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Vol. 19, Issue 10, 4122-4129, October 2008
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*Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802; and Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111
Submitted October 26, 2007;
Revised July 9, 2008;
Accepted July 18, 2008
Monitoring Editor: Yixian Zheng
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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90% 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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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 end-directed 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: >95% 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., 1987, 1988; Troutt and Burnside, 1988). More recently, the direction of movement of proteins that bind to growing microtubule plus ends was used to analyze axonal microtubule orientation in cultured mouse hippocampal and Purkinje neurons (Stepanova et al., 2003) and cultured Aplysia neurons (Erez et al., 2007). Using both assays, in sensory and central neurons, in organisms ranging from the invertebrate Aplysia to mammals, >95% of axonal microtubules have been found to be plus-end-out. Additionally, second harmonic generation microscopy has confirmed axonal microtubules in vivo and in vitro have uniform microtubule orientation (Dombeck et al., 2003). Uniform plus-end-out microtubule orientation thus seems to be a universal and evolutionarily conserved signature of axons.
Similarly, mixed orientation of microtubules has been considered a signature of dendrites (Alberts et al., 2002). However, dendrites are generally much more difficult to study, and their microtubule organization has been examined much less than that of axons. The hook method has been used to analyze dendritic microtubule orientation in one type of neuron with branched dendrites in vivo: frog mitral cells, which are interneurons. In these dendrites, approximately equal numbers of microtubules had plus and minus ends distal to the cell body throughout the length of the dendrite (Burton, 1988). Both hook labeling and microtubule plus end-binding protein dynamics have been used to analyze microtubule orientation in dendrites in cultured rodent interneurons. In proximal dendrites, both methods showed mixed microtubule orientation, with roughly equal numbers pointing in each direction. Close to dendrite growth cones, most microtubules had plus ends out (Baas et al., 1988; Stepanova et al., 2003). Thus, the prevailing model of microtubule orientation in vertebrate neurons is mixed in proximal dendrites, and plus-end-out in distal dendrites (Figure 1).
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). One simple way to generate this asymmetry would be to selectively transport proteins and organelles into dendrites with a minus end-directed motor, because minus-end-out microtubules are only present in dendrites (Black and Baas, 1989). Selective transport of several types of vesicles from the cell body into dendrites has been observed previously (Burack et al., 2000; Rosales et al., 2005). Dendrites also lose dendritic shape and organelles when minus-end-out microtubules are reduced (Yu et al., 2000). However, the role of minus-end-out microtubules in selective transport into dendrites has been called into question for several reasons. First, most organelles can move bidirectionally and so seem to have both plus- and minus-end–directed motors associated with them (Welte, 2004). Second, cultured neurons have a region at their tip in which minus-end-out microtubules are rare (Baas et al., 1988). Third, plus-end–directed kinesins have been found in association with neurotransmitter receptors (Setou et al., 2000, 2002). Thus, current models of directional transport into dendrites rely on specific targeting of kinesins or kinesin-cargo pairs to plus-end-out dendritic microtubules (Setou et al., 2004; Hirokawa and Takemura, 2005; Kennedy and Ehlers, 2006; Levy and Holzbaur, 2006), and the role of minus-end-out microtubules in polarized transport and neuronal polarity is not clear.
However, recent analysis of microtubule orientation in branched Drosophila sensory neurons raised the possibility that minus-end-out microtubules may actually be the most important component of the dendritic microtubule cytoskeleton. Using EB1-green fluorescent protein (GFP) dynamics to infer microtubule orientation, >95% of microtubules in dendritic arborization (da) dendrites were found to have minus ends distal to the cell body (Rolls et al., 2007). Several possible explanations exist for the difference between these results and those in vertebrate neurons: 1) dendrites of sensory neurons (examined in flies) have different arrangements of microtubules than interneurons (examined in vertebrates); 2) EB1-GFP dynamics revealed the orientation of a special subset of microtubules, and stable plus-end-out microtubules are present in Drosophila dendrites; or 3) all Drosophila dendrites have mostly minus-end-out microtubules (Figure 1).
To distinguish between these possibilities, we used EB1-GFP dynamics to generate a complete map of microtubule orientation in all major classes of Drosophila neurons: sensory neurons, interneurons, and motor neurons. We found that minus-end-out microtubules predominate in dendrites from all three types of neurons, and we propose that minus-end-out microtubules are a conserved signature of dendrites. Our map of microtubule orientation makes very specific predictions about the layout of microtubule tracks at dendrite branch points: that microtubules run between the cell body and dendrites, but not from one dendrite to the other. To test the completeness of our map, we analyzed the layout of stable microtubules and the paths taken by endosomes at dendrite branch points. Results from both methods agreed with predictions of the map based on EB1-GFP dynamics, and they were inconsistent with a set of stable plus-end-out microtubules in dendrites.
An even more striking test of our microtubule map was offered by unipolar neurons. Our microtubule map predicts that there are no continuous tracks for cargo transport between the cell body and dendrites of unipolar neurons. We confirmed this prediction by tracking endosomes in unipolar neurons. The absence of a direct route between the cell body and dendrites makes sense only in the context of our microtubule map.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). UAS:EB1-GFP lines were provided by Tadashi Uemura (Kyoto University, Kyoto, Japan).
Live Imaging of Da Neurons
Da neurons were imaged in whole, live second instar larvae. Larvae were either placed on a slide with halocarbon 27 oil (Sigma-Aldrich, St. Louis, MO), then covered with a coverslip and imaged within 10 min of mounting, or they were placed in a chamber slide (Kiehart et al., 1994) in halocarbon oil with gas-permeable membrane (YSI, Yellow Springs, OH) on one side and a coverslip on the other side. Images were collected on an LSM510 confocal microscope Carl Zeiss (Thornwood, NY). Z stacks were collected for larvae homozygous for tau-GFP to image stable microtubules. Single frames were collected every 2 s to image EB1-GFP dynamics at dendrite tips. Larvae heterozygous for the 109(2)80 Gal4 and UAS:EB1-GFP were used for this experiment.
Live Imaging of Central Neurons
Embryos either heterozygous for the RN2-Gal4 and UAS:EB1-GFP, or homozygous for both transgenes, and RN2-Gal4, UAS:EB1-GFP; UAS-Rab4-RFP homozygous embryos, were collected for 5 h on apple caps with yeast paste and aged for 2 d on the cap at room temperature. Larvae were then transferred to standard fly media for 1 d at room temperature. Brains were then dissected from these larvae and cultured in imaging chambers as described previously (Siller et al., 2005). Images in a single plane were collected every 1 or 2 s with a Zeiss LSM510 confocal microscope. Movies were made with ImageJ (http://rsbweb.nih.gov/ij/) and QuickTime.
Analysis of EB1-GFP and Rab4-RFP Dynamics
Moving structures were manually tracked in movies. Only structures that could be clearly observed in three consecutive frames were included. For statistical analysis, data were divided into sets based on order of acquisition; so, for example, if 16 brains were imaged as for Figure 7, the first five animals would be one group, the second five would be another group, and the last six would be the final group. This grouping allowed us to calculate standard deviations. The numbers of objects from single animals was too low to consider each animal separately. Information about numbers of animals in sets, and total numbers of fluorescent objects counted, is in the figure legends.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In that study, we analyzed microtubule orientation in proximal dendrites, and we grouped data from different subclasses of dendritic arborization neurons. To complete and refine the map of microtubule orientation in Drosophila sensory neurons, we analyzed EB1-GFP dynamics in both the tips, and main trunk, of dendrites in a single type of class I dendritic arborization neuron, ddaE (Figure 2 and Supplemental Movie 1). We expressed EB1-GFP in da neurons by using the pan-neuronal elav-Gal4 line and performed live imaging of EB1-GFP in whole larvae. EB1-GFP dots moved in one direction, and then disappeared, consistent with binding only to growing microtubule plus ends. Dots moving away from the cell body therefore indicated plus-end-out microtubules, and dots moving toward the cell body indicated minus-end-out microtubules.
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). However, distal to the last branch point, EB1-GFP dynamics indicated a more mixed population of microtubules, with only 55 ± 15% minus-end-out (Figure 2, B and C, arrows). This increase in plus-end-out microtubules at dendrite tips is very similar to that seen in dendrites of cultured vertebrate neurons.
Most Microtubules in Drosophila Interneuron Dendrites Have Minus Ends Distal to the Cell Body
Sensory dendrites are not postsynaptic; so, they could have a different arrangement of microtubules than central dendrites. To determine the arrangement of dendritic microtubules in central neurons, we imaged EB1-GFP dynamics in an interneuron in the larval brain. Live imaging of subcellular structures has not been performed previously in Drosophila central neuron dendrites. Central dendrites are generally embedded deep within the nervous system, and they have complex three-dimensional structures. To identify a central neuron with dendrites amenable to live imaging, we screened through Gal4 lines that drive expression in subsets of neurons. The RN2-Gal4 line expresses Gal4 under control of part of the eve enhancer (Fujioka et al., 2003). In 3-d-old larvae, it reproducibly expressed UAS-controlled EB1-GFP in a single isolated neuron per brain lobe. In larvae with one copy of RN2-Gal4 and UAS-EB1-GFP, expression was often seen in only one lobe (Figure 3A), whereas with two copies of each transgene, expression was seen in both lobes (data not shown). We could identify dendrites and axons of this neuron based on morphology: dendrites branched off the primary neurite on the same side of the brain as the cell body, and axons continued to the other brain lobe where they branched and often had large bulb-like terminals that likely house presynaptic specializations (Figure 3A). Unlike the da neurons, this neuron is unipolar. This arrangement of axons and dendrites branching from a single process, or primary neurite, that arises from the cell body is frequently seen in invertebrates (Craig and Banker, 1994; Sanchez-Soriano et al., 2005), but it can also be present in familiar systems used for neuronal polarity including cultured rodent hippocampal neurons (see, for example, Figure 4 in Burack et al., 2000).
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Minus-End-Out Microtubules Predominate in Drosophila Motor Neuron Dendrites
To determine whether all Drosophila neurons are likely to have a similar arrangement of microtubules, we extended our analysis of microtubule orientation to one more type of neuron. Motor neurons have been well characterized in Drosophila, and they have dendrites that are in many ways similar to vertebrate dendrites (Sanchez-Soriano et al., 2005). RN2-Gal4 drives expression of UAS-controlled transgenes in three neurons per hemisegment in the Drosophila embryonic ventral nerve cord. These cells have been identified as the motor neurons RP2 and aCC and the interneuron pCC (Fujioka et al., 2003). We were not able to image EB1-GFP dynamics in embryos, but expression in these cells persisted in larvae that were homozygous for RN2-Gal4 and UAS-EB1-GFP. In larvae, motor neuron dendrites were much longer and more branched than in embryos, and we could use brain explants that included the ventral ganglion to image EB1-GFP dynamics in them (Figure 4 and Supplemental Movie 3). In axons of RP2 or aCC motor neurons, most microtubules were plus-end-out as expected (Figure 4B). In dendrites, results were similar to those in da neurons and interneurons, with 88.0 ± 7% minus-end-out microtubules (Figure 4). We were unable to image EB1-GFP dynamics in the primary neurite, as other structures overlapped this part of the cell.
Because axonal and dendritic microtubules have a similar arrangement in all major types of Drosophila neurons (summarized in Figures 2D and 4C) based on EB1-GFP dynamics, we can eliminate neuron subtype differences (Figure 1B) as an explanation for the difference between mammalian and Drosophila dendritic microtubule orientation. We next tested whether EB1-GFP dynamics might have missed a population of stable plus-end-out dendritic microtubules.
Organization of Stable Microtubules at Dendrite Branch Points Is Consistent with a Predominantly Minus-End-Out Microtubule Orientation
Our map of microtubule orientation makes specific predictions about the layout of microtubules at dendrite branch points. We tested whether our map, which is based on dynamic microtubules, also represents the organization of stable microtubules in dendrites by using two methods: imaging stable microtubules at dendrite branch points, and imaging movements of cargo along microtubule tracks at dendrite branch points.
If almost all microtubules in dendrites have a minus-end-out orientation, then very few microtubules should extend from one dendrite to the other at a branch point; they should mostly extend between the cell body and dendrites (Figure 5A). To visualize stable microtubules clearly, we used a GFP protein trap line that contains the GFP coding sequence inserted into the genomic copy of the Drosophila tau gene (see Clyne et al., 2003 for a description of how these lines were generated). This insertion results in expression of tau-GFP from the endogenous promoter, and in larvae fluorescence is seen in da neurons, but not the surrounding tissues. Unlike mammalian tau, which binds only axonal microtubules, Drosophila tau is found on microtubules in both axons and dendrites, although in some neurons it is enriched in the first part of the axon (Rolls et al., 2007). This difference may be because tau is the only Drosophila member of the family of proteins that also includes the broadly expressed microtubule-associated protein (MAP) 4 and dendritic MAP2 in mammals.
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Endosome Movements at Dendrite Branches Follow Predicted Tracks
Like EB1-GFP, it is possible that tau-GFP gives an incomplete picture of dendritic microtubules; so, we also tested our predictions about microtubule layout with another method. We reasoned that tracking the movement of cargo along microtubules would give an additional readout of their arrangement. We labeled endosomes in neurons expressing EB1-GFP with Rab4-RFP and followed their movement at dendrite branch points (Figure 6 and Supplemental Movie 4). The Rab4-RFP transgene has not been extensively characterized, but its localization to punctate structures throughout neurons in our study and a previous study (Sweeney, 2006) is consistent with endosomal localization. Like other organelles (Welte, 2004), Rab4-RFP endosomes moved bidirectionally along microtubules. Overall, more moved toward than away from the cell body, consistent with net movement of endosomes from the periphery to the cell center. Endosomes that traveled completely through the branch were classified as moving between the dendrite and cell body (97.2 ± 1.7%) or from dendrite to dendrite (1.7 ± 2.9%). Consistent with our predictions about the layout of microtubule tracks, very few endosomes moved from dendrite to dendrite. This result suggests (although does not completely rule out) that there is not a stable population of plus-end-out microtubules that is invisible in experiments with EB1-GFP.
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To determine whether cargo follows tracks predicted by our microtubule map, or the more logical direct route between cell body and dendrites in unipolar neurons, we imaged Rab4-RFP-labeled endosomes in motor neurons at the primary neurite–dendrite–axon junction (Figure 7 and Supplemental Movie 5). Of endosomes that moved through the branch (as opposed to stopping in it), we found that 41.1 ± 8.4% traveled between cell body to axon (movement in both directions was pooled). However, only 5.6 ± 5.1% moved between the cell body and dendrite, whereas 53.3 ± 13.3% moved between the dendrite and axon. Thus, consistent with our prediction, many unipolar neurons in Drosophila do not seem to have tracks for direct transport between the cell body and dendrites. Instead, cargo must follow an indirect route, either via the axon, or a fusing and budding event with other membranes in the dendrite branch. This result would be very surprising in a context other than our microtubule map.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our general picture of microtubule orientation in Drosophila neurons (Figures 2D and 4C) is thus a more polarized version of vertebrate neuronal microtubule orientation (Figure 1). The predominance of minus-end-out microtubules in proximal dendrites, which ranged from 88 to 94% in different classes of neurons, suggests that minus-end-out microtubules, rather than mixed orientation microtubules, are the defining feature of the dendritic microtubule cytoskeleton.
In addition to analyzing microtubule orientation in axons and dendrites, we obtained results in the primary neurite of a unipolar interneuron. Models of neuronal compartmentalization generally divide neurons into two major regions: somatodendritic and axonal (Craig and Banker, 1994). Because axonal and dendritic cargoes must both pass through the primary neurite, the expected microtubule arrangement was not clear. We show that the primary neurite has the same arrangement of microtubules as the axon. This result generates the surprising prediction that cargo cannot take a direct route from the primary neurite into dendrites or back. We confirmed this prediction by imaging movements of endosomes at the branch point. In a previous study of vesicle movement in cultured rat hippocampal neurons, transport at a similar branch was analyzed. In these cells, vesicles could travel directly between the primary neurite and dendrite (Burack et al., 2000), consistent with the presence of mixed orientation microtubules in these dendrites.
The extreme polarity of microtubules in Drosophila neurons raises questions about how dendrite microtubule polarity is established, and it has important implications for models of polarized neuronal transport. One mechanism that contributes to dendritic microtubule organization has been described. The dendritic kinesin MKLP1 (Xu et al., 2006) is proposed to transport minus-end-out microtubules into dendrites along plus-end-out microtubules (Sharp et al., 1997; Yu et al., 2000). In Drosophila, other mechanisms must account for minus-end-out microtubules in dendrites because there are so few plus-end-out microtubules present. It will be extremely interesting to determine how a minus-end-out microtubule array is generated in proximal dendrites.
The predominance of minus-end-out microtubules in Drosophila dendrites suggests that, at least in this organism, minus-end–directed motors are the primary motors for anterograde dendritic traffic and that microtubule polarity could be key to polarized neuronal transport. The only essential minus-end–directed motor in Drosophila is dynein. If dynein is required for the bulk of transport into dendrites but not axons, one would expect mutations in dynein subunits to significantly affect dendrite, but not axon outgrowth. In fact, Drosophila neurons with mutations in cytoplasmic dynein or Lis1 exhibit specific reduction of dendrite growth. In clones of neurons with mutations in Lis1 or Dhc64C, dendrite growth was severely reduced, but axons were normal length (Liu et al., 2000). At the time these phenotypes were reported, the specific effect of dynein loss on dendrite growth was difficult to explain, and it was suggested to arise from sensitivity of a population of minus-end-out microtubules to loss of dynein, or greater overall structural changes of dendrites than axons during development (Liu et al., 2000). Our analysis of microtubule orientation in Drosophila dendrites offers an extremely simple interpretation for the selective effect of dynein mutations on dendrite growth: that dynein is the major motor for transport of membranes and proteins required for dendrite growth from the cell body into dendrites.
Cargo can be selectively transported into dendrites in cultured hippocampal neurons (Burack et al., 2000; Rosales et al., 2005), but the role of microtubule orientation in this, or any other, aspect of neuronal polarity is not clear. We expect that the very simple and highly polarized layout of microtubules in Drosophila neurons will allow the function and significance of microtubule orientation in neurons to be uncovered.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Melissa M. Rolls (mur22{at}psu.edu)
Abbreviations used: da, dendritic arborization.
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