Cyclin-dependent kinase 5 regulates the polarized trafficking of neuropeptide-containing dense-core vesicles in Caenorhabditis elegans motor neurons

Abstract

The polarized trafficking of axonal and dendritic proteins is essential for the structure and function of neurons. Cyclin-dependent kinase 5 (CDK-5) and its activator CDKA-1/p35 regulate diverse aspects of nervous system development and function. Here, we show that CDK-5 and CDKA-1/p35 are required for the polarized distribution of neuropeptide-containing dense-core vesicles (DCVs) in Caenorhabditis elegans cholinergic motor neurons. In cdk-5 or cdka-1/p35 mutants, the predominantly axonal localization of DCVs containing INS-22 neuropeptides was disrupted and DCVs accumulated in dendrites. Time-lapse microscopy in DB class motor neurons revealed decreased trafficking of DCVs in axons and increased trafficking and accumulation of DCVs in cdk-5 mutant dendrites. The polarized distribution of several axonal and dendritic markers, including synaptic vesicles, was unaltered in cdk-5 mutant DB neurons. We found that microtubule polarity is plus-end out in axons and predominantly minus-end out in dendrites of DB neurons. Surprisingly, cdk-5 mutants had increased amounts of plus-end-out microtubules in dendrites, suggesting that CDK-5 regulates microtubule orientation. However, these changes in microtubule polarity are not responsible for the increased trafficking of DCVs into dendrites. Genetic analysis of cdk-5 and the plus-end-directed axonal DCV motor unc-104/KIF1A suggest that increased trafficking of UNC-104 into dendrites cannot explain the dendritic DCV accumulation. Instead, we found that mutations in the minus-end-directed motor cytoplasmic dynein, completely block the increased DCVs observed in cdk-5 mutant dendrites without affecting microtubule polarity. We propose a model in which CDK-5 regulates DCV polarity by both promoting DCV trafficking in axons and preventing dynein-dependent DCV trafficking into dendrites.

Figure 1.

The unc-129 promoter controls expression in a subset of DA and DB motor neurons. A, Schematic diagram of DA and DB motor neurons under the control of the unc-129 promoter used in this study. DA and DB neurons have cell bodies located on the ventral side with dendrites in the VNC and axons in the DNC. DA neurons (red) project their processes toward the anterior, whereas DB neurons (blue) project their processes toward the posterior. The DA-rich and DB-only regions of the VNC and DNC imaged throughout this study are indicated. B, A photomontage of the VNC of transgenic animals expressing soluble mCherry under the control of the unc-129 promoter. Specific DA and DB neuron cell bodies and processes expressing mCherry are shown. Neuron identities were determined based on the position of their cell bodies and the projections of their commissures. Strong mCherry expression was observed in DA1–6 and DB4–7, whereas occasional, weak expression was seen in DB3, whose cell body is located just anterior to DA2 (see A). To isolate fluorescent signals derived only from DB neurons, neuronal processes were imaged in regions of the VNC and DNC posterior to the DA6 cell body (DB-only region). To enrich for fluorescent signals derived from DA neurons, processes were imaged in regions of the VNC and DNC anterior to the DA3 cell body (DA-rich region). The animals are oriented with anterior to the left and dorsal to the top.

Figure 2.

CDK-5 regulates the polarized distribution of DCVs in DB motor neurons. A, Schematic diagram of a DB motor neuron with axon in the DNC and dendrite in the VNC (top panel). This diagram is oriented with the anterior of the animal to the left for this and all subsequent figures. The boxed region denotes that the axon was imaged for data presented in A–C and G–I. Representative images of INS-22::Venus in DB axons of young adult wild-type, cdk-5(ok626), cdk-5(gm336), Punc-129::cdk-5;cdk-5(gm336) rescue, cdka-1(gm335), and Punc-129::cdka-1;cdka-1(gm335) rescue animals (bottom panels). B, C, Quantification of INS-22::Venus puncta intensity (B) and density (C) in axons of wild-type (n = 30), cdk-5(ok626) (n = 25), cdk-5(gm336) (n = 35), cdk-5 rescue (n = 19), cdka-1(gm335) (n = 23), and cdka-1 rescue (n = 20) animals. D, Schematic diagram of a DB motor neuron (top panel). The boxed region denotes that the dendrite was imaged for data presented in D–F and J–L. Representative images of INS-22::Venus in DB dendrites of wild-type, cdk-5(ok626), cdk-5(gm336), Punc-129::cdk-5;cdk-5(gm336) rescue, cdka-1(gm335), and Punc-129::cdka-1;cdka-1(gm335) rescue animals are shown. For this and all other dendrite images, the white asterisk (*) indicates the position of a motor neuron cell body. E, F, Quantification of INS-22::Venus puncta intensity (E) and density (F) in dendrites of wild-type (n = 27), cdk-5(ok626) (n = 19), cdk-5(gm336) (n = 19), cdk-5 rescue (n = 19), cdka-1(gm335) (n = 15), and cdka-1 rescue (n = 18) animals. G, Representative images of IDA-1::GFP in DB axons of young adult wild-type and cdk-5(gm336) mutant animals. H, I, Quantification of IDA-1::GFP puncta intensity (H) and density (I) in axons of wild-type (n = 27) and cdk-5(gm336) (n = 25) mutant animals. J, Representative images of IDA-1::GFP in DB dendrites of wild-type and cdk-5(gm336) mutant animals. K, L, Quantification of IDA-1::GFP puncta intensity (K) and density (L) in dendrites of wild-type (n = 23) and cdk-5(gm336) (n = 25) mutant animals. For this and all subsequent figures, error bars denote SEM. Values that differ significantly [Tukey–Kramer (B, C, E, F) and Student's t test (H, I, K, L)] from wild type (marked by asterisks above each bar) or from other genotypes (comparisons marked by brackets) are denoted on the graphs (^#p < 0.05; *p < 0.01; **p < 0.001), and values that do not differ significantly (p > 0.05) are denoted by n.s.

Figure 3.

The polarized distribution of axonal and dendritic markers in DB motor neurons is not affected in cdk-5 mutants. A, Schematic diagram of a DB motor neuron (top panel). The boxed region denotes that the axon was imaged for data presented in A–C, E–G, I, and K. Representative images of UNC-10::GFP in DB axons of wild-type and cdk-5 mutant animals (bottom panels). B, C, Quantification of UNC-10::GFP puncta intensity (B) and density (C) in axons of wild-type (n = 21) and cdk-5(gm336) (n = 20) mutant animals. D, Schematic diagram of a DB motor neuron (top panel). The boxed region denotes that the dendrite was imaged for data presented in D, H, J, and L. Representative images of UNC-10::GFP in DB dendrites of wild-type and cdk-5(gm336) mutant animals (bottom panels). E, Representative images of GFP::RAB-3 in DB axons of wild-type and cdk-5(gm336) mutant animals. F, G, Quantification of GFP::RAB-3 puncta intensity (F) and density (G) in axons of wild-type (n = 22) and cdk-5(gm336) (n = 21) mutant animals. H, Representative images of GFP::RAB-3 in DB dendrites of wild-type and cdk-5(gm336) mutant animals. Values that differ significantly from wild-type (Student's t test) are denoted on graphs (*p ≤ 0.01). I, Representative images of UNC-9::GFP in DB axons of wild-type and cdk-5(gm336) mutant animals. J, Representative images of UNC-9::GFP in DB dendrites of wild-type and cdk-5(gm336) mutant animals. K, Representative images of FBN-1::GFP in DB axons of wild-type and cdk-5(gm336) mutant animals. L, Representative images of FBN-1::GFP in DB dendrites of wild-type and cdk-5(gm336) mutant animals.

Figure 4.

CDK-5 regulates the orientation of microtubules in DB dendrites. A, Schematic diagram of a DB motor neuron (top panel). The boxed region denotes that the axon was imaged for data presented in A and B. Representative kymographs generated from a 25 s movie of EBP-1::GFP movement in the axon of wild-type and cdk-5(gm336) mutant animals (bottom panels). B, Quantification of percentage of plus-end- and minus-end-out microtubules in wild-type (n = 76 puncta) and cdk-5 (n = 93 puncta) mutant animals based on analysis of EBP-1::GFP movement in DB axons. C, Schematic diagram of a DB motor neuron (top panel). The boxed region denotes that the dendrite was imaged for data presented in C and D. Representative kymographs generated from a 25 s movie of EBP-1::GFP movement in the dendrite of wild-type and cdk-5(gm336) mutant animals (bottom panels). D, Quantification of percentage of plus-end- and minus-end-out microtubules in wild-type (n = 107 puncta) and cdk-5 (n = 195 puncta) mutant animals based on analysis of EBP-1::GFP movement in DB dendrites. Values that differ significantly from wild type (χ² test, Yates' correction) are denoted on the graphs (**p < 0.001).

Figure 5.

CDK-5 regulates DCV trafficking in DB motor neuron axons and dendrites. A, Schematic diagram of a DB motor neuron (top panel). The boxed region denotes that the axon commissure was imaged for data presented in A–C. Representative kymographs generated from 20 s movies of INS-22::Venus puncta movement in DB axon commissures of wild-type and cdk-5(gm336) mutant animals (bottom panels). B, Quantification of the average number of INS-22::Venus puncta moving anterogradely, retrogradely, or remaining stationary in each kymograph from wild-type (n = 23) and cdk-5 (n = 25) mutant axon commissures. C, Quantification of the direction of INS-22::Venus puncta movement as a percentage of total puncta, in wild-type (n = 257 puncta) and cdk-5 (n = 258) mutant axon commissures. D, Schematic diagram of a DB motor neuron (top panel). The boxed region denotes that the dendrite was imaged for data presented in D–H. Representative kymographs generated from 20 s movies of INS-22::Venus movement in DB dendrites of wild-type and cdk-5(gm336) mutant animals (bottom panels). E, Quantification of the average number of INS-22::Venus puncta moving anterogradely, retrogradely, or remaining stationary in each kymograph from wild-type (n = 26) and cdk-5 (n = 29) mutant dendrites. F, Quantification of the direction of INS-22::Venus puncta movement, as a percentage of total puncta, in wild-type (n = 238 puncta) and cdk-5 (n = 479) mutant dendrites. G, Histogram of INS-22::Venus puncta velocities in dendrites of wild-type and cdk-5 mutant animals. Positive velocities represent anterograde movements, and negative velocities represent retrograde movements. H, Histogram of mobile and stationary INS-22::Venus puncta intensity in dendrites of wild-type and cdk-5 mutant animals. Values that differ significantly [Student's t test (B, E) and χ² test (C)] from wild type are denoted on graphs (**p < 0.001; *p < 0.01; ^#p < 0.05).

Figure 6.

UNC-104/Kif1A is not required for the increase in dendritic DCVs in cdk-5 mutants. A, Schematic diagram of a DB motor neuron (top panel). The boxed region denotes that the axon was imaged for data presented in A. Representative images of INS-22::Venus in DB axons of wild-type and unc-104(e1265) mutant animals (bottom panels). B, Schematic diagram of a DB motor neuron (top panel). The boxed region denotes that the cell body was imaged for data presented in B and C. Representative images of INS-22::Venus in the DB6 motor neuron cell body of wild-type, cdk-5(gm336), unc-104(e1265), and cdk-5;unc-104 double-mutant animals (bottom panels). C, Quantification of INS-22::Venus fluorescence intensity in DB cell bodies of wild-type (n = 24), cdk-5 (n = 14), unc-104 (n = 11), and cdk-5;unc-104 (n = 14) double-mutant animals. D, Schematic diagram of a DB motor neuron (top panel). The boxed region denotes that the dendrite was imaged for data presented in D–F. Representative images of INS-22::Venus in DB dendrites of wild-type, cdk-5(gm336), unc-104(e1256), and cdk-5;unc-104 double-mutant animals (bottom panels). E, F, Quantification of INS-22::Venus puncta intensity (E) and density (F) in DB dendrites of wild-type (n = 27), cdk-5 (n = 19), unc-104 (n = 18), and cdk-5;unc-104 (n = 18) animals. Values that differ significantly (Tukey–Kramer test) from wild type (marked by asterisks above each bar) or from other genotypes (comparisons marked by brackets) are denoted on the graphs (**p < 0.001; n.s., p > 0.05).

Figure 7.

Cytoplasmic dynein is required for the increase in dendritic DCVs in cdk-5 mutant DB dendrites. A, Schematic diagram of a DB motor neuron (top panel). The boxed region denotes that the axon was imaged for data presented in A–C and G. Representative images of INS-22::Venus DB axons of wild-type, cdk-5(gm336), dhc-1(js319), and cdk-5;dhc-1 double-mutant animals (bottom panels). B, C, Quantification of INS-22::Venus puncta intensity (B) and density (C) in axons of wild-type (n = 30), cdk-5 (n = 35), dhc-1 (n = 22), cdk-5;dhc-1 (n = 20) animals. D, Schematic diagram of a DB motor neuron (top panel). The boxed region denotes that the dendrite was imaged for data presented in D–F and H. Representative images of INS-22::Venus in DB dendrites of wild-type, cdk-5(gm336), dhc-1(js319), and cdk-5;dhc-1 double-mutant animals (bottom panels). E, F, Quantification of INS-22::Venus puncta intensity (E) and density (F) in dendrites of wild-type (n = 27), cdk-5 (n = 19), dhc-1 (n = 12), and cdk-5;dhc-1 (n = 19) animals. G, Quantification of percentage plus-end-out and minus-end-out microtubules in DB axons of wild-type (n = 76 puncta), cdk-5(gm336) (n = 93), dhc-1(js319) (n = 36), and cdk-5;dhc-1(n = 61) double mutants based on analysis of EBP-1::GFP movement in axons. H, Quantification of percentage plus-end-out and minus-end-out microtubules in DB dendrites of wild-type (n = 107 puncta), cdk-5(gm336) (n = 195), dhc-1(js319) (n = 60), and cdk-5;dhc-1 (n = 218) double-mutant animals based on analysis of EBP-1::GFP movement in dendrites. Values that differ significantly [Tukey–Kramer test (B, C, E, F) and χ² test (G, H)] from wild type (marked by asterisks above each bar) or from other genotypes (comparisons marked by brackets) are denoted on the graphs (**p < 0.001; *p < 0.01; ^#p < 0.05; n.s., p > 0.05).

Figure 8.

Cytoplasmic dynein is required for DCV trafficking in DB motor neuron dendrites in wild-type and cdk-5 mutant animals. A, Schematic diagram of a DB motor neuron (top panel). The boxed region denotes that the dendrite was imaged for data presented in A. Quantification of the direction of INS-22::Venus puncta movement in DB dendrites of wild-type (n = 34), cdk-5(gm336) (n = 36), dhc-1(js319) (n = 21), and cdk-5;dhc-1 (n = 29) double-mutant dendrites (bottom panel). B, Schematic diagram of a DB motor neuron (top panel). The boxed region denotes that the distal dendrite tip was imaged for data presented in B and C. Representative images of GFP::DLI-1 accumulations at the distal tip of DB7 dendrites in wild-type and cdk-5(gm336) mutant animals (bottom panels). C, Quantification of GFP::DLI-1 puncta intensity at the tip of DB7 dendrites in wild-type (n = 29) and cdk-5 (n = 23) mutant animals. Values that differ significantly (Tukey–Kramer test) from wild type (marked by symbols above each bar) or from other genotypes (comparisons marked by brackets) are denoted on the graphs (**p < 0.001; ^#p < 0.05; ns, p > 0.05).