The nuclear protein Waharan is required for endosomal-lysosomal trafficking in Drosophila.

Here we report Drosophila Waharan (Wah), a 170-kD predominantly nuclear protein with two potential human homologues, as a newly identified regulator of endosomal trafficking. Wah is required for neuromuscular-junction development and muscle integrity. In muscles, knockdown of Wah caused novel accumulations of tightly packed electron-dense tubules, which we termed 'sausage bodies'. Our data suggest that sausage bodies coincide with sites at which ubiquitylated proteins and a number of endosomal and lysosomal markers co-accumulate. Furthermore, loss of Wah function generated loss of the acidic LysoTracker compartment. Together with data demonstrating that Wah acts earlier in the trafficking pathway than the Escrt-III component Drosophila Shrb (snf7 in Schizosaccharomyces pombe), our results indicate that Wah is essential for endocytic trafficking at the late endosome. Highly unexpected phenotypes result from Wah knockdown, in that the distribution of ubiquitylated cargos and endolysosomal morphologies are affected despite Wah being a predominant nuclear protein. This finding suggests the existence of a relationship between nuclear functions and endolysosomal trafficking. Future studies of Wah function will give us insights into this interesting phenomenon.

Context-specific requirements of functional domains of the Spectraplakin Short stop in vivo.

Spectraplakins are large multifunctional cytoskeletal interacting molecules implicated in various processes, including gastrulation, wound healing, skin blistering and neuronal degeneration. It has been speculated that the various functional domains and regions found in Spectraplakins are used in context-specific manners, a model which would provide a crucial explanation for the multifunctional nature of Spectraplakins. Here we tested this possibility by studying domain requirements of the Drosophila Spectraplakin Short stop (Shot) in three different cellular contexts in vivo: (1) neuronal growth, which requires dynamic actin-microtubule interaction; (2) formation and maintenance of tendon cells, which depends on highly stabilised arrays of actin filaments and microtubules, and (3) compartmentalisation in neurons, which is likely to involve cortical F-actin networks. Using these cellular contexts for rescue experiments with Shot deletion constructs in shot mutant background, a number of differential domain requirements were uncovered. First, binding of Shot to F-actin through the first Calponin domain is essential in neuronal contexts but dispensable in tendon cells. This finding is supported by our analyses of shot(kakP2) mutant embryos, which produce only endogenous isoforms lacking the first Calponin domain. Thus, our data demonstrate a functional relevance for these isoforms in vivo. Second, we provide the first functional role for the Plakin domain of Shot, which has a strong requirement for compartmentalisation in neurons and axonal growth, demonstrating that Plakin domains of long Spectraplakin isoforms are of functional relevance. Like the Calponin domain, also the Plakin domain is dispensable in tendon cells, and the currently assumed role of Shot as a linker of microtubules to the tendon cell surface may have to be reconsidered. Third, we demonstrate a function of Shot as an actin-microtubule linker in dendritic growth, thus shedding new light into principal growth mechanisms of this neurite type. Taken together, our data clearly support the view that Spectraplakins function in tissue-specific modes in vivo, and even domains believed to be crucial for Spectraplakin function can be dispensable in specific contexts.

Are dendrites in Drosophila homologous to vertebrate dendrites?

Dendrites represent arborising neurites in both vertebrates and invertebrates. However, in vertebrates, dendrites develop on neuronal cell bodies, whereas in higher invertebrates, they arise from very different neuronal structures, the primary neurites, which also form the axons. Is this anatomical difference paralleled by principal developmental and/or physiological differences? We address this question by focussing on one cellular model, motorneurons of Drosophila and characterise the compartmentalisation of these cells. We find that motorneuronal dendrites of Drosophila share with typical vertebrate dendrites that they lack presynaptic but harbour postsynaptic proteins, display calcium elevation upon excitation, have distinct cytoskeletal features, develop later than axons and are preceded by restricted localisation of Par6-complex proteins. Furthermore, we demonstrate in situ and culture that Drosophila dendrites can be shifted from the primary neurite to their soma, i.e. into vertebrate-like positions. Integrating these different lines of argumentation, we propose that dendrites in vertebrates and higher invertebrates have a common origin, and differences in dendrite location can be explained through translocation of neuronal cell bodies introduced during the evolutionary process by which arthropods and vertebrates diverged from a common urbilaterian ancestor. Implications of these findings for studies of dendrite development, neuronal polarity, transport and evolution are discussed.