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[
Curr Biol,
2016]
To establish and maintain their complex morphology and function, neurons and other polarized cells exploit cytoskeletal motor proteins to distribute cargoes to specific compartments [1]. Recent studies in cultured cells have used inducible motor protein recruitment to explore how different motors contribute to polarized transport and to control the subcellular positioning of organelles [2,3]. Such approaches also seem promising avenues for studying motor activity and organelle positioning within more complex cellular assemblies, but their applicability to multicellular in vivo systems has so far remained unexplored. Here, we report the development of an optogenetic organelle transport strategy in the in vivo model system Caenorhabditis elegans. We demonstrate that movement and pausing of various organelles can be achieved by recruiting the proper cytoskeletal motor protein with light. In neurons, we find that kinesin and dynein exclusively target the axon and dendrite, respectively, revealing the basic principles for polarized transport. In vivo control of motor attachment and organelle distributions will be widely useful in exploring the mechanisms that govern the dynamic morphogenesis of cells and tissues, within the context of a developing animal.
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[
Curr Biol,
2015]
The fundamental importance of neuronal connectivity for neural function has stimulated brain-mapping efforts in several organisms. In the nematode Caenorhabditis elegans an essentially complete wiring diagram has been available for nearly thirty years. To identify neglected, but well-connected neurons of C. elegans we used online activity and literature searches to rank 'popularity'. Cross-referencing this list with a topological analysis of the connectome, DVC emerged as the worm's most understudied hub neuron. We found that optogenetic activation of DVC promotes backward locomotion and that this response is dependent on the vesicular glutamate transporter, EAT-4.
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[
Curr Biol,
2011]
Sleep-like states occur in the life of all animals carefully studied and are characterized by reduced behavioral and neural activity as well as reduced responsiveness to stimulation [1]. How is reduced responsiveness to stimulation generated? We used calcium imaging to investigate a sleep-like state in larvae of the nematode Caenorhabditis elegans. We found that overall spontaneous neural activity was reduced during the sleep-like state in many neurons, including the mechanosensory neuron ALM. Stimulus-evoked calcium transients and behavior were reduced in ALM during the sleep-like state. Thus, reduced activity of ALM may contribute to reduce responsiveness during a sleep-like state.
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[
Nature,
1999]
Intraflagellar transport (IFT) is important in the formation and maintenance of many cilia, such as the motile cilia that drive the swimming of cells and embryos, the nodal cilia that generate left-right asymmetry in vertebrate embryos, and the sensory cilia that detect sensory stimuli in some animals. The heterotrimeric kinesin-II motor protein drives the anterograde transport of macromolecular complexes, called rafts, along microtubule tracks from the base of the cilium to its distal tip, whereas cytoplasmic dynein moves the rafts back in the retrograde direction. We have used fluorescence microscopy to visualize for the first time the intracellular transport of a motor and its cargo in vivo. We observed the anterograde movement of green fluorescent protein (GFP)-labelled kinesin-II motors and IFT rafts within sensory cilia on chemosensory neurons in living Caenorhabditis elegans.
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Kim Y, Kim S, Kim J, Lee S, Shong M, Chung J, Song S, Kim JM, Park J, Lee SB, Bae E
[
Nature,
2006]
Autosomal recessive juvenile parkinsonism (AR-JP) is an early-onset form of Parkinson''s disease characterized by motor disturbances and dopaminergic neurodegeneration. To address its underlying molecular pathogenesis, we generated and characterized loss-of-function mutants of Drosophila PTEN-induced putative kinase 1 (PINK1), a novel AR-JP-linked gene. Here, we show that PINK1 mutants exhibit indirect flight muscle and dopaminergic neuronal degeneration accompanied by locomotive defects. Furthermore, transmission electron microscopy analysis and a rescue experiment with Drosophila Bcl-2 demonstrated that mitochondrial dysfunction accounts for the degenerative changes in all phenotypes of PINK1 mutants. Notably, we also found that PINK1 mutants share marked phenotypic similarities with parkin mutants. Transgenic expression of Parkin markedly ameliorated all PINK1 loss-of-function phenotypes, but not vice versa, suggesting that Parkin functions downstream of PINK1. Taken together, our genetic evidence clearly establishes that Parkin and PINK1 act in a common pathway in maintaining mitochondrial integrity and function in both muscles and dopaminergic neurons.
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[
Nature,
2006]
Parkinson''s disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction has been implicated as an important trigger for Parkinson''s disease-like pathogenesis because exposure to environmental mitochondrial toxins leads to Parkinson''s disease-like pathology. Recently, multiple genes mediating familial forms of Parkinson''s disease have been identified, including PTEN-induced kinase 1 (PINK1; PARK6) and parkin (PARK2), which are also associated with sporadic forms of Parkinson''s disease. PINK1 encodes a putative serine/threonine kinase with a mitochondrial targeting sequence. So far, no in vivo studies have been reported for pink1 in any model system. Here we show that removal of Drosophila PINK1 homologue (CG4523; hereafter called pink1) function results in male sterility, apoptotic muscle degeneration, defects in mitochondrial morphology and increased sensitivity to multiple stresses including oxidative stress. Pink1 localizes to mitochondria, and mitochondrial cristae are fragmented in pink1 mutants. Expression of human PINK1 in the Drosophila testes restores male fertility and normal mitochondrial morphology in a portion of pink1 mutants, demonstrating functional conservation between human and Drosophila Pink1. Loss of Drosophila parkin shows phenotypes similar to loss of pink1 function. Notably, overexpression of parkin rescues the male sterility and mitochondrial morphology defects of pink1 mutants, whereas double mutants removing both pink1 and parkin function show muscle phenotypes identical to those observed in either mutant alone. These observations suggest that pink1 and parkin function, at least in part, in the same pathway, with pink1 functioning upstream of parkin. The role of the pink1-parkin pathway in regulating mitochondrial function underscores the importance of mitochondrial dysfunction as a central mechanism of Parkinson''s disease pathogenesis.