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.

Insights on UNC-104-dynein/dynactin interactions and their implications on axonal transport in Caenorhabditis elegans.

Bidirectional cargo transport in neurons can be explained by two models: the "tug-of-war model" for short-range transport, in which several kinesin and dynein motors are bound to the same cargo but travel in opposing directions, and by the "motor coordination model" for long-range transport, in which small adaptors or the cargo itself activates or deactivates opposing motors. Direct interactions between the major axonal transporter kinesin-3 UNC-104(KIF1A) and the dynein/dynactin complex remains unknown. In this study, we dissected and evaluated the interaction sites between UNC-104 and dynein as well as between UNC-104 and dynactin using yeast two-hybrid assays. We found that the DYLT-1(Tctex) subunit of dynein binds near the coiled coil 3 (CC3) of UNC-104, and that the DYRB-1(Roadblock) subunit binds near the CC2 region of UNC-104. Regarding dynactin, we specifically revealed strong interactions between DNC-6(p27) and the FHA-CC3 stretch of UNC-104, as well as between the DNC-5(p25) and the CC2-CC3 region of UNC-104. Motility analysis of motors and cargo in the nervous system of Caenorhabditis elegans revealed impaired transport of UNC-104 and synaptic vesicles in dynein and dynactin mutants (or in RNAi knockdown animals). Further, in these mutants UNC-104 clustering along axons was diminished. Interestingly, when dynamic UNC-104 motors enter a stationary UNC-104 cluster their dwelling times are increased in dynein mutants (suggesting that dynein may act as an UNC-104 activator). In summary, we provide novel insights on how UNC-104 interacts with the dynein/dynactin complex and how UNC-104 driven axonal transport depends on dynein/dynactin in C. elegans neurons.

Toxic Effects of Size-tunable Gold Nanoparticles on Caenorhabditis elegans Development and Gene Regulation.

We utilized size-tunable gold nanoparticles (Au NPs) to investigate the toxicogenomic responses of the model organism Caenorhabditis elegans. We demonstrated that the nematode C. elegans can uptake Au NPs coated with or without 11-mercaptoundecanoic acid (MUA), and Au NPs are detectable in worm intestines using X-ray microscopy and confocal optical microscopy. After Au NP exposure, C. elegans neurons grew shorter axons, which may have been related to the impeded worm locomotion behavior detected. Furthermore, we determined that MUA to Au ratios of 0.5, 1 and 3 reduced the worm population by more than 50% within 72 hours. In addition, these MUA to Au ratios reduced the worm body size, thrashing frequency (worm mobility) and brood size. MTT assays were employed to analyze the viability of cultured C. elegans primary neurons exposed to MUA-Au NPs. Increasing the MUA to Au ratios increasingly reduced neuronal survival. To understand how developmental changes (after MUA-Au NP treatment) are related to changes in gene expression, we employed DNA microarray assays and identified changes in gene expression (e.g., clec-174 (involved in cellular defense), cut-3 and fil-1 (both involved in body morphogenesis), dpy-14 (expressed in embryonic neurons), and mtl-1 (functions in metal detoxification and homeostasis)).

Uptake of TiO2 Nanoparticles into C. elegans Neurons Negatively Affects Axonal Growth and Worm Locomotion Behavior.

We employ model organism Caenorhabditis elegans to effectively study the toxicology of anatase and rutile phase titanium dioxide (TiO2) nanoparticles (NPs). The experimental results show that nematode C. elegans can take up fluorescein isothiocyanate-labeled TiO2 NPs and that both anatase and rutile TiO2 NPs can be detected in the cytoplasm of cultured primary neurons imaged by transmission electron microscopy. After TiO2 NP exposure, these neurons also grow shorter axons, which may be related to the detected impeded worm locomotion behavior. Furthermore, anatase TiO2 NPs did not affect the worm's body length; however, we determined that a concentration of 500 µg/mL of anatase TiO2 NPs reduced the worm population by 50% within 72 h. Notably, rutile TiO2 NPs negatively affect both the body size and worm population. Worms unable to enter the L4 larval stage explain a severe reduction in the worm population at TiO2 NPs LC50/3d. To obtain a better understanding of the cellular mechanisms involved in TiO2 NP intoxication, DNA microarray assays were employed to determine changes in gene expression in the presence or absence of TiO2 NP exposure. Our data reveal that three genes (with significant changes in expression levels) were related to metal binding or metal detoxification (mtl-2, C45B2.2, and nhr-247), six genes were involved in fertility and reproduction (mtl-2, F26F2.3, ZK970.7, clec-70, K08C9.7, and C38C3.7), four genes were involved in worm growth and body morphogenesis (mtl-2, F26F2.3, C38C3.7, and nhr-247), and five genes were involved in neuronal function (C41G6.13, C45B2.2, srr-6, K08C9.7, and C38C3.7).

Intramolecular regulation of presynaptic scaffold protein SYD-2/liprin-α.

SYD-2/liprin-α is a multi-domain protein that associates with and recruits multiple active zone molecules to form presynaptic specializations. Given SYD-2's critical role in synapse formation, its synaptogenic ability is likely tightly regulated. However, mechanisms that regulate SYD-2 function are poorly understood. In this study, we provide evidence that SYD-2's function may be regulated by interactions between its coiled-coil (CC) domains and sterile α-motif (SAM) domains. We show that the N-terminal CC domains are necessary and sufficient to assemble functional synapses while C-terminal SAM domains are not, suggesting that the CC domains are responsible for the synaptogenic activity of SYD-2. Surprisingly, syd-2 alleles with single amino acid mutations in the SAM domain show strong loss of function phenotypes, suggesting that SAM domains also play an important role in SYD-2's function. A previously characterized syd-2 gain-of-function mutation within the CC domains is epistatic to the loss-of-function mutations in the SAM domain. In addition, yeast two-hybrid analysis showed interactions between the CC and SAM domains. Thus, the data is consistent with a model where the SAM domains regulate the CC domain-dependent synaptogenic activity of SYD-2. Taken together, our study provides new mechanistic insights into how SYD-2's activity may be modulated to regulate synapse formation during development.

Tau/PTL-1 associates with kinesin-3 KIF1A/UNC-104 and affects the motor's motility characteristics in C. elegans neurons.

Tauopathies are neurodegenerative diseases based on pathological tau-aggregation including Alzheimer's disease, frontotemporal dementia (FTD) and Pick's disease. In general, cargo (e.g., ß-amyloid precursor protein, tau, neurofilaments) accumulation is a commonly observed phenomenon in degenerated neurons. Therefore, it is crucial to investigate the interaction between cargo, microtubule-binding proteins and molecular motors. We report the effect of tau/PTL-1 (protein with tau-like repeats) on the transport characteristics of the major axonal transporter kinesin-3 KIF1A/UNC-104 in the nervous system of Caenorhabditis elegans. Using confocal spinning disk time-lapse imaging we analyzed the motility of UNC-104::mRFP in ptl-1 knockout worms and found that predominantly retrograde moving characteristics are affected (rather than the motor's anterograde displacements). A similar motility pattern was observed for synaptobrevin-1-containing vesicles, a major cargo of UNC-104. Moreover, UNC-104 and PTL-1 colocalize and occasionally co-migrate. We further confirmed physical interactions between PTL-1 and UNC-104 in living animals using the bimolecular fluorescence complementation assay (BiFC) as well as in co-immunoprecipitation experiments. Though this study focuses on PTL-1/UNC-104 interactions, we extended our research on monitoring conventional kinesin-1 (UNC-116) as well as dynein motility pattern and found that in ptl-1 mutants retrograde displacements were also affected for UNC-116, while for dynein, interestingly, its anterograde movements were affected.

Synaptic scaffolding protein SYD-2 clusters and activates kinesin-3 UNC-104 in C. elegans.

Kinesin-3 motor UNC-104/KIF1A is essential for transporting synaptic precursors to synapses. Although the mechanism of cargo binding is well understood, little is known how motor activity is regulated. We mapped functional interaction domains between SYD-2 and UNC-104 by using yeast 2-hybrid and pull-down assays and by using FRET/fluorescence lifetime imaging microscopy to image the binding of SYD-2 to UNC-104 in living Caenorhabditis elegans. We found that UNC-104 forms SYD-2-dependent axonal clusters (appearing during the transition from L2 to L3 larval stages), which behave in FRAP experiments as dynamic aggregates. High-resolution microscopy reveals that these clusters contain UNC-104 and synaptic precursors (synaptobrevin-1). Analysis of motor motility indicates bi-directional movement of UNC-104, whereas in syd-2 mutants, loss of SYD-2 binding reduces net anterograde movement and velocity (similar after deleting UNC-104's liprin-binding domain), switching to retrograde transport characteristics when no role of SYD-2 on dynein and conventional kinesin UNC-116 motility was found. These data present a kinesin scaffolding protein that controls both motor clustering along axons and motor motility, resulting in reduced cargo transport efficiency upon loss of interaction.

Softness, strength and self-repair in intermediate filament networks.

One cellular function of intermediate filaments is to provide cells with compliance to small deformations while strengthening them when large stresses are applied. How IFs accomplish this mechanical role is revealed by recent studies of the elastic properties of single IF protein polymers and by viscoelastic characterization of the networks they form. IFs are unique among cytoskeletal filaments in withstanding large deformations. Single filaments can stretch to more than 3 times their initial length before breaking, and gels of IF withstand strains greater than 100% without damage. Even after mechanical disruption of gels formed by crossbridged neurofilaments, the elastic modulus of these gels rapidly recovers under conditions where gels formed by actin filaments are irreversibly ruptured. The polyelectrolyte properties of IFs may enable crossbridging by multivalent counterions, but identifying the mechanisms by which IFs link into bundles and networks in vivo remains a challenge.

The interaction of neurofilaments with the microtubule motor cytoplasmic dynein.

Neurofilaments are synthesized in the cell body of neurons and transported outward along the axon via slow axonal transport. Direct observation of neurofilaments trafficking in live cells suggests that the slow outward rate of transport is due to the net effects of anterograde and retrograde microtubule motors pulling in opposition. Previous studies have suggested that cytoplasmic dynein is required for efficient neurofilament transport. In this study, we examine the interaction of neurofilaments with cytoplasmic dynein. We used fluid tapping mode atomic force microscopy to visualize single neurofilaments, microtubules, dynein/dynactin, and physical interactions between these neuronal components. AFM images suggest that neurofilaments act as cargo for dynein, associating with the base of the motor complex. Yeast two-hybrid and affinity chromatography assays confirm this hypothesis, indicating that neurofilament subunit M binds directly to dynein IC. This interaction is blocked by monoclonal antibodies directed either to NF-M or to dynein. Together these data suggest that a specific interaction between neurofilament subunit M and cytoplasmic dynein is involved in the saltatory bidirectional motility of neurofilaments undergoing axonal transport in the neuron.