Cytoskeleton-based regulation of microtubule motors in intracellular transport.

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The morphology and mechanical properties of the cell are maintained by the cytoskeleton, a set of biopolymeric filaments. The cytoskeleton is highly dynamic, constantly remodeling to allow changes in cell shape required for essential functions like cell division and motility. At the same time, cytoskeletal filaments serve as tracks along which motor proteins carry vesicular cargoes to different locations in the cell. Directed transport is critical, particularly for long cells like neurons that can extend up to 1 m in humans. These dual structural and trafficking roles place competing demands on the cytoskeleton - to simultaneously act as a solid to maintain cell morphology, and as a fluid to allow efficient movement of vesicular cargoes and organelles through the cellular space. To meet a diverse array of demands, cytoskeletal filaments continually polymerize and depolymerize and crosslinking proteins form dynamic bonds between filaments and to the cell membrane, allowing mechanical properties to be tuned across various time and length scales. The cytoskeleton does not simply act as a passive track for motor proteins, instead it actively modulates their activity through its network architecture, post-translational modifications, and cytoskeletal-associated proteins. These cytoskeletal cues direct intracellular traffic to maintain the proper spatial and temporal localization of proteins, mRNA, organelles, and signaling molecules. This Discovery program seeks to (1) identify and characterize the molecular mechanisms that allow the cytoskeleton to dynamically adapt the cell’s mechanical properties, and (2) understand how the cytoskeleton modulates motor protein function to achieve the directed transport of intracellular cargoes. In the proposed Discovery program, we will develop advanced optical trapping techniques to perform high-resolution measurements of the frequency-dependent viscoelastic properties of the cellular environment, and examine the effect of perturbing the kinetics of cytoskeletal crosslinkers on the dynamic mechanical response. In addition, we will use novel in vitro reconstitution assays to examine the motility of isolated endogenous cargoes on native cytoskeletal networks. Through this program, two PhD students and two undergraduate students will become experts in biophysical, cell biological, and analytical techniques. The proposed studies will result in novel insights into how the cytoskeleton dynamically tunes its mechanical response and regulates motor proteins to direct intracellular transport, leading to advances in our fundamental understanding of cell biology, and the ability to design engineered nano-scale devices powered by biological motor proteins.