The mammalian brain is a complex organ, the result of a very specific and regulated differentiation process. Although there are many different cell types in the mammalian brain, neurons make up the bulk of the tissue. Neurons come from the divisions of radial glial progenitors (RGPs), which are columnar stem cells in the developing brain. These cells undergo two types of division: symmetric or asymmetric. Symmetric divisions expand the stem cell population, resulting in two new RGPs. Symmetric divisions are critical for ensuring the stem cell population is not depleted too quickly in development. Asymmetric divisions are neurogenic, producing one RGP and one cell that will either differentiate into one neuron, or an intermediate progenitor (IP) that will divide again and produce two to four neurons (Shitamukai, Konno, and Matsuzaki 2011). Several factors have been linked to this determination, including mitotic spindle orientation, centrosomal inheritance, and exposure to proliferative factors, like sonic hedgehog and Notch (Chenn and McConnell 1995; Gaiano and Fishell 2002; Han 2016). This work will focus on spindle orientation, which has been linked to cell fate in many contexts (Lancaster and Knoblich 2012; Williams and Fuchs 2013; Chenn and McConnell 1995). Spindle orientationmust be tightly controlled in order to expand the RGP cell population in early development, then, with more randomized spindles, to shift to producing neural precursors during cortical expansion (Götz and Huttner 2005). While the exact mechanism is still unknown, the orientation of the mitotic spindle relative to the ventricular surface at the time of division affects what type of division occurs (Lancaster and Knoblich 2012). A related process in RGP neural production is interkinetic nuclear migration (INM), in which the RGP nucleus travels apically and basally in a cell-cycle dependent manner (Noctor et al. 2001; Sauer 1935; Hu et al. 2013). The RGP only divides when the nucleus reaches the apical surface; why this occurs is still not known. INM ensures that only a small population of RGPs is dividing in a controlled manner, allowing for cells to interpret polarity cues and orientthe spindle while dividing. One protein that is important to multiple processes in neuronal development is Kif1A. Kif1A is a kinesin motor that has been shown to be critical for INM, in particular for transporting the nucleus basally after division. When Kif1A expression is reduced using shRNA, RGPs fail to migrate away from the ventricular surface, but continue to go through the cell cycle at a normal rate (Carabalona, Hu, and Vallee 2016). Additionally, RGPs that lack Kif1A also exhibit more horizontal and symmetric divisions. This indicates that Kif1a is involved in asymmetric, oblique divisions that produce neurons. Thus, without Kif1a, RGPs produce fewer neurons, instead expanding the RGP cell population. Another kinesin that may be involved in spindle orientation is Kif13B. Kif13B is in the same kinesin-3 subfamily as Kif1A. While structurally very similar to Kif1A, it does have distinct features. It contains a CAP-gly domain, used for binding to the plus end of microtubules. This domain is absent from other kinesin-3 family members, including the most closely related,Kif13A. Kif13B has been shown to be critical for spindle orientation in polarized Drosophila S2 cells, as well as in neuroblasts (Carabalona, Hu, and Vallee 2016; Siegrist and Doe 2005). Kif13B functions to anchor the mitotic spindle to other factors at the cell cortex during mitosis. This occurs through direct interaction with Discs large (Dlg1), which then connects to other factors at the cell membrane, including G?i, LGN, and NuMA. This is a critical process to ensure daughter cells are properly specified. Many of these factors, including LGN and NuMA have been identified as important spindle regulators in RGP divisions as well. Kif13B binds to Dlg1 and to 14-3-3 ?, which is bound to 14-3-3 ?, bound to NudE and Dynein, connecting the Kif13B to Dynein (Lu and Prehoda 2013). Kif13B, as a kinesin, moves along microtubules towards the plus end. Dynein moves in the opposite direction, towards the minus end. The connection of two opposing motors moving in opposite directions may serve to put tension on the spindle and prevent it from freely moving within the cell. When Kif13B is knocked down or removed in cells, the spindle orients randomly in the cell, not in line with LGN or NuMA at the cell cortex (Siegrist and Doe 2005; Lu and Prehoda 2013). This indicates that in mammalian systems, it likely is important for maintaining orientation, and its loss in RGPs would result in random orientation as well. This would result in more neurogenic divisions in RGPs, which is the opposite of the effect seen with Kif1a shRNA. By using in utero electroporation of embryonic rat brains as well as a mouse model ofKif13b knockout in RGPs, I have shown that Kif13B and Kif1A have opposing roles in neurogenesis. This difference can be traced to an alteration of IP production, which Kif1A shRNA decreases, and Kif13b shRNA increases. This can be further traced to the opposing effects on spindle orientation of dividing RGPs. Kif1a shRNA results in more horizontal spindle angles while Kif13b shRNA or deletion results in more random spindle angles. While the kinesin-3 family members are very similar in structure, there are key differences between them. Kif1A has a cargo binding domain at its C terminus, the pleckstrin homology (PH) domain. Kif13B contains a CAP-gly domain. This difference in tail domains would presumably allow Kif13B to bind to microtubule plus ends, while Kif1A would dissociate from the spindle. This difference, therefore, could explain why these two very similar kinesins appear to be performing the opposite roles in spindle orientation. This work provides evidence for a novel mechanism of regulation of neuron production in the mammalian cortex.

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Mitosis, or cell division, is the mechanism by which cells divide and is an intricate process requiring the action and control of numerous proteins. Such proteins serve either as structural entities within the mitotic spindle, or perform the "work" within the apparatus. In particular, Kinesin-5 motor proteins, a subset within the kinesin motor protein superfamily, are primarily responsible for organization of microtubules (MTs) within the mitotic apparatus, and are consequently vital for efficient mitosis. These proteins utilize energy from ATP hydrolysis in order to "walk" along antiparallel MTs, positioning them into the bipolar mitotic spindle. Loss of Kinesin-5 activity results in formation of a monoastral spindle and subsequent cell cycle arrest. Recently, a wide variety of small molecules have been identified that possess the ability to inhibit certain Kinesin-5 motors. Such compounds, including monastrol (the first Kinesin-5 inhibitor identified), have been employed to study Kinesin-5 activity. A thorough understanding of Kinesin-5 function, combined with the ability to specifically target these proteins with small molecules, may provide the capability to control cell division and may therefore have significant implications in anti-cancer therapies. The following dissertation describes research that utilizes small molecules to probe the function (ATPase activity and MT interactions) of various Kinesin-5 proteins and provides information that will lead to a better understanding of exactly how such proteins function in vivo. Further, a greater knowledge of Kinesin-5 protein activity as well as specific interactions with small-molecule compounds, may lead to the development of more potent, less toxic anti-cancer drugs. / Ph. D.

Non-claret disjunctional (Ncd) is a kinesin-14 microtubule motor protein involved in the assembly and stability of meiotic and mitotic spindles in Drosophila oocytes and early embryos, respectively. Ncd functions by cross-linking microtubules through the tail and motor domains. It was originally believed that the role of the Ncd tail domain was to only statically bind microtubules. However, the Ncd tail domain has recently been shown to have properties that stabilize and bundle microtubules, and contribute to the overall motility of the Ncd protein. Continued characterization of the Ncd tail domain is essential to understanding the complete role of Ncd in cell division. This work explored the regulatory function and microtubule binding properties of the Ncd tail domain. Ncd activity is regulated during interphase by nuclear sequestration. GFP-Ncd fusion proteins, containing full length Ncd, individual Ncd domains, or combinations of Ncd domains, were used to identify the presence of a nuclear localization signal (NLS) in the Ncd polypeptide. The nuclear localization of only the GFP fusion proteins containing the Ncd tail sequence indicates that the NLS is contained within the tail domain. Subsequent, experiments performed with GFP fusion proteins containing segments of the tail domain indicate that essential NLS amino acid segments may span the length of the tail domain. Attempts to characterize the microtubule binding properties of the Ncd tail domain, using bacterially expressed MBP-Ncd tail-stalk, were unsuccessful. MBP-Ncd tail-stalk proteins aggregated under binding assay conditions, preventing an accurate determination of the stoichiometric binding relationship between Ncd and the tubulin dimer. / Master of Science

Proteins have been optimized by evolution for billions of years to work on a nanometer scale. Therefore, they are extremely promising for nanotechnological applications. Cytoskeletal filaments propelled by surface-attached motor proteins have been recently established as versatile transport platforms for nano-sized cargo in molecular sorting and nano-assembly devices. However, in this gliding motility setup, cargo and motors share the filament lattice as a common substrate for their activity. Therefore, it is important to understand the influence of cargo-loading on transport properties. By performing single molecule stepping assays on biotinylated microtubules, it was shown that kinesin-1 motors first stop and then detach when they encounter a streptavidin obstacle on their path along the microtubule. Consequently, the deceleration of streptavidin coated microtubules in gliding assays could be attributed to an obstruction of kinesin-1's path on the microtubule rather than to "frictional" streptavidin-surface interactions. The insights gained by studying kinesin-1's behavior at obstacles were then used to demonstrate a novel sensing application: Using a mixture of two distinct microtubule populations that each bind a different kind of protein, the presence of these proteins was detected via speed changes in the respective microtubule populations. In future applications, this detection scheme could be combined with other recent advancements in the field, creating highly integrated lab-on-a-chip devices that use microtubule based transport to detect, sort and concentrate analytes. It has been envisioned that the kinesin-1-microtubule system could be used for even more complex appliances like nano-assembly lines. However, currently available control mechanisms for kinesin-1 based transport are not precise enough. Therefore, improved temporal control mechanisms for kinesin-1 were investigated: Using a polymer that changes its size in solution with temperature, starting and stopping of gliding microtubules was demonstrated. In combination with local heating by light, this effect could be used to control the gliding of single microtubules. Finally, a strategy to create photo-switchable kinesin-1 was developed and tested for feasibility using molecular modeling.

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Optical tweezers are a sensitive position and force transducer widely employed in physics and biology. In a focussed laser, forces due to radiation pressure enable to trap and manipulate small dielectric particles used as probes for various experiments. For sensitive biophysical measurements, microspheres are often used as a handle for the molecule of interest. The force range of optical traps well covers the piconewton forces generated by individual biomolecules such as kinesin molecular motors. However, cellular processes are often driven by ensembles of molecular machines generating forces exceeding a nanonewton and thus the capabilities of optical tweezers. In this thesis I focused, fifirst, on extending the force range of optical tweezers by improving the trapping e fficiency of the probes and, second, on applying the optical tweezers technology to understand the mechanics of molecular motors. I designed and fabricated photonically-structured probes: Anti-reflection-coated, high-refractive-index, core-shell particles composed of titania. With these probes, I significantly increased the maximum optical force beyond a nanonewton. These particles open up new research possibilities in both biology and physics, for example, to measure hydrodynamic resonances associated with the colored nature of the noise of Brownian motion. With respect to biophysical applications, I used the optical tweezers to study the mechanics of single kinesin-8. Kinesin-8 has been shown to be a very processive, plus-end directed microtubule depolymerase. The underlying mechanism for the high processivity and how stepping is affected by force is unclear. Therefore, I tracked the motion of yeast (Kip3) and human (Kif18A) kinesin-8s with high precision under varying loads. We found that kinesin-8 is a low-force motor protein, which stalled at loads of only 1 pN. In addition, we discovered a force-induced stick-slip motion, which may be an adaptation for the high processivity. Further improvement in optical tweezers probes and the instrument will broaden the scope of feasible optical trapping experiments in the future.

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.