Date of Award
Master of Science (MS)
School of Biological Sciences
Martin F. Engelke
Autoinhibition is a conserved mechanism regulating the motor proteins of the kinesin superfamily, which allows active motors to switch into an inactive state after cargo unloading. This regulation is generally believed to be an ATP conservation mechanism. Autoinhibition occurs when the motor molecule assumes a folded conformation in which the non-motor portions interact with, and thereby inhibit the activity of, the motor domains. This process is incompletely characterized in the heterotrimeric kinesin-2 motor (KIF3A/KIF3B), which is essential for mammalian anterograde intraflagellar transport and ciliogenesis. Furthermore, the physiological role that kinesin autoinhibition plays in cellular processes has only recently started to be elucidated. To delineate the mechanism and function of KIF3A/KIF3B autoinhibition, we expressed wild-type and engineered variants of mouse Kif3a and Kif3b genes in a Kif3a, Kif3b double knockout mouse embryonic fibroblast cell lines (NIH-3T3 cells). Since the double knockout cells cannot produce cilia, we have used them to develop a ciliogenesis rescue assay, in which we reexpress KIF3A/KIF3B motor variants to test if they rescue ciliogenesis and hence have wild-type functionality. To assay motor activity status, we utilize the peripheral accumulation assay, in which we examine the cellular localization of fluorescently-tagged variant motors. Diffuse distribution of motor proteins within the cell indicates intact autoinhibition, and a peripherally accumulated motor distribution indicates nonfunctional autoinhibition and thus a constitutively active motor. Using the assays described above, we find that deletion of the hinge renders KIF3A/KIF3B constitutively active. Moreover, motor constructs in which the stalk and motor domains of KIF3A and KIF3B have been swapped accumulate peripherally. This indicates that specific interactions between the motor and tail portions mediate autoinhibition. Interestingly, these motor constructs are unable to rescue ciliogenesis. Since the cargo-binding portion of these motor constructs is unchanged, this finding suggests that intact autoinhibition is indispensable for the KIF3A/KIF3B motor to drive ciliogenesis. Next, we expressed several motors with successive truncations in the tail regions of KIF3A and KIF3B. We find that the shortest truncations lose autoinhibition, indicating that a short stretch of amino acids in the N-terminal tail portion of KIF3A and KIF3B is required for autoinhibition. Lastly, expression of further chimeric motors suggests that the coiled-coiled domains might also be involved in autoinhibition, and that the stalk-tail portion of the motor facilitates autoinhibition by preferentially binding to the KIF3A motor domain. In summary, our data suggest that intact autoinhibition is not merely an energy conservation mechanism but is required for motors to drive specific processes such as ciliogenesis. Furthermore, our findings provide the groundwork for unraveling the molecular basis of KIF3A/KIF3B autoinhibition in cells and how this motor is regulated for intracellular transport and ciliogenesis.
Sawe, Caleb Ayooluwa, "Characterization of the Autoinhibition Mechanism of the Heterotrimeric Kinesin-2 Motor Protein Kif3a / Kif3 B" (2023). Theses and Dissertations. 1865.
Available for download on Sunday, January 11, 2026