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The kinesin-associated protein UNC-76 is required for axonal transport in the Drosophila nervous system.

ABSTRACT: Kinesin-I is essential for the transport of membrane-bound organelles in neural and nonneural cells. However, the means by which kinesin interacts with its intracellular cargoes, and the means by which kinesin-cargo interactions are regulated in response to cellular transport requirements are not fully understood. The C terminus of the Drosophila kinesin heavy chain (KHC) was used in a two-hybrid screen of a Drosophila cDNA library to identify proteins that bind specifically to the kinesin tail domain. UNC-76 is an evolutionarily conserved cytosolic protein that binds to the tail domain of KHC in two-hybrid and copurification assays, indicating that kinesin and UNC-76 form a stable complex in vivo. Loss of Drosophila Unc-76 function results in locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants, suggesting that UNC-76 is required for kinesin-dependent axonal transport. Unc-76 exhibits dosage-sensitive genetic relationships with Khc and Kinesin light chain mutations, further supporting the hypothesis that UNC-76 and kinesin-I work in a common transport pathway. Given the interaction of FEZ1, the mammalian homolog of UNC-76, with protein kinase Czeta, and the role of FEZ1 in axon outgrowth, we propose that UNC-76 helps integrate kinesin activity in response to transport requirements in axons.

SUBMITTER: Gindhart JG

PROVIDER: S-EPMC181572 | biostudies-literature | 2003 Aug

REPOSITORIES: biostudies-literature

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The kinesin-associated protein UNC-76 is required for axonal transport in the Drosophila nervous system.

Gindhart Joseph G JG Chen Jinyun J Faulkner Melissa M Gandhi Rita R Doerner Karl K Wisniewski Tiffany T Nandlestadt Aline A

Molecular biology of the cell 20030503 8

Kinesin-I is essential for the transport of membrane-bound organelles in neural and nonneural cells. However, the means by which kinesin interacts with its intracellular cargoes, and the means by which kinesin-cargo interactions are regulated in response to cellular transport requirements are not fully understood. The C terminus of the Drosophila kinesin heavy chain (KHC) was used in a two-hybrid screen of a Drosophila cDNA library to identify proteins that bind specifically to the kinesin tail ...[more]

PMID: 12925768

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Project description:BackgroundMembers of the evolutionary conserved Ser/Thr kinase Unc-51 family are key regulatory proteins that control neural development in both vertebrates and invertebrates. Previous studies have suggested diverse functions for the Unc-51 protein, including axonal elongation, growth cone guidance, and synaptic vesicle transport.Methodology/principal findingsIn this work, we have investigated the functional significance of Unc-51-mediated vesicle transport in the development of complex brain structures in Drosophila. We show that Unc-51 preferentially accumulates in newly elongating axons of the mushroom body, a center of olfactory learning in flies. Mutations in unc-51 cause disintegration of the core of the developing mushroom body, with mislocalization of Fasciclin II (Fas II), an IgG-family cell adhesion molecule important for axonal guidance and fasciculation. In unc-51 mutants, Fas II accumulates in the cell bodies, calyx, and the proximal peduncle. Furthermore, we show that mutations in unc-51 cause aberrant overshooting of dendrites in the mushroom body and the antennal lobe. Loss of unc-51 function leads to marked accumulation of Rab5 and Golgi components, whereas the localization of dendrite-specific proteins, such as Down syndrome cell adhesion molecule (DSCAM) and No distributive disjunction (Nod), remains unaltered. Genetic analyses of kinesin light chain (Klc) and unc-51 double heterozygotes suggest the importance of kinesin-mediated membrane transport for axonal and dendritic development. Moreover, our data demonstrate that loss of Klc activity causes similar axonal and dendritic defects in mushroom body neurons, recapitulating the salient feature of the developmental abnormalities caused by unc-51 mutations.Conclusions/significanceUnc-51 plays pivotal roles in the axonal and dendritic development of the Drosophila brain. Unc-51-mediated membrane vesicle transport is important in targeted localization of guidance molecules and organelles that regulate elongation and compartmentalization of developing neurons.

Project description:Here, we established the Drosophila Giant Fiber neurons (GF) as a novel model to study axonal trafficking of L1-type Cell Adhesion Molecules (CAM) Neuroglian (Nrg) in the adult CNS using live imaging. L1-type CAMs are well known for their importance in nervous system development and we previously demonstrated a role for Nrg in GF synapse formation. However, in the adult they have also been implicated in synaptic plasticity and regeneration. In addition, to its canonical role in organizing cytoskeletal elements at the plasma membrane, vertebrate L1CAM has also been shown to regulate transcription indirectly as well as directly via its import to the nucleus. Here, we intend to determine if the sole L1CAM homolog Nrg is retrogradley transported and thus has the potential to relay signals from the synapse to the soma. Live imaging of c-terminally tagged Nrg in the GF revealed that there are at least two populations of retrograde vesicles that differ in speed, and either move with consistent or varying velocity. To determine if endogenous Nrg is retrogradely transported, we inhibited two key regulators, Lissencephaly-1 (Lis1) and Dynactin, of the retrograde motor protein Dynein. Similar to previously described phenotypes for expression of poisonous subunits of Dynactin, we found that developmental knock down of Lis1 disrupted GF synaptic terminal growth and that Nrg vesicles accumulated inside the stunted terminals in both mutant backgrounds. Moreover, post mitotic Lis1 knock down in mature GFs by either RNAi or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) induced mutations, resulted in normal length terminals with fully functional GF synapses which also exhibited severe accumulation of endogenous Nrg vesicles. Thus, our data suggests that accumulation of Nrg vesicles is due to failure of retrograde transport rather than a failure of terminal development. Together with the finding that post mitotic knock down of Lis1 also disrupted retrograde transport of tagged Nrg vesicles in GF axons, it demonstrates that endogenous Nrg protein is transported from the synapse to the soma in the adult central nervous system in a Lis1-dependent manner.

Dataset Information

The kinesin-associated protein UNC-76 is required for axonal transport in the Drosophila nervous system.

Publications

The kinesin-associated protein UNC-76 is required for axonal transport in the Drosophila nervous system.

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