [36], [37], [38] to study the kinetics of the shocks that are also present in the discrete versions of the TASEP and ASEP. In this regard, it is natural to think that the considered initial conditions, namely perturbations to the motor density with respect to a steady state, can be created in the real systems simply by the interaction with the vesicle. It might then be the case also to propose that the geometry of the vesicle plays an important role to direct its own movement within intracellular environment. This seems to be, for example, an attractive alternative for explaining aspects of virus movement inside the cell.

Transport of intracellular cargo is an important and dynamic process required for cell maintenance and survival. Dynein is the motor protein that carries organelles and vesicles from the cell periphery to the cell center along the microtubule network. Dynactin is a protein that activates dynein for this transport process. Together, dynein and dynactin forms a motor protein complex that is essential for transport processes in all the vertebrate cells. Using fluorescent microscope based live cell imaging techniques and kymograph analyses, I studied dynein/dynactin disruptions on the intracellular transport in two different cell systems. In one set of experiments, effects of dynein heavy chain (DHC) mutations on the vesicular motility were characterized in the fungus model system Neurospora crassa. I found that many DHC mutations had a severe transport defect, while one mutation linked to neurodegeneration in mice had a subtle effect on intracellular transport of vesicles. In a different set of experiments in mammalian tissue culture CAD cells, I studied the effects of dynactin knockdown and dynein inhibition on mitochondrial motility. My results indicated that reductions in dynactin levels decrease the average number of mitochondrial movements and surprisingly, increase the mitochondrial run lengths. Also, I determined that the dynein inhibitory drug Ciliobrevin causes changes in mitochondrial morphology and decreases the number of mitochondrial movements inside cells. Overall, my research shows that distinct disruptions in the dynein and dynactin motor complex alters intracellular motility, but in different ways. So far, my studies have set the ground work for future experiments to analyze the motility mechanism of motor proteins having mutations that lead to neurodegenerative disorders.

Dans cette thèse, des méthodes de la physique statistique hors équilibre sont utilisées pour décrire deux processus intracellulaires. Le transport bidirectionnel sur les microtubules est décrit à l'aide d'un gaz sur réseau stochastique quasi-unidimensionnel. Deux espèces de particules sautent dans des directions opposées en interagissant par exclusion. La présence habituelle d'accumulations de particules peut être supprimée en rajoutant la dynamique du réseau, c'est-à-dire de la microtubule. Un modèle simplifié pour la dynamique du réseau produit une transition de phase vers un état homogène avec un transport très efficace dans les deux directions. Dans la limite thermodynamique, une propriété de l'état stationnaire limite la longueur maximale des accumulations. La formation de voies peut être causée par des interactions entre particules. Néanmoins, ces mécanismes s'avèrent peu robustes face à une variation des paramètres du modèle. Dans presque tous les cas, la dynamique du réseau a un effet positif et bien plus important sur le transport que la formation de voies. Par conséquent, la dynamique du réseau semble un point-clé pour comprendre la régulation du transport intracellulaire. La dernière partie introduit un modèle pour la dynamique d'une microtubule sous l'action d'une protéine qui favorise les sauvetages. Des phénomènes intéressants de vieillissement apparaissent alors, et devraient être observables dans des expériences. / This thesis uses methods and models from non-equilibrium statistical physics to describe intracellular processes. Bidirectional microtubule-based transport within axons is modeled as a quasi-one-dimensional stochastic lattice gas with two particle species moving in opposite directions under mutual exclusion interaction. Generically occurring clusters of particles in current models for intracellular transport can be dissolved by additionally considering the dynamics of the transport lattice, i.e., the microtubule. An idealized model for the lattice dynamics is used to create a phase transition toward a homogenous state with efficient transport in both directions. In the thermodynamic limit, a steady state property of the dynamic lattice limits the maximal size of clusters. Lane formation mechanisms which are due to specific particle-particle interactions turn out to be very sensitive to the model assumptions. Furthermore, even if some particle-particle interaction is considered, taking the lattice dynamics into account almost always improves transport. Thus the lattice dynamics seems to be the key aspect in understanding how nature regulates intracellular traffic. The last part introduces a model for the dynamics of a microtubule which is limited in its growth by the cell boundary. The action of a rescue-enhancing protein which is added to the growing tip of a microtubule and then slowly dissociates leads to interesting aging effects which should be experimentally observable.