Presynaptic components mislocalize to the dendrite of the DA9 motor neuron in wy575 and wy302 mutants
The cholinergic motor neuron DA9 elaborates a morphologically and molecularly distinct axon and dendrite, and is well suited for the study of mechanisms regulating the polarized localization of axonal and dendritic proteins in vivo
. We visualized both presynaptic components and dendritic proteins in DA9 using cell-specific promoters (itr-1 pB
) to express fluorescently tagged protein markers in vivo
. Consistent with electron microscopic reconstruction data (White et al., 1976
), synaptic vesicle proteins and active zone markers localize only to a discrete segment of the axon while several proteins localize selectively to the dendrite (Klassen and Shen, 2007
; Poon et al., 2008
To identify factors important for localizing presynaptic components, we visually screened randomly mutagenized animals for mutants with abnormal distribution patterns of presynaptic components. We isolated two mutants with similar and fully penetrant defects with presynaptic markers present in both axons and dendrites. In both wy575
mutants, synaptic vesicle-associated RAB-3, active zone component SYD-2/liprin-α, synaptic vesicle transmembrane proteins SNB-1/synaptobrevin and SNG-1/synaptogyrin mislocalize to the DA9 dendrite (, Figure S1A-L
). DA9 axonal and dendritic morphology is otherwise normal. Furthermore, the majority of the presynaptic proteins is still distributed in the axon, and these proteins colocalize with one another in the normal synaptic domain (). These results indicate that except for mislocalized presynaptic components, many aspects of DA9 development, including axon guidance, dendrite outgrowth, and synaptic target selection, are normal in these two mutants. Consistent with this notion, while both wy575
mutants have slightly lower brood sizes, they are fertile, active, and exhibit largely normal locomotion.
Synaptic Vesicle-Associated RAB-3 and Active Zone Protein SYD-2/Liprin-α Mislocalize to the DA9 Dendrite in wy575 or wy302 Mutants,.
wy575 and wy302 are mutant alleles of a cyclin-dependent kinase and a cyclin, respectively
Using single nucleotide polymorphism mapping, transgenic rescue, and sequence analysis, we mapped the wy575
mutation to pct-1
, which encodes a highly conserved Pctaire kinase of the cyclin-dependent kinase family (Figure S1U, V
). Pctaire kinases have been implicated in neurite outgrowth, exocytosis, and tau phosphorylation (Graeser et al., 2002
; Herskovits and Davies, 2006
; Liu et al., 2006
). cDNA sequences predict three isoforms of PCT-1 with common C-terminal kinase domains and variable N-terminal sequences. wy575
is a missense mutation of a highly conserved aspartic acid residue in the ATP-binding pocket of the kinase (D377N). We also examined the phenotypes of two different deletion alleles of pct-1
in DA9: one affects two of the three isoforms (ok1707)
while the other removes the kinase domain in all three isoforms and is a predicted null (tm2175). ok1707
mutants have no mislocalization defect, while tm2175
mutants are indistinguishable from wy575
mutants (), suggesting that the third isoform is sufficient and that both wy575
are likely null alleles.
PCT-1, CYY-1, CDK-5, and CDKA-1/p35 Act in Two Pathways in DA9.
Using similar methods, we mapped wy302
at the genetic locus ZK353.1. This gene encodes two highly similar isoforms containing a predicted cyclin domain. It is orthologous to mammalian cyclin Y that has not been studied previously (Figure S1X, Y
). We named the gene CYY-1 (Cy
). The wy302
allele results in a stop codon in the ninth amino acid and likely represents a null allele.
PCT-1, CYY-1, CDK-5, and CDKA-1/p35 act cell-autonomously in DA9 to ensure the polarized localization of presynaptic components
Since the phylogenetic analysis shows that PCT-1 is most closely related to CDK-5 (Figure S1U
), we further exam CDK-5 and other CDK mutants. While mutations in CDK-1, CDK-7, and CDK-8 have no effect (data not shown), null mutations in cdk-5
or its activator cdka-1/p35
lead to dendritic accumulation of presynaptic components (, Figure S1
). Like the pct-1
mutants, this mislocalization defect in cdk-5
mutants is fully penetrant. The mutant animals have normal DA9 guidance and morphology, and do not exhibit obvious defects in locomotory behavior.
We subsequently determined if these four genes are expressed endogenously in DA9. Cytoplasmic fluorophore under the control of their endogenous promoters, revealed fluorescence in DA9 as well as other ventral cord motor neurons (data not shown). CYY-1 is also expressed in other tissues such as the gut and gonad. Using the itr-1 pB promoter that drives expression specifically in DA9 in the tail, we asked if DA9-specific expression of each gene is sufficient to rescue the mislocalization defect of SVPs. For each gene, we generated two independent transgenic strains expressing the corresponding cDNA or genomic fragment in DA9. Expression of these four genes in DA9 almost completely rescues the mutant phenotype, suggesting that PCT-1, CYY-1, CDK-5, and CDKA-1/p35 act cell-autonomously in DA9 ().
PCT-1, CYY-1, CDK-5, and CDKA-1/p35 act in parallel pathways
Since pct-1, cyy-1, cdk-5,
mutants have similar defects, we investigated their genetic relationships. Using putative null mutants, we generated all six combinations of double mutants and compared them with the single mutants (). Quantitative analysis of GFP
RAB-3 distribution in the DA9 axon and dendrite showed that between 19-47% of total RAB-3 fluorescence is present in the dendrite of these single mutants compared to 0.3% in wild-type animals (). As cyy-1; pct-1
double mutant animals are indistinguishable from each single mutants, PCT-1 and CYY-1 likely function in the same genetic pathway. Likewise, cdka-1/p35 cdk-5
double mutant animals have a mislocalization defect similar to cdk-5
single mutants, a result one would expect if CDKA-1/p35 activates CDK-5.
The other four double mutants, on the other hand, exhibit dramatically enhanced mislocalization defects. Nearly all SVPs (86-98%) are present in the DA9 dendrite in the cdk-5; pct-1, cdka-1/p35; pct-1, cyy-1 cdk-5,
and cdka-1/p35 cyy-1
double mutants (). The active zone protein SYD-2/liprin-α is similarly dramatically mislocalized in these double mutants, suggesting that trafficking and localization of multiple presynaptic components are defective (Figure S2
). To address if these defects are specific to the cholinergic DA9 neuron, we examined the localization of synaptic vesicle-associated RAB-3 in other neurons. We found that the GABAergic DD motor neurons and the RIA interneurons are also severely affected in cyy-1 cdk-5
double mutants and GFP
RAB-3 mislocalizes to dendrites or dendritic segments (Figure S3
). Intriguingly, the localization of synaptic vesicle markers in the cholinergic DB motor neurons is only mildly affected in cyy-1 cdk-5
double mutants (data not shown), suggesting that these two pathways are required for many but not all neurons. Not surprisingly, these four double mutants are largely paralyzed, indicating a loss of functional synapses in motor neurons. These results are consistent with pct-1
acting in a pathway parallel to cdk-5
in multiple neurons. The observed drastic enhancement further highlights that these two pathways are essential for the polarized localization of presynaptic components.
To definitively understand the synaptic structural defects in the cyy-1 cdk-5 double mutants, we reconstructed segments (4-7μm) of the dorsal nerve cord using serial electron microscopy (EM) from three cyy-1 cdk-5 double mutant animals and three wild-type control animals. Consistent with our findings with fluorophore-tagged synaptic markers, we found that the number of synaptic vesicles and active zones in the DA and DD axons are significantly reduced whereas presynaptic specializations in the DB neurons appear normal ( and unpublished data, Watanabe and Shen). Since the reconstruction was performed where the DA1 to DA4 neurons form synapses, the synaptic vesicle localization defect in DA neurons is not limited to DA9, but is true for the anterior DA neurons as well. This EM analysis also revealed that the size and appearance of the synaptic vesicles in cyy cdk-5 mutants is indistinguishable from that in wild-type controls. Furthermore, the appearance of the muscle arms and their contacts with the motor neurons are largely normal, arguing that the wiring of the motor circuits remains intact in the mutant animals (data not shown).
The Number of Synaptic Vesicles and Active Zones is Reduced in the Dorsal Axons of DA Neurons in cyy-1 cdk-5 Double Mutants.
The close phylogenetic relationship, the similarity of their loss-of-function phenotypes, and the enhanced phenotype of the double mutants indicate that the PCT-1 and CDK-5 pathways might perform similar and redundant functions in DA9. To directly address this, we examined if over-activating one pathway could compensate for the loss of the other pathway. Indeed, we found that overexpression of PCT-1 in cdk-5 mutants significantly rescues their mislocalization defects, and likewise, overexpression of CDK-5 significantly rescues defects of pct-1 mutant animals ().
CYY-1 Activates PCT-1 and Possibly CDK-5.
CYY-1 acts as an essential activator of PCT-1
While it is well established that p35 directly binds to and activates CDK-5 (Lew et al., 1994
; Tsai et al., 1994
; Uchida et al., 1994
), the interaction between CYY-1 and PCT-1 has not been studied. No vertebrate cyclin is known to activate Pctaire kinases (Liu et al., 2006
). We therefore tested if CYY-1 associates with and activates PCT-1 using tagged recombinant CYY-1 and PCT-1 in 293T cells. We observed that CYY-1 and PCT-1 robustly coimmunoprecipitate with each other regardless of the tag we used to perform the pull-down experiment (). Furthermore, the kinase activity of PCT-1 is strictly dependent on the presence of CYY-1 (). Consistently, overexpression of functional PCT-1 completely fails to rescue the cyy-1
mutants, indicating that the function of PCT-1 depends on CYY-1 (). These results strongly suggest that CYY-1 directly binds and activates PCT-1 to regulate the localization of presynaptic components.
If PCT-1 is the only downstream factor of CYY-1, CYY-1 should have no activity in pct-1 null mutant animals. However, we observed that overexpression of CYY-1 significantly rescues the mislocalization defect in pct-1(tm2175) mutant animals (). This indicates that overexpression of CYY-1 can affect the localization of presynaptic components through a mechanism independent of PCT-1. A primary candidate is CDK-5 since it is closely related to PCT-1 and we observed that the CDK-5 transgene that robustly rescues pct-1 mutant animals only marginally rescues cyy-1 mutant animals (). As presented in , the CYY-1 transgene that rescues the mislocalization defect in pct-1 mutants completely fails to rescue the mislocalization defect in cdk-5; pct-1 or cdka-1/p35; pct-1 double mutants. Taken together, these genetic interaction studies indicate that CYY-1 acts upstream of PCT-1 and can also potentially activate the CDKA-1/p35-CDK-5 pathway.
To further test whether CYY-1 can directly activate CDK-5, we performed in vitro
kinase assays. We found that the kinase activity of CDK-5 can indeed be stimulated by CYY-1 suggesting that CYY-1 might also be an activator of CDK-5 (Figure S4A
Subcellular localization of PCT-1, CYY-1, CDK-5, and CDKA-1/p35
Next, we examined the subcellular localization of PCT-1, CYY-1, CDK-5, and CDKA-1/p35. We found that CDK-5
YFP predominantly localizes to presynaptic sites and is present as faint puncta in the dendrite (Figure S4C
YFP shows a presynaptic distribution in the axon and a punctate localization pattern in the dendrite (Figure S4E
). The localization of CDK-5 is completely dependent on UNC-104/KIF1A (data not shown). Consistent with the presynaptic localization of the vertebrate CDK-5 (Tomizawa et al., 2002
), these two distribution patterns suggest that CDK-5 and CDKA-1/p35 are likely to be associated with presynaptic components.
Interestingly, CDK-5 and PCT-1 have very different localization patterns. PCT-1
GFP is diffusely localized within DA9 and labels both the dendrite and axon evenly (Figure S4B
YFP is also present in both axons and dendrites, but is enriched in the dendrite and proximal axon (Figure S4D
). Taken together, if CYY-1 is required for the activity of PCT-1, one would imagine that the activity of PCT-1 is highest in the dendrite and lowest in the synaptic domain and distal axon.
Collectively, these data suggest that although both PCT-1 and CDK-5 affect similar biological processes, their exact cell biological functions in vivo might be different. However, they can substitute for each other when overexpressed in the single mutant background.
Dendritic mislocalization of presynaptic components in CDK mutants is unlikely to be caused by axon-dendrite specification or synapse assembly defects
After establishing the genetic and biochemical interactions between PCT-1, CYY-1, CDK-5, and CDKA-1/p35, we considered the following three possibilities that may lead to dendritic presynaptic components: 1, a defect in axon-dendrite specification; 2, a failure of local assembly at presynaptic specializations; 3, an impairment of intracellular transport of presynaptic components.
To address the first possibility, we examined DA9 axonal and dendritic guidance and outgrowth, trafficking of plus- and minus-end directed motors, dendritic proteins, and a non-synaptic axonal cargo. Both the timing and directionality of axonal and dendritic outgrowth are normal in cyy-1 cdk-5
double mutants (Figure S5A-D
, data not shown). The overall MT organization in both the axon and dendrite is generally unaltered in the absence of the CDK pathways assayed by the subcellular distribution of MT-based motors (Figure S5E-H
). Futhermore, we examined four dendritic proteins including CAM-1/ROR receptor tyrosine kinase, UNC-9/innexin, DYS-1/dystrophin, and fibrillin (Poon et al., 2008
; Sieburth et al., 2005
) and they localize nomally to dendrites in cyy-1 cdk-5
mutant animals (Figure S5I-P
). Glutamate receptor localizes to dendrite of another neuron RIA, further indicating that trafficking of dendritic cargo is unaffected (Figure S3H, K
). Lastly, we investigated if other axonal cargo are trafficked normally. TOM20, a subunit of the mitochondrial translocase complex, remains present in the axon in cyy-1 cdk-5
mutant animals (Figure S5Q-R
), suggesting that non-presynaptic axonal cargo continues to be trafficked appropriately to the axon. Taken together, our data suggest that general axon and dendrite specification is normal in the absence of the CDK pathways and that the CDKs specifically affect localization of presynaptic components and not other axonal or dendritic cargo.
Is the failure of local assembly at presynaptic specializations the direct cause for dendritic mislocalization of presynaptic components in cdk-5
mutant? Since cdk-5
single mutants have a relatively normal accumulation of RAB-3 in the presynaptic region compared with the syd-1; syd-2/liprin-α
double mutants that have severe presynaptic assembly defects in DA9 whereas cdk-5
single mutants exhibit much more severe accumulation of RAB-3 in the dendrites, there is no correlation between defective presynaptic assembly and the accumulation of stable RAB-3 puncta in the dendrite (Figure S5S-T
). It is thus unlikely that the assembly defect alone can fully account for the dramatic mislocalization of presynaptic components in cyy-1 cdk-5
We then considered the third possibility that the CDK pathways regulate intracellular transport of presynaptic components. Since intracellular transport occurs continuously throughout the life of the neuron, we expect the CDKs to be required continuously if they regulate transport. Using a temperature-dependent silencing strategy (Poon et al., 2008
), we observed that disruption of PCT-1 or CDK-5 late in development is sufficient to mislocalize SVPs to the dendrite. In addition, rescuing PCT-1 or CDK-5 late in development is sufficient to partially remove ectopic SVPs from the dendrite (Figure S6A-C
). These findings suggest that both CDKs are required to maintain the polarized localization of SVPs and that this defect is reversible.
PCT-1 and CDK-5 genetically interact with the kinesin motor UNC-104/KIF1A
To further understand how the CDK pathways regulate molecular transport, we examined the primary motor responsible for SVP transport, UNC-104/KIF1A, in greater depth. Consistent with previous reports (Hall and Hedgecock, 1991
), we found that RAB-3 is completely absent from the axon of unc-104
mutants, suggesting that UNC-104 is the key anterograde motor responsible for the axonal transport of SVPs. Surprisingly, we also found that SVPs dramatically accumulate in the DA9 cell body and dendrite of unc-104
mutants, a phenotype similar to that observed in cyy-1 cdk-5
double mutants (). This distribution pattern indicates that there might be another motor that transports SVPs into the dendrite in the absence of the UNC-104 motor. The similar phenotypes caused by disrupting both CDK pathways or by mutations in UNC-104 suggest that they might be involved in the same biological process. Consistent with this notion, the unc-104; cyy-1 cdk-5
triple mutants are indistinguishable from the unc-104
single mutant ().
The CDKs Affect Axonal Transport of Presynaptic Components.
To further understand the relationship between UNC-104 and the CDK pathways, we overexpressed UNC-104 in the absence of both CDK pathways. As seen in , UNC-104 expression leads to a partial suppression of the dendritic RAB-3 phenotype. In about 30% of the animals, the dendritic RAB-3 signal is completely absent, suggesting that overproduction of UNC-104 can partially compensate for the loss of both CDK pathways by trafficking SVPs to the axon (). However, despite the absence of dendritic RAB-3 in these animals, their dorsal axonal RAB-3 distribution is still significantly different from that in wild-type animals (). There are fewer and dimmer RAB-3 puncta, which are spread over a larger than normal segment of the dorsal axon. Though these results suggest that the CDKs are likely also involved in events distinct from UNC-104-mediated anterograde transport, they are consistent with the CDK pathways affecting intracellular transport of presynaptic components.
PCT-1 and CDK-5 inhibit retrograde transport of SVPs
To further elucidate how the CDK pathways affect intracellular transport, we performed time-lapse imaging experiments to visualize transporting SVPs in DA9 in vivo. We found that there are numerous small RAB-3 puncta in the axonal segment between the cell body and the dorsal synaptic domain (). These dim puncta are often motile during imaging sessions that last up to a minute, unlike synaptic RAB-3 puncta that remain stable over several days (data not shown). These motile RAB-3 puncta are missing from the axon in unc-104 mutants, suggesting that trafficking utilizes the same molecular motor as the synaptic vesicles. Hence, we hypothesize that these small puncta represent motile SVPs during axonal transport.
The CDKs inhibit retrograde transport of presynaptic components.
Time-lapse imaging experiments were performed on the dorsal asynaptic segment of the axon immediately proximal to the synaptic region of DA9 (). Measurements in wild-type animals revealed that 76% of the movements are toward the distal axon (anterograde), while 24% of the moving events are retrograde. The average speed of anterograde events is 1.9 μm/s, similar to the speed of dense core vesicles measured in vivo
in C. elegans
(Zahn et al., 2004
). The average speed of the retrograde movements is 2.3 μm/s, similar to the speed of the retrograde motor dynein in living Dictyostelium
cells (Ma and Chisholm, 2002
). In order to understand if UNC-104 mediates anterograde movements, we measured the speed of anterograde events in a hypomorphic allele unc-104(e1265wy565)
. We found that only the speed of anterograde movements, but not retrograde events, is drastically reduced, suggesting that UNC-104 mediates anterograde movements ().
Besides the speed, we also measured the absolute number of anterograde and retrograde movement events. In unc-104(e1265wy565)
mutants, the absolute number of anterograde events is drastically reduced, consistent with a weakened anterograde motor. Retrograde movement is also reduced, but to a lesser extent, probably due to the reduction of available SVPs to be measured in the mutant axon. Unlike unc-104(e1265wy565)
mutants, the number of anterograde events is not significantly altered in cyy-1 cdk-5
double mutants. Instead, we observed a dramatic increase in retrograde events (194% compared with wild type
, ). Other dynamic parameters such as the run length and the pause rate remain unaffected in the cyy-1 cdk-5
double mutant (data not shown). Though unc-104(e1265wy565)
mutants exhibit a severe impairment in anterograde SVP transport judging from the speed and number of anterograde events (), there is still significantly more RAB-3 signal in the unc-104(e1265wy565)
mutant axon compared with the cyy-1 cdk-5
double mutant (Figure S6D-E
). Based on these data, it is more likely that the dramatic increase in retrograde events in cyy-1 cdk-5
double mutants leads to an imbalance between anterograde and retrograde trafficking, ultimately resulting in failed assembly of presynaptic specializations in the axon and ectopic accumulation of presynaptic components in the dendrite. Similar dynamic measurements in cdk-5
single mutants also reveal imbalanced trafficking with an abnormally high ratio of retrograde events. The amplitude of the defect in single mutants is smaller compared with that of the double mutants (Figure S6F
). Hence, we hypothesize that the CDK pathways promote axonal transport by inhibiting retrograde transport. The most direct prediction of this hypothesis is that mutations in a retrograde motor should suppress the mislocalization defect in the CDK mutants.
PCT-1 and CDK-5 likely regulate SVP transport by inhibiting the cytoplasmic dynein complex
To further identify other downstream components of the CDK pathways, we conducted an unbiased cdk-5
suppressor screen and isolated five mutants with little or no dendritic RAB-3 in the cdk-5
mutant background. Four belong to a single complementation group and we mapped one of the four, wy622
, which turned out to be a mutant allele of DHC-1
, which encodes C. elegans
ortholog of the heavy chain of the retrograde motor dynein (). Sequence analysis revealed that wy622
represents a point mutation in a conserved leucine residue in N-terminal region 2, while the previously characterized but unsequenced loss-of-function allele js319
represents a splice acceptor mutation at the end of intron 12 in the C-terminal conserved region () (Koushika et al., 2004
). We generated dhc-1; cdk-5
and dhc-1; pct-1
double mutants with the js319
allele. Similar to the wy622
allele, the js319
allele completely suppresses the dendritic accumulation of SVPs in either cdk-5
single mutants (). The dhc-1(js319)
mutant also strongly suppresses the severe dendritic RAB-3 defect and reduces the number of retrogradely moving SVPs in cyy-1 cdk-5
double mutant animals (, ). These results suggest the CDK pathways promote axonal transport by inhibiting dynein-mediated retrograde movement in wild-type animals.
Loss-of-Function Mutations in Dynein Suppress the Mislocalization Defect in the CDK Mutants.
NUD-2 likely acts downstream of CDK-5 and PCT-1
To further establish the mechanistic link between the CDK-5 pathways and the cytoplasmic dynein complex, we considered NUD-2 as a direct target. NUD-2 is the worm ortholog of Nudel, a component of the cytoplasmic dynein complex that has been demonstrated as a substrate of CDK5 in vertebrate system (Niethammer et al., 2000
; Sasaki et al., 2000
). Indeed, we found that the null nud-2
) allele completely suppresses the dendritic localization of SVPs in cdk-5
single mutants, and significantly suppresses the mislocalization phenotype in the cyy-1 cdk-5
double mutants (). This suppression by nud-2
is indistinguishable compared with the suppression by dhc-1
. These results indicate that both DHC-1 and NUD-2 function downstream of the CDK-5/PCT-1 pathways and that CDK-5 and PCT-1 inhibit the activity of the cytoplasmic dynein complex ().