Download or read book Mechanochemical Basis of Functional Diversity in the Kinesin Superfamily written by Keith Mickolajczyk and published by . This book was released on 2018 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Kinesin motor proteins drive numerous active processes the cell, including vesicle transport, DNA and organelle repositioning, intraflagellar transport, and mitotic spindle organization. There are 45 kinesin genes in the human genome, each of which encodes an isoform that is optimized to drive some processes, but is incapable of driving others. Years of in vitro total internal reflection fluorescence microscopy (TIRFM) and optical trapping assays have helped to characterize biophysical metrics like velocity, run length, and stall force for various kinesin family members. Coupled with structural and cellular work, the kinesin field has produced a solid understanding of what different kinesins are capable of. What remains misunderstood, however, is how the 14 kinesin families are capable of performing such diverse jobs overall, but such specific jobs individually. For example, it is unknown why a kinesin-1 knockdown is embryonic lethal despite the fact that kinesins -1, -2, and -3 are all ubiquitously expressed transporters and are all capable of attaching to similar cargoes. Understanding the functional diversity of kinesins is important because it is fundamental to cell biology, and it provides insights into how kinesin dysfunction leads to disease states such as Charcot-Marie-Tooth disease, hereditary spastic paraplegia, hereditary sensory and autonomic neuropathy type II, Alzheimers disease, and various cancers. The goal of this dissertation is to determine the mechanistic underpinnings of functional diversity across the kinesin superfamily. This is done by carrying out comparative studies of motors in the kinesin-1 and kinesin-2 families to determine how critical structural differences in these transport motors tune transition rates between conformational intermediates in the mechanochemical cycle. This kinetic tuning describes exactly how the aforementioned biophysical metrics arise and provides insight into the structure-function relationship underlying family-specific emergent behaviors.Answering cornerstone questions regarding the mechanistic differences between kinesins has to date been hampered by the resolution barriers of single-molecule techniques. Dimeric transport kinesins take discrete 8-nm steps along microtubules in a hand-over-hand fashion, hydrolyzing one ATP per step. Kinesins thus use a single ATP molecule to transition from a state with both heads bound to the microtubule, to a one-head-bound state where the rear head has been lifted off the microtubule, and back to a two-heads-bound state where the front and rear heads have now switched positions. Because kinesins take tens to hundreds of 8-nm steps per second, measuring steps under the microscope requires very high spatiotemporal resolution. Measuring the transitions between the two- and one-head-bound states (substeps) that subdivide each step requires higher resolution still. TIRFM, the ubiquitously used microscopy technique for single-molecule studies is fundamentally incapable of achieving the spatiotemporal resolution necessary to measure kinesin substeps at physiological stepping rates.This dissertation introduces two new breakthrough superresolution microscopy techniques, total internal reflection dark-field microscopy and interferometric scattering microscopy, both of which are capable of measuring the position of active proteins with 1-nm and 1-ms resolution. These new microscopes are first described in detail, including instructions on how to build them, and then are applied to studying kinesin mechanochemistry. By tracking single kinesin motors with unprecedented spatiotemporal resolution, the first measurement of substeps are made. This exciting discovery opens up the entirely new paradigm of measuring structural kinetics from single-molecule tracking experiments.Beyond the introduction of new microscopy techniques, this dissertation provides four major contributions to motor protein cell and molecular biology. First, the mechanochemical cycles of kinesin-1 and -2 are solved, providing a complete and quantitative description of how these motors transduce chemical energy into mechanical motion. Second, the generalized mechanochemical basis of processivity is elucidated, providing a detailed strategy for mapping structural differences between kinesin isoforms to differences in their ability to transport cargoes long distances. Third, the mechanochemical basis of kinesin sidestepping is elucidated, providing a means of mapping structural differences between isoforms to their ability to navigate microtubules crowded with other proteins, especially in disease states like Alzheimers. Finally, the mechanochemical basis of motor-motor coordination in multimotor transport is uncovered, providing a clear description of why both kinesins -1 and -2 are needed for effective cargo transport. Overall, this dissertation provides engineering design principles and instructions for two new exciting superresolution microscopy techniques, and leverages those techniques to answer critical biological questions about how functional diversity is achieved in the kinesin superfamily.