unc-51 genetically interacts with unc-76 in axonal transport. SGNs of wild type or the indicated mutants immunostained with anti-CSP at the third instar larval stage. Arrows show CSP-positive aggregates. Bar, 20 μm. The number of aggregates found per 50 μm of nerve shaft was scored for each genotype (graph). Statistical significance evaluated by Student t test. (**) P < 0.01. Error bars show ±SEM. Genotypes are w^- (A), unc-51³/+ (B), unc-76^Df(1)107/+ (C), and unc-76^Df(1)107/+;unc-51³/+ (D).

Disorganized localization of SV and motor complexes in unc-51 mutants. (A) SGNs of unc-51 mutants (unc-51³/unc-51²⁵) or wild-type third instar larvae immunostained with anti-UNC-76 (red) and anti-Syt-1 (green). Red arrows and arrowheads point to aberrant localization of the indicated proteins. The right panel shows enlarged image (arrowheads, UNC-76; arrows, Syt-1). The numbers of puncta positive for UNC-76 and/or Syt-1 in unc-51 mutant SGNs were scored and the percentages of single-positive or double-positive puncta are shown in the graph. SGNs are oriented from the proximal side (top) toward the distal (bottom). (B) SGNs of unc-51 mutants or wild-type larvae immunostained with anti-UNC-76 (red) and anti-KHC (green). Red arrows point to aberrant localization of the indicated proteins. The right panel shows enlarged image (asterisks, KHC/UNC-76 double-positive aggregates). The numbers of puncta positive for UNC-76 and/or KHC in unc-51 mutant SGNs were scored and the percentages of single-positive or double-positive puncta are shown (graph). (C) SGNs of unc-51 mutants or wild-type larvae immunostained with anti-CSP (red) and anti-KHC (green). Red arrows and arrowheads point to aberrant localization of the indicated proteins. Right panel shows enlarged image (arrowheads, CSP; arrows, KHC). (Graph) The numbers of puncta positive for CSP and/or KHC in unc-51 mutant SGNs were scored and the percentages of single-positive or double-positive puncta are shown. (D) SGNs of unc-51 mutant or wild-type larvae immunostained with anti-CSP (red) and anti-UNC-76 (green) and observed with confocal microscopy (with 100× objective; single optical section taken at 0.7-μm interval). SGNs are oriented from the proximal side (left) toward the distal (right). Areas positive for CSP, UNC-76 or both signals were calculated based on the confocal data using an image analysis program, and schematically represented on the graph. Bars, 10 μm.

UNC-51 regulates the affinity between UNC-76 and Syt-1. (A) UNC-51-mediated association of Syt-1 and UNC-76. Myc- or HA-tagged UNC-51 (full-length), UNC-76 (full-length), and Syt-1 (full-length) were coexpressed in HEK293T cells as indicated. Cell lysates were immunoprecipitated by anti-HA followed by immunoblot with anti-myc. (B) Phosphorylation-dependent association of Syt-1 and UNC-76. HA-UNC-76, myc-Syt-1, and myc-UNC-51 or UNC-51/K38A were expressed in HEK293T cells as indicated. Cell lysates were immunoprecipitated with anti-HA. The immunecomplexes were incubated in the presence (+) or absence (−) of alkaline phosphatase (CIAP) and analyzed by immunoblot using anti-myc. (C) Syt-1–UNC-76 interaction does not require Ca²⁺. HA-UNC-76, myc-Syt-1, and myc-UNC-51 or UNC-51/K38A were expressed in HEK293T cells as indicated. Cell lysates were immunoprecipitated with anti-HA. The immune complexes were incubated in the presence of EGTA or CaCl₂ and analyzed by immunoblot using anti-myc. (D) Syt-1–UNC-76 interaction detected by FRET. COS7 cells were transfected with Syt-1-CFP, YFP-UNC-76, YFP-UNC-76/S143D, YFP-UNC-76/S143A, or UNC-51/K38A in combination as indicated. For each combination, initial (red) and fluorescence after acceptor photobleaching spectra (green) were plotted for a range of wavelengths indicated. Increase in donor fluorescence after acceptor bleaching indicates FRET (green arrows). Quantification of FRET (graph). (**) Statistical significance (P < 0.01). (E) KHC/UNC-76/Syt-1 complex formation. GST alone (G) and GST fused with KHC (amino acids 675–975) (GK) were produced in E. coli, purified on Glutathione-Sepharose beads, and incubated with myc-UNC-76 and myc-Syt-1 expressed in HEK293T cells. Proteins that bound to the beads were eluted and analyzed by immunoblot using anti-myc.

unc-51 functions in axonal transport. (A) Molecular structures of the unc-51-null mutant alleles. P-element EP(3)3348 is inserted within the first exon of the unc-51 gene, 539 bp upstream of the initiation ATG codon. Two unc-51-null alleles, unc-51³ and unc-51²⁵, were generated by imprecise excision. PCR amplification of these unc-51 alleles (blue arrows indicate locations of primers) and subsequent sequencing analyses confirmed that unc-51³ has an 804 bp deletion and unc-51²⁵ has a 1787-bp deletion. Both alleles lack the start codon, and did not affect the expression unit of CG17667 located within intron 2. (B) SGNs of unc-51-null mutant (unc-51³/unc-51²⁵) or wild-type third instar larvae immunostained with anti-Syt-1 (red) and anti-CSP (green). Syt-1-positive aggregates colocalized with those positive for CSP in unc-51 mutants. Bar, 10 μm. (C) Ultrastructure of SV aggregates in unc-51 mutants. Electron micrograph of a cross-section of an unc-51 mutant SGN immunostained with anti-Syt-1. Arrowheads indicate SVs positive for anti-Syt-1. (Table) The number of SV aggregates (defined as a cluster of >20 SVs per aggregate) was scored in electron-micrographs from unc-51 mutants (n = 34) and wild type (n = 23). Bar, 200 nm. (D) Genetic rescue of axonal transport defect in unc-51 mutants by unc-51 transgene. CSP-positive SV aggregation phenotype in unc-51 mutants was rescued by pan-neuronal expression of unc-51/wild-type transgene (elav^c155 > UAS-unc-51^WT; unc-51³/unc-51²⁵), but not by a kinase-deficient unc-51 (elav^c155 > UAS-unc-51^K38A; unc-51³/unc-51²⁵). Numbers of CSP-positive puncta per 50 μm SGN (mean ± SEM) were plotted for each genotype (graph). (**) Statistically significant rescue (P < 0.01, Student t test). (n.s.) Not significant (P = 0.843). Bar, 10 μm.

Physical interaction of UNC-51, UNC-76, and KHC. (A) Myc-tagged UNC51 (full-length) and HA-tagged UNC-76 (truncation mutants) were coexpressed in HEK293T cells as indicated. Cell lysates were immunoprecipitated by anti-HA followed by immunoblot with anti-myc. (*) Nonspecific band (22 kDa) detected by anti-HA antibody. (B) UNC-51 binds UNC-76 in vitro. GST alone or GST fused with full-length UNC-76 was produced in E. coli, purified on Glutathione-Sepharose beads, and incubated with myc-tagged UNC-51 C′-terminal domain (amino acids 557–855) produced by in vitro translation (IVT). Proteins that bound to the beads were eluted and analyzed by immunoblot using anti-myc. (C) Schematic representation of the domains involved in UNC-51−UNC-76 binding. Black bars show deletion constructs that bind the other in coimmunoprecipitation assays. Constructs shown in gray did not bind. Binding of all deletion constructs except for UNC-76 (118−340 amino acids) were tested by coimmunoprecipitation assays in A and Supplemental Figure S5, whereas no binding of UNC-76 (118−340 amino acids) with UNC-51 was confirmed by GST pull-down assay (data not shown). The UNC-76 and UNC-51 functional domains are indicated within each protein. (D) UNC-51 forms a complex with KHC via UNC-76. HEK293T cell extracts containing heterologously expressed UNC-51 (full-length), UNC-76 (full-length), and KHC (full-length), as indicated, were analyzed by immunoprecipitation with anti-HA followed by immunoblot with the indicated antibodies.

Phosphorylation of UNC-76 Ser¹⁴³ by UNC-51 is necessary for axonal transport. (A) In vitro phosphorylation of UNC-76 by UNC-51 kinase. HA-tagged UNC-51 or UNC-51/K38A expressed in HEK293T cells was immunoprecipitated and incubated with His-UNC-76 in the presence of Mg²⁺ and [γ-³²P]-ATP. Phosphoproteins were analyzed by autoradiograph. Black arrow indicates UNC-51 autophosphorylation. Red arrowhead indicates UNC-76 phosphorylation. (B) Myc-tagged UNC-76 and HA-tagged UNC-51 were coexpressed in HEK293T cells. Cell lysates were incubated for an indicated period of time with (+) or without (−) alkaline phosphatase (CIAP) and analyzed by immunoblot using anti-myc. Red bracket indicates mobility shifts of UNC-76. Black bracket indicates nonshifted UNC-76. (C) Myc-tagged wild type and Ser/Thr → Ala mutant UNC-76 were expressed in HEK293T cells with UNC-51 or UNC-51/K38A. Cell lysates were analyzed by immunoblot using anti-myc. Red bracket indicates mobility shifts of UNC-76. (D) Extracts from wild-type and unc-51 mutant larvae were analyzed by immunoblot using an anti-phospho-UNC-76 antibody. Anti-actin and anti-UNC-76 were used as loading controls. (E) Genetic rescue of axonal transport defect in unc-76 mutants by unc-76 transgene. CSP-positive aggregation (arrows) and KHC aggregation (arrowheads) in unc-76 mutants were rescued by unc-76^WT (top) or unc-76^S143D (middle), but not by unc-76^S143D transgene (bottom). Transgene expression was driven by elav. unc-76^DF(1)107 hemizygous males were used as unc-76-null mutants. Third instar larvae for each genotype were stained with anti-KHC (green), anti-UNC-76 (red), and anti-CSP (blue). Note the equivalent expression levels of UNC-76 transgenes in VNC and SGN. (**) Statistical significance (P < 0.01, Student t test). (n.s.) Not significant (P = 0.18). Bars: red, 50 μm; white, 20 μm.

Schematic model for UNC-51 kinase-mediated motor–cargo assembly Our results suggest a model in which UNC-51 kinase functions in axonal transport via the kinesin-1-dependent pathway. When phosphorylated by UNC-51 kinase, UNC-76, a KHC adaptor and a potential activator of kinesin-1, displays an increased affinity to SV membrane proteins such as Syt-1. UNC-51 likely phosphorylates additional substrates that are essential for axonal transport (dashed arrow). Loss or attenuation of UNC-51 kinase activity would result in a lower affinity of UNC-76 to SV membrane proteins and dissociation of SV cargoes from the motor complexes, as might be the case at NMJ. Kinesin-3 (imac/UNC-104) is also responsible for SV transport in a large fraction and is important for carrying SVs from neuronal soma to axons and synapses (red arrow).