| Description |
Intracellular cargos are shuttled around the cell via molecular motor proteins along their respective filament substrates. For decades, biophysicists have taken advantage of in vitro techniques to study fine details of the molecular motor machinery. Today, thanks in large part to in vitro experimentation, a great deal is known about the structure-function relationship of various motors, including kinesin-1. The field is now shifting to investigate how multiple motors work together to transport cargos around the cell's complex microtubule (MT) network. Due to the complexity of the cell's complex biochemical makeup and the heterogeneity of its three-dimensional (3D) MT network, this topic is virtually impossible to address quantitatively in the native cellular environment. Instead, in vitro experiments must be used to ensure full control over all relevant variables to study how geometry alone, impacts cargo transport. Traditional in vitro bead assays cannot faithfully model the cell's 3D MT network, and thus cannot be used to test how MT network geometry (orthogonal filament separation, or crossing angle) affects cargo transport. To remedy this, we developed a novel in vitro method to manipulate individual MT filaments in 3D with nanometer precision. With this technique, we constructed MT-MT crossings with various geometries to test how separation distance and angle between MT filaments impact transport behaviors of artificial model cargos driven by kinesin-1. We find that variable separation distance and angle influence cargo navigational behaviors at MT-MT crossings. We also use our experimental data to constrain a 3D simulation to probe aspects of the overall transport system that are not possible to assay experimentally. We propose detailed mechanisms that underlie the MT network's influence on cargo transport. |
