Administrative Supplement for Award 93818 (R01GM144578)

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PROJECT SUMMARY This supplement requests a Vitrobot 5 plunge freezer for the parent grant (GM144578), which will allow to apply a new approach, cryo-electron microscopy (cryo-EM) to the parent grant, which investigates how the cell nucleus is bi-directionally transported and positioned in a cell cycle specific manner, a process that is important for cell cycle control as well as brain and muscle development. The importance of nuclear positioning for brain and muscle development is underscored by the fact that human disease mutations of proteins engaged in the transport of the nucleus cause severe brain and muscle development diseases, including spinal muscular atrophy, which is the most common genetic cause of death in infants. Yet, it is unknown how teams of opposing motor complexes collaborate to achieve correct timing, directionality and velocity of transport. We plan: 1) To establish how motility of the motor complex dynein is modulated by dynein adapter/cargo complexes and by the opposite polarity motor complex kinesin-1. 2) to establish a structural basis for recognition of the cell nucleus as cargo by dynein adapters. 3) To establish whether the opposite polarity motors dynein and kinesin-1 are recruited in a cooperative manner to the nucleus. Cryo-EM was already proposed in the parent grant as an alternative approach for structure determination and will enable the PI to expand her structural studies to larger complexes including full-length dynein adapter proteins and intact motor complexes. With the current approach, that combines X-ray crystallography and NMR for structure determination, the PI has been limited to structural studies of minimal complexes with truncated fragments. Cryo-EM will increase the impact of all three aims of the parent grant since structures of intact complexes will reveal a greater level of mechanistic insights compared to truncated complexes. This is particularly important for the proposed aims which plan to establish the activation and cargo-recognition mechanism of BicD2, as well as how bi-directional transport of the opposite polarity motors kinesin-1 and dynein is regulated by the adapter proteins. Our study serves as a model system to understand how cargo adapters regulate the motility and directionality of cargo transported bi-directionally by both dynein and kinesin-1, which is important as these motors facilitate a vast number of cellular transport events that are essential for chromosome segregation, signal transmission at synapses, brain and muscle development. More specifically, results will establish how correctly timed bi- directional transport of the nucleus is regulated, which is crucial for cell cycle control, muscle and brain development. Mutations of proteins of these pathways cause devastating neuromuscular diseases, and results will help devise therapies for these diseases.