Loss of intermediate filament IFB-1 reduces mobility, density, and physiological function of mitochondria in Caenorhabditis elegans sensory neurons.

Mitochondria and intermediate filament (IF) accumulations often occur during imbalanced axonal transport leading to various types of neurological diseases. It is still poorly understood whether a link between neuronal IFs and mitochondrial mobility exist. In Caenorhabditis elegans, among the 11 cytoplasmic IF family proteins, IFB-1 is of particular interest as it is expressed in a subset of sensory neurons. Depletion of IFB-1 leads to mild dye-filling and significant chemotaxis defects as well as reduced life span. Sensory neuron development is affected and mitochondrial transport is slowed down leading to reduced densities of these organelles. Mitochondria tend to cluster in neurons of IFB-1 mutants likely independent of the fission and fusion machinery. Oxygen consumption and mitochondrial membrane potential is measurably reduced in worms carrying mutations in the ifb-1 gene. Membrane potential also seems to play a role in transport such as carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone treatment led to increased directional switching of mitochondria. Mitochondria co-localize with IFB-1 in worm neurons and appear in a complex with IFB-1 in pull-down assays. In summary, we propose a model in which neuronal IFs may serve as critical (transient) anchor points for mitochondria during their long-range transport in neurons for steady and balanced transport.

Characterization of TAG-63 and its role on axonal transport in C. elegans.

Model organisms are increasingly used to study and understand how neurofilament (NF)-based neurological diseases develop. However, whether a NF homolog exists in C. elegans remains unclear. We characterize TAG-63 as a NF-like protein with sequence homologies to human NEFH carrying various coiled coils as well as clustered phosphorylation sites. TAG-63 also exhibits features of NFL such as a molecular weight of around 70 kD, the lack of KSP repeats and the ability to form 10 nm filamentous structures in transmission electron micrographs. An anti-NEFH antibody detects a band at the predicted molecular weight of TAG-63 in Western blots of whole worm lysates and this band cannot be detected in tag-63 knockout worms. A transcriptional tag-63 reporter expresses in a broad range of neurons, and various anti-NFH antibodies stain worm neurons with an overlapping expression of axonal vesicle transporter UNC-104(KIF1A). Cultured neurons grow shorter axons when incubating with drugs known to disintegrate the NF network and rhodamine-labeled in vitro reconstituted TAG-63 filaments disintegrate upon drug exposure. Speeds of UNC-104 motors are diminished in tag-63 mutant worms with visibly increased accumulations of motors along axons. UNC-104/TAG-63 and SNB-1/TAG-63 not only colocalize in neurons but also revealed positive BiFC (bimolecular fluorescence assay) signals. In summary, we identified and characterized TAG-63 in C. elegans, and demonstrate that lack of this protein limits axonal transport efficiencies. Additionally, this study would aid in developing NF-related disease models in the future.

PTP-3 phosphatase promotes intramolecular folding of SYD-2 to inactivate kinesin-3 UNC-104 in neurons.

UNC-104 is the Caenorhabditis elegans homolog of kinesin-3 KIF1A known for its fast shuffling of synaptic vesicle protein transport vesicles in axons. SYD-2 is the homolog of liprin-α in C. elegans known to activate UNC-104; however, signals that trigger SYD-2 binding to the motor remain unknown. Because SYD-2 is a substrate of PTP-3/LAR PTPR, we speculate a role of this phosphatase in SYD-2-mediated motor activation. Indeed, coimmunoprecipitation assays revealed increased interaction between UNC-104 and SYD-2 in ptp-3 knockout worms. Intramolecular FRET analysis in living nematodes demonstrates that SYD-2 largely exists in an open conformation state in ptp-3 mutants. These assays also revealed that nonphosphorylatable SYD-2 (Y741F) exists predominately in folded conformations, while phosphomimicking SYD-2 (Y741E) primarily exists in open conformations. Increased UNC-104 motor clustering was observed along axons likely as a result of elevated SYD-2 scaffolding function in ptp-3 mutants. Also, both motor velocities as well as cargo transport speeds were visibly increased in neurons of ptp-3 mutants. Lastly, epistatic analysis revealed that PTP-3 is upstream of SYD-2 to regulate its intramolecular folding.