Abstract
Neurons have two types of processes: axons and dendrites. Axons have an active disassembly program activated by severing. It has not been tested whether dendrites have an analogous program. We sever Drosophila dendrites in vivo and find that they are cleared within 24 hours. Morphologically this clearance resembles developmental dendrite pruning, and, to some extent, axon degeneration. Like axon degeneration, both injury-induced dendrite degeneration and pruning can be delayed by expression of Wld(s) or UBP2. We therefore hypothesized that they use common machinery. Surprisingly, comparison of dendrite pruning and degeneration in the same cell demonstrated that none of the specific machinery used to prune dendrites is required for injury-induced dendrite degeneration. In addition, we show that the rapid program of dendrite degeneration does not require mitochondria. Thus dendrites do have a rapid program of degeneration, as do axons, but this program does not require the machinery used during developmental pruning.
Keywords: Wallerian degeneration, Wld(s), ubiquitin-proteasome system, dendrite degeneration, dendrite pruning, miro
Introduction
It is now well-established that axons have an active program of degeneration that disassembles them after they are severed from the cell body (Raff et al., 2002; Ehlers, 2004; Luo and O'Leary, 2005; Saxena and Caroni, 2007). This program, which is known as Wallerian degeneration, facilitates clearance of badly damaged axons after injury, perhaps in preparation for regeneration (Vargas and Barres, 2007). The fact that distal regions of axons can be stabilized after severing by overexpression of proteins including Wld(s) led to the idea that endogenous machinery actively disassembles the axon, somewhat analogous to the way that caspases disassemble apoptotic cells (Raff et al., 2002; Ehlers, 2004; Luo and O'Leary, 2005; Saxena and Caroni, 2007).
It is known that dendrites can also be damaged, but it has not been clearly established whether they have a program of degeneration in the same way that axons do. Dendrite beading has been described during excitotoxicity and after ischemia (Oliva et al., 2002; Greenwood et al., 2007; Zeng et al., 2007; Li and Murphy, 2008; Murphy et al., 2008). However, this beading may not be related to that seen after axon severing as it is reversible in at least some cases (Oliva et al., 2002; Greenwood et al., 2007; Zeng et al., 2007; Li and Murphy, 2008; Murphy et al., 2008).
Both axons and dendrites have programs of developmental disassembly known as pruning. Many axons generate extra projections that are removed during development (Luo and O'Leary, 2005; Saxena and Caroni, 2007). Large-scale remodeling of axons and dendrites also takes place during metamorphosis in insects including Drosophila melanogaster (Luo and O'Leary, 2005; Williams and Truman, 2005). Morphologically, axon and dendrite pruning appear similar to injury-induced axon degeneration. Moreover, axon degeneration is characterized by involvement of the ubiquitin-proteasome system (UPS) (Zhai et al., 2003) and axon and dendrite pruning require the UPS and can be inhibited by expression of the ubiquitin protease, UBP2 (Watts et al., 2003; Kuo et al., 2005). Thus most researchers treat pruning of axons and dendrites and injury-induced axon degeneration interchangeably. Indeed pruning is often used as a model for studying injury-induced axon degeneration (Raff et al., 2002; Zhai et al., 2003; Ehlers, 2004; Kuo et al., 2005; Luo and O'Leary, 2005; Saxena and Caroni, 2007; Schoenmann et al., 2010). Only one study has suggested that injury-induced degeneration and pruning use different pathways. In this study, the Wld(s) protein was shown to block injury-induced degeneration, but not developmental pruning in mammals or Drosophila (Hoopfer et al., 2006). Even in this work, the authors surmise that the two “may converge on a common execution pathway” (Hoopfer et al., 2006). Moreover, the generality of the conclusion that Wld(s) distunguishes developmental pruning and injury-induced degeneration has recently been challenged by the finding that Wld(s) does in fact block developmental pruning of dendrites in Drosophila (Schoenmann et al., 2010). Thus the simplest hypothesis, and prevailing view in the field, is that there exists a common disassembly pathway that can remove axons after injury and axons and dendrites during developmental pruning.
In this study, we test whether dendrites also have an injury-induced degeneration program that could feed into this common disassembly pathway. We show that dendrites do have an rapid program of degeneration, but that it uses different machinery than dendrite pruning.
Materials and Methods
Systems to study dendrite degeneration and pruning
To study the injury-induced degeneration, we expressed fluorescent proteins in ddaE and ddaC neurons in Drosophila larvae. ddaE neurons are visualized through EB1-GFP and mCD8-GFP driven by 221-Gal4. mCD8-GFP driven by ppk-Gal4 was used to monitor ddaC neurons. Responses to severing were studied in ddaE and ddaC, and pruning was studied in the ddaC neuron.
Dendrite severing was performed by aiming a pulsed UV laser (Photonic Instruments, St. Charles, IL) on a region of the dendrite fairly close to the cell body. Images were acquired right after severing to make sure dendrites were completely cut and 18h after severing to exam successful removal of the transected part. All experiments were performed on an LSM510 confocal microscope Carl Zeiss, Thornwood, NY. In time course experiment, the same larva was mounted on slides every 3h. In between imaging, larvae were returned to normal Drosophila media as described (Stone et al., 2010)
In the mitochondrial involvement assay, mitochondrial were labeled with mito-GFP (Pilling et al., 2006) driven by elav Gal4 while membranes were labeled by mCD8-RFP (Ye et al., 2007). To image the ddaC neuron during pupal stages, the pupal cases need to be removed as described (Williams et al., 2006) before mounting on slides. ImageJ software was employed to analyze and assemble images (http://rsb.info.nih.gov/ij/; NIH). Overviews of neurons are maximum projection Z stacks from confocal images.
Drosophila stocks and RNAi
The tester lines for RNAi experiments were as follows: 1. UAS-dicer2; 221-Gal4, UAS-EB1-GFP, UAS-dicer2; 2. UAS-dicer2; ppk-Gal4, mCD8-GFP/TM6, 3. UAS-mCD8-mRFP, UAS-dicer2/CyO; elav-Gal4, UAS-mito-GFP/TM6. Dicer-2 was included in all RNAi experiments to increase the effectiveness of neuronal RNAi (Dietzl et al., 2007). Tester lines for overexpression studies were similar, but did not have dicer2.
RNAi or overexpression experiments were performed by crossing the tester lines to the following transgenic fly strains: UAS-dmiro-RNAi (VDRC 106683), UAS-Dronc-RNAi (VDRC 23035), UAS- Katanin-p60L1-RNAi (VDRC 31598), UAS-IK2-RNAi (VDRC 103748), pUAST-Wld(s) (MacDonald et al., 2006), UAS-UBP2 (DiAntonio et al., 2001), UAS-p35 (Bloomington Drosophila Stock Center, Bloomington, IN), UAS-DIAP1 (Bloomington Drosophila Stock Center, Bloomington, IN). Crosses to UAS-rtnl2-RNAi (VDRC 33318) and yw flies were used as controls.
Results
Dendrites undergo beading and clearance within 24 hours after severing
We used Drosophila larval multidendritic neurons as a model system. These neurons lie under the cuticle and are responsible for mechanosensation and nociception (Hughes and Thomas, 2007; Song et al., 2007; Zhong et al., 2010). They are multipolar neurons with several dendrites and a single axon that emerge from the cell body, and they have been used very successfully to study dendrite pruning, development and branching (Gao et al., 1999; Grueber et al., 2002; Gao and Bogert, 2003; Sugimura et al., 2003; Grueber and Jan, 2004; Kuo et al., 2005; Kuo et al., 2006; Williams et al., 2006; Kirilly et al., 2009; Schoenmann et al., 2010)
To test whether dendrites initiate a rapid degeneration program after severing from the cell body, we used a pulsed UV laser to sever dendrites of two different types of multidendritic cells in Drosophila larvae. We chose to use the class I cell ddaE because we have previously studied axon regeneration in this cell (Stone et al., 2010), and we used the class IV ddaC cell because it has been used as a model system in which to study dendrite pruning (Kuo et al., 2005; Kuo et al., 2006; Williams et al., 2006; Kirilly et al., 2009; Schoenmann et al., 2010). The class refers to the complexity of dendrite branching pattern: class I multidendritic neurons have the simplest dendritic arbors and class IV have the most complex (Grueber et al., 2002). A pulsed UV laser can very effectively sever axons or dendrites of these cells (Sugimura et al., 2003; Stone et al., 2010). We labeled class I and class IV neurons by expressing a GFP-tagged membrane marker, mCD8-GFP, or the microtubule and soluble marker EB1-GFP, selectively in these cells with class I and class IV-specific Gal4 drivers.
In both cell types all dendrite fragments distal to the cut site were cleared within 24 hours (Figure 1). To determine the time course of clearance we analyzed morphology of ddaC (Figure 1) and ddaE (Figure S1) at multiple time points after dendrite severing. The degeneration process of both cells appeared similar.
Figure 1.
Dendrites degenerate rapidly after severing. A. The multidendritic neuron ddaC was labeled with mCD8-GFP. The binary Gal4-UAS system was used to express the fluorescent protein in a subset of neurons. Whole larvae were mounted on a microscope slide and a dendrite was severed with a pulsed UV laser. The cell was imaged after severing, and at additional later timepoints. Between imaging sessions the larva was recovered to food. The orange arrow indicates the site of severing and the bracket indicates the region of the dendrite separated from the cell body by the cut. B. The ddaC cell was labeled with mCD8-GFP, but the axon was severed rather than the dendrite. As in A, the larva was remounted at different times after severing to track degeneration. Arrow and brackets are as in A. C. White prepupae expressing mCD8-GFP in ddaC under control of ppk-Gal4 were collected and aged until the indicated times after puparium formation (APF). At 14h dendrites were disconnected from the cell body and fragmented. In the same animal, dendrites were completely cleared at 18h APF. D. Closeup images of ddaE or ddaC neurons expressing mCD8-GFP at different times after dendrite severing or after pupariation. Movies 1–5 illustrate different aspects of dendrite degeneration. Movie 6 shows a late stage in axon degeneration.
Until 3–4 hours after severing the dendrite remained continuous and microtubules labeled with EB1-GFP continued to grow in the severed part of the dendrite (Movie 1). Between 3 and 6 hours dendrite morphology began to change, and at 6h after injury, dendrite branches often had swollen regions and a beaded appearance. At 9 or 12 hours the region previously occupied by the degenerating dendrite was completely clear (Figures 1 and S1). The same general events were observed with both EB1-GFP and mCD8-GFP.
In addition to the swelling and beading, we also observed extensive membrane protrusion before the dendrite fragmented. Around 4–5 hours after severing, blebs were seen to emerge from the dendrite shaft with both the mCD8-GFP marker (Figure 1 D and Movie 2) and EB1-GFP (Movies 3 and 4). At later timepoints, bright internalized membranes were observed (Figure 1D and Movie 5). As the membrane protrusion seen at 4–5 hours has not been described during axon degeneration (for example (Kerschensteiner et al., 2005), but looks similar to events during dendrite pruning (Kirilly et al., 2009), we wished to directly compare axon degeneration, dendrite degeneration and dendrite pruning in the same cell. Membrane protrusion, swelling and beading were all observed during dendrite pruning of ddaC (Figure 1 C and D). In ddaC axons we observed swelling, beading (Figure 1B) and internalized membranes similar to those seen at late stages of dendrite degeneration (Movie 6), however, we did not observe membrane protrusion. The timecourse of degeneration in the ddaC axon was slightly slower than in the dendrites. Axons could remain intact until 6–7 hours (Figure 1B), but were almost always fragmented by 12 hours after severing. Clearance of the severed pieces was more variable than that of dendrites. Most often all fragments were cleared by 24 hours after severing, but in some cases they could persist until 48–72 hours (not shown).
We conclude that some morphological features are shared between axon and dendrite degeneration, and that dendrite degeneration and dendrite pruning are morphologically indistinguishable at this level of resolution. Both axon degeneration and dendrite pruning are known to be active processes; we therefore hypothesized that dendrite degeneration after injury is an active process similar to Wallerian degeneration. As dendrite degeneration appears extremely similar to dendrite pruning, we also hypothesized that they may use the same machinery.
Dendrite degeneration is blocked by overexpression of Wld(s) or UBP2
As a first test of whether injury-induced dendrite degeneration might use a mechanism similar to Wallerian degeneration, we tested whether it could be blocked by expression of the Wld(s) protein. Expression of Wld(s) in mammalian or Drosophila neurons delays axon degeneration by days or even weeks after axon severing (Luo and O'Leary, 2005; Hoopfer et al., 2006; MacDonald et al., 2006). In control ddaC cells approximately 98% of animals completely cleared all debris from severed dendrites by 18h after injury (Figure 2A and D). Two different genotypes were used as controls: one was a cross of our tester line (UAS-dicer2; ppk-Gal4, UAS-mCD8-GFP) to yw flies, and one was a cross of the same tester line to UAS-rtnl2 hairpin RNA flies. yw was used as a control strain for crosses because it does not contain any transgenes, but is a background in which many transgenics are made. RNAi to rtnl2 was used as we have not observed any phenotypes in cells that express this hairpin RNA. Results from both control genotypes were very similar. The cross to yw resulted in 2/111 animals with some dendrite remnants present at 18h after severing, and the cross to rtnl2 RNAi resulted in no animals out of 25 with any traces of dendrites left 18h after severing. In all figures rtnl2 RNAi images are shown, and the numbers in the tables as controls are crosses to yw.
Figure 2.
Expression of Wld(s) or UBP2 blocks dendrite degeneration. Whole larvae expressing mCD8-GFP under control of the class IV da neuron driver ppk-Gal4 were mounted and subjected to dendrite severing with a pulsed UV laser. Animals were then recovered to food, and remounted for imaging at later time points. In A larvae expressed a hairpin RNA directed against rtnl2 (a control); in B larvae also expressed UAS-controlled Wld(s) (MacDonald et al., 2006), and in C they expressed UAS-controlled UBP2. For quantitation (D), WT larvae expressing only mCD8-GFP were assayed for the presence of any dendrite traces at 18h after severing. Similarly, larvae expressing mCD8-GFP and Wld(s) or UBP2 in the ddaC cell were assayed for dendrite traces 18h after severing. Both transgenes were extremely effective at blocking dendrite degeneration.
In ddaC neurons that expressed the Wld(s) protein, severed dendrites were present for days after injury (Figure 2B). For quantitation of the effect, we assayed animals 18h after severing, as in control animals, and found that all animals still had dendrites present 18h after severing (Figure 2D). We conclude that, as for axon degeneration, the Wld(s) protein can dramatically delay dendrite degeneration.
To further test whether dendrites have a degeneration program similar to that in axons, we overexpressed the ubiquitin protease, UBP2. This manipulation has previously been shown to block both axon and dendrite pruning (Watts et al., 2003; Kuo et al., 2005). Expression of UBP2 also resulted in dendrites that remained for many days after severing (Figure 2C). In this case 83% of animals still had distal dendrites 18h after severing. Thus dendrites likely have an active program of degeneration that involves the ubiquitin proteasome system (UPS).
Dendrite degeneration is independent of mitochondria
As the UPS is required for both dendrite pruning and axon degeneration (Watts et al., 2003; Zhai et al., 2003; Kuo et al., 2005), and Wld(s) can also block both, we hypothesized that all three processes may use the same disassembly machinery. Dendrite pruning has been shown to involve caspases (Kuo et al., 2006; Williams et al., 2006), and trophic factor withdrawal also induces a form of axon degeneration that is caspase-dependent (Schoenmann et al., 2010), although it is not clear that injury-induced axon degeneration uses caspases (Finn et al., 2000).
To test whether dendrite degeneration might use a mitochondria-dependent pathway similar to apoptosis, we wished to determine whether dendrites completely devoid of mitochondria would degenerate with a similar timing to normal dendrites. Mitochondria are typically distributed throughout axons and dendrites, so we needed to reduce overall levels of mitochondria in dendrites to be able to find a dendrite without any mitochondria. The miro protein is required for both anterograde and retrograde mitochondrial transport (Russo et al., 2009), so we reasoned that it might also be required for transport of mitochondria into dendrites. Indeed, when we targeted the miro transcript with a hairpin RNA in the ddaE cell, levels of mitochondria were reduced by about 50% in the dorsal comb-like dendrite (Figure S2).
In neurons expressing mito-GFP, which brightly labels mitochondria, and mCD8-RFP to label membranes as well as the hairpin RNA to target miro, we identified dendrites that lacked mitochondria in distal regions. These were then severed and followed over time. Dendrites lacking mitochondria were completely gone by 12–18h as usual (Figure 3). We performed this experiment in 20 ddaE neurons. In each case we identified a region of the neuron completely devoid of mitochondria and severed it from the cell body as shown in Figure 3. In all instances the distal dendrite was completely cleared by 18h after severing.
Figure 3.
Mitochondria are not required locally for dendrite degeneration. The number of mitochondria in dendrites was reduced by expression of a hairpin RNA targeting the miro transcript. A pan-neuronal Gal4 driver (elav-Gal4) was used to express this hairpin, mito-GFP and mCD8-RFP in neurons. As ddaE cells are relatively separate from other da neurons, and also have shorter dendrites, we could identify dendrites that completely lacked mitochondria in this cell. Two examples (out of 20) are shown. Severed regions of dendrites that lack mitochondria are indicated by brackets; cut sties are indicated by orange arrows. In both cases the severed regions of the dendrite were cleared within the normal time frame. The “f” in the top middle panel indicates a region of the dendrite that is in a different focal plane.
We conclude that dendrite degeneration does not require local mitochondria. This suggests that the program of dendrite degeneration may not use apoptotic components, even though these are known to play a role in other forms of pruning and degeneration.
Dendrite degeneration is independent of apoptosis machinery
It has not been directly tested whether dendrite pruning is dependent on mitochondria, but dendrite pruning is known to depend on caspases (Kuo et al., 2006; Williams et al., 2006; Schoenmann et al., 2010). We therefore also wished to determine whether injury-induced dendrite degeneration was caspase dependent. To make sure that the manipulations we performed to alter the apoptotic pathway were effective, we established that we could assay dendrite pruning in the ddaC cell as shown in other studies. We were able to observe complete clearance of ddaC dendrites by 18h APF (Figure 1C and 4). We could also see that clearance of dendrites was delayed by expression of either Wld(s) or UBP2 in about 50% of animals (Figure 4). Expression of either Wld(s) or UBP2 blocked degeneration of ddaC dendrites more effectively than either blocked pruning in the same cell (Figure 4), so we were reassured that we would be able to determine whether other machinery was shared between degeneration and pruning using this type of comparison.
Figure 4.
Pruning of ddaC is delayed by Wld(s) and UBP2. Larvae expressing mCD8-GFP in ddaC were aged until 18h APF and then mounted for imaging. One ddaC cell per animal was scored for the presence of dendrite remnants; any dendrite fragments were scored as a positive. Dendrite fragments were frequently observed in larvae expressing UBP2 or Wld(s), but not control larvae. Quantitation is shown as black bars in the graph. Grey bars are the data from Figure 2 for comparison of the effectiveness of Wld(s) and UBP2 in pruning and degeneration.
We used three previously tested manipulations to block caspase activity in ddaC cells, and then compared their effects on dendrite degeneration and pruning. Overexpression of the effector caspase inhibitor p35, the Drosophila inhibitor of apoptosis, DIAP1, or RNAi targeting the initiator caspase, dronc, all very effectively delayed clearance of dendrites during pruning (Figure 5). However, in the same cell, none of these manipulations had any effect on degeneration of dendrites after severing (Figure 5). We conclude that apoptotic machinery is not involved in the program of dendrite degeneration.
Figure 5.
Apoptosis machinery is required for dendrite pruning, but not dendrite degeneration. Animals of the same genotypes were assayed for both dendrite degeneration after severing and dendrite pruning during metamorphosis. Control genotypes are as in Figure 2. In addition to mCD8-GFP, experimental animals expressed Gal4-controlled p35, DIAP1 or a hairpin RNA to target the dronc transcript. For severing experiments larvae were mounted and a dendrite from ddaC was severing with a pulsed UV laser. After 18h recovery on food the presence of dendrite remnants was scored. For pruning experiments, animals were mounted for imaging 18h APF, and presence of ddaC dendrite remnants was scored.
The pruning machinery is not required for injury-induced dendrite degeneration
Several additional proteins that contribute to removal of dendrites during pruning have been identified. The putative microtubule severing protein Kat-60L1 is required for an early step in pruning; the disconnection of dendrites from the cell body (Lee et al., 2009). The kinase IK2 seems to play a role in the same pathway (Lee et al., 2009), and large multidomain protein Mical seems to function at a similar step in dendrite pruning (Kirilly et al., 2009). These pruning studies were performed in the ddaC cell, so we confirmed that in our hands RNAi to target each of these players would delay ddaC pruning. In all cases we found a strong effect (Figure 6). When we severed dendrites in the same cells during larval life, dendrite degeneration proceeded as usual (Figure 6). Thus, while both dendrite pruning and dendrite degeneration can be blocked by expression of Wld(s) or UBP2, the specific machinery used to disassemble dendrites in these two cases seems to be entirely different. We conclude that dendrites have an rapid program of degeneration uses a different set of machinery from dendrite pruning.
Figure 6.
Specific players required for dendrite pruning are not required for dendrite degeneration. The same strategy shown in Figure 5 was used to test the role of IK2, Kat-60L1 and Mical in dendrite degeneration, again with comparison to pruning in cells of the same genotype. RNAi to reduce each of these proteins was performed by expressing UAS-controlled hairpin RNAs. The presence of dendrite remnants in ddaC was scored 18h after severing or 18h APF.
Discussion
While it is well-established that axons have an active program of degeneration; it has not been directly tested whether dendrites have a similar program. We show that after severing, dendrites are rapidly cleared, and that they undergo morphological changes similar to those seen during dendrite pruning. Moreover, like dendrite pruning, and axon degeneration, dendrite degeneration can be blocked by overexpression of Wld(s) or UBP2.
Because axon degeneration, dendrite pruning and dendrite degeneration involve similar morphological changes and can all be blocked by overexpression of Wld(s) or UBP2, it seems logical that they might all involve the same disassembly machinery. Indeed it has previously been suggested that pruning might be a good model system to use to understand injury-induced degeneration (Raff et al., 2002; Zhai et al., 2003; Ehlers, 2004; Kuo et al., 2005; Luo and O'Leary, 2005; Saxena and Caroni, 2007; Schoenmann et al., 2010). We have rigorously tested this idea by comparing dendrite pruning and injury-induced dendrite degeneration in the same cell. We confirm that pruning uses caspases, IK2, Kat-60L1 and Mical, but none of these proteins is required for injury-induced dendrite degeneration. Mitochondria are also dispensable for injury-induced dendrite degeneration. Mitochondria have previously been implicated in the protective effect of Wld(s) or nmnat protection of axons from degeneration (Yahata et al., 2009). It is therefore possible that the protective effect of Wld(s) does not arise from blocking the intrinsic degeneration machinery, but rather by activating a different pathway.
We propose that at least two pathways exist to disassemble axons and dendrites. One pathway is caspase-dependent, and is activated during pruning. Recent studies have also shown that caspases are involved in axon degeneration after trophic-factor withdrawal (Schoenmann et al., 2010), so pruning and loss of trophic support could activate the same disassembly machinery. In contrast, none of the specific machinery used for dendrite pruning is used for injury-induced dendrite degeneration. Thus an entirely different, and as yet unidentified, set of machinery must be used in this process. Injury-induced axon degeneration may share this machinery as it also seems to be a caspase-independent process (Finn et al., 2000). But testing this idea will first require identifying specific proteins required for either axon or dendrite degeneration after injury.
All disassembly pathways can be blocked by Wld(s) or UBP2. It is still unclear how Wld(s) protects axons or dendrites, but this may involve changes in NAD+ (Coleman and Freeman, 2010). These changes must have a very general blocking effect as they inhibit both dendrite pruning and dendrite degeneration, which seem to be separate pathways. The UPS also seems to play a role in both pathways as UBP2 can inhibit both pruning and degeneration. The UPS could be a common effector of both pathways, or different specific substrates could be targeted by the UPS in each pathway.
IK2, Kat-60L1 and Mical seem to be involved in a specific early step in pruning in which the dendrite is clipped from the cell body (Kirilly et al., 2009; Lee et al., 2009). Typically dendrites are first disconnected from the cell body at their base (see Figure 5), and then the distal region fragments. When caspases are inhibited this early step can still occur (Figure 5), but when IK2, Kat-60L1 or Mical is targeted, dendrites typically remain connected to the cell body (Figure 6 and (Kirilly et al., 2009; Lee et al., 2009)). It is unclear whether machinery is needed to fulfill this early disconnection function after dendrite injury, as the injury itself may replace this step. However, some other machinery must substitute for the later caspase-dependent steps of fragmentation.
In the current study we show that at least two disassembly pathways exist in the same compartment of a single cell, and that one is used to remove dendrites developmentally and one is used to remove them after injury. Additional pathways may exist in the axon of this cell or in dendrites and axons of different cells. Until now it has been assumed that all active disassembly of axons or dendrites would use ubiquitous machinery. As this does not seem to be the case, it will be extremely important to identify the specific machinery activated in each type of disassembly.
Supplementary Material
Movie 1. Live imaging of EB1-GFP dynamics in ddaC 4.5 hours after dendrite severing. Frames were acquired every 2s; lookup table was inverted for the movie. Growing microtubules appear as dark spots that move in consecutive frames, and several examples are indicated with stars. The frame width in the zoomed in part of the movie is 105 microns.
Movie 2. Live imaging of mCD8-GFP in ddaC 4 hours after dendrite severing. Images were acquired every 2s. Some mCD8-GFP was also expressed in epithelial cells in this animal, and in some parts of the movie edges of these hexagonal cells can be observed. Two forming membrane blebs are indicated with stars. The frame width in the zoomed in part of the movie is 134 microns.
Movie 3. Live imaging of EB1-GFP in ddaC 3 hours after dendrite severing. Frames were acquired every 2s. Diffuse EB1-GFP labels the cytoplasm, and a protrusion can be seen extending from the side of one of the dendrite branches (star). The width of the movie is 210 microns.
Movie 4. Live imaging of EB1-GFP in ddaC 9 hours after dendrite severing. Frames were acquired every 2s. A dramatic example of blebbing (arrows) can be seen near the cut site in this dendrite. Frame width is 210 microns.
Movie 5. Membranes of ddaC labeled with mCD8-GFP are shown 9 hours after dendrite severing. Frames were acquired every 2 seconds. In contrast to mCD8-GFP labeled membranes at earlier time points, the brightest structures are now vesicles that look like they are contained internally. The width of the movie frame is 80 microns.
Movie 6. Live imaging of mCD8-GFP in ddaC 12 hours after axon severing. Frames were acquired every 2 seconds. Bright internal membranes are visible in the disintegrating axon. The width of the frame in the zoomed in part of the movie is 50 microns.
Figure S1. Dendrites of ddaE neurons degenerate rapidly after severing. The multidendritic neuron ddaE was labeled with EB1-GFP. The binary Gal4-UAS system was used to express the fluorescent protein in a subset of neurons. The 221-Gal4 expresses at high levels in class I dendritic arborization (da) neurons including ddaE. Whole larvae were mounted on a microscope slide and the dorsal dendrite of the ddaE neuron was severed with a pulsed UV laser. The cell was imaged immediately after severing. The larva was then recovered to food and remounted for imaging at 3h intervals. The orange arrow indicates the site of severing and the bracket indicates the region of the dendrite separated from the cell body by the cut.
Figure S2. Targeting miro by RNAi reduces the number of mitochondria in dendrites. Animals expressed hairpin RNAs to target either rtnl2 (control) or miro in all neurons under control of elav-Gal4. Mito-GFP and mCD8-RFP were also expressed in all neurons by elav-Gal4. Total numbers of mitochondria in the dorsal comb-shaped dendrite of the ddaE neuron were counted (n=5 for control and n=4 for miro RNAi). The difference between the average number of mitochondria in this dendrite between control and miro RNAi was statistically significant using an unpaired t-test (p< 0.05).
Acknowledgements
We are very grateful to Wes Grueber, Bing Ye, Marc Freeman, the Bloomington Drosophila Stock Center, and the Vienna Drosophila RNAi Center (VDRC) for Drosophila lines. This work was supported by R21 NS066216. MMR is a Pew Scholar in the Biomedical Sciences.
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Associated Data
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Supplementary Materials
Movie 1. Live imaging of EB1-GFP dynamics in ddaC 4.5 hours after dendrite severing. Frames were acquired every 2s; lookup table was inverted for the movie. Growing microtubules appear as dark spots that move in consecutive frames, and several examples are indicated with stars. The frame width in the zoomed in part of the movie is 105 microns.
Movie 2. Live imaging of mCD8-GFP in ddaC 4 hours after dendrite severing. Images were acquired every 2s. Some mCD8-GFP was also expressed in epithelial cells in this animal, and in some parts of the movie edges of these hexagonal cells can be observed. Two forming membrane blebs are indicated with stars. The frame width in the zoomed in part of the movie is 134 microns.
Movie 3. Live imaging of EB1-GFP in ddaC 3 hours after dendrite severing. Frames were acquired every 2s. Diffuse EB1-GFP labels the cytoplasm, and a protrusion can be seen extending from the side of one of the dendrite branches (star). The width of the movie is 210 microns.
Movie 4. Live imaging of EB1-GFP in ddaC 9 hours after dendrite severing. Frames were acquired every 2s. A dramatic example of blebbing (arrows) can be seen near the cut site in this dendrite. Frame width is 210 microns.
Movie 5. Membranes of ddaC labeled with mCD8-GFP are shown 9 hours after dendrite severing. Frames were acquired every 2 seconds. In contrast to mCD8-GFP labeled membranes at earlier time points, the brightest structures are now vesicles that look like they are contained internally. The width of the movie frame is 80 microns.
Movie 6. Live imaging of mCD8-GFP in ddaC 12 hours after axon severing. Frames were acquired every 2 seconds. Bright internal membranes are visible in the disintegrating axon. The width of the frame in the zoomed in part of the movie is 50 microns.
Figure S1. Dendrites of ddaE neurons degenerate rapidly after severing. The multidendritic neuron ddaE was labeled with EB1-GFP. The binary Gal4-UAS system was used to express the fluorescent protein in a subset of neurons. The 221-Gal4 expresses at high levels in class I dendritic arborization (da) neurons including ddaE. Whole larvae were mounted on a microscope slide and the dorsal dendrite of the ddaE neuron was severed with a pulsed UV laser. The cell was imaged immediately after severing. The larva was then recovered to food and remounted for imaging at 3h intervals. The orange arrow indicates the site of severing and the bracket indicates the region of the dendrite separated from the cell body by the cut.
Figure S2. Targeting miro by RNAi reduces the number of mitochondria in dendrites. Animals expressed hairpin RNAs to target either rtnl2 (control) or miro in all neurons under control of elav-Gal4. Mito-GFP and mCD8-RFP were also expressed in all neurons by elav-Gal4. Total numbers of mitochondria in the dorsal comb-shaped dendrite of the ddaE neuron were counted (n=5 for control and n=4 for miro RNAi). The difference between the average number of mitochondria in this dendrite between control and miro RNAi was statistically significant using an unpaired t-test (p< 0.05).