RNA interference (RNAi) is a broadly used reverse genetics method in C. elegans. Unfortunately, RNAi does not inhibit all genes. We show that loss of function of a putative RNA-directed RNA polymerase (RdRP) of C. elegans, RRF-3, results in a substantial enhancement of sensitivity to RNAi in diverse tissues. This is particularly striking in the nervous system; neurons that are generally refractory to RNAi in a wild-type genetic background can respond effectively to interference in an rrf-3 mutant background. These data provide the first indication of physiological negative modulation of the RNAi response and implicate an RdRP-related factor in this effect. The rrf-3 strain can be useful to study genes that, in wild-type, do not show a phenotype after RNAi, and it is probably the strain of choice for genome-wide RNAi screens.
Rim1 was previously identified as a Rab3 effector localized to the presynaptic active zone in vertebrates. Here we demonstrate that C. elegans unc-10 mutants lacking Rim are viable, but exhibit behavioral and physiological defects that are more severe than those of Rab3 mutants. Rim is localized to synaptic sites in C. elegans, but the ultrastructure of the presynaptic densities is normal in Rim mutants. Moreover, normal levels of docked synaptic vesicles were observed in mutants, suggesting that Rim is not involved in the docking process. The level of fusion competent vesicles at release sites was reduced fivefold in Rim mutants, but calcium sensitivity of release events was unchanged. Furthermore, expression of a constitutively open form of syntaxin suppressed the physiological defects of Rim mutants, suggesting Rim normally acts to regulate conformational changes in syntaxin. These data suggest Rim acts after vesicle docking likely via regulating priming.
Structural DNA nanotechnology seeks to build synthetic molecular machinery from DNA. DNA nanomachines are artifi cially designed assemblies that switch between defi ned conformations in response to an external cue. Though it has proved possible to create DNA machines and rudimentary walkers, the function of such autonomous DNA-based molecular devices has not yet been achieved inside living organisms. Here we demonstrate the operation of a pH-triggered DNA nanomachine inside the nematode Caenorhabditis elegans . The nanomachine uses fl uorescence resonance energy transfer to effectively map spatiotemporal pH changes associated with endocytosis in wild type as well as mutant worms, demonstrating autonomous function within the organismal milieu in a variety of genetic backgrounds. From this fi rst demonstration of the independent functionality of a DNA nanomachine in vivo , we observe that rationally designed DNA-based molecular devices retain their in vitro functionality with quantitative precision. This positions DNA nanodevices as exciting and powerful tools to interrogate complex biological phenomena.
The polarized distribution of neuronal proteins to axons and dendrites relies upon microtubule-binding proteins such as CRMP, directed motors such as kinesin UNC-104/Kif1A, and diffusion barriers such as ankyrin. The causative relationships between these molecules are unknown. We show here that Caenorhabditis elegans CRMP (UNC-33) acts early in neuronal development, together with ankyrin (UNC-44), to organize microtubule asymmetry and axon-dendrite sorting. In unc-33 and unc-44 mutants, axonal proteins are present in dendrites and vice versa, suggesting bidirectional failures of axon-dendrite identity. UNC-33 protein is localized to axons by unc-44, and enriched in a region that resembles the axon initial segment. unc-33 and unc-44 establish the asymmetric dynamics of axonal and dendritic microtubules; in their absence, microtubules are disorganized, the axonal kinesin UNC-104 invades dendrites, and inappropriate UNC-104 activity randomizes axonal protein sorting. We suggest that UNC-44 and UNC-33 direct polarized sorting through their global effects on neuronal microtubule organization.
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