Microtubules are organized independently of the centrosome in Drosophila neurons

<p>Abstract</p> <p>Background</p> <p>The best-studied arrangement of microtubules is that organized by the centrosome, a cloud of microtubule nucleating and anchoring proteins is clustered around centrioles. However, noncentrosomal microtubule arrays are common in many differentiated cells, including neurons. Although microtubules are not anchored at neuronal centrosomes, it remains unclear whether the centrosome plays a role in organizing neuronal microtubules. We use <it>Drosophila </it>as a model system to determine whether centrosomal microtubule nucleation is important in mature neurons.</p> <p>Results</p> <p>In developing and mature neurons, centrioles were not surrounded by the core nucleation protein γ-tubulin. This suggests that the centrioles do not organize functional centrosomes in <it>Drosophila </it>neurons <it>in vivo</it>. Consistent with this idea, centriole position was not correlated with a specific region of the cell body in neurons, and growing microtubules did not cluster around the centriole, even after axon severing when the number of growing plus ends is dramatically increased. To determine whether the centrosome was required for microtubule organization in mature neurons, we used two approaches. First, we used <it>DSas-4 </it>centriole duplication mutants. In these mutants, centrioles were present in many larval sensory neurons, but they were not fully functional. Despite reduced centriole function, microtubule orientation was normal in axons and dendrites. Second, we used laser ablation to eliminate the centriole, and again found that microtubule polarity in axons and dendrites was normal, even 3 days after treatment.</p> <p>Conclusion</p> <p>We conclude that the centrosome is not a major site of microtubule nucleation in <it>Drosophila </it>neurons, and is not required for maintenance of neuronal microtubule organization in these cells.</p

Patronin-mediated minus end growth is required for dendritic microtubule polarity.

Microtubule minus ends are thought to be stable in cells. Surprisingly, in Drosophila and zebrafish neurons, we observed persistent minus end growth, with runs lasting over 10 min. In Drosophila, extended minus end growth depended on Patronin, and Patronin reduction disrupted dendritic minus-end-out polarity. In fly dendrites, microtubule nucleation sites localize at dendrite branch points. Therefore, we hypothesized minus end growth might be particularly important beyond branch points. Distal dendrites have mixed polarity, and reduction of Patronin lowered the number of minus-end-out microtubules. More strikingly, extra Patronin made terminal dendrites almost completely minus-end-out, indicating low Patronin normally limits minus-end-out microtubules. To determine whether minus end growth populated new dendrites with microtubules, we analyzed dendrite development and regeneration. Minus ends extended into growing dendrites in the presence of Patronin. In sum, our data suggest that Patronin facilitates sustained microtubule minus end growth, which is critical for populating dendrites with minus-end-out microtubules

Functional Interactions between the erupted/tsg101 Growth Suppressor Gene and the DaPKC and rbf1 Genes in Drosophila Imaginal Disc Tumors

BACKGROUND: The Drosophila gene erupted (ept) encodes the fly homolog of human Tumor Susceptibility Gene-101 (TSG101), which functions as part of the conserved ESCRT-1 complex to facilitate the movement of cargoes through the endolysosomal pathway. Loss of ept or other genes that encode components of the endocytic machinery (e.g. synatxin7/avalanche, rab5, and vps25) produces disorganized overgrowth of imaginal disc tissue. Excess cell division is postulated to be a primary cause of these 'neoplastic' phenotypes, but the autonomous effect of these mutations on cell cycle control has not been examined. PRINCIPAL FINDINGS: Here we show that disc cells lacking ept function display an altered cell cycle profile indicative of deregulated progression through the G1-to-S phase transition and express reduced levels of the tumor suppressor ortholog and G1/S inhibitor Rbf1. Genetic reductions of the Drosophila aPKC kinase (DaPKC), which has been shown to promote tumor growth in other fly tumor models, prevent both the ept neoplastic phenotype and the reduction in Rbf1 levels that otherwise occurs in clones of ept mutant cells; this effect is coincident with changes in localization of Notch and Crumbs, two proteins whose sorting is altered in ept mutant cells. The effect on Rbf1 can also be blocked by removal of the gamma-secretase component presenilin, suggesting that cleavage of a gamma-secretase target influences Rbf1 levels in ept mutant cells. Expression of exogenous rbf1 completely ablates ept mutant eye tissues but only mildly affects the development of discs composed of cells with wild type ept. CONCLUSIONS: Together, these data show that loss of ept alters nuclear cell cycle control in developing imaginal discs and identify the DaPKC, presenilin, and rbf1 genes as modifiers of molecular and cellular phenotypes that result from loss of ept

Two Drosophila model neurons can regenerate axons from the stump or from a converted dendrite, with feedback between the two sites

Abstract Background After axon severing, neurons recover function by reinitiating axon outgrowth. New outgrowth often originates from the remaining axon stump. However, in many mammalian neurons, new axons initiate from a dendritic site when the axon is injured close to the cell body. Methods Drosophila sensory neurons are ideal for studying neuronal injury responses because they can be injured reproducibly in a variety of genetic backgrounds. In Drosophila, it has been shown that a complex sensory neuron, ddaC, can regenerate an axon from a stump, and a simple sensory neuron, ddaE, can regenerate an axon from a dendrite. To provide a more complete picture of axon regeneration in these cell types, we performed additional injury types. Results We found that ddaE neurons can initiate regeneration from an axon stump when a stump remains. We also showed that ddaC neurons regenerate from the dendrite when the axon is severed close to the cell body. We next demonstrated if a stump remains, new axons can originate from this site and a dendrite at the same time. Because cutting the axon close to the cell body results in growth of the new axon from a dendrite, and cutting further out may not, we asked whether the initial response in the cell body was similar after both types of injury. A transcriptional reporter for axon injury signaling, puc-GFP, increased with similar timing and levels after proximal and distal axotomy. However, changes in dendritic microtubule polarity differed in response to the two types of injury, and were influenced by the presence of a scar at the distal axotomy site. Conclusions We conclude that both ddaE and ddaC can regenerate axons either from the stump or a dendrite, and that there is some feedback between the two sites that modulates dendritic microtubule polarity