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 C. 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 where CDK-5 regulates DCV polarity by both promoting DCV trafficking in axons and preventing dynein-dependent DCV trafficking into dendrites.

A Novel CaM Kinase II Pathway Controls the Location of Neuropeptide Release fromCaenorhabditis elegansMotor Neurons

“…In C. elegans DA and DB motor neurons, the commissures extend from the cell somas on the ventral side of the animal across the body to the synaptic region of the axon in the dorsal nerve cord ( Figure 6A). The concentration of DCVs in a wild-type commissure is much lower than in the synaptic region, and time-lapse imaging studies suggest that many fluorescently tagged neuropeptide puncta within commissures represent individual vesicles or packets of cotransported vesicles (Goodwin et al 2012; also supported by Figure 10 in this study).…”

“…In C. elegans DA and DB motor neurons, the commissures extend from the cell somas on the ventral side of the animal across the body to the synaptic region of the axon in the dorsal nerve cord ( Figure 6A). The concentration of DCVs in a wild-type commissure is much lower than in the synaptic region, and time-lapse imaging studies suggest that many fluorescently tagged neuropeptide puncta within commissures represent individual vesicles or packets of cotransported vesicles (Goodwin et al 2012; also supported by Figure 10 in this study).We compared the number of detectable fluorescent puncta containing soluble and aggregated neuropeptide cargos in wild-type and unc-43 lf mutant commissures. In the ventral commissure region, which is the closest region to the cell soma, the number of detectable puncta containing soluble cargo and aggregated neuropeptide cargo in an unc-43 lf mutant was reduced by 63 and 82%, respectively (Figure 6B).…”

“…The soluble DCV cargo NLP-21-Venus is reduced by 60% in unc-43 lf mutant somas ( Figure 2C), and the aggregated neuropeptide cargos INS-22-Venus and unprocessed NLP-21-Venus are reduced by 45 and 80%, respectively ( Figure 7A). Prior studies have found that mutants that affect the polarity of DCV trafficking, such as cdk-5 (cyclin-dependent kinase-5) and syd-2 (Liprin-a) mutants, have lower levels of DCV cargos in their DB motor neuron axons, but the missing cargos can be found in dendrites (Goodwin et al 2012;Goodwin and Juo 2013). However, that is not the case here, since unc-43 mutant dendrites also have lower levels of the DCV cargos ( Figure 7A).…”

“…The fluorescent signal comes from NLP-21-Venus neuropeptide in dense core vesicles expressed from the integrated transgene ceIs56. Time-lapse imaging demonstrates that most of these puncta represent individual vesicles (Goodwin et al 2012). (B and C) Representative, identically scaled images and quantification of soluble NLP-21-Venus cargo, expressed from the integrated transgene ceIs56 (first pair of bars), or unprocessed NLP-21-Venus cargo (aggregated neuropeptide cargo; Figure S1) ), expressed from the ceIs56 transgene in an egl-3 null mutant background (second pair of bars) in DA/DB motor neuron axons in animals with the indicated genotypes.…”

Cyclin-dependent kinase 5 regulates the proliferation, motility and invasiveness of lung cancer cells through its effects on cytoskeletal remodeling

“…Toll-like receptor 4 induces remodeling via phosphorylation of the myosin light chain for the release of microparticles, thus regulating tumor inflammatory microenvironment (17). Goodwin et al (18) reported that, in motor neuron cells, CDK-5 regulates microtubule orientation and in cancer, Strock et al (8) reported that inhibiting the activity of CDK5 resulted in changes in the microtubule cytoskeleton, loss of cellular polarity and loss of motility in prostate cancer. In the present study, inhibition of CDK5 resulted in failed microtubule assembly, thus leading to impaired mobility of the A549 lung cancer cells.…”