In this study, we investigated how microtubule motors organize microtubules in Drosophila neurons. We showed that, during the initial stages of axon outgrowth, microtubules display mixed polarity and minus-end-out microtubules push the tip of the axon, consistent with kinesin-1 driving outgrowth by sliding antiparallel microtubules. At later stages, the microtubule orientation in the axon switches from mixed to uniform polarity with plus-end-out. Dynein knockdown prevents this rearrangement and results in microtubules of mixed orientation in axons and accumulation of microtubule minus-ends at axon tips. Microtubule reorganization requires recruitment of dynein to the actin cortex, as actin depolymerization phenocopies dynein depletion, and direct recruitment of dynein to the membrane bypasses the actin requirement. Our results show that cortical dynein slides ‘minus-end-out’ microtubules from the axon, generating uniform microtubule arrays. We speculate that differences in microtubule orientation between axons and dendrites could be dictated by differential activity of cortical dynein.DOI: http://dx.doi.org/10.7554/eLife.10140.001

Summary Recently, we demonstrated that kinesin-1 can slide microtubules against each other providing the mechanical force required for initial neurite extension in Drosophila neurons. This sliding is only observed in young neurons actively forming neurites and is dramatically downregulated in older neurons. The downregulation is not caused by the global shut-down of kinesin-1, as the ability of kinesin-1 to transport membrane organelles is not diminished in mature neurons, suggesting that microtubule sliding is regulated by a dedicated mechanism [1]. Here, we have identified the “mitotic” kinesin Pavarotti (Pav-KLP) as an inhibitor of kinesin-1-driven microtubule sliding. Depletion of Pav-KLP in neurons strongly stimulated the sliding of long microtubules and neurite outgrowth, while its ectopic overexpression in the cytoplasm blocked both of these processes. Furthermore, postmitotic depletion of Pav-KLP in Drosophila neurons in vivo reduced embryonic/larval viability, with only a few animals surviving to the third instar larval stage. A detailed examination of motor neurons in the surviving larvae revealed the overextension of axons and mistargeting of neuromuscular junctions, resulting in uncoordinated locomotion. Taken together, our results identify a new role for Pav-KLP as a negative regulator of kinesin-1 driven neurite formation. These data suggest an important parallel between long microtubule-microtubule sliding in anaphase B and sliding of interphase microtubules during neurite formation.

a b s t r a c tIntracellular protein aggregates formed under severe thermal stress can be reactivated by the concerted action of the Hsp70 system and Hsp100 chaperones. We analyzed here the interaction of DnaJ/ DnaK and ClpB with protein aggregates. We show that aggregate properties modulate chaperone binding, which in turn determines aggregate reactivation efficiency. ClpB binding strictly depends on previous DnaK association with the aggregate. The affinity of ClpB for the aggregate-DnaK complex is low (K d = 5-10 lM), indicating a weak interaction. Therefore, formation of the DnaK-ClpB bichaperone network is a three step process. After initial DnaJ binding, the cochaperone drives association of DnaK to aggregates, and in the third step, as shown here, DnaK mediates ClpB interaction with the aggregate surface.

On a global basis, peatlands are a major reserve of carbon (C). Hydrological changes can affect the decomposition processes in peatlands and in turn can alter their C balance. Since 1959, a groundwater extraction plant has generated a water-level gradient at our study site that has gradually changed part of the wet fen into a dry peatland forest. The average water-level drawdown of the gradient (from a pristine 9 cm to 26 cm in the dry end) is close to an estimate predicted by an increase in mean global temperature of 3 degrees C. We studied the total microbial community of the aerobic surface peat in four locations along the gradient through phospholipid fatty acid and PCR-DGGE methods. Additionally, field measurements of soil respiration showed a threefold increase in the C-emission rate at the driest location compared with the wettest one, indicating enhanced decomposition. Also, both fungal and bacterial biomass increased in the drier locations. At the species level, the fungal community changed due to water-level drawdown whereas actinobacteria were less sensitive to drying. The majority of fungal sequences were similar to ectomycorrhizal (ECM) fungi, which dominated throughout the gradient. Our results indicate that ECM fungi might act as important facultative decomposers in organic-rich environments such as peatlands.