title: Identification and Characterization of LIN-2(CASK) as a Regulator of Kinesin-3 UNC-104(KIF1A) Motility and Clustering in Neurons.
abstract: Kinesin-3 UNC-104(KIF1A) is the major axonal transporter of synaptic vesicles. Employing yeast two-hybrid and co-immunoprecipitation (Co-IP) assays, we characterized a LIN-2(CASK) binding site overlapping with that of reported UNC-104 activator protein SYD-2(Liprin-α) on the motor's stalk domain. We identified the L27 and GUK domains of LIN-2 to be the most critical interaction domains for UNC-104. Further, we demonstrated that the L27 domain interacts with the sterile alpha motifs (SAM) domains of SYD-2, while the GUK domain is able to interact with both the coiled coils and SAM domains of SYD-2. LIN-2 and SYD-2 colocalize in Caenorhabditis elegans neurons and display interactions in bimolecular fluorescence complementation (BiFC) assays. UNC-104 motor motility and Synaptobrevin-1 (SNB-1) cargo transport are largely diminished in neurons of LIN-2 knockout worms, which cannot be compensated by overexpressing SYD-2. The absence of the motor-activating function of LIN-2 results in increased motor clustering along axons, thus retaining SNB-1 cargo in cell bodies. LIN-2 and SYD-2 both positively affect the velocity of UNC-104, however, only LIN-2 is able to efficiently elevate the motor's run lengths. From our study, we conclude that LIN-2 and SYD-2 act in a functional complex to regulate the motor with LIN-2 being the more prominent activator.
authors.affiliation: Institute of Molecular and Cellular Biology & Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan (ROC).
authors.forename: Gong-Her
authors.initials: GH
authors.lastname: Wu
authors.affiliation: Institute of Molecular and Cellular Biology & Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan (ROC).
authors.forename: Muniesh
authors.initials: M
authors.lastname: Muthaiyan Shanmugam
authors.affiliation: Institute of Molecular and Cellular Biology & Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan (ROC).
authors.forename: Prerana
authors.initials: P
authors.lastname: Bhan
authors.affiliation: Institute of Molecular and Cellular Biology & Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan (ROC).
authors.forename: Yu-Hsin
authors.initials: YH
authors.lastname: Huang
authors.affiliation: Institute of Molecular and Cellular Biology & Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan (ROC).
authors.forename: Oliver Ingvar
authors.initials: OI
authors.lastname: Wagner
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism; D001370:Axonal Transport / Q000502:physiology*; D001369:Axons / Q000378:metabolism*; D017173:Caenorhabditis elegans / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement / Q000502:physiology*; D015801:Helminth Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism*
chemical_list: D029742:Caenorhabditis elegans Proteins; D015801:Helminth Proteins; C097978:Lin-2 protein, C elegans; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; C423186:UNC-104 protein, C elegans
keywords: BiFC; C. elegans; CASK; KIF1A; LIN-2; Liprin-α; MAGUK; SNB-1; SYD-2; Synaptobrevin-1; UNC-104