Delineating The Molecular Mechanisms That Regulate The Motor Protein Kinesin-2
Intracellular transportation is vitally important to cell function: without the help of motor proteins carrying molecular cargo across the vast distances of the cell’s cytosol, few cellular mechanisms would be able to take place. We study the heterodimeric motor protein kinesin-2 (KIF3A/KIF3B), which is of particular interest due to its implication in several human genetic diseases. KIF3A/KIF3B drives cilia formation as well as transport, so it is expressed in many cell types. Trucks consume gas to travel highways. Kinesins need ATP to walk along microtubules, or “cell highways.” The resources of a cell must be allocated economically. To save energy, motors must only run when needed to transport cargo. We aimed to investigate how KIF3A/KIF3B regulates its own movement through a process called “autoinhibition.” Previous kinesin research provides a framework for possible autoinhibition mechanisms. The autoinhibition of many kinesins is mediated through hinges in certain regions that allow the protein to interact with itself, folding back and inhibiting the microtubule-binding motor domains. These interactions are thought to block cargo-kinesin and microtubule-kinesin interactions and thereby inhibit microtubule-based movement and ATP consumption. The specific regions of tail and motor domain that interact in KIF3A/KIF3B to facilitate autoinhibition are, however, not known. We hypothesize that heterodimeric kinesin-2 is also autoinhibited by specific interactions between tail-, stalk-, and motor domains and that by disrupting these interactions the motor will be rendered constitutively active. To test this hypothesis, we generated a fluorescentlytagged, engineered motor in which we fused the motor domain of KIF3A to the stalk and tail domain of KIF3B and vice versa. Fluorescence microscopy revealed that unlike the wild-type motor which was diffusely distributed across the cytosol, this engineered motor strongly accumulated in the cellular periphery. This accumulation is a hallmark phenotype for a kinesin that has lost regulation by autoinhibition. We are moving forward with this project by constructing kinesin-2 constructs that have successively longer truncations of these regions of interest. Analyzing the intracellular localization of these motors will allow us to map the interactions that mediate the autoinhibition of the KIF3A/KIF3B motor.
Murarus, Alexandra, "Delineating The Molecular Mechanisms That Regulate The Motor Protein Kinesin-2" (2021). Biology. 23.