"The class IX myosin is a member of the myosin superfamily and found in variety of tissues. Myosin IX is quite unique among the myosin superfamily in that the tail region contains a GTPase activating protein (GAP) domain for the small GTP-binding protein, Rho. Recently it was reported that myosin IX shows processive movement that travels on an actin filament for a long distance. This was an intriguing discovery, because myosin IX is a “single-headed” myosin unlike other processive myosins which have “double-headed” structure. It has been thought that “processive” motors walk on their track with their two heads, thus traveling for a long distance. Therefore, it is reasonable to expect that the processive movement of single headed myosin IX is based on the unique feature of myosin IX motor function. In this study, I investigated the mechanism of processive movement of single-headed myosins by analyzing the mechanism of ATPase cycle of myosin IX that is closely correlated with the cross-bridge cycle (the mechanical cycle of actomyosin). In the first part, I performed the transient enzyme kinetic analysis of myosin IX using the motor domain construct to avoid the complexity raised by the presence of the tail domain. It was revealed that the kinetical characteristics of myosin IX ATPase is quite different from other processive myosins. It was particularly notable that the affinity of the weak actin binding state of Myosin IX was extremely high comparing with known myosins. It is thought that the high affinity for actin throughout the ATPase cycle is a major component to explain the processive movement of myosin IX. In the second part of this study, I cloned full length human myosin IX construct to further investigate the regulation of motor activity of myosin IX. It was revealed that the basal ATPase activity but not the actin dependent ATPase activity of myosin IX is inhibited by its tail region. Furthermore full-length myosin IX is regulated by calcium, presumably due to the calcium binding to the CaM light chain. These result suggest that the tail domain serves as a regulatory component of myosin IX."

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Biological transport is an essential phenomenon for the living systems. A mechanistic investigation of biological transport processes is highly important for the characterization of physiological and cellular events, the design and functioning of several biomedical devices and the development of new therapies. To investigate the physical-chemical details of this phenomenon, concerted efforts of both experiments and theory are necessary. Motor proteins constitute a major portion of the active transport in the living cell. However, the actual mechanism of how chemical energy is converted into their directed motion has still remained obscure. Recent experiments on motor proteins have been producing exciting results that have motivated theoretical studies. In order to provide deep insight onto motor protein's mechanochemical coupling we have used stochastic modeling based on discrete-state chemical kinetic model. Such models enable us to (1) resolve the contradiction between experimental observations on heterodimeric kinesins and highly popular hand-over-hand mechanism, (2) take into account the free energy landscape modification of individual motor domains due to interdomain interaction, (3) recognize the effect of spatial fluctuations on biochemical properties of molecular motors, and (4) calculate the dynamical properties such as velocities, dispersions of complex biochemical pathways. We have also initiated the investigation of the dynamics of coupled motor assemblies using stochastic modeling. Furthermore, an extensive Monte Carlo lattice simulation based study on facilitated search process of DNA-binding proteins is presented. This simulation shows that the accelerated search compared to pure Smoluchowski limit can be achieved even in the case where the one-dimensional diffusion is order of magnitude slower than the three-dimensional diffusion. We also show that facilitated search is not only the manifestation of dimensionality reduction but correlation times play a crucial role in the overall search times. Finally, a more general field of stochastic processes, namely first-passage time process is investigated. Explicit expressions of important properties, such as splitting probailities and mean first-passage times, that are relevant to (but not limited to) biological transport, are derived for several complex systems.

Cytoskeletal motor proteins are essential to the function of a wide range of intra-cellular mechano-systems. The biophysical characterization of the movement of motor proteins along their filamentous tracks is therefore of large importance. Towards this end, in vitro stepping motility assays are commonly used to determine the motor’s velocities and runlengths. However, comparing results from such experiments has proved difficult due to influences from variations in the experimental setups, the experimental conditions and the data analysis methods. This work describes a novel unified method to evaluate traces of fluorescently-labeled, processive dimeric motor proteins and proposes an algorithm to correct the measurements for finite filament length as well as photobleaching. Statistical errors of the proposed evaluation method are estimated by a bootstrap method. Numerical simulation and experimental data from GFP-labeled kinesin-1 motors stepping along immobilized microtubules was used to verify the proposed approach and it was shown (i) that the velocity distribution should be fitted by a t location-scale probability density function rather than a normal distribution, (ii) that the temperature during the experiments should be controlled with a precision well below 1 K, (iii) that the impossibility to measure events shorter than the image acquisition time needs to be accounted for, (iv) that the motor’s runlength can be estimated independent of the filament length distribution, and (v) that the dimeric nature of the motors needs to be considered when correcting for photobleaching. This allows for a better statistical comparison of motor proteins influenced by other external factors e.g. ionic strength, ATP concentration, or post-translational modifications of the filaments. In this context, the described method was then applied to experimental data to investigate the influence of the nucleotide state of the microtubule on the motility behavior of the kinesin-1 motor proteins. Here, a small but significant difference in the velocity measurements was found, but no significant difference in the runlength and interaction time measurements. Consequently, this work provides a framework for the evaluation of a wide range of experiments with single fluorescently-labeled motor proteins.

Motor proteins are microscopic biological machines that convert chemical energy into mechanical motion and work. They power a diverse range of biological processes, for example the swimming and crawling motion of bacteria, intracellular transport, and muscle contraction. Understanding the physical basis of these processes is interesting in its own right, but also has an interesting potential for applications in medicine and nanotechnology. The ongoing rapid developments in single molecule experimental techniques make it possible to probe these systems on the single molecule level, with increasing temporal and spatial resolution. The work presented in this thesis is concerned with physical modeling of motor proteins on the molecular scale, and with theoretical challenges in the interpretation of single molecule experiments. First, we have investigated how a small groups of elastically coupled motors collaborate, or fail to do so, when producing strong forces. Using a simple model inspired by the motor protein PilT, we find that the motors counteract each other if the density becomes higher than a certain threshold, which depends on the asymmetry of the system. Second, we have contributed to the interpretation of experiments in which the stepwise motion of a motor protein is followed in real time. Such data is naturally interpreted in terms of first passage processes. Our main conclusions are (1) Contrary to some earlier suggestions, the stepping events do not correspond to the cycle completion events associated with the work of Hill and co-workers. We have given a correct formulation. (2) Simple kinetic models predict a generic mechanism that gives rise to correlations in step directions and waiting times. Analysis of stepping data from a chimaeric flagellar motor was consistent with this prediction. (3) In the special case of a reversible motor, the chemical driving force can be extracted from statistical analysis of stepping trajectories. / QC 20100820

A major challenge in nanotechnology is the spatially controlled transport of cargo on the nanometer scale. The use of a nanoscale transport system based on molecular motors and filaments of the cytoskeleton proved as a promising approach to this problem. Therefore, the objective of this work was to pattern planar surfaces with motor proteins in a way that allows controlled and guided movement of microtubule-shuttles. The first part of the work was in particular focused on generating nanometer–sized tracks of motor proteins on unstructured surfaces. Specifically, microtubules themselves were used as biological templates for the stamping and alignment of motor proteins. Compared to other soft lithography techniques like microcontact printing this approach circumvented protein denaturation due to drying and conformational changes caused by mechanical stress. Given the large persistence length of microtubules their encounters with the boundaries of the nanotracks are limited to shallow approach angles. This way, the generated structures proved very efficient for the guiding of microtubules without topographical barriers. Topography-free guiding, as demonstrated in this work, is expected to significantly ease the design and fabrication of microtubule-transport systems and opens up the possibility to transport cargo of unlimited size, i.e. without any constraints by the dimensions of topographic guiding channels. Moreover, the biotemplated patterning is a promising tool for in vitro studies on the individual and cooperative action of motor proteins. In particular it might be helpful for the reconstitution of complex subcellular machineries in synthetic environments. As an example, microtubule-microtubule sliding by the biomolecular motor ncd has been shown to induce directional sliding between antiparallel microtubules and static cross-linking between parallel ones. The second part of the work explored an in-situ patterning technique for motor proteins to enable user-defined pattern designs, and investigated the achievable resolution. Photothermal patterning, based on localized light-to-heat conversion combined with a thermoresponsive polymer layer, was presented as a novel method. Specifically, the conformation of poly(N-isopropylacrylamide) (PNIPAM) molecules in aqueous solution was switched between the swollen state at T < 30°C (protein-repelling conformation) to the collapsed state at T > 33°C (protein-binding conformation) by optical signals of visible light to generate heat in a highly-localized manner. Upon heating of a light-absorbing layer on the substrate, the surface-grafted PNIPAM molecules collapsed locally and allowed motor proteins in solution to bind in the illuminated areas. To confirm the successful patterning of kinesin-1 molecules and their functionality microtubule-based gliding motility assays were performed. It was shown that the microtubules bind to the patterned kinesin-1 molecules and are transported exclusively in the patterned areas. While the achieved pattern sizes were currently in the range of ten micrometers, finite element modeling (implemented in COMSOL) showed that increased optical intensities possibly combined with cooling of the sample allow to significantly scale down the pattern dimensions. The produced patterns can be reversibly activated and deactivated at high and low temperature, respectively. Moreover, sequential patterning of multiple kinds of proteins on the same surface will be possible in a similar way without the need for specific linker molecules or elaborate surface preparation. Another advantage of the method is the use of visible light, which is versatile as any wavelength can be applied. In addition visible light is in comparison to other UV-based photopatterning techniques non-damaging to proteins. / Der räumlich kontrollierte Transport von nanoskaligen Objekten ist eine große Herausforderung auf dem Gebiet der Nanotechnologie. Ein auf molekularen Motoren und Filamenten des Zellskeletts basierendes Nanotransportsystem hat sich dabei als ein viel versprechender Ansatz erwiesen. Das Ziel der vorgelegten Arbeit war es daher, ebene Oberflächen so mit Motorproteinen zu strukturieren, dass eine kontrollierte und geführte Bewegung von Mikrotubuli-Transportern ermöglicht wird. Der erste Teil der Arbeit war insbesondere darauf fokussiert, Motorprotein-Spuren im Nanometerbereich zu erzeugen. Im zweiten Teil der Arbeit wurde eine Strukturierungsmethode zur Realisierung von benutzerdefinierten Musterdesigns untersucht und die erreichbare Auflösung bestimmt. Für die Nanometerstrukturierung von Oberflächen mit funktionalen Motorproteinen wurde ein neuer Ansatz demonstriert. Mit der Anwendung von Biotemplaten, wie hier der Mikrotubuli, konnte ein hoch-lokalisiertes und orientiertes Anbinden von Proteinen an Oberflächen sowie gleichzeitig geringer Proteindenaturierung erreicht werden. Durch spezifisches Stempeln beziehungsweise Binden von Motoren wurden Muster aus funktionellen Proteinen mit hoher Oberflächendichte hergestellt. Die erzeugten Motor-Spuren haben gezeigt, dass Nanometerstrukturierungen möglich sind und ohne topographische Barrieren zu zuverlässiger Führung von Mikrotubuli führen können. Bisher konnte die nicht-topographische Strukturierung von Oberflächen mit Kinesin-1-Motoren nur im Mikrometerbereich demonstriert werden. Wegen der hohen Steifigkeit der Mikrotubuli war die thermische Energie des Systems in diesen Fällen nicht ausreichend, um die führende Spitze der Mikrotubuli zurück auf das Gebiet mit den strukturierten Motoren zu biegen. Dieses Problem wird durch die kleine Breite der hier demonstrierten Motor-Nanospuren verhindert, da das Auftreffen der Mikrotubuli mit den Grenzlinien auf extrem flache Winkel begrenzt ist. Interessanterweise haben sich Spuren des nicht-prozessiven Motors Kinesin-14 für das Führen und den Transport im Nanometerbereich als noch zuverlässiger herausgestellt als Kinesin-1-Spuren. Es ist zu erwarten, dass nicht-topographisches Führen, wie es in dieser Arbeit gezeigt wurde, das Design und die Herstellung von Mikrotubuli-Transportsystemen deutlich vereinfacht und die Möglichkeit eröffnet, Cargo mit unlimitierter Größe, d.h. ohne Einschränkungen durch die Abmessungen der topographischen Führungskanäle, zu transportieren. Zusätzlich ist die biotemplierte Strukturierung ein viel versprechendes Werkzeug um das individuelle und das kooperative Arbeiten von Motorproteinen in vitro untersuchen und komplexe subzelluläre Maschinerien in synthetischer Umgebung rekonstituieren zu können. Dies wurde am Beispiel des gerichteten Gleitens des biomolekularen Motors Kinesin-14 gezeigt, der ein gerichtetes Gleiten zwischen antiparallelen Mikrotubuli und statisches Vernetzen zwischen parallelen Mikrotubuli hervorruft. Mit dem Ansatz des biotemplierten Strukturierens ist es jedoch nicht einfach möglich, benutzerdefinierte Spuren zu erzeugen. Daher wurde die photothermische Proteinstrukturierung als eine neue Methode für die frei programmierbare, hochauflösende und schnelle Herstellung von strukturierten Proteinoberflächen eingeführt. Auf diese Weise wurden Kinesin-1-Muster durch licht-induziertes Heizen einer licht-absorbierenden Substratschicht erzeugt. Die thermisch schaltbaren poly(N-isopropylacrylamid) (PNIPAM) Moleküle auf der Oberfläche kollabierten lokal und erlaubten es den Motorproteinen, in den beleuchteten Gebieten aus der Lösung an die Oberfläche zu binden. Die Bewegung gleitender Mikrotubuli bestätigte anschließend die erfolgreiche Strukturierung der Kinesin-1-Motoren und deren Funktionalität, da die Mikrotubuli an die strukturierten Motoren banden und ausschließlich in den strukturierten Gebieten transportiert wurden. Neben der Proteinstrukturierung wurde die lokalisierte Licht-zu-Wärme-Umwandlung kombiniert mit einer thermisch schaltbaren Polymerschicht auch für die lokale Aktivierung von Kinesin-1-Motoren auf der Oberfläche genutzt. Ein Vorteil der photothermischen Proteinstrukturierung ist die Möglichkeit, sichtbares Licht zu verwenden, da jede beliebige Wellenlänge angewendet werden kann und sichtbares Licht, im Vergleich zu anderen UV-basierten Photostrukturierungsmethoden, Proteine nicht schädigt. Modellierungen mit Hilfe der Finite-Elemente-Methode (implementiert in der Software COMSOL) haben gezeigt, dass die Lichtintensität und die Oberflächentemperatur speziell eingestellt werden müssen, um definierte Strukturgrößen zu erzielen. Während die derzeitig erzeugten Muster Größen im Bereich von zehn Mikrometern hatten, könnten durch höhere optische Intensitäten kombiniert mit Kühlung der Probe die Größenordnungen signifikant reduziert werden. Die reale experimentelle Auflösung wird jedoch auch von der Schaltcharakteristik des Polymers und der Proteinbindungsdynamik abhängen. Die hergestellten Muster können reversibel bei hohen beziehungsweise niedrigen Temperaturen aktiviert und deaktiviert werden. Zusätzlich können auf die gleiche Weise verschiedene Proteinsorten sequentiell auf einer Oberfläche strukturiert werden, ohne dass spezifische Bindungsmoleküle oder aufwändige Oberflächenpräparationen notwendig wären. Die Möglichkeit, Proteine reversibel an die Oberfläche zu binden, um geschriebene Muster wieder löschen zu können, wäre eine Weiterentwicklung und würde die Anwendungsmöglichkeiten der photothermischen Strukturierungsmethode erweitern. Außerdem würden optisch schaltbare Polymere das direkte Strukturieren von Motoren mit Licht ermöglichen und daher die Methode vereinfachen.

The accurate regulation of cytoplasmic dynein-1 (dynein) is very important since dynein performs multiple functions in cells. In interphase, dynein is responsible for the correct positioning of membrane organelles, such as the Golgi complex and lysosomes. Previous work suggests that dynein's accessory proteins NudEL/Nde1/LIS1¬ may be involved in regulating dynein-dependent organelle movement. This study focuses on how NudEL regulates dynein-driven membrane movement. By using various NudEL fragments, this work presents the first evidence that NudEL is involved in the regulation of dynein-driven ER movement in vitro. Moreover, the in vivo organelle positioning assays also indicate additional regulatory function of NudEL.NudEL fragment (1-157 aa) which contains both the dynein and LIS1 binding domains is sufficient to activate dynein-driven membrane movement, since NudEL1-157 aa activates ER motility in vitro and enhances clustering of the Golgi complex and lysosomes in the peri-nuclear region in vivo. On the other hand, NudEL 96-206 aa containing the LIS1 binding domain alone inhibits ER motility in vitro and causes scattering of the Golgi complex and lysosomes in vivo, indicating an inhibition of dynein-dependent organelle movement. The activation of dynein activity requires the recruitment of LIS1 to the dynein complex by NudEL, since NudEL 1-157 aa has strong binding affinity to both LIS1 and dynein whereas NudEL 96-206 aa binds to LIS1 but not dynein which suggests the sequestering of LIS1 from the dynein complex. Interestingly, NudEL 1-206 aa, which also contains both the dynein and LIS1 binding domains, causes the dispersal of the Golgi complex and lysosomes in vivo, but to a lesser extent than NudEL 96-206 aa. The putative NudEL regulatory domain (157 -242 aa, which contains various phosphorylation sits and is less conserved between NudEL and Nde1) in NudEL 1-206 aa may regulate the interaction of LIS1 and the dynein complex, since NudEL 1-206 aa has strong binding affinity to LIS1 and weak binding affinity to dynein. However, further work is needed to understand the exact mechanism by which this putative NudEL domain regulates dynein activity.

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.

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