Intracellular transport of membrane-bound vesicles and organelles is a process fundamental for many cellular functions including cell morphogenesis and signaling. The transport is mediated by ensembles of motor proteins, such as kinesins, walking on microtubule tracks. When transporting membrane-bound cargo inside a cell, the motors are linked to diffusive lipid bilayers either directly or via adaptor molecules. The fluidity of the lipid bilayers induces loose inter-motor coupling which is likely to impact the collective motor dynamics and may induce cooperativity. Here, we investigate the influence of loose coupling of kinesin motors on its transport characteristics. In the first part of this thesis, we used truncated kinesin-1 motors with a streptavidin-binding-peptide (SBP) tag and performed gliding motility assays on streptavidin-loaded biotinylated supported lipid bilayers (SLBs), so called ‘membrane-anchored’ gliding motility assays. We show that the membrane-anchored motors act cooperatively; the microtubule gliding velocity increases with increasing motor density. This is in contrast to the transport behavior of multiple motors rigidly bound to a substrate. There, the motility is either insensitive to the motor density or shows negative interference at higher motor density, depending on the structure of the motors. The cooperativity in transport driven by membrane-anchored motors can be explained as following: while stepping on a microtubule, membrane-anchored motors slip backwards in the viscous membrane, thus propelling the microtubule in the solution at a velocity, given by the difference of the motor stepping velocity and the slipping velocity. The motor stepping on the microtubule occurs at maximal stepping velocity because the load on the membrane-anchored motors is minute. Thus, the slipping velocity of membrane-anchored motors determines the microtubule gliding velocity. At steady state, the drag force on the microtubule in the solution is equal to the collective drag force on the membrane-anchored motors slipping in the viscous membrane. As a consequence, at low motor density, membrane-anchored motors slip back faster to balance the drag force of the microtubule in the solution. This results in a microtubule gliding velocity significantly lower than the maximal stepping velocity of the individual motors. In contrast, at high motor density, the microtubules are propelled faster with velocities equal to the maximal stepping velocity of individual motors. Because, in this case, the collective drag force on the motors even at very low slipping velocity, is large enough to balance the microtubule drag in the solution. The theoretical model developed based on this explanation is in good agreement with the experimental data of gliding velocities at different motor densities. The model gives information about the distance that the diffusing motors can isotropically reach to bind to a microtubule, which for membrane-anchored kinesin-1 is ~0.3 µm, an order of magnitude higher as compared to rigidly bound motors, owing to the lateral mobility of motors on the membrane. In addition, the model can be used to predict the number of motors involved in transport of a microtubule based on its gliding velocity. In the second part of the thesis, we investigated the effect of loose inter-motor coupling on the transport behavior of KIF16B, a recently discovered kinesin motor with an inherent lipid-binding domain. Recent studies based on cell biological and cell extract experiments, have postulated that cargo binding of KIF16B is required to activate and dimerize the motor, making it a superprocessive motor. Here, we demonstrate that recombinant full-length KIF16B is a dimer even in the absence of cargo or additional proteins. The KIF16B dimers are active and processive, which demonstrates that the motors are not auto-inhibited in our experiments. Thus, in cells and cell extracts Kif16B may be inhibited by additional factors, which are removed upon cargo binding. Single molecule analysis of KIF16B-GFP reveals that the motors are not superprocessive but exhibit a processivity similar to kinesin-1 indicating that additional factors are most likely necessary to achieve superprocessivity. Transport on membrane-anchored KIF16B motors exhibited a similar cooperative behavior as membrane-anchored kinesin-1 where the microtubule gliding velocity increased with increasing motor density. Taken together, our results demonstrate that the loose coupling of motors via lipid bilayers provides flexibility to cytoskeletal transport systems and induces cooperativity in multi-motor transport. Moreover, our ‘membrane-anchored’ gliding motility assays can be used to study the effects of lipid diffusivity (e.g. the presence of lipid micro-domains and rafts), lipid composition, and adaptor proteins on the collective dynamics of different motors.:Abstract vii 1 Introduction 1 1.1 Intracellular transport driven by motor proteins 2 1.2 Attachment of motor proteins to cargo 13 1.3 In vitro approaches to study transport by motor proteins 16 1.4 Aim of this study 23 2 Transport by kinesin-1 anchored to supported lipid bilayers 24 2.1 Formation and characterization of biotinylated SLBs 26 2.2 Anchoring kinesin-1 to biotinylated SLBs 28 2.3 Gliding motility of microtubules by kinesin-1 linked to SLBs 34 2.4 Theoretical description of gliding motility on diffusing motor proteins 40 2.5 Comparison of the gliding velocity between experiment and theory 46 2.6 Gliding motility on phase-separated SLBs 53 2.7 Discussion 55 3 Transport by KIF16B with an inherent lipid-binding domain 62 3.2 Biophysical characterization of KIF16B 70 3.3 Gliding motility of microtubules by KIF16B linked to SLBs 78 3.4 Transport of SUVs and lipid-coated beads attached to KIF16B 87 3.5 Discussion 90 4 Conclusion and outlook 96 5 Materials and methods 99 5.1 Reagents and solutions 99 5.2 Molecular biology 100 5.3 Protein expression and purification 104 5.4 In vitro motility assays 110 5.5 Image acquisition and data analysis 118 References 126 List of figures 141 List of tables 143 Abbreviations and symbols 144 Acknowledgements 147

Endocytosis is an evolutionary conserved process of internalization of cargo from the extracellular environment, be they ligands, nutritional and signaling or pathogens into cells. Following their entry, cargo is received into vesiculo-tubular network of early endosomal compartments from where they are sorted and routed to appropriate cellular destinations through transport along the endocytic network. Recycling cargo is sorted away from other cargo resident in early endosomes through tubulation resulting in fission of recycling vesicles, while those to be degraded are progressively concentrated in early endosomes to be degraded in lysosomes. Early endosomes are dynamic organelles that have been shown to move centripetally following the internalization of cargo into at the cell periphery. Their motility from the cell periphery to the juxtanuclear location of the cell involves convoluted trajectories that include directed motility, bi-directional switches, saltatory behavior and stalls. This complex motility presumably contributes toward the cargo sorting, duration of cargo residence and spatio-temporal signaling by early endosomes. How the different regimes of motility, and nature and number of molecular motors involved in early endosome motility contribute toward endosome function is not understood. The aim of this study was to probe into the regulation of endosome motility and understand how transport organizes early endosome network. Towards this end, live cell time-lapse movies of Rab5 endosomes were analyzed to derive motility properties contributing to organization of early endosomes. Consistent and significant bias toward the cell centre (minus end motility) in kinetic parameters such as speed, displacement and duration of motility contribute to centripetal flux of Rab5 early endosomes. A phenomenological property of early endosome motility is its saltatory behavior that produces saturation curves in Mean Square Displacement (MSD) plots. This phase of motility is descriptive, with no understanding of its mechanism or function. Live cell candidate RNAi screen and cytoskeletal perturbation analysis were performed to identify molecules regulating saltatory motility. To this end, cellular microtubule perturbation and RNAi knock down of several Kinesin motor candidates showed a loss in saturation behavior. Potential candidates identified have to be tested for their effect on endosome function through cargo sorting and kinetic assays to gain insights into the role of saltatory motility in endosome function. Molecular motors mediate Rab5 motility. Therefore, understanding regulation of motility requires identifying number and nature of molecular motors involved in their transport. Towards this end, a functional cargo (LDL) degradation RNAi screen targeting molecular motors was performed. The Ambion Select technology was used with 3 siRNAs targeting every gene in the library. Analysis of screen produced by lack of phenotype consistency between the multiple siRNAs targeting the same gene. Hence, a search for technology with better target specificity was initiated. Technologies tested were Ambion Select, Ambion Silencer Select, Dharmacon ON-TARGET Plus, esiRNA and Invitrogen Stealth. Invitrogen Stealth technology was found to produce the least off-targets and was most specific in terms of consistency of phenotypes produced by multiple siRNAs silencing the same target gene. Assay conditions were also found to influence the silencing specificities to a significant extent. Hence, a systematic assay optimization exercise was performed in terms of the concentration of siRNA used for transfection and time window of assay to maximize specificity of siRNA silencing. Insights obtained from methodologies developed herein not only provide invaluable guidelines in choosing RNAi commercial libraries for screens, but also underscore the importance of establishing optimal assay conditions to minimize off-targets and improve specificity of silencing target genes. The motor screen was repeated with RNAi library from Invitrogen Stealth. Several potentially interesting candidates have been identified. Also, correlation analyses of phenotypes produced in the screen have indicated toward potential regulatory motor complexes, all of which await biochemical validation.

Microtubule associated molecular motors are involved in a multitude of fundamental cellular processes such as intracellular transport and spindle positioning. During these movements multiple motor proteins often work together and are, therefore, able to exert high forces. Thus force generation and sensing are common mechanisms for controlling motor driven movement. These mechanisms play a pivotal role when motor proteins antagonize each other, e.g. to facilitate oscillations of the spindle or the nucleus. Single motor proteins have been characterized in depth over the last two decades, our understanding of the collective behavior of molecular motors remains, however, poor. Since motor proteins often cooperate while they walk along microtubules, it is necessary to describe their collective reaction to a load quantitatively in order to understand the mechanism of many motor-driven processes. I studied the antagonistic action of many molecular motors (of one kind) in a gliding geometry. For this purpose I crosslinked two microtubules in an antiparallel fashion, so that they formed \"doublets\". Then I observed the gliding motility of these antiparallel doublets and analyzed the gliding velocity with respect to the relative number of motors pulling or pushing against each other. I observed that the antiparallel doublets gliding on conventional kinesin-1 (from Drosophila melanogaster) as well as cytoplasmic dynein (from Saccharomyces cerevisae) exhibited two distinct modes of movement, slow and fast, which were well separated. Furthermore I found a bistability, meaning, that both kinds of movement, slow and fast, occurred at the same ratio of antagonizing motors. Antiparallel doublets gliding on the non-processive motor protein Ncd (the kinesin-14 from D. melanogaster) showed, however, no bistability. The collective dynamics of all three motor proteins were described with a quantitative theory based on single-motor properties. Furthermore the response of multiple dynein motors towards an external, well-defined load was measured in a gliding geometry by magnetic tweezing. Examples of multi-motor force-velocity relationships are presented and discussed. I established, furthermore, a method for counting single surface immobilized motors to guide the evaluation of the tweezing experiments.:1 Introduction to the functions of molecular motors 1 1.1 How molecular motors move 1 1.1.1 Of muscles and molecules 1 1.1.2 Kinesin-1, the working horse of single-molecule research 3 1.1.3 Kinesin-14, an unusual kinesin with a new twist 6 1.1.4 Cytoplasmic dynein, the molecule with many qualities 7 1.2 Structure and function of microtubules 8 1.3 The directionality of molecular motors 9 1.4 Force regulation in cell biology via molecular motors 10 1.4.1 Bidirectional cargo transport 10 1.4.2 Dynein drives intracellular oscillations 13 1.4.3 Control of spindle length 15 2 Introduction to the collective behavior of molecular motors in vitro 19 2.1 Cooperativity of molecular motors 19 2.2 How multiple motors work against a load 21 2.2.1 Theoretical concepts 21 2.2.2 Optical tweezing of multiple motors 22 2.2.3 Alternative experimental approaches 23 2.2.4 Membrane tube dynamics 24 2.3 Antagonizing molecular motors 25 2.3.1 Competition between dissimilar motors 25 2.3.2 Competition between identical motors 26 2.4 Aim of the project 28 3 Characterization of molecular motors 31 3.1 Results: The run length of processive motors 31 3.1.1 Run length of kinesin-1 at different ATP concentrations 31 3.1.2 The run length of cytoplasmic dynein 34 3.2 Results for multi-motor gliding assays 37 3.2.1 The effect of ATP on the gliding motility 37 3.2.2 The effect of temperature on the gliding motility 39 3.2.3 Bead transport does not influence gliding motility 42 3.3 Discussion 43 4 Magnetic tweezing of multiple molecular motors 45 4.1 Concepts of the magnetic tweezing setup 45 4.1.1 Theoretical concepts 45 4.1.2 Implementation 48 4.1.3 Calibration 51 4.2 Results of multi-motor force measurements 53 4.2.1 External force leads to microtubule re-orientation 53 4.2.2 Cytoplasmic dynein is able to withstand high opposing loads 55 4.2.3 Force-velocity curves at very low motor densities 56 4.2.4 Averaging of multi-motor force-velocity relationships 58 4.3 Discussion 60 5 Reconstitution of antagonizing motor activity 63 5.1 The doublet assay 63 5.2 Experimental results of the doublet assay 65 5.2.1 Kinesin-1 driven doublets move in discrete velocity regimes 65 5.2.2 Velocity affects the shape of the bistability curve 68 5.2.3 Dynein\'s processivity allows bistability at low velocity 69 5.2.4 Ncd does not exhibit a bistability curve 70 5.3 Theoretical results of the doublets assay 71 5.3.1 General concepts 71 5.3.2 Theory for processive motors 73 5.3.3 Theory for non-processive motors 75 5.3.4 The emergence of bistability 78 5.3.5 Model for single-motor force-velocity relationships 81 5.4 Comparison between theoretical and experiment results 83 5.5 Discussion 87 6 Materials and Methods 91 6.1 List of chemicals and equipment 91 6.2 Buffer recipes 92 6.3 Protein purification 93 6.4 Preparation of microtubules 95 6.5 Preparation of flow cells 96 6.6 Fluorescence microscopy 98 6.7 Errors computation 100 6.8 Software 100 7 References 103 8 Acknowledgement 113

This dissertation presents the results of interdisciplinary research, at the crossing of chemistry and physics. Rational design strategies have been developed towards the synthesis of novel molecular machines able to produce a mechanical output at the nanoscale. LT-UHV STM measurements have been performed to manipulate these molecules using electrical voltage pulses as input. Two classes of molecule with different properties have been chosen as core of the designs; azulene due to the high dipole moment when an appropriate substitution pattern is selected and DMBI (1,3 dimethyl 2 phenyl 2,3 dihydro 1 H benzoimidazol) because of the zwitterionic nature. One can assume that these two distinct key features might facilitate a distinct and controllable molecule response to the external electrical field of the STM tip.

We investigate the relaxation dynamics of two distinct non-equilibrium processes: relaxation of three-dimensional antiferromagnetic lattice spin models with Heisenberg interaction following a critical quench, and a one-dimensional exclusion process inspired by the gear-like motion of molecular motors. In a system of three-dimensional Heisenberg antiferromagnets the non-conserved staggered magnetization components couple non-trivially to the conserved magnetization densities inducing fully reversible terms that enter the Langevin dynamic equation. We simulate the exact microscopic dynamics of such a system of antiferromagnets by employing a hybrid simulation algorithm that combines the reversible spin precession implemented by the fourth-order Runge-Kutta integration method with the standard relaxational dynamics at finite temperatures using Monte Carlo updates. We characterize the dynamic universality class of this system by probing the early temporal window where the system exhibits aging scaling properties. We also verify an earlier renormalization group prediction that the temporal decay exponent in the two-time spin autocorrelation function exhibits non-universality, specifically it depends on the width of the initial spin orientation distribution. We employ a similar numerical technique to study the critical dynamics of an anisotropic Heisenberg antiferromagnet in the presence of an external field. The phase diagram of this system exhibits two critical lines that meet at a bicritical point. We study the aging scaling dynamics for the model C critical line, probe the model F critical line by investigating the system size dependence of the characteristic spin-wave frequencies near criticality, and measure the dynamic critical exponents for the order parameter including its aging scaling at the bicritical point. We introduce a one-dimensional non-equilibrium lattice gas model representing the processive motion of dynein molecular motors over the microtubule. We study both dynamical and stationary state properties for the model consisting of hardcore particles hopping on the lattice with variable step sizes. We find that the stationary state gap-distribution exhibits striking peaks around gap sizes that are multiples of the maximum step size, for both open and periodic boundary conditions, and verify this using a mean-field calculation. For open boundary conditions, we observe intriguing damped oscillator-like distribution of particles over the lattice with a periodicity equal to the maximum step size. To characterize transient dynamics, we measure the mean square displacement that shows weak superdiffusive growth with exponent γ≈ 1.34 for periodic boundary and ballistic growth ( γ≈ 2) for open boundary conditions at early times. We also study the effect of Langmuir dynamics on the density profile. / Doctor of Philosophy / Most systems found in nature are out of equilibrium. In this dissertation we investigate the relaxation dynamics of two such non-equilibrium systems: 1. We investigate a three-dimensional antiferromagnetic system relaxing towards equilibrium from an initial state that is driven far away from equilibrium at the point in the parameter space where the system undergoes a second-order phase transition. We devise a novel simulation method that captures emerging dynamic universal features and scaling features at these points of continuous phase transition in the early times of relaxation when the system is still far away from equilibrium. 2. Cytoplasmic dyneins are one of three kinds of motor proteins that move on tubular structures called microtubules carrying and transporting cellular cargo inside the cells. Unlike the other molecular motors that move forward with fixed step sizes, the dyneins have been experimentally observed to vary their step size depending on the amount of cargo they are carrying. We model an exclusion process in a one-dimensional lattice inspired by the motion of the dynein molecular motors where the motors can hop from one to four steps depending on their internal states. We study the effect of this variable step size on the dynamics of a collection of dyneins. We observe intriguing oscillating density profiles and discrete peaks in the distribution of empty sites. Our results suggest self-organization among the motors and the empty sites.

No description available.

No description available.

Para estudar os mecanismos celulares que levam à mudança de cor cromomotora em crustáceos investigamos os cromatóforos ovarianos vermelhos do camarão de água doce Macrobrachium olfersi. A natureza do receptor do hormônio agregador de pigmento vermelho (RPCH) localizado na membrana plasmática é desconhecida. Muitos eventos das cascatas de sinalização induzidas por Ca2+ e GMPc, assim como os tipos de motores moleculares por elas ativados, são ainda obscuros. Avaliamos, farmacologicamente, pela perfusão in vitro dos cromatossomos com pigmentos inicialmente dispersos, possíveis funções do receptor acoplado à proteína G (GPCR), de receptores de glutamato não-NMDA (rGlu), da óxido nítrico sintase (NOS), da proteína cinase G (PKG), da cinase (MLCK) e da fosfatase (MLCP) da cadeia leve da miosina, da protéina cinase Rho (ROCK) e da miosina II não-muscular no mecanismo que induz a translocação pigmentar. Também investigamos a presença de microfilamentos de actina, microtúbulos, miosinas, cinesina e dineína, por microscopia de fluorescência. A inibição do GPCR com GDP--S (10 µM) não tem efeito significativo, mas com AntPG (5 µM) a agregação induzida por RPCH é inibida em 50%, e tem velocidade máxima de 13,3 ± 2,1 m/min (= RPCH-controle, 16,7 ± 1,6 m/min, P=0,85), seguida de dispersão espontânea. A inibição de rGlu com CNQX (50 µM) causa sutil hiperdispersão e inibe 25% da agregação induzida por RPCH, com velocidade máxima de 16 ± 1,5 µm/min (= RPCH-controle, P=0,95). A estimulação de rGlu com AMPA (30 µM) causa forte hiperdispersão (115%) e não afeta a agregação em relação ao RPCH-controle (velocidade máxima de 16,3 ± 1,8 µm/min, P=0,86). Com a inibição da NOS por L-NAME (5 mM), a agregação induzida por RPCH dura 14 min e chega aos 43,5 ± 10% de dispersão, com velocidade máxima de 11,1 ± 1,3 µm/min (= RPCH-controle, P=0,38). Com a PKG inibida por rp-sGMPc-trietilamina (3 µM), a agregação induzida por RPCH chega aos 36,2 ± 5,6% de dispersão em 12 min, com velocidade máxima de 16,9 ± 1,8 µm/min (= RPCH-controle, P=0,626), seguida de dispersão espontânea. A inibição da MLCP com cantaridina (10 µM) acelera a fase rápida da agregação induzida pelo RPCH (25,1 ± 2,6 µm/min, P= 0,017) e inibe sutilmente sua fase final (9,2 ± 5,1% após 30 min). A inibição da MLCK com ML-7 (10 µM) não afeta significativamente a agregação induzida pelo RPCH, que atinge 8,7 ± 3,14% de dispersão com velocidade máxima de 14,1 ± 1,6 µm/min (= RPCH-controle, P= 0,277). As inibições da ROCK com Y-27632 a 3 µM e H-1152 a 50 nM afetam a agregação pigmentar induzida por RPCH em 15,4 ± 4,8% e 32,8 ± 14,3%, e as velocidades máximas são similares ao RPCH-controle, de 18 ± 3,5 m/min (P=0,86) e 13,9 ± 2,3 m/min (P=0,9), respectivamente. Com H-1152 ocorre dispersão espontânea; e com ambos os compostos a dispersão durante a lavagem do RPCH é acelerada. A inibição da miosina II não-muscular com blebistatina reduz a resposta ao RPCH, havendo agregação até os 47 ± 6,2% em 16 min, com velocidade máxima de 9,1 ± 1,5 µm/min, (= RPCH-controle, P= 0,007), seguida de dispersão espontânea; a dispersão com a lavagem do RPCH ocorre normalmente. Por microscopia de fluorescência foram identificados microtúbulos, presentes nas extensões celulares com o pigmento agregado; microfilamentos de actina, aparentemente formandos trilhos aos grânulos pigmentares; miosina II não-muscular, em associação ao citoesqueleto; miosina esquelética e muscular, cinesina e dineína, em associação aos grânulos pigmentares. Evidenciamos que o receptor do RPCH pode ser do tipo GPCR. Os receptores pGlu não parecem ter papel na transdução de sinal deste neuropeptídeo. A NOS, a PKG, a MLCP e a ROCK têm papéis importante na agregação pigmentar, mas a MLCK aparentemente não. Sugerimos que o RPCH se acopla a um receptor associado à proteína G0 na membrana plasmática, e concomitantemente à elevação da concentração intracelular de Ca2+, desencadeia a ativação da NOS, que produz NO, estimulando da GC-S a liberar GMPc. Este segundo mensageiro ativa a PKG, que fosforila um sítio de ativação da miosina. O movimento da miosina é impulsionado por ciclos de fosforilação/defosforilação em um sítio regulatório de suas cadeias leves, catalizados pela MLCP e pela ROCK. Um dos tipos de miosina ativada pela PKG pode ser a miosina II não-muscular, que parece efetuar principalmente a fase lenta da agregação pigmentar. Outras miosinas e a dineína possivelmente também participam da agregação, enquanto que a cinesina parece ter papel na dispersão pigmentar. / To study the cellular mechanisms that lead to cromomotor color changes in crustaceans, we investigated the red ovarian chromatophores of the freshwater shrimp Macrobrachium olfersi. The nature of the receptor for red pigment concentrating hormone (RPCH) in the plasma membrane is unknown. Many events of the induced Ca2+ and GMPc signaling cascades, as well types of molecular motors activated are still obscure. We evaluated, using pharmacological perfusions in vitro of chromatossomes with initially dispersed pigments, putative functions of a G protein coupled receptor (GPCR), non-NMDA glutamate receptors (rGlu), nitric oxide sintase (NOS), protein kinase G (PKG), myosin light chain kinase (MLCK) and phosphatase (MLCP), Rho protein kinase (ROCK) and non-muscular myosin II in the mechanism that induces pigment translocation. We also investigated by fluorescence microscopy the presence of myosins, kinesin, dinein, actin microfilaments and microtubules. GPCR inhibition with 10 µM GDP--S has no significant effect, but 5 µM PGAnt inhibits 50% of RPCH-triggered aggregation, that has maximum velocity of 13,3 ± 2,1 m/min (= RPCH-control, 16,7 ± 1,6 m/min, P=0,85), followed by spontaneous dispersion. rGlu inhibition with 50 µM CNQX causes subtle hyperdispersion and inhibits 25% RPCH induced aggregation, with a maximum velocity of 16 ± 1,5 µm/min (= RPCH-control, P=0,95). rGlu stimulation with 30 µM AMPA causes strong pigment hyperdispersion (115%) but does not affect aggregation compared to RPCH-control (16,3 ± 1,8 µm/min maximum velocity, P=0,86). NOS inhibition with 5 mM L-NAME affects RPCH-triggered aggregation, that lasts 14 min and reaches 43,5 ± 10% dispersion, with maximum velocity of 11,1 ± 1,3 µm/min (= RPCH-control, P=0,38). PKG inhibition with 3 µM rp-cGMPs-thrietylamine affects RPCH-triggered aggregation, that lasts 2 min and reaches 36,2 ± 5,6% dispersion with maximum velocity of 16,9 ± 1,8 µm/min (= RPCH-control, P=0,626), followed by spontaneous dispersion. MLCP inhibition with 10 µM cantharidin accelerates the RPCH-triggered aggregation fast phase (25,1 ± 2,6 µm/min, P= 0,017) and subtly inhibits final aggregation (9,2 ± 5,1% after 30 min). MLCK inhibition with 10 µM ML-7 does not significantly affect RPCH-induced aggregation, that reaches 8,7 ± 3,14% dispersion with a maximum velocity of 4,1 ± 1,6 µm/min (= RPCH-control, P= 0,277). ROCK inhibition with 3µM Y-27632 or 50 nM H-1152 decreases RPCH-triggered pigment aggregation by 15,4 ± 4,8% and 32,8 ± 14,3%; maximum velocities are similar to RPCH-control, 18 ± 3,5 m/min (P=0,86) and 13,9 ± 2,3 m/min (P=0,9), respectively. H-1152 induces spontaneous dispersion; dispersion during RPCH washout is accelerated by both Y-27632 and H-1152. Non-muscular myosin II inhibited with blebbistatin reduces the response to RPCH, aggregation reaching 47 ± 6,2% in 16 min, with a maximum velocity of 9,1 ± 1,5 µm/min (= RPCH-control, P= 0,007), followed by spontaneous dispersion; RPCH washout leads to normal dispersion. Microtubules are present in the cellular extensions in chromatophores with aggregated pigments; actin microfilaments, apparently form trails to associate with pigment granules; non-muscular myosin II is associated with the cytoskeleton; skeletal and muscular myosin, kinesin and dinein, associated with the granules, were revealed by fluorescence microscopy. We showed that the RPCH receptor may be a GPCR. A pGlu receptor does not seem to be present and play a role in signal transduction. NOS, PKG, MLCP and ROCK play important roles in pigment aggregation, although MLCK apparently does not. We suggest that RPCH binds to a G0 protein coupled receptor in the plasma membrane, and together with cytosolic [Ca2+] increase, triggers NOS activation, producing NO, that stimulates GC-S to release cGMP. This second messenger activates PKG, that phosphorylates an activation site on myosin, whose movements are driven by a phosphorylation/dephosphorylation cycle at a regulatory site on the myosin light chain, catalyzed by MLCP and ROCK. One of the PKG activated myosins may be non-muscular myosin II, which seems to effect mainly the slow phase of pigment aggregation. Other myosins and dinein possibly also participate in pigment aggregation, while kynesin seems to play a role in pigment dispersion.

Faithful chromosome segregation is essential for all living organisms. Bacterial chromosome segregation utilizes highly conserved directional SpoIIIE/FtsK translocases to move large DNA molecules between spatially separated compartments. These translocases employ an accessory DNA-interacting domain (gamma) that dictates the direction of DNA transport by recognizing specific DNA sequences. To date it remains unclear how these translocases use DNA sequence information as a trigger to expend chemical energy (ATP turnover) and thereby power mechanical work (DNA movement). In this thesis, I undertook a mechanistic study of directional DNA movement by SpoIIIE from the Gram-positive model bacterium Bacillus subtilis. Specifically, I was interested in understanding the information transfer within the protein from sequence recognition, to ATP turnover, and ultimately to chromosome translocation. How do DNA sequences trigger directional chromosome movement?

Para estudar os mecanismos celulares que levam à mudança de cor cromomotora em crustáceos investigamos os cromatóforos ovarianos vermelhos do camarão de água doce Macrobrachium olfersi. A natureza do receptor do hormônio agregador de pigmento vermelho (RPCH) localizado na membrana plasmática é desconhecida. Muitos eventos das cascatas de sinalização induzidas por Ca2+ e GMPc, assim como os tipos de motores moleculares por elas ativados, são ainda obscuros. Avaliamos, farmacologicamente, pela perfusão in vitro dos cromatossomos com pigmentos inicialmente dispersos, possíveis funções do receptor acoplado à proteína G (GPCR), de receptores de glutamato não-NMDA (rGlu), da óxido nítrico sintase (NOS), da proteína cinase G (PKG), da cinase (MLCK) e da fosfatase (MLCP) da cadeia leve da miosina, da protéina cinase Rho (ROCK) e da miosina II não-muscular no mecanismo que induz a translocação pigmentar. Também investigamos a presença de microfilamentos de actina, microtúbulos, miosinas, cinesina e dineína, por microscopia de fluorescência. A inibição do GPCR com GDP--S (10 µM) não tem efeito significativo, mas com AntPG (5 µM) a agregação induzida por RPCH é inibida em 50%, e tem velocidade máxima de 13,3 ± 2,1 m/min (= RPCH-controle, 16,7 ± 1,6 m/min, P=0,85), seguida de dispersão espontânea. A inibição de rGlu com CNQX (50 µM) causa sutil hiperdispersão e inibe 25% da agregação induzida por RPCH, com velocidade máxima de 16 ± 1,5 µm/min (= RPCH-controle, P=0,95). A estimulação de rGlu com AMPA (30 µM) causa forte hiperdispersão (115%) e não afeta a agregação em relação ao RPCH-controle (velocidade máxima de 16,3 ± 1,8 µm/min, P=0,86). Com a inibição da NOS por L-NAME (5 mM), a agregação induzida por RPCH dura 14 min e chega aos 43,5 ± 10% de dispersão, com velocidade máxima de 11,1 ± 1,3 µm/min (= RPCH-controle, P=0,38). Com a PKG inibida por rp-sGMPc-trietilamina (3 µM), a agregação induzida por RPCH chega aos 36,2 ± 5,6% de dispersão em 12 min, com velocidade máxima de 16,9 ± 1,8 µm/min (= RPCH-controle, P=0,626), seguida de dispersão espontânea. A inibição da MLCP com cantaridina (10 µM) acelera a fase rápida da agregação induzida pelo RPCH (25,1 ± 2,6 µm/min, P= 0,017) e inibe sutilmente sua fase final (9,2 ± 5,1% após 30 min). A inibição da MLCK com ML-7 (10 µM) não afeta significativamente a agregação induzida pelo RPCH, que atinge 8,7 ± 3,14% de dispersão com velocidade máxima de 14,1 ± 1,6 µm/min (= RPCH-controle, P= 0,277). As inibições da ROCK com Y-27632 a 3 µM e H-1152 a 50 nM afetam a agregação pigmentar induzida por RPCH em 15,4 ± 4,8% e 32,8 ± 14,3%, e as velocidades máximas são similares ao RPCH-controle, de 18 ± 3,5 m/min (P=0,86) e 13,9 ± 2,3 m/min (P=0,9), respectivamente. Com H-1152 ocorre dispersão espontânea; e com ambos os compostos a dispersão durante a lavagem do RPCH é acelerada. A inibição da miosina II não-muscular com blebistatina reduz a resposta ao RPCH, havendo agregação até os 47 ± 6,2% em 16 min, com velocidade máxima de 9,1 ± 1,5 µm/min, (= RPCH-controle, P= 0,007), seguida de dispersão espontânea; a dispersão com a lavagem do RPCH ocorre normalmente. Por microscopia de fluorescência foram identificados microtúbulos, presentes nas extensões celulares com o pigmento agregado; microfilamentos de actina, aparentemente formandos trilhos aos grânulos pigmentares; miosina II não-muscular, em associação ao citoesqueleto; miosina esquelética e muscular, cinesina e dineína, em associação aos grânulos pigmentares. Evidenciamos que o receptor do RPCH pode ser do tipo GPCR. Os receptores pGlu não parecem ter papel na transdução de sinal deste neuropeptídeo. A NOS, a PKG, a MLCP e a ROCK têm papéis importante na agregação pigmentar, mas a MLCK aparentemente não. Sugerimos que o RPCH se acopla a um receptor associado à proteína G0 na membrana plasmática, e concomitantemente à elevação da concentração intracelular de Ca2+, desencadeia a ativação da NOS, que produz NO, estimulando da GC-S a liberar GMPc. Este segundo mensageiro ativa a PKG, que fosforila um sítio de ativação da miosina. O movimento da miosina é impulsionado por ciclos de fosforilação/defosforilação em um sítio regulatório de suas cadeias leves, catalizados pela MLCP e pela ROCK. Um dos tipos de miosina ativada pela PKG pode ser a miosina II não-muscular, que parece efetuar principalmente a fase lenta da agregação pigmentar. Outras miosinas e a dineína possivelmente também participam da agregação, enquanto que a cinesina parece ter papel na dispersão pigmentar. / To study the cellular mechanisms that lead to cromomotor color changes in crustaceans, we investigated the red ovarian chromatophores of the freshwater shrimp Macrobrachium olfersi. The nature of the receptor for red pigment concentrating hormone (RPCH) in the plasma membrane is unknown. Many events of the induced Ca2+ and GMPc signaling cascades, as well types of molecular motors activated are still obscure. We evaluated, using pharmacological perfusions in vitro of chromatossomes with initially dispersed pigments, putative functions of a G protein coupled receptor (GPCR), non-NMDA glutamate receptors (rGlu), nitric oxide sintase (NOS), protein kinase G (PKG), myosin light chain kinase (MLCK) and phosphatase (MLCP), Rho protein kinase (ROCK) and non-muscular myosin II in the mechanism that induces pigment translocation. We also investigated by fluorescence microscopy the presence of myosins, kinesin, dinein, actin microfilaments and microtubules. GPCR inhibition with 10 µM GDP--S has no significant effect, but 5 µM PGAnt inhibits 50% of RPCH-triggered aggregation, that has maximum velocity of 13,3 ± 2,1 m/min (= RPCH-control, 16,7 ± 1,6 m/min, P=0,85), followed by spontaneous dispersion. rGlu inhibition with 50 µM CNQX causes subtle hyperdispersion and inhibits 25% RPCH induced aggregation, with a maximum velocity of 16 ± 1,5 µm/min (= RPCH-control, P=0,95). rGlu stimulation with 30 µM AMPA causes strong pigment hyperdispersion (115%) but does not affect aggregation compared to RPCH-control (16,3 ± 1,8 µm/min maximum velocity, P=0,86). NOS inhibition with 5 mM L-NAME affects RPCH-triggered aggregation, that lasts 14 min and reaches 43,5 ± 10% dispersion, with maximum velocity of 11,1 ± 1,3 µm/min (= RPCH-control, P=0,38). PKG inhibition with 3 µM rp-cGMPs-thrietylamine affects RPCH-triggered aggregation, that lasts 2 min and reaches 36,2 ± 5,6% dispersion with maximum velocity of 16,9 ± 1,8 µm/min (= RPCH-control, P=0,626), followed by spontaneous dispersion. MLCP inhibition with 10 µM cantharidin accelerates the RPCH-triggered aggregation fast phase (25,1 ± 2,6 µm/min, P= 0,017) and subtly inhibits final aggregation (9,2 ± 5,1% after 30 min). MLCK inhibition with 10 µM ML-7 does not significantly affect RPCH-induced aggregation, that reaches 8,7 ± 3,14% dispersion with a maximum velocity of 4,1 ± 1,6 µm/min (= RPCH-control, P= 0,277). ROCK inhibition with 3µM Y-27632 or 50 nM H-1152 decreases RPCH-triggered pigment aggregation by 15,4 ± 4,8% and 32,8 ± 14,3%; maximum velocities are similar to RPCH-control, 18 ± 3,5 m/min (P=0,86) and 13,9 ± 2,3 m/min (P=0,9), respectively. H-1152 induces spontaneous dispersion; dispersion during RPCH washout is accelerated by both Y-27632 and H-1152. Non-muscular myosin II inhibited with blebbistatin reduces the response to RPCH, aggregation reaching 47 ± 6,2% in 16 min, with a maximum velocity of 9,1 ± 1,5 µm/min (= RPCH-control, P= 0,007), followed by spontaneous dispersion; RPCH washout leads to normal dispersion. Microtubules are present in the cellular extensions in chromatophores with aggregated pigments; actin microfilaments, apparently form trails to associate with pigment granules; non-muscular myosin II is associated with the cytoskeleton; skeletal and muscular myosin, kinesin and dinein, associated with the granules, were revealed by fluorescence microscopy. We showed that the RPCH receptor may be a GPCR. A pGlu receptor does not seem to be present and play a role in signal transduction. NOS, PKG, MLCP and ROCK play important roles in pigment aggregation, although MLCK apparently does not. We suggest that RPCH binds to a G0 protein coupled receptor in the plasma membrane, and together with cytosolic [Ca2+] increase, triggers NOS activation, producing NO, that stimulates GC-S to release cGMP. This second messenger activates PKG, that phosphorylates an activation site on myosin, whose movements are driven by a phosphorylation/dephosphorylation cycle at a regulatory site on the myosin light chain, catalyzed by MLCP and ROCK. One of the PKG activated myosins may be non-muscular myosin II, which seems to effect mainly the slow phase of pigment aggregation. Other myosins and dinein possibly also participate in pigment aggregation, while kynesin seems to play a role in pigment dispersion.