© 2016. Published by The Company of Biologists Ltd | Journal of Science (2016) 129, 1635-1648 doi:10.1242/jcs.184929

RESEARCH ARTICLE The effects of ER morphology on synaptic structure and function in melanogaster James B. Summerville1,*, Joseph F. Faust1,*, Ethan Fan1, Diana Pendin2, Andrea Daga3, Joseph Formella1, Michael Stern1,‡ and James A. McNew1,‡

ABSTRACT (Di Sano et al., 2012). The nature of most of these secondary Hereditary spastic paraplegia (HSP) is a set of genetic diseases functions remains to be revealed. caused by mutations in one of 72 that results in age-dependent The large ER network also maintains luminal and membrane corticospinal degeneration accompanied by spasticity and continuity throughout the . This interconnected nature paralysis. Two genes implicated in HSPs encode that of the ER network is required for ER function and is maintained regulate (ER) morphology. Atlastin 1 (ATL1, by the ER membrane fusion GTPase atlastin (Orso et al., 2009), also known as SPG3A) encodes an ER membrane fusion GTPase which is a member of the fusion dynamin-related family and 2 (RTN2, also known as SPG12) helps shape ER tube (fusion DRP) (McNew et al., 2013; Moss et al., 2011; Pendin et al., formation. Here, we use a new fluorescent ER marker to show that the 2011). ER within wild-type Drosophila motor nerve terminals forms a network The ER is closely associated with and functionally connected to of tubules that is fragmented and made diffuse upon loss of the the plasma membrane. This connection is often associated with 2+ atlastin 1 ortholog atl. atl or Rtnl1 loss decreases evoked transmitter the management of Ca stores in the ER lumen. The ER protein release and increases arborization. Similar to other HSP proteins, Atl STIM1 diffuses through the ER membrane to find binding inhibits bone morphogenetic protein (BMP) signaling, and loss of atl partners in the plasma membrane including the Orai channel – causes age-dependent locomotor deficits in adults. These results (Jozsef et al., 2014; Soboloff et al., 2012). This set of protein 2+ demonstrate a crucial role for ER in neuronal function, and identify protein associations works to restore ER Ca through the store- 2+ – mechanistic links between ER morphology, neuronal function, BMP operated Ca channel system. ER plasma-membrane contact sites signaling and adult behavior. are also generated by the association of ER-integral extended synaptotagmins (E-Syt) with phospholipids in the plasma KEY WORDS: Atlastin, ER, Reticulon, Neuron membrane (Fernández-Busnadiego et al., 2015; Giordano et al., 2013; Schauder et al., 2014) as well as proteins like junctophillins INTRODUCTION in certain cell types (Helle et al., 2013; Stefan et al., 2013). The function of intracellular is tightly coordinated with Most recently, the ER has been found to be stably associated with location within the cytoplasm. The endoplasmic reticulum (ER) is endosomal structures (Raiborg et al., 2015a; Rowland et al., 2014). an interconnected network of narrow tubes and flattened cisternae In this circumstance, the specific proteins on each surface that or sheets (Hu et al., 2011; Shibata et al., 2009, 2006; Terasaki interact remain to be precisely defined, but the consequence of the et al., 2013; Westrate et al., 2015). In most cells, the ER is the interaction is functional segregation of certain cargoes within the most abundant subcellular and extends elaborate endosome that permits regulated sorting into membrane processes throughout the cytoplasm. The ER membrane is subdomains prior to an ER-directed membrane fission event formed into its tubular architecture by the action of structural (Raiborg et al., 2015a,b; Rowland et al., 2014). proteins within the reticulon, REEP and DP1 family (English and ER structure appears to be crucially important for cell function Voeltz, 2013; Hu et al., 2008; Shibata et al., 2009; Voeltz et al., given that disease results when components that control this 2006; Yang and Strittmatter, 2007). The members of this diverse structure are compromised by mutation. The hereditary spastic family of proteins share a common protein motif called the paraplegias (HSPs) are a group of related genetic disorders caused reticulon homology domain (RHD). The hydrophobic ∼200- by mutations in any of more than 70 genes, denoted SPG1 to SPG72 amino-acid RHD likely forms a helical hairpin structure that (Lo Giudice et al., 2014; Noreau et al., 2014). Lower limb weakness intercalates four hydrophobic helical segments into the outer and spasticity represent two prominent clinical features of these leaflet of the ER membrane to induce curvature and maintain a diseases, which occur as a consequence of dysfunction or tubular shape (Voeltz et al., 2006; Zurek et al., 2011). Many degeneration of the upper motor neurons (Blackstone et al., members of the Reticulon, REEP and DP1 family also contain an 2010). The observation that atlastin 1 (ATL1) and reticulon 2 extended N-terminal segment ranging from a few hundred to a (RTN2) are HSP genes responsible for SPG3A and SPG12, thousand amino acids that likely provides additional functionality respectively, implicates ER morphology in the neuronal dysfunction that causes HSPs. The properties of three additional HSP genes, spartin (SPG20), 1Department of BioSciences, Program in Biochemistry and Cell Biology, Rice 2 SPAST SPG4 NIPA1 University, Houston, TX 77005, USA. CNR, Neuroscience Institute, 35121 Padova, spastin ( , also known as ) and (also known as Italy. 3E. Medea Scientific Institute, 31015 Conegliano, Italy. spichthyin or SPG6), implicate receptor trafficking through the *These authors contributed equally to this work endocytic system in HSP neuronal dysfunction. For example, loss of ‡ Authors for correspondence ([email protected]; [email protected]) spartin attenuates both ligand-stimulated EGF receptor uptake (Bakowska et al., 2007) as well as depolarization-stimulated FM1-

Received 15 December 2015; Accepted 17 February 2016 43 uptake (Nahm et al., 2013), whereas loss of spastin increases Journal of Cell Science

1635 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 1635-1648 doi:10.1242/jcs.184929 endosome tubule number and alters transferrin receptor sorting altered ER structure in all tissues that were examined (Fig. 1). Upon (Allison et al., 2013). NIPA1 is also located in endosomes and fixation, ER tubules appeared to fragment into discrete punctae and promotes the endocytosis of receptors for bone morphogenetic the uniformly labeled tubules seen in live imaging converted into a protein (BMP) (Tsang et al., 2009). ‘beads on a string’ morphology consistent with ruptured tubules. It Phenotypic analysis of mutations in the HSP orthologs of model is possible that the ER tubules are maintained under stress by systems has provided additional clues to the cellular function of association with the underlying cytoskeletal network and that these proteins. In Drosophila, loss of spastin, spartin and chemical fixation disrupts this mechanical tension. Regardless of spichthyin confers a similar, but not identical, set of phenotypes the mechanism, these observations reveal the disruptive effect of including stabilized microtubules, increased synaptic bouton tissue fixation on the ER in vivo. For this reason, all subsequent ER number and decreased evoked transmitter release at the larval imaging exhibited here was performed on live cells. neuromuscular junction (NMJ), age-dependent locomotor deficits and increased BMP signaling at the larval NMJ (Nahm et al., BiP–sfGFP–HDEL marks the ER 2013; Ozdowski et al., 2011; Sherwood et al., 2004; Wang et al., We investigated ER anatomy in larval motor nerve terminals in 2007). These shared phenotypes might reflect disruption of a more detail. To orient the BiP–sfGFP–HDEL signal with the plasma common pathway in endocytic receptor trafficking in these mutants. membrane, we co-expressed BiP–sfGFP–HDEL with the plasma Some of these phenotypes, such as stabilized microtubules, age- membrane marker myr::tdTomato. We found that BiP–sfGFP– dependent locomotor deficits and increased synaptic bouton HDEL was located immediately adjacent to the myr::tdTomato number, are also observed in flies lacking atlastin (atl) (Lee et al., signal (Fig. 2A–C), indicating that the ER baskets in motor nerve 2009, 2008). terminals directly underlie the plasma membrane. We also co- Here, we extend this phenotypic analysis of altered atl and expressed BiP–sfGFP–HDEL along with the ER membrane marker reticulon-like 1 (Rtnl1) activities in Drosophila. We show that the tdTomato–Sec61β. As expected, these two markers showed ER in motor nerve terminals from wild-type larvae forms a network extensive overlap (Fig. 2D–F), thus validating both transgenes as of tubules that resembles a ‘basket’, but is diffuse in larvae lacking ER markers. atl. We find that neuronal RNA interference (RNAi)-mediated knockdown of either atl or Rtnl1 increases arborization at the larval The atl2 null mutation alters ER structure in motor and neuromuscular junction and decreases evoked transmitter release presynaptic boutons from larval motor nerve terminals, and that elevated bath [Ca2+] atl encodes an ER fusion GTPase (Orso et al., 2009), and thus we fully rescues these transmitter release phenotypes. We also show anticipated that, by preventing ER fusion, the null mutation atl2 that atl is required only in motor neurons to affect transmitter would fragment the ER. To test this prediction, we compared ER release, whereas Rtnl1 is required additionally in the target muscle morphology in motor neuron cell bodies, motor axon initial and peripheral glia. Finally, we show that loss of atl increases BMP segments, and motor nerve terminals in control and atl2 signaling in larval motor neurons and causes age-dependent (Fig. 3A). We found little effect on ER morphology in cell bodies locomotor deficits in adults. Thus, loss of atl and Rtnl1 confers (Fig. 3Bi versus Fig. 3Bii), but in axon initial segments, we found phenotypes similar, but not identical, to each other as well as to that the ER crossbridges found in wild-type were almost completely mutants defective in spartin, spastin and spichthyin. Our results eliminated in atl2 cells, and that the long straight ER tubules demonstrate specific mechanistic links between ER morphology observed in wild-type became wavy (Fig. 3Biii,iv). In motor nerve and several aspects of neuronal anatomy and function. terminals, we found that atl2 eliminated the basket structures and caused a diffuse ER signal, which we attribute to ER fragmentation RESULTS (Fig. 3Ci,ii). 2 Chemical fixation disrupts ER morphology in Drosophila To quantify the effects of atl on ER morphology in nerve 2 motor neurons, muscles and S2 cells terminals, we reasoned that the BiP–sfGFP–HDEL signal in atl ER structure has been difficult to examine in Drosophila.To larvae would be uniformly distributed across the bouton and thus a address this issue, we developed improved reagents for in vivo frequency histogram for the pixel intensities within each terminal imaging of the ER. First, we utilized a mutant GFP, termed would exhibit a Gaussian distribution. In contrast, we anticipated Superfolder GFP (sfGFP) (Aronson et al., 2011; Costantini et al., that pixel intensities in wild-type nerve terminals would comprise a 2015; Pédelacq et al., 2006), that has been found to fold properly in small number of bright pixels indicating the tubules, and a larger oxidizing environments such as the ER lumen. This protein traffics number of dark pixels indicating voids between tubules. Thus, we to the ER because of addition of the Drosophila BiP signal sequence predicted that a frequency histogram for the pixel intensities from and is retained by addition of the HDEL ER retention signal from wild-type would be positively skewed. We compared frequency calreticulin (BiP–sfGFP–HDEL). Next, we introduced an ER histograms of pixel intensities, normalized to mean intensity, membrane marker containing Sec61β linked to the fluorescent between wild-type and atl2 and found, as predicted, a strong positive marker tdTomato. In Fig. 1, we show the ER labeled by BiP– skew in wild-type (Fig. 3Ciii) but a more Gaussian distribution in sfGFP–HDEL expressed in the motor neuron cell body (Fig. 1A), atl2 (Fig. 3Civ). Furthermore, the mode and median pixel intensities the neuromuscular junction (NMJ) (Fig. 1B), larval body wall were close to the mean in atl2, consistent with a Gaussian muscle (Fig. 1C) and cultured S2 cells (Fig. 1D). distribution, but were less than the mean in wild-type, consistent Previous results have suggested that organelle structure in vivo is with a positive skew (Fig. 3D). In addition, we compared line scans labile to fixation (Johnson et al., 2015), and therefore an accurate across nerve terminals in atl2 and wild-type. Whereas wild-type line depiction of ER structure might require imaging in live cells. To scans showed multiple peaks, corresponding to tubules within the determine whether ER structures in Drosophila larvae or S2 cells baskets, atl2 line scans were uniform, consistent with a diffuse ER were similarly labile to fixation, we compared ER structure in live signal (Fig. 3Cv,vi). cells with the fixed tissue. We found that very mild fixation (4% Next, we determined whether the atl2 ER fragmentation could be formaldehyde for 5 min with no permeabilization) significantly recapitulated by targeted atl knockdown in motor neurons. Thus, we Journal of Cell Science

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Fig. 1. Chemical fixation disrupts the ER network in motor neurons, muscles, and S2 cells. Representative confocal slices through the center (A) and periphery (A′) of live and fixed wild-type motor neuron (MN) cell bodies in the ventral nerve cord. (B) ER in wild-type larval boutons under live and fixed conditions. (B′) Magnification of boxed regions in B. (C) ER in wild-type muscle 6 from segment A3 under live and fixed conditions. (C′) Magnification of boxed regions in C. (D) ER in S2 cells under live and fixed conditions. (D′) Magnification of boxed regions in D. Chemical fixation was achieved by a 5 min exposure to 4% paraformaldehyde. ER was imaged using BiP–sfGFP–HDEL. Scale bars: 5 µm, except B′ (2 µm). expressed both an atl RNAi transgene and the dominant-negative Impaired evoked transmitter release in atl2 and Rtnl11 null K51A atl allele (Orso et al., 2009) in neurons and found a similar mutants decrease in ER tubule crossbridges in axon initial segments Altered levels of several HSP proteins, including Spartin and (Fig. 4D–F) and a similar diffuse ER signal at motor nerve Spastin, alter transmitter release from Drosophila motor nerve terminals (Fig. 4G–I). These results demonstrate that maintenance terminals (Nahm et al., 2013; Ozdowski et al., 2011; Sherwood of the correct ER structure in motor neurons requires atlastin et al., 2004). We tested the possibility that atl2 and Rtnl11 might activity. similarly affect transmitter release. We used the larval In contrast to atl2, the null Rtnl11 allele conferred no detectable neuromuscular preparation (Jan and Jan, 1976; Stewart et al., abnormalities in ER structure in motor neuron cell bodies, axons or 1994) to measure synaptic transmission and found that each nerve terminals (data not shown). mutation indeed decreased evoked transmitter release, using the Journal of Cell Science

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Fig. 2. The ER lumen marker BiP–sfGFP– HDEL colocalizes with the ER membrane marker tdTomato–Sec61β. Third-instar larval motor nerve terminals from segment A2 muscle 4. (A–C) Representative confocal z-projections showing the plasma membrane marker myr:: tdTomato (A), the ER marker BiP–sfGFP–HDEL (B) and the merged signals (C). (D–F) z-projections showing the ER membrane marker tdTomato–Sec61β (D), the ER lumen marker BiP–sfGFP–HDEL (E) and the merged signals (F). All transgenes were driven by the motor neuron driver OK371-Gal4. Scale bars: 5 µm.

consequent muscle depolarization termed excitatory junctional release of individual vesicles of transmitter, were not substantially potential (EJP) as a readout. Fig. 5A and B show averaged EJPs in affected in atl2 or Rtnl11 larvae (Fig. 5G). The modest, albeit wild-type versus atl2, respectively, at the four indicated bath [Ca2+]. significant, increase in mini EJP amplitude observed in atl2 is likely a At a bath [Ca2+] of 0.6 mM, both atl2 and Rtnl11 decrease genetic background effect unrelated to the atl genotype, as mini EJP transmitter release almost two-fold (Fig. 5C). amplitude is not significantly affected by arm-Gal4-driven expression To confirm that these transmitter release phenotypes were caused of the atl+ transgene (data not shown), and elav-Gal4-driven by loss of atl and Rtnl1, we determined whether expression of UAS- expression of atl RNAi does not affect mini EJP amplitude (Fig. 5H). atl+ and UAS-Rtnl1+ transgenes would be sufficient to restore wild- It has been previously observed that atlastin and reticulon confer type transmitter release to atl2 and Rtnl11. We found that the atl2 opposite effects on ER morphology. In particular, whereas atl2 transmitter release phenotype was fully rescued by expression of fragments the ER (Orso et al., 2009), Rtnl1 knockdown by RNAi UAS-atl+ driven by the weak ubiquitous Gal4 driver arm-Gal4 (O’Sullivan et al., 2012) converts ER tubes into sheets. We found a (Fig. 5D). In contrast, the Rtnl11 transmitter release phenotype was mutual suppression of the evoked transmitter release phenotype in the rescued only partially by arm-Gal4-driven UAS-Rtnl1+; this partial Rtnl1;atl2 double mutant (Fig. 5C,E), which supports the possibility rescue most likely reflected weak expression of the Rtnl1+ transgene that atlastin and reticulon have antagonistic effects on ER morphology. because, as shown below, driving UAS-Rtnl1+ expression with the We examined synapse architecture in atl2 and Rtnl11 mutants by stronger Da-Gal4 driver elicited complete rescue (see Fig. 7B). performing immunocytochemistry with directed at the Previous reports have indicated that nerve terminal ER is capable presynaptic active zone protein Bruchpilot and the postsynaptic of controlling transmitter release by influencing cytoplasmic [Ca2+] neurotransmitter receptor GluRII. We found no obvious structural (Emptage et al., 2001; Liang et al., 2002; Llano et al., 2000). We or organizational defects in the number or size of active zones or in wondered whether the decreased evoked transmitter release in atl2 the pattern of GluRII immunoreactivity (data not shown). and Rtnl11 might reflect attenuation of the increased cytoplasmic [Ca2+] caused by nerve stimulation. If so, then we predicted that this atl acts in the motor neuron to control transmitter release decreased transmitter release would be rescued by elevated bath Next, we wished to identify the tissues in which Atl and Rtnl1 are [Ca2+], at which Ca2+ is less limiting for transmitter release (Wong required to control neurotransmitter release. Because atl2 and Rtnl11 et al., 2014). For both atl2 and Rtnl11, we found that as we increased exhibited the greatest deficit in neurotransmitter release at the lowest bath [Ca2+], transmitter release became progressively similar to that bath [Ca2+] (Fig. 5), we performed the next set of measurements at in wild type (Fig. 5E, which shows quantal content, corrected for the low bath [Ca2+] of 0.1 mM, in a solution (HL3.1) conducive for nonlinear summation and normalized to wild type). Elevated bath low [Ca2+] recordings. [Ca2+] completely rescued the Rtnl11 transmitter release phenotype, Inhibiting atl by driving atl RNAi expression with the motor but rescued the atl2 phenotype only partially. In addition, we found neuron driver D42-Gal4 (Fig. 6A, left, green diamonds) or the pan- that RNAi-mediated knockdown of either atl or Rtnl1 in neurons neuronal driver elav-Gal4 (Fig. 6A, left, green squares) significantly similarly failed to decrease transmitter release at elevated bath decreased EJP amplitude. Similarly, inhibiting atl by expressing the [Ca2+] (Fig. 5F; efficacy of the atl and Rtnl1 RNAis is demonstrated dominant-negative atlK51A with either D42-Gal4 (Fig. 6A, left, in experiments described below). It is not known why elevated bath green inverted triangles) or the pan-neuronal driver nSyb-Gal4 [Ca2+] rescues the atl RNAi knockdown phenotype completely, but (Fig. 6A, left, green triangles) also significantly decreased EJP atl2 only partially. However, it is possible that atl acts in non- amplitude. These results indicate that atl activity is required in the neuronal tissues to affect transmitter release at elevated bath [Ca2+]. motor neuron for wild-type evoked transmitter release. In contrast to the strong effects of altered atl and Rtnl1 on EJP We confirmed that the decrease in EJP amplitude represented amplitude, the amplitudes of ‘mini’ EJPs, generated by spontaneous decreased transmitter release rather than decreased muscle Journal of Cell Science

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Fig. 3. Loss of atl disrupts the tubular ER network in motor axons and motor nerve terminals of third-instar larvae. (A) Schematic of the regions of the motor neuron from which images were collected. (B) Representative central confocal slices of motor neuron cell bodies in control [OK371>BiP–sfGFP–HDEL] (Bi) and atl2 [atl2, OK371>BiP–sfGFP–HDEL] (Bii) larvae. z-projections of motor axons within the ventral nerve cord in wild-type (control) (Biii) and atl2 (Biv) larvae. (C) z-projections of neuromuscular junctions from muscle 4 at segment A6 in wild-type (Ci) and atl2 (Cii) larvae. Average frequency histograms of pixel intensities taken from regions of interest around boutons for control (Ciii) and atl2 (Civ) (w1118 n=5, atl2 n=6; red and blue dashed lines represent the mode and median, respectively, of the histogram and are color coded with the analysis in D). Boxed regions in Ci and Cii are expanded in Ci′ and Cii′, respectively. Dashed lines in Ci′ and Cii′ represent the positions used for the linescans of pixel intensities for wild-type (Cv) and atl2 (Cvi). (D) Mode and median of pixel intensity frequency histograms (mean±s.e.m.) are shown for five control and six atl2 images. P-values shown represent an unpaired Student’s t-test performed with KaleidaGraph. Scale bars: 5 µm, except for Ci′ and Cii′ (2 µm). responsiveness to transmitter by analyzing the frequency of synaptic Overexpression of atl in the motor neuron impairs transmission failures. In the low [Ca2+] bath utilized in Fig. 6, the neurotransmitter release motor neuron responds to nerve stimulation with release of either Expression of UAS-atl+ in Drosophila motor neurons causes no transmitter, which is called a failure, or one or two vesicles of excessive fusion and expansion of the and ER transmitter, which is called a success. The decreased transmitter membrane, and defects in the secretory pathway in both the release in the atl knockdown larvae was accompanied by a neuron cell body and presynaptic terminal (Orso et al., 2009). decreased frequency of successes (Fig. 6A, right), indicating that Using the BiP–sfGFP–HDEL imaging reagent described above, atl knockdown decreases transmitter release. we found that atl+ overexpression caused accumulation of large Transmitter release at the Drosophila neuromuscular junction ER punctae in motor neuron cell bodies and axons (Fig. S1A). can be affected by the target muscle or the neighboring peripheral To determine whether atl+ overexpression affected transmitter glia (Huang and Stern, 2002; Kerr et al., 2014; McCabe et al., 2003; release, we compared EJP amplitudes in atl+ overexpressing Schmidt et al., 2012). RNAi-mediated atl knockdown in muscle or and control larvae at both low (0.1 mM) and high (1.5 mM) bath glia did not affect transmitter release or frequency of synaptic [Ca2+]. We found that atl+ overexpression significantly decreased failures (Fig. 6B, left, green squares; Fig. 6B, left, green diamonds). EJP amplitudes and the frequency of synaptic successes at low Journal of Cell Science

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RNAi Fig. 4. Expression of UAS-atl or UAS- K51A atl disrupts the tubular ER network in motor axons and presynaptic boutons. (A–C) ER in motor neuron (MN) cell bodies for control [nSyb>BiP-sfGFP-HDEL] (A), atl RNAi [nSyb>BiP-sfGFP-HDEL, atlRNAi] (B), and dominant-negative Atl [nSyb>BiP-sfGFP- HDEL, atlK51A] (C). (D–F) ER in motor axons for control (D), atl RNAi (E) and dominant-negative (DN) atlK51A (F). (G–I) ER in presynaptic boutons for control (G), atl RNAi (H) and dominant-negative atlK51A (I). (G′–I′) Magnification of boxed regions for wild- type (G′), atl RNAi (H′), and dominant-negative atlK51A (I′). Scale bars: 5 µm, except for G′,H′ and I′ (2 µm).

bath [Ca2+] (Fig. S1B), and decreased corrected quantal content mediated Rtnl1 knockdown in either muscle (Fig. 7A, left, blue at high bath [Ca2+](Fig.S1C).Rtnl1+ overexpression also diamonds) or glia (Fig. 7A, left, blue triangles) also decreased transmitter release but to a lesser extent than atl+.We significantly decreased evoked EJP amplitude and synaptic conclude that maximum levels of evoked transmitter release success rate (Fig. 7A, right). Thus, Rtnl1 activity is required in require specific ratios of Atl and Rtnl1 protein. all three cell types of the tripartite synapse for proper evoked transmitter release. Rtnl1 regulates transmitter release from multiple tissues at We next determined whether Rtnl1 activity in these three the NMJ cell types was sufficient for proper evoked transmitter release. We used both tissue-specific transgenic rescue and RNAi- First, we verified effectiveness of the Rtnl1+ rescue construct by mediated knockdown to identify the tissue(s) within which determining that ubiquitous expression of the Rtnl1+ transgene, Rtnl1 functions to control transmitter release. First, we found driven by the Da-Gal4 driver, was sufficient to rescue both that driving Rtnl1 RNAi expression with the pan-neuronal evoked EJP amplitude (Fig. 7B, left) and frequency of synaptic driver elav-Gal4 (Fig. 7A, left, blue squares) significantly successes of Rtnl11 (Fig. 7B, right). As expected from the RNAi decreased both evoked EJP amplitude and percentage of results described above, expression of Rtnl1+ driven pan- synaptic successes (Fig. 7A, right). This result demonstrates neuronally, in the motor neuron alone, the muscle alone, or that Rtnl1 is required in the motor neuron to affect transmitter both the motor neuron and muscle, was not sufficient to rescue release. We then determined whether Rtnl1 is required in the the EJP amplitude or synaptic success phenotypes of Rtnl11 other cell types of the tripartite synapse and found that RNAi- (Fig. 7B and data not shown). However, simultaneous expression Journal of Cell Science

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2 1 Fig. 5. atl and Rtnl1 decrease evoked neurotransmitter release. Average EJP traces for wild- type (w1118) (A) and atl2 (B) larvae at 0.4, 0.6, 1.0, and 1.5 mM Ca2+. In ascending [Ca2+], for w1118 n=7, 17, 7, and 7 and for atl2 n=7, 23, 7, and 7. (C) Mean±s.e.m. EJP amplitude for w1118, atl2, Rtnl11, and Rtnl11;atl2. P-values shown represent a one-way ANOVA using a Fisher’s LSD post hoc test performed with KaleidaGraph. (D) Mean± s.e.m. EJP amplitude for atl2; arm>UAS-atl+, Rtnl11; arm>UAS-Rtnl1+, and their respective controls using the ubiquitous arm-Gal4 driver. P-values shown represent an unpaired Student’s t-test performed with KaleidaGraph. Recordings for C and D were performed at 0.6 mM Ca2+. (E) Mean±s.e.m. corrected quantal content for w1118, Rtnl11, atl2, and Rtnl11;atl2. In ascending [Ca2+], w1118 n=7, 17, 7, and 7; atl2 n=7, 23, 7, and 7; Rtnl11 n=7, 10, 7, and 7; Rtnl11;atl2 n=7, 7, 7, and 7. Resting membrane potentials (RMPs) were not significantly different among the four genotypes at bath [Ca2+] of 0.4 mM or 1.5 mM. At bath [Ca2+] of 0.6 mM or 1.0 mM, RMPs of wild-type and atl2 were significantly different (P=0.0266, wild-type more hyperpolarized, at 0.6 mM [Ca2+], and P=0.0316, atl2 more hyperpolarized, at 1.0 mM [Ca2+]). RMP of Rtnl11; atl2 was significantly different from other genotypes at a bath [Ca2+] of 1.0 mM, Rtnl11;atl2 more hyperpolarized. (F) Mean± s.e.m. corrected quantal content for elav>dcr, elav>dcr, atlRNAi, and elav>dcr, Rtnl1RNAi at 1.5 mM [Ca2+]. (G) Mean±s.e.m. mEJP amplitudes for w1118, atl2, Rtnl11, and Rtnl11;atl2, pooled from all four [Ca2+]. (H) Mean± s.e.m. mEJP amplitudes for the indicated genotypes collected at 1.5 mM bath [Ca2+]. P-values in D, F,G represent an unpaired Student’s t-test performed with KaleidaGraph. Recordings were performed in HL3 from muscle 6 in segment A6. Each scattergram data point is the average of the first 20 EJP responses (successes and failures) recorded for each larva.

+ 2 of Rtnl1 in the motor neuron, muscle and peripheral glia was atl increases BMP signaling in larval motor neurons sufficient to significantly restore proper EJP amplitude and BMPs are a family of secreted ligands that control a wide variety of synaptic success frequency to Rtnl11 (Fig. 7B). We conclude that organismal functions. Following binding to membrane receptors, Rtnl1 activity in the motor neuron, target muscle and BMPs trigger the phosphorylation and activation of the Mad neighboring peripheral glia is sufficient as well as necessary to transcription factor (Miyazono et al., 2010), which is assayed with maintain proper synaptic transmission. antibodies specific to the active phosphorylated (p)Mad. In addition Journal of Cell Science

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Fig. 6. Neuronal expression of UAS- RNAi K51A atl and UAS-atl decreases evoked neurotransmitter release. (A) Mean±s.e.m. EJP amplitude and corresponding percentage success for wild- type (w1118), atl2, UAS-atlRNAi and UAS- atlK51A. Drivers include the pan-neuronal elav-Gal4 and nSyb-Gal4 and the motor neuronal D42-Gal4. (B) Mean±s.e.m. EJP amplitude and percentage success for muscle (Mef2-Gal4) and glial (Gli-Gal4) expression of UAS-atlRNAi and UAS-atlK51A. Recordings for A and B were made in HL3.1 at 0.1 mM Ca2+ from muscle 6 in segment A6. Each scattergram data point is the average of the first 20 EJP responses (successes and failures) recorded for each larva. P-values shown represent an unpaired Student’s t-test performed with KaleidaGraph.

to developmental functions, Drosophila BMP ligands released from increased in atl2 NMJs, and that this increase is rescued by muscle muscle, peripheral glia or the motor neuron itself regulate but not neuronal atl+ expression. We found that pan-neuronal transmitter release and synaptic growth in motor neurons knockdown of either atl or Rtnl1 significantly increased bouton (Fuentes-Medel et al., 2012; McCabe et al., 2003). Many HSP number, and that this increase was suppressed when both genes proteins, including Drosophila Spichthyin and Spartin, mammalian were knocked down simultaneously (Fig. S2). These results suggest spastin and atlastin, inhibit BMP signaling (Fassier et al., that Atl and Rtnl1 are required within the motor neuron to restrain 2010; Nahm et al., 2013; Wang et al., 2007), possibly through axon outgrowth. altered BMP receptor trafficking. To determine whether Atl might similarly inhibit BMP signaling, we measured nuclear pMad within Neuronal knockdown of atl causes age-dependent 2 motor neuron nuclei of atl third-instar larvae. We observed a locomotor deficits significant increase in nuclear pMad in atl2, which was rescued by HSP patients share a characteristic increase in spasticity and both weak ubiquitous (Fig. 8C, green squares) or motor neuron- weakening of the lower extremities with age. Drosophila mutant for specific expression of atl+ (Fig. 8C, green diamonds). Thus, Atl any of several HSP orthologues similarly display behavioral deficits behaves similarly to other HSP proteins as an inhibitor of BMP (Lee et al., 2008; Nahm et al., 2013; Sherwood et al., 2004). We signaling. used two assays to determine whether pan-neuronal atl inhibition generated locomotor deficits. First, we measured the time required RNAi Neuronal loss of atl and Rtnl1 causes axon terminal for elav>Dcr, atl adults to climb 6 cm, and second, we used the overgrowth iFly tracking system (Kohlhoff et al., 2011) to measure walking Mutations in the Drosophila HSP orthologs described above velocity. We observed age-dependent locomotor deficits with each increase synaptic bouton number at the third-instar larval NMJ. In assay (Fig. S3). It is unlikely that these locomotor deficits result addition, Lee et al. (2009) have reported that bouton number is also directly from the deficits in larval synaptic transmission that we have Journal of Cell Science

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Fig. 7. Rtnl1 affects neurotransmitter release from multiple tissues at the NMJ. (A) Mean±s.e.m. EJP amplitude and corresponding percentage success for wild-type (w1118), Rtnl11, and UAS-Rtnl1RNAi expression in neurons, muscles and glia using elav-Gal4, Mef2-Gal4 and Gli-Gal4, respectively. (B) Mean±s.e.m. EJP amplitude and corresponding percentage success for w1118, Rtnl11 and expression of UAS-Rtnl1+ in Rtnl11 mutants. UAS-Rtnl1+ was expressed ubiquitously, in motor neurons and in muscles using da-Gal4, D42-Gal4 and Mef2-Gal4, respectively. OK371, Mef2 and Gli-Gal4 denote concurrent expression of UAS-Rtnl1+ from motor neurons, muscles and glia. Recordings for A and B were made in HL3.1 at 0.1 mM Ca2+ from muscle 6 in segment A6. Each scattergram data point is the average of the first 20 EJP responses (successes and failures) recorded for each larva. P-values shown represent an unpaired Student’s t-test performed with KaleidaGraph. documented. Rather, these locomotor deficits might reflect age- use Drosophila to evaluate the nervous system deficits caused by dependent degeneration of specific populations of neurons as has altered atl and Rtnl1 activity. Using a new fluorescent ER imaging been reported previously for an atl hypomorph (Lee et al., 2008). reagent, we show that the ER in wild-type motor nerve terminals is present as a network of tubules, termed ‘baskets’, underlying the DISCUSSION plasma membrane, and that these baskets are eliminated in larvae The role of ER morphology in nervous system function and lacking or overexpressing atl. We also show that loss of either atl or anatomy Rtnl1 increases arborization and decreases evoked transmitter Mutations in two genes that affect ER morphology, atlastin (ATL1) release, and that evoked release is restored to normal by elevated and reticulon 2 (RTN2), cause two forms of hereditary spastic bath [Ca2+]. Finally, we show that atl loss increases signaling paraplegia (HSP), which result in progressive limb weakness, through the bone morphogenetic protein (BMP) pathway and causes spasticity and degeneration of the longest motor axons (Blackstone age-dependent declines in adult locomotion. This set of phenotypes et al., 2010). These observations suggest that altered ER is also exhibited by Drosophila mutant for the HSP orthologs of morphology is causal for motor axon dysfunction, but the spartin, spastin and spichthyin, as well as for spinster and nervous mechanisms underlying these dysfunctions are unclear. Here, we wreck, which encode regulators of receptor trafficking through Journal of Cell Science

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2 Fig. 8. atl increases BMP signaling in motor neurons. Representative confocal images of w1118 (A) and atl2 (B) motor neuron nuclei from third-instar larvae labeled with anti-pMad . Scale bar: 10 µm. (C) Mean±s.e.m. pMad levels for w1118, atl2, atl2; arm>+, atl2; arm>atl+, atl2; OK6>+, and atl2; OK6>atl+ in motor neuron nuclei. Each scattergram data point is the average nuclear signal intensity per area for a single larva. P-values shown represent an unpaired Student’s t-test performed with KaleidaGraph. endosomes (Nahm et al., 2013; O’Connor-Giles et al., 2008; Given the role of atlastin and the as ER-shaping Ozdowski et al., 2011; Sherwood et al., 2004; Sweeney and Davis, molecules, effects on ER Ca2+ release would be the most direct 2002; Wang et al., 2007). The possible involvement of the ER in this explanation for this Ca2+ phenotype. ER-localized Ca2+ release receptor trafficking pathway will be discussed. channels such as the inositol 1,4,5-trisphosphate (IP3) receptor, the ryanodine receptor and the TRPV1 channel play key roles in evoked Effects of atl loss or overexpression on ER morphology in neurotransmitter release (Emptage et al., 2001; Liang et al., 2002; motor neurons Llano et al., 2000; Wong et al., 2014). In addition, dominant- We made two adjustments to improve visualization of the ER. First, negative mutations in the Drosophila ER-localized Ca2+ pump we introduced into flies a transgene that encoded an ER-localized SERCA decrease evoked transmitter release by ∼50% (Sanyal et al., superfolder GFP, which was optimized for efficient folding in the 2005), which is consistent with the possibility that ER-derived Ca2+ ER. Second, based on previous results indicating that the ER as well contributes significantly to the Ca2+ required to trigger transmitter as the lysosomal tubule network is labile to fixation (Johnson et al., release. 2015), we imaged ER in live tissues. Using these approaches, we showed that the ER is present within the axon initial segments of Rtnl1 affects evoked neurotransmitter release from multiple motor neurons as a polygonal structure with numerous crossbridges tissues (three-way junctions), and in motor nerve terminals as a network of Unlike Atl, which appears to affect evoked transmitter release from tubules that we term ‘baskets’, which underlie the plasma neurons alone, Rtnl1 is required in neurons, muscles and peripheral membrane. We also showed that these structures are disrupted glia for correct evoked transmitter release. This finding is consistent by either loss of or overexpression of atl. In particular, atl with previous data demonstrating that proper synaptic transmission overexpression causes the aberrant appearance of large punctae in requires intercellular signaling among these three cell types. In motor neuron cell bodies or axon initial segments. In contrast, atl particular, loss of activity within the peripheral glia of the kinesin loss decreases the number of crossbridges in the axon initial heavy chain or the inebriated-encoded neurotransmitter segment, leading to excessively long tubules. A similar appearance transporter alters evoked transmitter release (Huang and Stern, was noted previously (Hu et al., 2009; Orso et al., 2009) and 2002; Schmidt et al., 2012). In addition, the peripheral glia secrete at attributed to deficits in fusion of orthogonal ER membranes. Loss of least two proteins, the TGF-β ligand Maverick and Wingless/Wnt, atl also disrupts nerve terminal baskets and appears to cause ER that regulate synaptic function (Fuentes-Medel et al., 2012; Kerr fragmentation. It is possible that the transition from tubules to et al., 2014). The muscle, in turn, secretes the BMP ligand Gbb to baskets as the ER moves from the interbouton region to boutons regulate both evoked transmitter release and motor neuron occurs through ER fragmentation followed by Atl-dependent arborization (McCabe et al., 2003). It is possible that loss of Rtnl1 reassembly. In this view, loss of atl would prevent this affects transmitter release from glia or muscle by perturbing reassembly, thus causing the fragmented ER that we observe. secretion of these or other regulators.

Deficits in evoked transmitter release in larvae lacking atl or Pan-neuronal atl knockdown causes progressive adult Rtnl1 are rescued by elevated bath [Ca2+] locomotor deficits during aging Evoked transmitter release deficits in both atl2 and Rtnl11 mutants, The most prominent clinical symptom in HSP patients is and in pan-neuronal atl or Rtnl1 knockdown larvae, were rescued progressive, age-dependent locomotor difficulties. Drosophila partially or completely by elevated bath [Ca2+]. These results mutant for any of several HSP orthologs, including spartin, indicate that loss of atl or Rtnl1 decreases evoked transmitter release spastin, atl and Rtnl1, as well as spinster, exhibit similar age- at low bath [Ca2+] through causing deficits in evoked increases in dependent locomotor deficits or lifespan deficits (Dermaut et al., cytoplasmic [Ca2+]. Insufficient Ca2+ influx could result from 2005; Lee et al., 2008; Nahm et al., 2013; O’Sullivan et al., 2012; attenuated action potentials, which would decrease the opening of Orso et al., 2009; Sweeney and Davis, 2002). Here, we show voltage-gated Ca2+ channels, decreases in number of plasma locomotor impairment in adults with neuronal-specific atl 2+ 2+ membrane Ca channels or decreased Ca release from the ER. knockdown. These results indicate a requirement for atl in Journal of Cell Science

1644 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 1635-1648 doi:10.1242/jcs.184929 neurons for proper locomotion but do not rule out crucial roles for driver line was provided by the Drosophila Genetic Resource Center. UAS- RNAi atl in other tissues as well. Rtnl1 (#7866) was obtained from the Vienna Drosophila Resource Center. OK6-Gal4 (Aberle et al., 2002) was provided by Hermann Aberle (Heinrich Heine University, Düsseldorf, Germany). D42-Gal4 (Yeh et al., A potential role for the ER in endocytic receptor trafficking 1995) was provided by Thomas Schwarz (Children’s Hospital Boston, F. M. Drosophila Mutants in orthologs of several HSP genes, including Kirby Neurobiology Center, Boston, MA). Gli-Gal4 (Sepp and Auld, 1999) spartin, spastin and spichthyin, and the additional related genes was provided by Vanessa Auld (The University of British Columbia, spinster and nervous wreck share a common set of nervous system Department of Zoology, Vancouver, British Columbia, Canada). UAS-atl+, phenotypes, including increased arborization and BMP signaling at UAS-atlK51A, and UAS-atlRNAi were as described previously (Orso et al., 2 1 the larval NMJ, decreased evoked transmitter release and locomotor 2009). atl (Lee et al., 2009) and Rtnl1 (Wakefield and Tear, 2006) were as + deficits (Nahm et al., 2013; O’Connor-Giles et al., 2008; Ozdowski described previously. UAS-Rtnl1 was provided by Andrea Daga. et al., 2011; Sherwood et al., 2004; Sweeney and Davis, 2002; All fly stocks were maintained on cornmeal and agar medium (6% Wang et al., 2007) (note that not all phenotypes have been reported dextrose, 6.8% cornmeal, 1.2% , 0.72% agar, 2% methyl 4- hydroxybenzoate) at room temperature (∼23°C) or 25°C. for each mutant). The encoded proteins localize to various compartments within the endocytic receptor trafficking pathway (Allison et al., 2013; Edwards et al., 2009; O’Connor-Giles et al., Plasmid construction To construct pJM952 (pAc5/BiP-sfGFP-HDEL), Superfolder GFP (sfGFP) 2008; Sweeney and Davis, 2002; Wang et al., 2007). In fact, the in pEGFP-N1 (Aronson et al., 2011)] was provided by Erik Snapp (Albert increased BMP signaling in several of these mutants has been Einstein College of Medicine, Department of Anatomy & Structural attributed to trafficking defects of the BMP receptor Wishful Biology, Bronx, NY). The Drosophila BiP signal sequence thinking (Wit). We have shown that atl loss confers these same (MKLCILLAVVAFVGLSLG-RS) was then fused to sfGFP with a C- phenotypes, raising the possibility that atl acts in the endocytic terminal ER retention signal (-HDEL) from Drosophila Calreticulin (Smith, receptor trafficking pathway as well. Although the ER is not known 1992) in pAc5.1/V5-His A (Invitrogen). To construct pJM1033 (UAS-BiP- to play prominent roles in this pathway, a recent report has sfGFP-HDEL), pUASTattB (Bischof et al., 2007) was provided by Konrad demonstrated that the ER is required for endosome fission in COS Basler (University of Zürich, Institute of Molecular Life Sciences, Zürich, cells, and, in fact, the ER selects the location of fission (Rowland Switzerland). The BiP-sfGFP-HDEL cassette from pJM952 was then moved to pUASTattB. To construct pJM1072 (UAS-tdTomato-dSec61β), et al., 2014). In addition, it has been found that this process is Drosophila β Drosophila Rtn4a atl tdTomato was fused to Sec61 ( Genomics Resource inhibited by overexpression of , which, similarly to loss, Center cDNA clone #RE18615) in a 20X-UAS-IVS vector derived from elongates ER tubules and inhibits formation of crossbridges pJFRC7 (Addgene #26220). (Rowland et al., 2014). Thus, loss of atl could impact on the receptor trafficking pathway in Drosophila nerve terminals by Drosophila stock construction similarly preventing endosome fission. pJM1033 and pJM1072 were injected into attP2 and VK37 embryos by The variety of phenotypes exhibited in common by the mutants Genetivision (Houston, TX). Injected flies were backcrossed twice to w1118 described above raises the possibility that certain phenotypes might flies and stocks carrying insertions on two (VK37) and three have causal relationships with others. The subcellular locations of (attP2) were established. The UAS-Rtnl1 line was produced with the Rtnl1- these proteins suggest that they might directly affect receptor PB isoform cDNA (LD14068). The cDNA was subcloned in-frame with an trafficking. If so, then the increased BMP signaling, as a HA tag in the pUAST vector, and transgenic lines were generated by consequence of altered Wit trafficking, might be the direct cause of standard microinjection. the increased arborization and locomotor deficits. The phenotypes conferred by direct activation of the BMP pathway in neurons are Live larval imaging consistent with this possibility (McCabe et al., 2003; Nahm et al., Wandering third-instar larvae, reared at 25°C, were dissected in HL6 buffer 2013). However, the increased BMP signaling is unlikely to cause the (Macleod et al., 2002), a complex buffer designed for imaging metabolically decreased transmitter release, as decreased BMP signaling, rather active fly larvae, with 0 mM CaCl2 and 7 mM monosodium glutamate on 35-mm petri dish lids completely filled with Sylgard 184 (Electron than increased BMP signaling, decreases evoked transmitter release Microscopy Sciences). Minutien pins (0.1-mm diameter, Fine Science (McCabe et al., 2003). We suggest that trafficking of receptors in Tools), bent to 90° and trimmed, were used to secure larvae to the Sylgard. addition to Wit are altered in the mutants described above, and it is the Round 25-mm coverslips (#1 thickness) were cut in half with a diamond altered signaling of these additional receptors that is at least partly knife and attached to the Sylgard on either side of dissected larvae to form a responsible for the transmitter release phenotype. Drosophila motor channel. Square 22-mm coverslips (#1.5 thickness) were placed over the nerve terminals express a cholecystokinin-like receptor (CCKLR), a larvae and secured to the bottom coverslips with nail polish. Larvae were toll-like receptor, a metabotropic glutamate receptor (mGluRA) and imaged on a Zeiss LSM 710 inverted confocal microscope using a Plan- likely the insulin receptor (Ballard et al., 2014; Bogdanik et al., 2004; Apochromat 63× (1.40 NA) oil immersion objective. GFP was excited with Chen and Ganetzky, 2012; Howlett et al., 2008). Loss of mGluRA a 488-nm argon laser and emitted light between 493 and 522 nm was increases evoked transmitter release (Bogdanik et al., 2004; Howlett collected. tdTomato was excited with a 543-nm helium neon laser and emitted light between 552 and 691 nm was collected. Images were adjusted et al., 2008), raising the possibility that increased mGluRA signaling for brightness and contrast in ImageJ. Regions of interest (ROIs) were might decrease transmitter release, which could explain the decreased manually drawn around boutons in ImageJ. ROI pixel intensities were transmitter release observed in these receptor trafficking mutants. extracted and processed with a custom Python script (available upon request). Pixel intensities were scaled by dividing each value by the mean MATERIALS AND METHODS pixel intensity. These data were binned into 50 equally spaced bins ranging Drosophila stocks and medium from 0 to 8 to generate a frequency histogram. The median and mode of pixel The following lines were obtained from the Bloomington Drosophila Stock intensities were calculated for each image and graphed with KaleidaGraph. Center at Indiana University: w1118 (#3605), arm-Gal4 (#1560), elav-Gal4 (#458), elav-Gal4; UAS-Dcr-2 (#25750), nSyb-Gal4 (#51635), OK371- Fixed larval imaging Gal4 (#26160), Mef2-Gal4 (#27390), UAS-Dcr-2 (#24650), UAS-Dcr-2 Wandering third-instar larvae were reared and dissected as described for live

(#24651), and UAS-myr::tdTomato (#32221). The da G32 (#108252) Gal4 imaging. Larvae were then fixed with 4% formaldehyde in HL6 buffer for Journal of Cell Science

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5 min, washed with HL6 and mounted in Vectashield (Vector Labs). Slides trials. If a fly failed to cross the 6-cm mark in 60 s, the time was recorded as were imaged as described for live larvae. 60 s. The minimum time for each fly was compiled and averaged as the time for that day. Results represent a minimum of ten flies. S2 cell imaging pJM952 was transfected into S2R+ cells (Drosophila Genomics Resource Velocity tests Center) using Fugene HD (Promega) according to the manufacturer’s Adult flies that were 0–2 days old were reared at 25°C and anesthetized with protocols. Cells were adhered to Concanavalin-A-coated glass-bottom carbon dioxide. Groups of 6–10 males were aged until 3, 10, 15, 25 and dishes (Mat-tek) overnight. Fixed cells were exposed to 4% formaldehyde in 30 days (±1 day). Flies were transferred, without anesthesia, into vials with HL3 for 5 min and washed with PBS. Cells were imaged on a Nikon A1-Rsi 10 ml of 1% agar and allowed to rest for 10 min. Flies were placed in a laser scanning confocal microscope using a CFI Plan Apo VC 60× (1.4 NA) white, translucent Plexiglas chamber with a digital video camera focused on oil immersion objective. GFP was excited with a 488-nm argon laser and the vial. Two mirrors placed behind the vial at 40° angles produce two emitted light between 500 and 550 nm was collected. reflections visible in the video. Vials were banged to knock flies to the agar surface and climbing-behavior videos were captured. Three trials of 30 s Electrophysiology each were recorded in succession for each group. Average velocity for each Wandering third-instar larvae were dissected in HL3 (Stewart et al., 1994) vial was measured using the iFly system (Jahn et al., 2011; Kohlhoff et al., for data collected at 0.4 mM, 0.6 mM, 1.0 mM and 1.5 mM [Ca2+], or 2011). HL3.1 (Feng et al., 2004) for data collected at 0.1 mM [Ca2+]. Recordings were performed as described previously (Howlett et al., 2008). Evoked Acknowledgements The authors would like to thank various members of the Drosophila community as response traces for larvae were recorded for at least 20 s. Quiescent traces of well as the Bloomington Stock Center for providing fly lines. Damian Crowther and 60 s were recorded for mEJP frequency and amplitude analysis. EJP and Eleonora Khabirova provided software and assistance with the iFly system. Enrique mEJP analysis was accomplished using Synaptosoft Mini Analysis Martinez assisted with the S2 imaging, Justin Lee assisted in determining EJP Program. Frequency and amplitude of mEJPs were analyzed using the amplitudes with Synaptosoft, Zach Wright assisted with imaging active zone ‘non-stop analysis’ function. For this analysis, the mEJP amplitude proteins, Miguel Betancourt-Solis assisted with the sfGFP constructions and Justin threshold was set at 0.7 mV with a LoPass Butterworth filter and a cut-off Vincent assisted with analysis of synaptic bouton number. frequency of 200 Hz. EJP amplitude was determined by manually selecting the peaks, which corresponded to the first 20 stimulations within a trace. Competing interests Failures within the first 20 stimulations of a trace were denoted as zeroes and The authors declare no competing or financial interests. contributed toward average EJP data. No threshold minimum was employed −1 Author contributions for EJPs. Corrected quantal content was calculated using m=ν/ν1(1-ν/V0) , where ν=EJP amplitude, ν =mEJP amplitude and V =resting potential J.A.M., M.S., J.B.S. and J.E.F. designed the experiments. M.S., J.B.S. and J.E.F. 1 0 generated reagents, D.P. constructed the UAS-Rtnl1 line while in the lab of A.D., minus a correction factor of 15 mV (Martin, 1955). J.B.S. and J.E.F. performed the experiments. J.B.S., J.E.F., E.F. and J.F. analyzed data. J.A.M., M.S., J.B.S. and J.F. wrote the manuscript. pMad quantification Wandering third-instar larvae, reared at room temperature, were dissected in Funding S2 medium (Invitrogen) and fixed immediately (4% formaldehyde in S2 E.F. was supported by a George J. Schroepfer, Jr. Undergraduate Summer medium) for 15 min. Larvae were washed with PBS-T (PBS plus 0.1% Fellowship; M.S. was supported by a grant from the Hamill Foundation; J.A.M. is Triton X-100) and transferred to Sylgard-coated 24-well plates. Larvae were supported by the National Institutes of Health [grant number GM101377]; the incubated with anti-p-Smad1/5 (Ser463/465) rabbit monoclonal antibody Simmons Family Foundation; and the Hamill Foundation. Deposited in PMC for release after 12 months. (Cell Signaling, 41D10, 1:50 in PBS-T) overnight (4°C) with gentle orbital shaking, washed with PBS-T, incubated with goat Alexa-Fluor-488- Supplementary information conjugated anti-rabbit IgG antibody (Invitrogen, A-11008, 1:1000 in Supplementary information available online at PBS-T) for 90 min at room temperature, washed with PBS, and mounted in http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.184929/-/DC1 vectashield. For each experiment, at least three w1118 larvae were included, and all larvae were exposed to the same antibody dilution. Ventral ganglia References from at least eight larvae from two independent experiments were analyzed. Aberle, H., Haghighi, A. P., Fetter, R. D., McCabe, B. D., Magalhaes,̃ T. R. and Larvae were imaged as described for live larvae. ROIs were drawn around Goodman, C. S. (2002). wishful thinking encodes a BMP type II receptor that motoneuronal nuclei in the ventral ganglia using ImageJ. Mean pixel regulates synaptic growth in Drosophila. Neuron 33, 545-558. intensity was divided by area. ROIs were summed and averaged for each Allison, R., Lumb, J. H., Fassier, C., Connell, J. W., Ten Martin, D., Seaman, larva, and normalized to the w1118 internal control. M. N. J., Hazan, J. and Reid, E. (2013). An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome. J. Cell Biol. 202, 527-543. Aronson, D. E., Costantini, L. M. and Snapp, E. L. (2011). Superfolder GFP is Arborization fluorescent in oxidizing environments when targeted via the Sec translocon. Wandering third-instar larvae reared at 25°C were dissected in HL3, Traffic 12, 543-548. fixed in 4% formaldehyde (in HL3) and washed with PBS-T. Pelts Bakowska, J. C., Jupille, H., Fatheddin, P., Puertollano, R. and Blackstone, C. were placed in sylgard-coated 24-well plates and exposed to goat Alexa- (2007). Troyer syndrome protein spartin is mono-ubiquitinated and functions in Fluor-488-conjugated anti-horseradish-peroxidase antibody (Jackson EGF receptor trafficking. Mol. Biol. Cell 18, 1683-1692. ImmunoResearch, 123-545-021, 1:500) overnight at 4°C with gentle Ballard, S. L., Miller, D. L. and Ganetzky, B. (2014). Retrograde neurotrophin orbital shaking, washed in PBS, and mounted in Vectashield. Boutons at signaling through Tollo regulates synaptic growth in Drosophila. J. Cell Biol. 204, 1157-1172. muscles 6 and 7 in segment A3 were imaged as described for live larvae. Bischof, J., Maeda, R. K., Hediger, M., Karch, F. and Basler, K. (2007). An Boutons were counted in ImageJ. optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc. Natl. Acad. Sci. USA 104, 3312-3317. Climb tests Blackstone, C., O’Kane, C. J. and Reid, E. (2010). Hereditary spastic paraplegias: Crosses were made with ten mating pairs in half-pint bottles at 25°C. membrane traffic and the motor pathway. Nat. Rev. Neurosci. 1, 31-42. Bogdanik, L., Mohrmann, R., Ramaekers, A., Bockaert, J., Grau, Y., Broadie, K. Progeny were collected 2 days after eclosion, and flies were aged for 0 and – and Parmentier, M.-L. (2004). The Drosophila metabotropic glutamate receptor 2 days post eclosion. Males were placed into vials in groups of 10 12 flies DmGluRA regulates activity-dependent synaptic facilitation and fine synaptic and aged at 25°C. Adult male flies were separated into individual empty morphology. J. Neurosci. 24, 9105-9116. vials, tapped to the bottom and timed to see how long it took them to climb 6 Chen, X. and Ganetzky, B. (2012). A neuropeptide signaling pathway regulates cm vertically. Flies experienced three time trials with 5 min of rest between synaptic growth in Drosophila. J. Cell Biol. 196, 529-543. Journal of Cell Science

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Costantini, L. M., Baloban, M., Markwardt, M. L., Rizzo, M., Guo, F., Verkhusha, Liang, Y., Yuan, L. L., Johnston, D. and Gray, R. (2002). Calcium signaling at V. V. and Snapp, E. L. (2015). A palette of fluorescent proteins optimized for single mossy fiber presynaptic terminals in the rat hippocampus. J. Neurophysiol. diverse cellular environments. Nat. Commun. 6, 7670. 87, 1132-1137. Dermaut, B., Norga, K. K., Kania, A., Verstreken, P., Pan, H., Zhou, Y., Callaerts, Llano, I., González, J., Caputo, C., Lai, F. A., Blayney, L. M., Tan, Y. P. and Marty, P. and Bellen, H. J. (2005). Aberrant lysosomal carbohydrate storage A. (2000). Presynaptic calcium stores underlie large-amplitude miniature IPSCs accompanies endocytic defects and neurodegeneration in Drosophila and spontaneous calcium transients. Nat. Neurosci. 3, 1256-1265. benchwarmer. J. Cell Biol. 170, 127-139. Lo Giudice, T., Lombardi, F., Santorelli, F. M., Kawarai, T. and Orlacchio, A. 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