The CC1-FHA dimer is essential for KIF1A-mediated axonal transport of synaptic vesicles in C. elegans
Introduction
Kinesins are a superfamily of microtubule-based molecular motors responsible for long-range intracellular transport of membrane vesicles/organelles and protein complexes in polarized cells such as neurons [1], [2], [3]. Kinesins have been divided into 14 sub-families (kinesin-1 to kinesin-14) [4]. KIF1A, a member of kinesin-3 motors, was identified from a screening of murine brain cDNAs that are essential for neuronal transport [5]. Interestingly, UNC-104, KIF1A homolog in Caenorhabditis elegans, was also demonstrated to be responsible for neuronal transport, i.e., anterograde transport of synaptic vesicle (SVs) precursors in axon [6], [7], and loss of function unc-104(e1265) mutant showed accumulation of SVs in the cell bodies [8]. Based on the previous functional characterizations, KIF1A/UNC-104 was regarded as a kinesin motor dedicated for the fast anterograde axonal transport of SVs in neurons [8].
Compared to the conventional kinesin motors, which can form processive dimers for transport, kinesin-3 KIF1A was surprisingly found to adopt a monomeric form in vitro and thus denoted as an unconventional “single-headed” kinesin [6]. However, recent several lines of studies have demonstrated that, similar to the conventional kinesin motors, kinesin-3 KIF1A can exist as processive dimers in vivo, and the dimerization of the motor is largely mediated by the neck coil (NC), a short coiled-coil immediately following the motor domain (MD) [9], [10]. Moreover, kinesin-73, another kinesin-3 motor from Drosophila, can form processive dimers in vivo as well [11], [12]. Thus, more and more data support the view that kinesin-3 family motors may adopt “two-headed” dimers but not “single-headed” monomers for the processive movement [13].
In KIF1A/UNC-104, a motor regulatory region immediately follows the N-terminal MD and NC, which includes a number of short non-continuous coiled-coil domains (CC1 to CC3) and a FHA domain (Fig. 1A). CC1 can directly sequester NC to prevent the NC-mediated dimerization of the motor, while CC2 can fold back to directly interact with the FHA domain to interfere with the motor activity [14]. Recently, we have found that the CC1-FHA tandem of KIF1A forms a stable dimer, which can promote the formation of motor dimers for the processive movement [15]. The structural studies of the CC1-FHA tandem further revealed that the linker between CC1 and the FHA domain unexpectedly forms a β-finger structure, which integrates the two domains forming a domain-swapped homo-dimer (Fig. 1B). Since both CC1 and the β-finger are essential for the motor inactivation, formation of the CC1-FHA dimer can directly sequester these two domains to prevent the CC1/β-finger-mediated motor inhibition [15]. Thus, in addition to facilitating the dimerization of KIF1A, the CC1-FHA tandem plays a critical role in regulating the motor activation, denoted as a central hub for controlling the dimerization and activation of KIF1A [15]. 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.
In this study, 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 ([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 CC1 and the β-finger, (L508Q/Y510Q-KIF1A)) was unable to rescue this locomotion defect. Moreover, dissociation of the CC1-FHA dimer with the mutation exposing CC1 and the β-finger also significantly impaired the axonal transport of SVs leading to the accumulation of SVs in the cell bodies. Together with previous 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.
Section snippets
C. elegans transformation
mkif1a:: gfp with various mutations were PCR amplifed from pGW1-mkif1a plasmid [16] and cloned into pD95.75 vector containing Punc-104 promoter. Germline transformation was performed by a standard microinjection method [17]. 80 ng/μl [Punc104::mkif1a::RFP] plasmid was injected into unc104(e1265); ceIs62[Punc-129:ANF::Venus, Punc-129:RFP::SNB-1, Pttx-3::RFP] worms. Multiple transgenic lines for each transgene were examined for fluorescence expression.
Behavioral assays
All behavioral assays were performed with
KIF1A/UNC-104 is broadly expressed in neurons of C. elegans
Given the short life cycle, compact genome, hermaphroditic reproduction and accessibility to genetic manipulation, C. elegans is an ideal model system for biological research in terms of genetic, cellular and molecular dissection of organism behavior [20], [21]. We set out to use this model system to explore the in vivo function of the CC1-FHA dimer for KIF1A-mediated neuronal activities. Based on the crystal structure of the CC1-FHA dimer (Fig. 1B), we selected two types of mutants for the
Discussion
Kinesin-3 KIF1A/UNC-104 is one of the key kinesin motors for the axonal transport in neurons, and it is debatable about its monomeric or dimeric conformation for the processive movement on microtubule tracks. We have demonstrated that the CC1-FHA tandem of KIF1A can form a stable dimer in solution, which can facilitate the dimerizaiton of the motor in vitro and thus support the view that kinesin-3 KIF1A is a dimeric processive motor. More intriguingly, besides the motor dimerization, the
Acknowledgments
We thank Dr. Kenneth G. Miller for providing ceIs62[Punc-129:ANF::Venus, Punc-129:RFP::SNB-1, Pttx-3::RFP] strains; Drs. Jae-Ran Lee and Eunjoon Kim for the full-length mouse KIF1A cDNA; Caenorhabditis Genetic Center for unc-104 (e1265) strain. This work was supported by grants from the National Major Basic Research Program of China (2010CB833701 and 2011CB910503), and the National Natural Science Foundation of China (31190062, 31070657, 31130065 and 31128010).
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