AbstractsEngineering

Microtubules are cytoskeletal filaments essential for multiple cell functions, including motility of microorganisms and cell division. Of particular interest is how these biological polymers generate the forces required for movement of chromosomes during mitosis and for formation of cilia and flagella. Defective microtubule-based force generation can lead to various pathological complications; therefore, an understanding of microtubule force generation is important for cancer research and biotechnology. The mechanism by which elongating microtubules generate force is unknown. Several proteins, including End-Binding Protein 1 (EB1) and adenomatous polyposis coli (APC), specifically localize to microtubule elongating ends where the microtubule is tightly bound to a motile object and generating force. The role of these end-tracking proteins is not fully understood, but they likely modulate microtubule-motile surface interactions, and may aid in force production. The objective of my research is to elucidate the role of polymerizing microtubules and end-binding proteins, specifically EB1, in force-dependent processes by formulating a model that explains their interaction and role in force generation. The commonly assumed Brownian Ratchet model describing the forces caused by elongating microtubules cannot easily explain force generation during rapid elongation and strong attachment of the microtubule to the motile object. I propose a novel mechanism in which EB1 proteins behave as end-tracking motors that have a higher affinity for guanosine triphosphate-bound tubulin than guanosine diphosphate-bound tubulin, thereby allowing them to convert the chemical energy of microtubule-filament hydrolysis to mechanical work. These microtubule end-tracking motors are predicted to provide the required forces for cell motility and persistent attachment between the motile surface and polymerizing microtubules. I have developed mechanochemical models that characterize the kinetics of these molecular motors based on experimentally determined binding parameters and thermodynamic constraints. These models account for the association of EB1 to tethered and untethered elongating microtubule ends, in the absence or presence of force, and with or without EB1 binding to solution-phase tubulin. These models explain the observed exponential profile of EB1 on untethered filaments and predict that affinity-modulated end-tracking motors should achieve higher stall forces than with the Brownian Ratchet system, while maintaining a strong, persistent attachment to the motile object.