The CC1-FHA Tandem Controlls The Dimerization And Activation Of KIF1A

Kinesin-3motors play prominent roles in the transport of synaptic vesicles and other membrane-bound organelles in neuronal cells.In the absence of cargo, kinesin motors are kept inactive to prevent futile ATP hydrolysis and motility. Two working models have been proposed for how the activity of kinesin motors is suppressed in the absence of cargo. The first model posits that the dimeric motors are regulated by an autoinhibitory mechanism. The second model states that motor activity is regulated by the transition from the monomeric to dimeric states. The current evidence shows that the Kinesin-3motor KIF1A adopts a monomeric form in vitro but act as a dimer in vivo.Here, we show that the expressed KIF1A is in the dimeric inactive statein vivo. KIF1Ais regulated by the autoinhibitory mechanism. We demonstrate that CC1-FHA tandem of KIF1A exists as a stable dimer. The structure of CC1-FHA reveals that the linker between CC1and FHA unexpectedly forms a β-finger hairpin, which integrates CC1with FHA assembling a CC1-FHA homodimer. More importantly, dissociation of the CC1-FHA dimer unleashes CC1and the β-finger, which are both essential for the motor inhibiton. Thus, dimerization of the CC1-FHA tandem not only promotes the KIF1A dimer formation but also may trigger the motor activity via sequestering the CC1/β-finger region. The CC1-FHA tandem likely functions as a hub for controlling the dimerization and activation of KIF1A, which may represent a new paradigm for the kinesin regulation shared by other Kinesin-3motors.However, it remains to be determined whether the CC1-FHA dimer biochemically and structurally characterized in vitro is essential for the KIF1A-mediated axonal cargo transport in vivo. We used C. elegans as the model organism to explore the potential role of the CC1-FHA dimer for the KIF1A-mediated axonal transport in vivo. Exogenous expression of either KIF1A or the mutant with deletion of the β-finger (Δ[474-486]-KIF1A) could largely rescue the locomotion defect of unc-104(e1265)mutant, whereas the mutant with point mutations that dissociate the CC1-FHA dimer (exposing CC1and the β-finger,(L508Q/Y510Q-KIF1A)) was unable to rescue this locomotion defect. Moreover, dissociation of the CC1-FHA dimer with the mutation exposing CC1and the β-finger also significantly impaired the axonal transport of SVs leading to the accumulation of SVs in the cell bodies. Together with the above in vitro biochemical studies, the in vivo data shown here firmly establish the essential role of the CC1-FHA dimer for KIF1A-mediated axonal SV transport.During recent years, new quantitative and mechanistic insights into the biomolecular function have been derived from the single-molecule technique. However, the current live cell, single-molecule imaging is most readily possible with events occurring on the plasma membranes. Whereas analysis of cytoplasmic events has been limited to some specialized labeling techniques. To analyze cytoplasmic events, it is required for single-molecule imagingto follow each individual molecule in the cytoplasm by fluorescent protein-based probes, which can maintain biomolecular functionality and also avoid artifacts associated with the external uptake. Here, we label the motor molecule Kinesin-3with mEos3.2(a photo-convertible fluorescent proteins) and demonstrate that it is possible to track the movement of single protein molecules in the cytoplasm of live cells with high temporal and spatial resolution. Moreover, we hypothesize that the various velocities of Kinesin-3motorsare determined by the catalytic domain, the motor domain.