Wednesday, March 12, 2025
3/12/2025
PROJECT SUMMARY 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 amyotrophic lateral sclerosis (ALS), hereditary spastic paraplegia and 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. Notably, the interactions of opposing motors with cargoes have not been characterized by biochemical or structural methods as proposed here. The nuclear pore complex protein Nup358 provides recruitment sites at the nuclear envelope for the opposing motor complexes dynein and kinesin-1, which can bind simultaneously and facilitate bi-directional positioning of the nucleus along microtubules. This pathway is important for faithful chromosome segregation and essential for a fundamental process in brain development that is required for brain progenitor cells to differentiate to neurons and other cell types. Dynein adapters such as Bicaudal D2 (BicD2) have key roles in transport, as they select cargoes and are required to activate dynein for processive motility; however, the underlying molecular mechanism is unknown. We plan: 1) To establish how dynein motility is modulated by dynein adapter/cargo complexes and by kinesin- 1. 2) to establish a structural basis for recognition of the cell nucleus as cargo by dynein adapters. 3) To establish whether dynein and kinesin-1 are recruited in a cooperative manner to Nup358 at the nucleus, i.e. whether binding of the first motor changes the affinity for the second motor. We plan to establish how BicD2/cargo complexes activate dynein for processive motility. Furthermore we plan to assess how kinesin-1 modulates motility of the dynein/BicD2/Nup358 complex. Our approach combines NMR spectroscopy, X-ray crystallography and biophysical methods, which are integrated with single-molecule processivity assays with intact dynein and kinesin-1 motors. Results will establish a structural basis for cargo selection by BicD2. 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.
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