In vertebrate neurons, axons have a uniform arrangement of microtubules with plus ends distal to the cell body (plus-end-out), and dendrites have equal numbers of plus-and minus-end-out microtubules. To determine whether microtubule orientation is a conserved feature of axons and dendrites, we analyzed microtubule orientation in invertebrate neurons. Using microtubule plus end dynamics, we mapped microtubule orientation in Drosophila sensory neurons, interneurons, and motor neurons. As expected, all axonal microtubules have plus-end-out orientation. However, in proximal dendrites of all classes of neuron, ϳ90% of dendritic microtubules were oriented with minus ends distal to the cell body. This result suggests that minus-end-out, rather than mixed orientation, microtubules are the signature of the dendritic microtubule cytoskeleton. Surprisingly, our map of microtubule orientation predicts that there are no tracks for direct cargo transport between the cell body and dendrites in unipolar neurons. We confirm this prediction, and validate the completeness of our map, by imaging endosome movements in motor neurons. As predicted by our map, endosomes travel smoothly between the cell body and axon, but they cannot move directly between the cell body and dendrites. INTRODUCTIONMany differentiated cells have highly polarized arrays of microtubules that likely play a large role in establishing their specialized architecture and function. Neurons are strikingly polarized and initially seemed that they would be the clearest example of cells in which microtubule orientation formed the basis of directional transport and cell polarity (Black and Baas, 1989). Most neurons have a cell body in which the bulk of proteins are synthesized, dendrites that are specialized to receive signals, and axons that are specialized to send them. Where examined, microtubules in vertebrate dendrites have mixed orientation, and in axons they have uniform orientation with all plus ends distal to the cell body. Thus, the simplest model for selective transport from the cell body to dendrites is use of a minus end-directed motor. However, current models of transport into dendrites rely on plus enddirected motors (Setou et al., 2004;Hirokawa and Takemura, 2005;Kennedy and Ehlers, 2006;Levy and Holzbaur, 2006). These models raise the question: are minus-end-out microtubules important for directional transport or neuronal polarity?Axonal microtubule orientation has been examined in a variety of neurons, all with the same result: Ͼ95% of plus ends are oriented away from the cell body (plus-end-out). Original studies on axonal microtubule orientation relied on decoration of microtubules with exogenous tubulin, which forms curved hooks on the sides of existing microtubules, and analysis by electron microscopy. The direction of hook curvature indicates microtubule polarity. This method was used to determine axonal microtubule orientation in many different types of vertebrate neurons (Burton and Paige, 1981;Heidemann et al., 1981;Baas et al., 1987Baas et al...

We look inside neurons in vivo and identify major cytoskeletal rearrangements that allow a dendrite to become a regenerating axon.

We describe a modified Agrobacterium-mediated method for the efficient transformation of Agaricus bisporus. Salient features of this procedure include cocultivation of Agrobacterium and fruiting body gill tissue and use of a vector with a homologous promoter. This method offers new prospects for the genetic manipulation of this commercially important mushroom species.

Summary Background In many differentiated cells microtubules are organized into polarized noncentrosomal arrays, yet few mechanisms that control these arrays have been identified. For example, mechanisms that maintain microtubule polarity in the face of constant remodeling by dynamic instability are not known. Drosophila neurons contain uniform polarity minus-end-out microtubules in dendrites, which are often highly branched. As undirected microtubule growth through dendrite branch points jeopardizes uniform microtubule polarity, we have used this system to understand how cells can maintain dynamic arrays of polarized microtubules. Results We find that growing microtubules navigate dendrite branch points by turning the same way, towards the cell body, 98% of the time, and that growing microtubules track along stable microtubules towards their plus ends. Using RNAi and genetic approaches, we show that kinesin-2, and the +TIPS EB1 and APC, are required for uniform dendrite microtubule polarity. Moreover, the protein-protein interactions and localization of Apc2-GFP and Apc-RFP to branch points suggests these proteins work together at dendrite branches. The functional importance of this polarity mechanism is demonstrated by the failure of neurons with reduced kinesin-2 to regenerate an axon from a dendrite. Conclusions We conclude that microtubule growth is directed at dendrite branch points, and that kinesin-2, APC and EB1 are likely to play a role in this process. We propose that is recruited to growing microtubules by +TIPS, and that the motor protein steers growing microtubules at branch points. This represents a newly discovered mechanism to maintain polarized arrays of microtubules.

Summary Axon injury triggers regeneration through activation of a conserved kinase cascade that includes the dual leucine zipper kinase (DLK). While dendrites are damaged during stroke, traumatic brain injury and seizure, it is not known whether mature neurons monitor dendrite injury and initiate regeneration. We probed the response to dendrite damage using model Drosophila neurons. Two larval neuron types regrew dendrites in distinct ways after all dendrites were removed. Dendrite regeneration was also triggered by injury in adults. We next tested whether dendrite injury was initiated with the same machinery as axon injury. Surprisingly, DLK, JNK and fos were dispensable for dendrite regeneration. Moreover, this MAP kinase pathway was not activated by injury to dendrites. Thus neurons respond to dendrite damage and initiate regeneration without using the conserved DLK cascade that triggers axon regeneration.