Chemical modification is a powerful technique for probing functionally important amino acids. N-ethylmaleimide (NEM) reacts readily with exposed sulfhydryl groups, and has previously been shown to inhibit the activity of MT-motor proteins and tubulin assembly. This project seeks to investigate the mechanisms by which NEM affects motor function and inhibits MT minus end assembly. Recombinant motor domains of Drosophila kinesin (DK350 and DK375), Ncd (MC1), and squid kinesin (p181) were modified by NEM. NEM treatment was shown to affect the binding of MC1, but not recombinant kinesin proteins to MTs in the co-sedimentation assay. NEM treatment decreased the MT-stimulated ATPase rates of MC1 and DK350 in an NEM-concentration dependent manner, but did not affect the rate of DK375. Observed effects with DK375, p181, and MC1 were correlated with the number of labeled cysteines determined with [3H]NEM. As previously known, when NEM-treated tubulin was combined with untreated tubulin at certain ratios, assembly occurred only at the MT plus end. To investigate the mechanism by which NEM affects the polarity of tubulin assembly, tubulin was treated with NEM and assembly was analyzed using video-enhanced differential interference contrast microscopy. [3H]NEM was used to follow the time course of modification and to determine the number of modified sites per tubulin subunit. After 10 minutes, one cysteine was labeled on both a and b tubulin and this was sufficient to inhibit minus end assembly. Additionally, having one subunit labeled out of five tubulin subunits was sufficient to observe this effect. Protein digestion methods were used to aid in elimination of cysteines, to characterize potential critical cysteines in MC1, a, and b tubulin. / Master of Science

Die vorliegende Doktorarbeit stammt aus (ist in) dem Bereich der NanoBioTechnologie. Ihr Ziel ist es, das Motorprotein Kinesin und Mikrotubuli einzusetzen, um DNS-Moleküle in einem synthetischen Umgebung zu manipulieren. Diese Doktorarbeit setzt sich aus fünf Kapiteln zusammen. In der Einführung wird die makromolekulare Struktur der Zelle beschrieben, z.B. das Zytoskelett und Kinesin, eins der Motorproteine, die auf Mikrotubuli entlang laufen können. Der Schwerpunkt dieses Kapitels liegt auf der Nützlichkeit biologischer Motoren für den Aufbau und die Organisation von Strukturen im technischen Umfeld. Das zweite Kapitel zeigt, wie Kinesin und Mikrotubulis in einem synthetischen Umfeld für den Transport verschiedener Frachten, z.B. Streptavidin, Quantum dots oder DNS-Molekülen, genutzt werden können. Hier liegt der Schwerpunkt auf der Manipulation der DNS-Moleküle durch motor-gesteuerte Mikrotubulis und wie dieser Fracht-Transport-Mechanismus prinzipiell als Basis für die Entwicklung neuer Konzepte im Bereich des Bioingenieurwesens dienen kann. Ein Beispiel für ein solches Konzept ist die auf DNS basierende Molekularelektronik, bei der die Bindung und Streckung von DNS-Molekülen zwischen leitfähigen Oberflächen notwendig ist. Das dritte Kapitel beschreibt den Einfluß der Oberflächeneigenschaften auf die DNS-Anbindung. Es bietet Antworten darauf, wie diese Eigenschaften erforscht, spezifisch gestaltet und vorbereitet werden können, so daß sie der wissenschaftlichen Zielsetzung angemessen sind. Auf die Betrachtung von komplexen Musteranordnungen, wie sie in der Nanoelektronik genutzt werden können, wird im vierten Kapitel eingegangen. Hier wird auf praktische Art und Weise deutlich gemacht, wie DNS-Moleküle an leitfähige Oberflächen gebunden und dort durch Motorproteine und Mikrotubulis manipuliert werden können. Die Vorteile der motor-basierten Manipulation gegenüber den konventionellen Methoden wie AFM oder der optischen Pinzette werden diskutiert. Das fünfte und letzte Kapitel zeigt, wie man das Kinesin-Mikrotubuli-System nutzen kann, um daraus Informationen über DNS-Moleküle abzuleiten. Dafür wurde das Verhalten der Mikrotubulis in Beziehung auf die von gebundenen DNS-Molekülen ausgeübten Kräfte untersucht. Zusammenfassend habe ich experimentelle Untersuchungen und Färbeprotokolle entwickelt, um den gesamten Manipulationsprozeß zu detektieren, visualisieren und kontrollieren. Weiterhin untersuchte ich seine Implikationen auf theoretische Analysen, sowie auf praktische Anwendungen im Nano-Ingenieurwesen. Meine Daten demonstrieren, das DNS-Moleküle im synthetischen Umfeld so manipuliert werden können, daß kontrollierte DNS-Bioschnittstellen entstehen; Schnittstellen, die sowohl für weitere nanoelektronische Anwendungen als auch für topologische DNS-Studien genutzt werden können. Es wird weiterhin erwartet, daß das Kinesin-Mikrotubuli-System für die 3D-Anordnung auf biomolekularer Ebene im technischen Umfeld eine ebenso wichtige Rolle spielen wird. Die Fähigkeit, Vorlagen von Biomolekülen und/oder Anordungen mit definierten Eigenschaften zu schaffen und gleichzeitig ihre biologische Aktivität zu erhalten, kann als Beweis dienen, daß biologische Motoren für die molekulare Fertigung genutzt werden können. - (Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: QuickTimeMovies (ca. 86 MB)- Übersicht über Inhalte siehe Dissertation S. IX - XIII) / The work described in this thesis is in the field of NanoBioTechnology. Its goal is to leverage the motor protein kinesin and its microtubule track to manipulate DNA molecules in synthetic environment. This thesis contains five chapters. The first chapter describes macromolecular structures of the cell: i. e. the cytoskeleton and one of the motor proteins that move along it, kinesin. Emphasized is how biological motors might prove useful for organizing structures in engineered environments. The second chapter demonstrates how kinesin and microtubules can be used in synthetic environments to transport different cargos: i.e. streptavidin, quantum dots and DNA molecules. Special emphasis is placed on the manipulation of DNA molecules by the motor-driven microtubules. This cargo transport mechanism serves as a proof-of-principle for new bioengineering concepts such as DNA-based molecular electronics. The third chapter describes the influences of the surface properties on the DNA attachment and offers answers as how surface characteristics can be investigated, specifically designed and prepared so that they can serve the desired scientific purpose. The fourth chapter describes the manner in which DNA molecules can be attached to conductive surfaces and manipulated with motor proteins and microtubules. The complex DNA pattern formation that can be used for nanoelectronics is demonstrated. The advantages of motor-based manipulation over the conventional "one-by-one" methods (AFM, optical tweezers etc.) are discussed. The fifth and last chapter shows how one can use the kinesin-microtubule system to derive information about DNA molecules. For this, the response of the microtubules to forces exerted by attached DNA molecules has been studied. In summary, I have generated experimental assays and staining procedures to detect, visualize and control the entire manipulation process and to investigate its implications for theoretical analysis as well as for practical nano-engineered applications. My data demonstrated that DNA molecules can be manipulated in synthetic environment by kinesin and microtubules in such a way that controlled DNA biointerfaces can be generated. These biointerfaces can then be used for nanoelectronical application as well as for DNA topological studies. The kinesin-microtubule system is also expected to be equally important for 3D biomolecular assembly in engineered environments. The ability to generate templates of biomolecules and/or bioassemblies with well-defined features while maintaining their bioactivity, serves as proof-of-principle that biological motors can be used for molecular manufacturing. - (The pressure copies contain in each case a CD-ROM as component: QuickTimeMovies (ca. 86 MB)- To overview of contents see thesis P. IX - XIII)

Die vorliegende Doktorarbeit stammt aus (ist in) dem Bereich der NanoBioTechnologie. Ihr Ziel ist es, das Motorprotein Kinesin und Mikrotubuli einzusetzen, um DNS-Moleküle in einem synthetischen Umgebung zu manipulieren. Diese Doktorarbeit setzt sich aus fünf Kapiteln zusammen. In der Einführung wird die makromolekulare Struktur der Zelle beschrieben, z.B. das Zytoskelett und Kinesin, eins der Motorproteine, die auf Mikrotubuli entlang laufen können. Der Schwerpunkt dieses Kapitels liegt auf der Nützlichkeit biologischer Motoren für den Aufbau und die Organisation von Strukturen im technischen Umfeld. Das zweite Kapitel zeigt, wie Kinesin und Mikrotubulis in einem synthetischen Umfeld für den Transport verschiedener Frachten, z.B. Streptavidin, Quantum dots oder DNS-Molekülen, genutzt werden können. Hier liegt der Schwerpunkt auf der Manipulation der DNS-Moleküle durch motor-gesteuerte Mikrotubulis und wie dieser Fracht-Transport-Mechanismus prinzipiell als Basis für die Entwicklung neuer Konzepte im Bereich des Bioingenieurwesens dienen kann. Ein Beispiel für ein solches Konzept ist die auf DNS basierende Molekularelektronik, bei der die Bindung und Streckung von DNS-Molekülen zwischen leitfähigen Oberflächen notwendig ist. Das dritte Kapitel beschreibt den Einfluß der Oberflächeneigenschaften auf die DNS-Anbindung. Es bietet Antworten darauf, wie diese Eigenschaften erforscht, spezifisch gestaltet und vorbereitet werden können, so daß sie der wissenschaftlichen Zielsetzung angemessen sind. Auf die Betrachtung von komplexen Musteranordnungen, wie sie in der Nanoelektronik genutzt werden können, wird im vierten Kapitel eingegangen. Hier wird auf praktische Art und Weise deutlich gemacht, wie DNS-Moleküle an leitfähige Oberflächen gebunden und dort durch Motorproteine und Mikrotubulis manipuliert werden können. Die Vorteile der motor-basierten Manipulation gegenüber den konventionellen Methoden wie AFM oder der optischen Pinzette werden diskutiert. Das fünfte und letzte Kapitel zeigt, wie man das Kinesin-Mikrotubuli-System nutzen kann, um daraus Informationen über DNS-Moleküle abzuleiten. Dafür wurde das Verhalten der Mikrotubulis in Beziehung auf die von gebundenen DNS-Molekülen ausgeübten Kräfte untersucht. Zusammenfassend habe ich experimentelle Untersuchungen und Färbeprotokolle entwickelt, um den gesamten Manipulationsprozeß zu detektieren, visualisieren und kontrollieren. Weiterhin untersuchte ich seine Implikationen auf theoretische Analysen, sowie auf praktische Anwendungen im Nano-Ingenieurwesen. Meine Daten demonstrieren, das DNS-Moleküle im synthetischen Umfeld so manipuliert werden können, daß kontrollierte DNS-Bioschnittstellen entstehen; Schnittstellen, die sowohl für weitere nanoelektronische Anwendungen als auch für topologische DNS-Studien genutzt werden können. Es wird weiterhin erwartet, daß das Kinesin-Mikrotubuli-System für die 3D-Anordnung auf biomolekularer Ebene im technischen Umfeld eine ebenso wichtige Rolle spielen wird. Die Fähigkeit, Vorlagen von Biomolekülen und/oder Anordungen mit definierten Eigenschaften zu schaffen und gleichzeitig ihre biologische Aktivität zu erhalten, kann als Beweis dienen, daß biologische Motoren für die molekulare Fertigung genutzt werden können. - (Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: QuickTimeMovies (ca. 86 MB)- Übersicht über Inhalte siehe Dissertation S. IX - XIII) / The work described in this thesis is in the field of NanoBioTechnology. Its goal is to leverage the motor protein kinesin and its microtubule track to manipulate DNA molecules in synthetic environment. This thesis contains five chapters. The first chapter describes macromolecular structures of the cell: i. e. the cytoskeleton and one of the motor proteins that move along it, kinesin. Emphasized is how biological motors might prove useful for organizing structures in engineered environments. The second chapter demonstrates how kinesin and microtubules can be used in synthetic environments to transport different cargos: i.e. streptavidin, quantum dots and DNA molecules. Special emphasis is placed on the manipulation of DNA molecules by the motor-driven microtubules. This cargo transport mechanism serves as a proof-of-principle for new bioengineering concepts such as DNA-based molecular electronics. The third chapter describes the influences of the surface properties on the DNA attachment and offers answers as how surface characteristics can be investigated, specifically designed and prepared so that they can serve the desired scientific purpose. The fourth chapter describes the manner in which DNA molecules can be attached to conductive surfaces and manipulated with motor proteins and microtubules. The complex DNA pattern formation that can be used for nanoelectronics is demonstrated. The advantages of motor-based manipulation over the conventional "one-by-one" methods (AFM, optical tweezers etc.) are discussed. The fifth and last chapter shows how one can use the kinesin-microtubule system to derive information about DNA molecules. For this, the response of the microtubules to forces exerted by attached DNA molecules has been studied. In summary, I have generated experimental assays and staining procedures to detect, visualize and control the entire manipulation process and to investigate its implications for theoretical analysis as well as for practical nano-engineered applications. My data demonstrated that DNA molecules can be manipulated in synthetic environment by kinesin and microtubules in such a way that controlled DNA biointerfaces can be generated. These biointerfaces can then be used for nanoelectronical application as well as for DNA topological studies. The kinesin-microtubule system is also expected to be equally important for 3D biomolecular assembly in engineered environments. The ability to generate templates of biomolecules and/or bioassemblies with well-defined features while maintaining their bioactivity, serves as proof-of-principle that biological motors can be used for molecular manufacturing. - (The pressure copies contain in each case a CD-ROM as component: QuickTimeMovies (ca. 86 MB)- To overview of contents see thesis P. IX - XIII)

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Kinesins are motor proteins responsible for the anterograde transport on microtubules. Kinesin-1 is the first characterized kinesin, and it consists of two heavy chains and two light chains. KIF5B is a form of Kinesin-1 heavy chains that is ubiquitously expressed in mammals. The head domain of KIF5B is responsible for ATP-dependent mechanical movement along microtubules, while the tail region is well-known for its interaction with cell specific cargos. Recent studies reveal a second microtubule binding site in the tail, suggesting special functions of KIF5B in microtubule sliding and bundling. To understand the role of KIF5B in long bone development, a conditional knockout mouse model was generated, in which Kif5b is deleted in early limb mesenchyme using Prx1-cre/LoxP mediated recombination. Unlike Col2a1-cre directed Kif5b knockout in chondrocytes, the expression of Prx1-cre in limb mesenchyme results in Kif5b knockout in both chondrocyte and osteoblast lineages. The Prx1-cre mediated Kif5b conditional knockout mice develop malformed long bones characterized by their bowed shape, shortened length and multiple fractures, which reflects a combination of defects in bone matrix and growth plate. The mutant mice demonstrate impaired bone matrix formation, as indicated by both collagen density reduction and collagen matrix disorganization. Also, the growth plate does not retain its normal organization, and the hypertrophic zone is absent. The KIF5B deficient chondrocytes not only lose planar cell polarity, but also undergo early apoptosis and fail in terminal differentiation. Interestingly, the binucleation rate is significantly increased in these chondrocytes, suggesting a severe cytokinesis defect. Besides, the intracellular retention of extracellular matrix (ECM) molecules and the uneven distribution of ECM in the cartilage imply both blockage and inappropriate direction of secretion. Cytokinetic defect in chondrocytes is closely associated with growth plate abnormality and growth retardation. In Kif5b knockout chondrocytes, cytokinetic defect is also one of the earliest and principal phenotypes. Therefore the underlying mechanism of cytokinetic defect was further investigated at cellular level. Since Kif5b knockout chondrocytes cannot survive in primary culture, RNA interference approach was adopted to generate a Kif5b-knockdown chondrogenic cell line. As expected, the Kif5b knockdown cells demonstrate cytokinetic defects characterized by increased binucleation rate and prolonged cytokinesis phase. In control cells, KIF5B becomes concentrated in the midbody during cytokinesis, and the midbody organization is disrupted in Kif5b knockdown cells. Furthermore, transient expression of full-length KIF5B significantly reduces the binucleation rate of these KIF5B deficient cells, whereas over-expression of a truncated KIF5B (without microtubule binding sites in tail region) cannot rescue the defect. Additionally, KIF5B is found to interact with midbody components PRC1 and Aurora B kinase by GST pull-down assay. This study demonstrates the multiple functions of KIF5B in long bone development and emphasizes its significant role as a key modulator in chondrocyte cytokinesis. More importantly, the study also brings new insights into the mechanisms of cytokinesis: we propose that KIF5B may participate in cytokinesis by regulating the midbody organization and stability via microtubule bundling and transporting or anchoring important components to the midbody. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

A number of viruses exhibit microtubule-based bidirectional transport following cell entry. This behavior raises three questions: First, what mediates their transport along microtubules? Second, how do viruses recruit the motor proteins? Finally, how do they go to the right place by bidirectional transport in a variety of cell types with different microtubule organizations? We studied these questions with Adenovirus 5 (Ad5), a virus with well characterized, dynein-mediated minus transport mechanism. One form of plus end directed motor, Kif5C, has been reported to disrupt Ad5 capsids at the Nuclear Pore Complexes(NPC), but the mechanisms and roles of microtubule plus end-directed Ad5 transport prior to this stage are largely unknown. Here we performed a RNAi screen of 38 microtuble plus end-directed kinesins, which implicated Kif5B (kinesin-1 family) in plus-end directed Ad5 transport, along with several other forms of kinesin. Kif5B knockdown caused an accumulation of Ad5 particles near the centrosomes in human pulmonary epithelial A549 cells. This effect was strongly enhanced by blocking Ad5 nuclear pore targeting with Leptomycin B and supports a role for Kif5B in Ad5 transport prior to NPC docking. Kif5B RNAi was rescued by expression of any of the three Kif5 orthologues. We also found that Ad5 directly interacts with kinesin-1 via the capsid subunit Penton Base in a PH-independent manner. Together with our earlier studies, these findings reveal that Ad5 has evolved distinct recruitment mechanisms for cytoplasmic dynein and at least one form of kinesin-1 during early infection. Despite clear evidence for short-range linear microtubule-associated Ad5 transport, we found the overall behavior of most Ad5 particles to be stochastic at a larger time scale, by mean-square-displacement (MSD) analysis. We named this behavior "assisted diffusion''. In consistent with this mechanism, Ad5 was able to maintain a normal nuclear targeting after we displaced centrosomes away from the nucleus by inhibiting CDK1 in late G2 cells. We also directly observed Ad5 switching from microtubule based transport to nuclear targeting from a microtubule near the nucleus. Kif5B RNAi dramatically inhibited this novel microtubule-based random-walk/“assisted-diffusion” mechanism. By super resolution microscopy, we found a more local distribution of NPC attached Ad5 over the entire nuclear surface under conditions of Kif5B knock down. We propose that adenovirus uses independently-recruited kinesin and dynein to fully explore the cytoplasm to search for and dock at the nucleus, a mechanism of potential importance for physiological cargoes as well.

The inside of a eukaryotic cell is a highly organized microscale factory that shuttles components that are created or obtained in one place for use or further modification in another. Diffusion cannot accomplish the feat of translocating an object in the cytoplasm to a particular location that is a micron or more away in a timely fashion, so cells rely instead on processive motor proteins. Microtubule motor proteins are enzymes that harness the chemical energy from ATP hydrolysis to produce force and carry vesicles, membrane-bound organelles, and other cargos along paths in the cell's microtubule filament network to their destinations in the cytoplasm. These proteins recognize the polarity of the microtubule, and different classes of motors walk in different directions with respect to this polarity, giving the cell control over the direction in which a cargo is carried. It has been observed experimentally that many cargos are carried by more than one motor simultaneously, and that these multiple-motor systems can consist both of motors of the same type and of varying numbers of motors of different types. Multiple-motor systems present the possibilities of both enhanced transport performance and of tunable behavior, where the number, type, and arrangement of motors on a group of cargos can be modulated by the cell like an analog-style control to induce those cargos to arrive at a particular distribution of locations in the cytoplasm. In order to resolve the mechanisms by which these things might occur, the combination of experimental and theoretical studies in this thesis focus on the relationship between the basic biophysical properties of the constituent motors in small multiple-motor systems and the degree and nature of the cooperation observed, from the standpoint of several relevant metrics. The results highlight the importance of both the mechanochemistry of the motors and the geometry of the system itself, and offer substantial new insights into why different classes of motors cooperate to different extents, with broad implications.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 98-102).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 90-103).

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy