A novel parabolic prism-type TIR microscope to study gold nanoparticle-loaded kinesin-1 motors with nanometer precision

Movement of motor proteins along cytoskeletal filaments is fundamental for various cellular processes ranging from muscle contraction over cell division and flagellar movement to intracellular transport. Not surprisingly, the impairment of motility was shown to cause severe diseases. For example, a link between impaired intracellular transport and neurodegenerative diseases, such as Alzheimer’s, has been established. There, the movement of kinesin-1, a neuronal motor protein transporting vesicles along microtubules toward the axonal terminal, is thought to be strongly affected by roadblocks leading to malfunction and death of the nerve cell. Detailed information on how the motility of kinesin-1 deteriorates in the presence of roadblocks and whether the motor has a mechanism to circumvent such obstructions is scarce. In this thesis, kinesin-1 motility was studied in vitro in the presence of rigor kinesin-1 mutants, which served as permanent roadblocks, under controlled single-molecule conditions. The 25 nm wide microtubule track, consisting of 13 individual protofilaments, resembles a multi-lane environment for transport by processive kinesin-1 motors. The existence of multiple traffic-lanes, allows kinesin-1 to utilize different paths for cargo transport and potentially also for the circumvention of roadblocks. However, direct observation of motor encounters with roadblocks has been intricate in the past, mainly due to limitations in both, spatial and temporal resolution. These limitations, intrinsic to fluorescent probes commonly utilized to report on the motor positions, originate from a low rate of photon generation (low brightness) and a limited photostability (short observation time). Thus, studying kinesin-1 encounters with microtubule-associated roadblocks requires alternative labels, which explicitly avoid the shortcomings of fluorescence and consequently allow for a higher localization precision. Promising candidates for replacing fluorescent dyes are gold nanoparticles (AuNPs), which offer an enormous scattering cross-section due to plasmon resonance in the visible part of the optical spectrum. Problematic, however, is their incorporation into conventionally used (fluorescence) microscopes, because illumination and scattered light have the same wavelength and cannot be separated spectrally. Therefore, an approach based on total internal reflection (TIR) utilizing a novel parabolically shaped quartz prism for illumination was developed within this thesis. This approach provided homogenous and spatially invariant illumination profiles in combination with a convenient control over a wide range of illumination angles. Moreover, single-molecule fluorescence as well as single-particle scattering were detectable with high signal-to-noise ratios. Importantly, AuNPs with a diameter of 40 nm provided sub-nanometer localization accuracies within millisecond integration times and reliably reported on the characteristic 8 nm stepping of individual kinesin-1 motors moving along microtubules. These results highlight the potential of AuNPs to replace fluorescent probes in future single-molecule experiments. The newly developed parabolic prism-type TIR microscope is expected to strongly facilitate such approaches in the future. To study how the motility of kinesin-1 is affected by permanent roadblocks on the microtubule lattice, first, conventional objective-type TIRF microscopy was applied to GFP-labeled motors. An increasing density of roadblocks caused the mean velocity, run length, and dwell time to decrease exponentially. This is explained by (i) the kinesin-1 motors showing extended pausing phases when confronted with a roadblock and (ii) the roadblocks causing a reduction in the free path of the motors. Furthermore, kinesin-1 was found to be highly sensitive to the crowdedness of microtubules as a roadblock decoration as low as 1 % sufficed to significantly reduce the landing rate. To study events, where kinesin-1 molecules continued their runs after having paused in front of a roadblock, AuNPs were loaded onto the tails of the motors. When observing the kinesin-1 motors with nanometer-precision, it was interestingly found that about 60 % of the runs continued by movements to the side, with the left and right direction being equally likely. This finding suggests that kinesin-1 is able to reach to a neighboring protofilament in order to ensure ongoing transportation. In the absence of roadblocks, individual kinesin-1 motors stepped sideward with a much lower, but non-vanishing probability (0.2 % per step). These findings suggest that processive motor proteins may possess an intrinsic side stepping mechanism, potentially optimized by evolution for their specific intracellular tasks