Single-molecule measurements of Kinesin motor proteins
von André Düselder
Datum der mündl. Prüfung:2013-12-11
Betreuer:Prof. Dr. Christoph F. Schmidt
Gutachter:Prof. Dr. Christoph F. Schmidt
Gutachter:Prof. Dr. Sarah Köster
EnglischThis thesis covers the developments and new findings on different regulatory processes in Kinesin-5 motor proteins. The first two chapters give a general introduction into motor proteins and some of the well established experiments to study them. In Chapter 3, a hypothesis is studied that tries to explain how processive motion in molecular motors is possible. The assumption was that intra-molecular communication is facilitated by the transmission of tension by the neck linker of a Kinesin. We studied a set of Kinesin constructs with different length of the neck linker which we expected to influence the motors’ properties. We were able to show that the simple model is, at least for our constructs, not true. At the same time, we found evidence that supports a different explanation and might indicate a conserved mechanism in all Kinesins. In recent studies, the ability of Kinesin-5 motors from Saccharomyces cerevisiae to switch their direction of movement was reported. This is contradicting the previous observation that processive Kinesin motors exclusively move to the plus end of microtubules. The mechanism, by which this is achieved, is completely unknown. In Chapter 4, we hypothesize that the interaction of the tail domains of a Kinesin-5 with the adjacent head groups might be able to regulate the directionality. To test this, different constructs of the S. cerevisiae motors without tail domains were designed. Chapter 5 is dedicated to the design and application of a new assay that mimics the environment of the mitotic spindle. So far, the motility of mitotic motors was most of the time investigated by using single-molecule techniques that either were in a load free environment, like single-molecule fluorescence assays, or in a completely artificial environment, as in single-bead motility assays. To generate a situation that is as closely related to the mitotic spindle, I developed a new optical trap based assay. With this technique, I was able to measure the native Kinesin-5 Eg5 as well as a tetrameric Kinesin-1/Kinesin-5 chimera in a loaded environment. Not only could I show that small angles between the filaments do not influence the native protein but I was also able to observe previously unknown characteristics of tetrameric motors that are only visible in this new assay. The last chapter of this thesis, Chapter 6, summarizes the results of this work and tries to offer some points for future studies to begin. This thesis covers examples of the regulatory processes that enable Kinesin motors to exhibit a vast range of different properties, even though they are structurally similar. The results, presented in the Chapters 3 to Chapter 5, provide some additional puzzle pieces that hopefully will help to complete our understanding of Kinesin-5 motility in particular and molecular motors in general.
Keywords: Optical trap; Kinesin; Motor protein