Kinetochore protein Spindly controls microtubule polarity in Drosophila axons.

Microtubule polarity in axons and dendrites defines the direction of intracellular transport in neurons. Axons contain arrays of uniformly polarized microtubules with plus-ends facing the tips of the processes (plus-end-out), while dendrites contain microtubules with a minus-end-out orientation. It has been shown that cytoplasmic dynein, targeted to cortical actin, removes minus-end-out microtubules from axons. Here we have identified Spindly, a protein known for recruitment of dynein to kinetochores in mitosis, as a key factor required for dynein-dependent microtubule sorting in axons of Drosophila neurons. Depletion of Spindly affects polarity of axonal microtubules in vivo and in primary neuronal cultures. In addition to these defects, depletion of Spindly in neurons causes major collapse of axonal patterning in the third-instar larval brain as well as severe coordination impairment in adult flies. These defects can be fully rescued by full-length Spindly, but not by variants with mutations in its dynein-binding site. Biochemical analysis demonstrated that Spindly binds F-actin, suggesting that Spindly serves as a link between dynein and cortical actin in axons. Therefore, Spindly plays a critical role during neurodevelopment by mediating dynein-driven sorting of axonal microtubules.

Conserved role for Ataxin-2 in mediating endoplasmic reticulum dynamics.

Ataxin-2, a conserved RNA-binding protein, is implicated in the late-onset neurodegenerative disease Spinocerebellar ataxia type-2 (SCA2). SCA2 is characterized by shrunken dendritic arbors and torpedo-like axons within the Purkinje neurons of the cerebellum. Torpedo-like axons have been described to contain displaced endoplasmic reticulum (ER) in the periphery of the cell; however, the role of Ataxin-2 in mediating ER function in SCA2 is unclear. We utilized the Caenorhabditis elegans and Drosophila homologs of Ataxin-2 (ATX-2 and DAtx2, respectively) to determine the role of Ataxin-2 in ER function and dynamics in embryos and neurons. Loss of ATX-2 and DAtx2 resulted in collapse of the ER in dividing embryonic cells and germline, and ultrastructure analysis revealed unique spherical stacks of ER in mature oocytes and fragmented and truncated ER tubules in the embryo. ATX-2 and DAtx2 reside in puncta adjacent to the ER in both C. elegans and Drosophila embryos. Lastly, depletion of DAtx2 in cultured Drosophila neurons recapitulated the shrunken dendritic arbor phenotype of SCA2. ER morphology and dynamics were severely disrupted in these neurons. Taken together, we provide evidence that Ataxin-2 plays an evolutionary conserved role in ER dynamics and morphology in C. elegans and Drosophila embryos during development and in fly neurons, suggesting a possible SCA2 disease mechanism.

Microtubule Dynamics, Kinesin-1 Sliding, and Dynein Action Drive Growth of Cell Processes.

Recent experimental studies of the role of microtubule sliding in neurite outgrowth suggested a qualitative model, according to which kinesin-1 motors push the minus-end-out microtubules against the cell membrane and generate the early cell processes. At the later stage, dynein takes over the sliding, expels the minus-end-out microtubules from the neurites, and pulls in the plus-end-out microtubules that continue to elongate the nascent axon. This model leaves unanswered a number of questions: why is dynein unable to generate the processes alone, whereas kinesin-1 can? What is the role of microtubule dynamics in process initiation and growth? Can the model correctly predict the rates of process growth in control and dynein-inhibited cases? What triggers the transition from kinesin-driven to dynein-driven sliding? To answer these questions, we combine computational modeling of a network of elastic dynamic microtubules and kinesin-1 and dynein motors with measurements of the process growth kinetics and pharmacological perturbations in Drosophila S2 cells. The results verify quantitatively the qualitative model of the microtubule polarity sorting and suggest that dynein-powered elongation is effective only when the processes are longer than a threshold length, which explains why kinesin-1 alone, but not dynein, is sufficient for the process growth. Furthermore, we show that the mechanism of process elongation depends critically on microtubule dynamic instability. Both modeling and experimental measurements show, surprisingly, that dynein inhibition accelerates the process extension. We discuss implications of the model for the general problems of cell polarization, cytoskeletal polarity emergence, and cell process protrusion.

Allosteric communication between the nucleotide binding domains of caseinolytic peptidase B.

ClpB is a hexameric chaperone that solubilizes and reactivates protein aggregates in cooperation with the Hsp70/DnaK chaperone system. Each of the identical protein monomers contains two nucleotide binding domains (NBD), whose ATPase activity must be coupled to exert on the substrate the mechanical work required for its reactivation. However, how communication between these sites occurs is at present poorly understood. We have studied herein the affinity of each of the NBDs for nucleotides in WT ClpB and protein variants in which one or both sites are mutated to selectively impair nucleotide binding or hydrolysis. Our data show that the affinity of NBD2 for nucleotides (K(d) = 3-7 µm) is significantly higher than that of NBD1. Interestingly, the affinity of NBD1 depends on nucleotide binding to NBD2. Binding of ATP, but not ADP, to NBD2 increases the affinity of NBD1 (the K(d) decreases from ≈160-300 to 50-60 µm) for the corresponding nucleotide. Moreover, filling of the NBD2 ring with ATP allows the cooperative binding of this nucleotide and substrates to the NBD1 ring. Data also suggest that a minimum of four subunits cooperate to bind and reactivate two different aggregated protein substrates.

Ataxin-2 is essential for cytoskeletal dynamics and neurodevelopment in Drosophila.

Ataxin-2 (Atx2) is a highly conserved RNA binding protein. Atx2 undergoes polyglutamine expansion leading to amyotrophic lateral sclerosis (ALS) or spinocerebellar ataxia type 2 (SCA2). However, the physiological functions of Atx2 in neurons remain unknown. Here, using the powerful genetics of Drosophila, we show that Atx2 is essential for normal neuronal cytoskeletal dynamics and organelle trafficking. Upon neuron-specific Atx2 loss, the microtubule and actin networks were abnormally stabilized and cargo transport was drastically inhibited. Depletion of Atx2 caused multiple morphological defects in the nervous system of third instar larvae. These include reduced brain size, impaired axon development, and decreased dendrite outgrowth. Defects in the nervous system caused loss of the ability to crawl and lethality at the pupal stage. Taken together, these data mark Atx2 as a major regulator of cytoskeletal dynamics and denote Atx2 as an essential gene in neurodevelopment, as well as a neurodegenerative factor.

A quantitative analysis of the effect of nucleotides and the M domain on the association equilibrium of ClpB.

ClpB is a hexameric molecular chaperone that, together with the DnaK system, has the ability to disaggregate stress-denatured proteins. The hexamer is a highly dynamic complex, able to reshuffle subunits. To further characterize the biological implications of the ClpB oligomerization state, the association equilibrium of the wild-type (wt) protein and of two deletion mutants, which lack part or the whole M domain, was quantitatively analyzed under different experimental conditions, using several biophysical [analytical ultracentrifugation, composition-gradient (CG) static light scattering, and circular dichroism] and biochemical (ATPase and chaperone activity) methods. We have found that (i) ClpB self-associates from monomers to form hexamers and higher-order oligomers that have been tentatively assigned to dodecamers, (ii) oligomer dissociation is not accompanied by modifications of the protein secondary structure, (iii) the M domain is engaged in intersubunit interactions that stabilize the protein hexamer, and (iv) the nucleotide-induced rearrangement of ClpB affects the protein oligomeric core, in addition to the proposed radial extension of the M domain. The difference in the stability of the ATP- and ADP-bound states [ΔΔG(ATP-ADP) = -10 kJ/mol] might explain how nucleotide exchange promotes the conformational change of the protein particle that drives its functional cycle.

Ser/Thr kinase Trc controls neurite outgrowth in Drosophila by modulating microtubule-microtubule sliding.

Correct neuronal development requires tailored neurite outgrowth. Neurite outgrowth is driven in part by microtubule-sliding - the transport of microtubules along each other. We have recently demonstrated that a 'mitotic' kinesin-6 (Pavarotti in Drosophila) effectively inhibits microtubule-sliding and neurite outgrowth. However, mechanisms regulating Pavarotti itself in interphase cells and specifically in neurite outgrowth are unknown. Here, we use a combination of live imaging and biochemical methods to show that the inhibition of microtubule-sliding by Pavarotti is controlled by phosphorylation. We identify the Ser/Thr NDR kinase Tricornered (Trc) as a Pavarotti-dependent regulator of microtubule sliding in neurons. Further, we show that Trc-mediated phosphorylation of Pavarotti promotes its interaction with 14-3-3 proteins. Loss of 14-3-3 prevents Pavarotti from associating with microtubules. Thus, we propose a pathway by which microtubule-sliding can be up- or downregulated in neurons to control neurite outgrowth, and establish parallels between microtubule-sliding in mitosis and post-mitotic neurons.

Unconventional Roles of Cytoskeletal Mitotic Machinery in Neurodevelopment.

At first look, cell division and neurite formation seem to be two different, essential biological processes. However, both processes require extensive reorganization of the cytoskeleton, and especially microtubules. Remarkably, in recent years, independent work from several groups has shown that multiple cytoskeletal components previously considered specific for the mitotic machinery play important roles in neurite initiation and extension. In this review article, we describe how several cytoplasmic and mitotic microtubule motors, components of mitotic kinetochores, and cortical actin participate in reorganization of the microtubule network required to form and maintain axons and dendrites. The emerging similarities between these two biological processes will certainly generate new insights into the mechanisms generating the unique morphology of neurons.

Responses of aerobic microbial communities and soil respiration to water-level drawdown in a northern boreal fen.

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.

The small molecule AMBMP disrupts microtubule growth, ciliogenesis, cell polarity, and cell migration.

2-Amino-4-(3,4-[methylenedioxy]benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) is a small molecule that has been previously reported to be both a Wnt agonist and a microtubule (MT) regulator. Here we report a detailed analysis of AMBMPs effects on MTs and on MT associated cellular processes including cell polarity, ciliogenesis, and cell migration. Specifically, treatment of Xenopus embryos with AMBMP leads to defects similar to the MT depolymerizing drug nocodazole, including a failure to generate or polarize cilia (depending on the timing of treatment) and a loss of the cell movements associated with radial intercalation. The dramatic effect AMBMP has on basic MT based cellular functions suggests that its usefulness as a Wnt regulator is questionable. Moreover, it may be an important new tool for experimental or pharmacological manipulation of MTs.

Effects of iron on Vitamin C/copper-induced hydroxyl radical generation in bicarbonate-rich water.

The aim of this study was to evaluate whether iron, like copper, could support Vitamin C mediated hydroxyl radical formation in bicarbonate-rich water. By using the hydroxyl radical indicator coumarin-3-carboxylic acid, we found that iron, in contrast to copper, was not capable to support Vitamin C induced hydroxyl radical formation. However, when 0.2 mg/l iron and 0.1 mg/l copper were both added to bicarbonate supplemented Milli-Q water, the Vitamin C induced formation of 7-hydroxycoumarin, as measured by HPLC analysis, was inhibited by 47.5%. The inhibition of hydroxyl radical formation by iron was also evident in the experiments performed on copper contaminated bicarbonate-rich household drinking water samples. In the presence of 0.2 mg/l of ferric iron the ascorbic acid induced hydroxyl radical formation was inhibited by 36.0-44.6%. This inhibition was even more significant, 47.0-59.2%, when 0.8 mg/l of ferric iron was present. None of the other redox-active metals, e.g. manganese, nickel or cobalt, could support ascorbic acid induced hydroxyl radical formation and did not have any impact on the ascorbic acid/copper-induced hydroxyl radical generation. Our results show, that iron cannot by itself produce hydroxyl radicals in bicarbonate rich water but can significantly reduce Vitamin C/copper-induced hydroxyl radical formation. These findings might partly explain the mechanism for the iron-induced protective effect on various copper related degenerative disorders that earlier has been observed in animal model systems.

Interplay between kinesin-1 and cortical dynein during axonal outgrowth and microtubule organization in Drosophila neurons.

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.

Sedimentation Equilibrium Analysis of ClpB Self-Association in Diluted and Crowded Solutions.

ClpB belongs to the Hsp100 family of ring-forming heat-shock proteins involved in degradation of unfolded/misfolded proteins and in reactivation of protein aggregates. ClpB monomers reversibly associate to form the hexameric molecular chaperone that, together with the DnaK system, has the ability to disaggregate stress-denatured proteins. Here, we summarize the use of sedimentation equilibrium approaches, complemented with sedimentation velocity and composition-gradient static light scattering measurements, to study the self-association properties of ClpB in dilute and crowded solutions. As the functional unit of ClpB is the hexamer, we study the effect of environmental factors, i.e., ionic strength and natural ligands, in the association equilibrium of ClpB as well as the role of the flexible N-terminal and M domains of the protein in the self-association process. The application of the nonideal sedimentation equilibrium technique to measure the effects of volume exclusion, reproducing in part the natural crowded conditions inside a cell, on the self-association and on the stability of the oligomeric species of the disaggregase will be described. Finally, the biochemical and physiological implications of these studies and future experimental challenges to eventually reconstitute minimal disaggregating machineries will be discussed.

Pavarotti/MKLP1 regulates microtubule sliding and neurite outgrowth in Drosophila neurons.

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 shutdown 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. Here, we have identified the "mitotic" kinesin-6 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 and 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.

Organelle transport in cultured Drosophila cells: S2 cell line and primary neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Nucleotide utilization requirements that render ClpB active as a chaperone.

ClpB is a member of the AAA+ superfamily that forms a ring-shaped homohexamer. Each protomer contains two nucleotide binding domains arranged in two rings that hydrolyze ATP. We extend here previous studies on ClpB nucleotide utilization requirements by using an experimental approach that maximizes random incorporation of different subunits into the protein hexamer. Incorporation of one subunit unable to bind or hydrolyze ATP knocks down the chaperone activity, while the wt hexamer can accommodate two mutant subunits that hydrolyze ATP in only one protein ring. Four subunits seem to build the functional cooperative unit, provided that one of the protein rings contains active nucleotide binding sites.

DnaK-mediated association of ClpB to protein aggregates. A bichaperone network at the aggregate surface.

Intracellular 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 microM), 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.