SUMMARY
Autophagy is spatially compartmentalized in neurons, with autophagosome biogenesis occurring in the axon and degradation in the cell body. The mechanisms that coordinate autophagosome formation, trafficking and degradation across the polarized structure of the neuron are not well understood. Here we use genetic screens and in vivo imaging in single neurons of C. elegans to demonstrate that specific steps of autophagy are differentially required in distinct subcellular compartments of the neuron. We demonstrate that completion of autophagosome biogenesis and closure at the synapse are necessary for dynein-mediated retrograde transport. We uncover a role for UNC-16/JIP3/Sunday Driver in facilitating autophagosome retrograde transport. Through forward genetic screens we then determine that autophagosome maturation and degradation in the cell body depend on removal of LGG-1/Atg8/GABARAP from autophagosomes by the protease ATG-4.2. Our studies reveal that regulation of distinct ATG4 proteases contributes to the coordination of autophagy across subcellular regions of the neuron.
HIGHLIGHTS and eTOC Blurb
Autophagosome closure, but not maturation, occurs locally at presynaptic sites
Retrograde transport of autophagosomes requires the motor adaptor UNC-16/JIP3
The autophagy protease ATG-4.2, but not the related ATG-4.1, is required for autophagosome maturation and degradation
Defects in retrograde transport and maturation genetically interact and enhance accumulation of autophagosomes in presynaptic regions
INTRODUCTION
Macroautophagy (also termed autophagy) is a cellular degradation process capable of removing bulk cytoplasm or organelles from cells. Autophagy is well conserved from yeast to mammalian cells, and is best known for its roles in cellular homeostasis (Yin et al., 2016; Zhang and Baehrecke, 2015). Neurons are especially vulnerable to defects in autophagy (Liang and Sigrist, 2017; Son et al., 2012; Vijayan and Verstreken, 2017), as they are post-mitotic cells incapable of diluting defective proteins through repeated cell divisions. Disruption of autophagy is associated with neuronal dysfunction, neurodegeneration and disease (Nah et al., 2015; Nixon, 2013; Wong and Cuervo, 2010).
Neurons are highly polarized cells in which autophagy is spatially compartmentalized. In primary neurons, autophagosomes preferentially form at the distal end of the axon, indicating compartmentalization of autophagosome biogenesis in neurites (Maday et al., 2012). Local formation of autophagosomes is also observed at synapses (Maday and Holzbaur, 2014; Soukup et al., 2016; Stavoe et al., 2016). Synaptic autophagy can be enhanced under conditions of prolonged neuronal activity, suggesting a physiological link between neuronal activity and local autophagosome biogenesis (Shehata et al., 2012; Soukup et al., 2016; Wang et al., 2015). In Drosophila neuromuscular junctions, autophagosome biogenesis occurs at presynaptic sites even when motor nerves are severed from the cell bodies, indicating local production of autophagosomes (Soukup et al., 2016). Studies in cultured neurons reveal that local autophagosome biogenesis requires the ordered recruitment of assembly factors to the distal axon (Maday and Holzbaur, 2014; Maday et al., 2012). Similarly, in intact neurons of C. elegans, where autophagosomes are observed to form near presynaptic sites, biogenesis depends on the local transport of transmembrane protein ATG-9 to synapses (Stavoe et al., 2016). Together, these studies indicate that autophagy can be locally regulated to result in compartmentalized autophagosome biogenesis in neurons.
Autophagosomes are degraded upon fusion with late endosomes or lysosomes, a process which involves the small GTPase Rab-7 and SNARE machinery (Gutierrez et al., 2004; Hyttinen et al., 2013; Itakura et al., 2012; Jager et al., 2004). In neurons, where lysosomes are preferentially enriched in the cellular soma, autophagosomes in the distal axon are transported towards the soma for degradation (Hollenbeck, 1993; Kaasinen et al., 2008). Retrograde transport of autophagosomes is tightly controlled through both the recruitment and regulation of the dynein motor complex (Ikenaka et al., 2013; Katsumata et al., 2010; Maday et al., 2012). Dynein is recruited to autophagosomes through fusion of autophagosomes with late endosomes (Cheng et al., 2015), and robust retrograde transport is facilitated by the motor scaffolding protein JIP1 (Fu et al., 2014). The importance of these regulated processes in neuronal autophagy is perhaps best exemplified by the consequences of their disruption, including autophagosome accumulation at presynaptic terminals, Alzheimer’s disease-like autophagic stress and axonal pathology (Ikenaka et al., 2013; Lee et al., 2011; Nixon et al., 2005; Takats et al., 2013; Tammineni et al., 2017).
While it is known that autophagy is spatially compartmentalized in neurons, and that regulation of this compartmentalization is important for neuronal physiology, less is known about how the stepwise progression of autophagy maps to the cell biology of the neuron, particularly in vivo. Where are the different enzymes of this multi-step process required in the context of the polarized neuron? What mechanisms regulate integration of the distinct steps of autophagy across neuronal regions?
In this study we use forward and reverse genetics, combined with in vivo imaging, to systematically dissect the cell biology of autophagy in the neuron. We visualize autophagosomes at single-neuron resolution by examining the localization of the autophagosome-associated protein LGG-1/Atg8/GABARAP (Manil-Segalen et al., 2014; Melendez et al., 2003; Stavoe et al., 2016; Zhang et al., 2015). We find that genes required for autophagosome closure, atg-2 and epg-6/WIPI3/4, are also required for autophagosomes to leave the synapse, but that the autophagosome maturation/acidification gene epg-5 is not. We identify a new role for the motor adaptor protein, UNC-16/JIP3, in facilitating robust retrograde transport of autophagosomes. To then uncover mechanisms that coordinate transport and clearance of autophagosomes from the neurite, we perform an enhancer screen in unc-16/jip3 mutant animals and identify a novel dominant enhancer mutation in the poorly understood autophagy gene atg-4.2. Mutations in atg-4.2;unc-16 double mutants display dramatic accumulation of autophagosomes in the neurite, while atg-4.2 single mutants accumulate autophagosomes in the cell body, phenotypes not observed for the other atg-4 gene in C. elegans, atg-4.1. We observe that accumulated autophagosomes in atg-4.2 mutants fail to mature and degrade, supporting a model in which the cysteine protease atg-4.2 is specialized to remove LGG-1 from autophagosomes and enable their fusion with lysosomes and degradation. Our studies uncover novel mechanisms that regulate transport and clearance of autophagosomes in neurites and provide a framework to understand how these molecular pathways are coordinated across sites in the neuron in vivo.
RESULTS
Autophagosomes at C. elegans synapses are transported towards and acidified near the cell body
To examine the cell biology of autophagy in neurons of living animals, we imaged LGG-1 in the AIY interneurons of C. elegans (schematic, Figure 1A and as previously described in (Stavoe et al., 2016)). LGG-1, a homologue of Atg8 (in yeast) and GABARAP (in mammals), associates with immature and mature autophagosomal structures and is a marker used to track autophagosomes in living cells ((Alberti et al., 2010; Manil-Segalen et al., 2014; Melendez et al., 2003) for validation and controls of the autophagosome marker in AIY interneurons, please see (Stavoe et al., 2016) and Experimental Procedures). Through cell-specific expression in AIY interneurons we visualized autophagosome biogenesis and transport in intact cells and in vivo under physiological conditions.
Autophagosomes in AIY are present in the neurite (Figures 1B-1D) and in the cell body (Figure 1E; (Stavoe et al., 2016)). In the neurite, the majority of autophagosome biogenesis events occur near synaptic regions. We observe that in living animals the number of synaptic autophagosomes in AIY predictably varies depending on the firing state of the neuron, which we can manipulate by altering physiological stimuli that promote AIY responses (Figures S1A-S1C, and S1F), by genetically inhibiting synaptic transmission (Figures S1D, S1E, and S1G) or by chemo-genetically altering the response state of the neuron (Figure S1H). Our findings are consistent with and extend observations that the state of neuronal activity is linked to autophagosome formation at the synapse (Shehata et al., 2012; Soukup et al., 2016; Wang et al., 2015).
Autophagosomes in the distal axon of cultured cells are transported, acidified through fusion with late endosomes or lysosomes and cleared through degradation (Cheng et al., 2015; Maday et al., 2012). Impairment of autophagosome degradation in vertebrate neurons results in accumulation of toxic protein aggregates, axon pathology and neurodegenerative disease (Tammineni et al., 2017). The acidification and degradation of synaptic autophagosomes has not been documented in vivo. To better understand the mechanistic regulation of biogenesis, maturation, and degradation of synaptic autophagosomes, we imaged the acidification state of autophagosomes in AIY neurites by employing a tandem label strategy (Chang et al., 2017; Kimura et al., 2007) in which LGG-1 is fused to both mCherry and GFP (Figure 1F). Immature autophagosomes are labeled with both GFP and mCherry, but because GFP is preferentially quenched in an acidic environment, mature structures lose their GFP signal and display solely mCherry signal (schematic, Figure 1G). Using this approach in vivo and in single neurons, we observed that 100% (n=14) of synaptic LGG-1 labeled structures retained GFP signal, suggesting that autophagosomes at synapses are not yet mature. In contrast, examination of LGG-1 labeled structures in the asynaptic region (proximal to the cell body) or in the cell body of the neuron demonstrated that over 50% of the structures are positive for mCherry, but not for GFP (57% in asynaptic zone 1 (n=7 puncta); 54% in the cell body (n=245 puncta); from 31 neurons; Figures 1F-1N). Lysosomes promote acidification of autophagosomes, and consistent with synaptic autophagosomes being transported towards the cell body for acidification and degradation, we observe lysosomes preferentially localize to the cell body in AIY (Figures 1O and 1P).
Our findings indicate that while autophagosome biogenesis occurs at the synapse, mature and acidified autolysosomes are preferentially present at or near the cell body. Our in vivo observations for synaptic autophagosomes are consistent with studies in cultured vertebrate neurons that demonstrate that fusion of axonal autophagosomes with late endosomes/lysosomes promote retrograde transport, and with studies that demonstrate that autophagosomes are progressively more acidic closer to the cell body (Cheng et al., 2015; Maday et al., 2012). Importantly our in vivo system provides a platform to understand the mechanisms that regulate the integration of the distinct steps of autophagy across the neuronal cell.
Autophagosome closure, but not maturation, is necessary for retrograde transport of synaptic autophagosomes
To map where within the neuron the distinct steps of autophagy are required, we recorded and tracked individual synaptic autophagosomes in wild type and autophagy mutant animals. We found that for wild type animals, autophagosome biogenesis events in the synaptic region average 12 minutes in duration from the first frame of detection until trafficking (with a range of 6 to 23 minutes, n=7). Moreover, all observed LGG-1-containing synaptic structures present at the start of imaging left the synaptic region within 30 minutes (n=12) (Figure 2F). Our findings indicate that following biogenesis at the synapse, autophagosomes are efficiently engaged in retrograde transport towards the cell body.
We then examined if later steps of autophagy, such as autophagosome maturation, were necessary for retrograde transport. ATG-2 and EPG-6/WIP3/4 are necessary for autophagosome closure (Lu et al., 2011; Velikkakath et al., 2012), while EPG-5 is required for autophagosome maturation (Wang et al., 2016; Zhao et al., 2013) (schematic in Figure 2A). We observe that, unlike wild type animals, 76% of LGG-1-containing structures in atg-2 mutants (n=37) and 92% in epg-6 mutants (n=39) remained at the synapse after 30 minutes (Figures 2C, 2D, 2F and Supplemental Movies S1-S3), suggesting that completion of autophagosome closure is necessary for initiation of retrograde transport. However, epg-5 mutant animals, in which autophagosomes are able to close but not be acidified (Zhao et al., 2013), phenocopied wild type animals (n=18) (Figures 2E, 2F and Supplemental Movie S4). Our findings indicate that while autophagosome closure is necessary for efficient retrograde transport of synaptic autophagosomes, autophagosome maturation is not. Our genetic findings are consistent with our cell biological observations that maturation of autophagosomes occurs near the neuronal cell body, and support the model that different steps of autophagy occur at distinct subcellular compartments in neurons. Our findings also extend our understanding of the differential requirement of these late steps of autophagy in the context of the cell biology of the neuron, and link regulated closure of autophagosomes with retrograde transport, maturation and removal.
Dynactin and JIP3/UNC-16 promote autophagosome retrograde transport and retention in the cell body
To investigate the removal of synaptic autophagosomes, we first examined the molecular mechanisms underlying the trafficking of autophagosomes from the synapse in vivo. In C. elegans, as in vertebrate neurons, retrograde transport of autophagosomes depends on the dynein complex (Cheng et al., 2015; Ikenaka et al., 2013; Katsumata et al., 2010; Maday et al., 2012) (Figures 3A-3C). We therefore examined known regulators of transport to investigate their specific requirement in autophagosome trafficking.
We observed that while jip-1/Mapk8Ip1, unc-116/kinesin-1 and unc-14/RUN mutants did not display defects in autophagosome accumulation at synapses (Figure 3G), loss of JIP3/UNC-16/Sunday Driver resulted in a significant accumulation of autophagosomes in the neurite, but not in the cell body (Figures 3E-3G, and 6G). The unc-16/jip3 mutant phenotype was similar to the phenotype seen for dnc-1/p150 dynactin complex subunit mutants (which affect the dynein complex) ((Ikenaka et al., 2013) and Figures 3B-3C). JIP3/UNC-16/Sunday Driver is a conserved adaptor protein that binds to kinesin and dynein (Arimoto et al., 2011; Cavalli et al., 2005) to regulate early endosome and lysosome transport (Brown et al., 2009; Byrd et al., 2001; Drerup and Nechiporuk, 2013; Edwards et al., 2015; Edwards et al., 2013; Gowrishankar et al., 2017). Three independent alleles of unc-16 (ju146, e109, and n730) (Figure 3D) result in an increase from 34% (in wild type) to 100% (in unc-16 alleles) of animals with autophagosomes in the neurites. Moreover, while wild type animals average less than one autophagosome per neurite, unc-16 mutants average ∼2 autophagosomes in the presynaptic regions (Figure 3G), indicating abnormal accumulation of autophagosomes. Analyses of the subcellular localization of LGG-1-containing structures in unc-16/jip3 mutants also revealed a higher probability for autophagosomes to be present in the distal synaptic Zone 3 region (Figures 3H and 3I). The distal accumulation of autophagosomes observed for unc-16 mutants was not due to a rearrangement of microtubule polarity, as we observed that the microtubule plus-end-binding protein EBP-2/EB1 (Baas and Lin, 2011; Maniar et al., 2011) is similarly oriented (plus-end-out) in wild type and unc-16/jip3 mutant animals (Figure S2). Our findings indicate that unc-16/jip3 is necessary to prevent the abnormal accumulation of autophagosomes in the neurite, and are consistent with a requirement for unc-16/jip3 in autophagosome retrograde transport.
Previous reports have implicated UNC-16/JIP3/Sunday Driver in the regulation of retrograde transport and retention of lysosomes, early endosomes and Golgi in the cell body ((Brown et al., 2009; Byrd et al., 2001; Edwards et al., 2013) and Figures S3). The role of UNC-16/JIP3 in transport of autophagosomes has not been examined. To directly investigate if unc-16/jip3 is required for retrograde transport of autophagosomes, we performed time-lapse imaging of individual autophagosomes in wild type neurons (n=71) and unc-16 mutant neurons (n=73). We observed that in wild type animals 54% of autophagosomes in neurites (n=57 autophagosomes) traffic in the retrograde direction towards the cell body, while in unc-16/jip3 mutants only 7% (n=482 autophagosomes) traffic in the retrograde direction (Figures 3K-3M). Instead unc-16/jip3 mutant animals display increased anterograde trafficking of autophagosomes (from 2% in wild type to 17% in unc-16 mutants) and increased number of paused autophagosomes (defined as autophagosomes that do not traffic within a 5 minute observation window; from 44% in wild type to 76% in unc-16 mutants) (Figures 3K-3M). In wild type animals, autophagosomes in the cell body are sometimes transported in an anterograde fashion into the neurite, but 85% of those autophagosomes (n=13 autophagosomes) return to the cell body via retrograde transport within the 5-minute examination window. In contrast, in unc-16 mutant neurites only 17% of autophagosomes (n=29 autophagosomes) that leave the cell body return within 5 minutes (Figure 3J). Our findings are consistent with previous reports that demonstrate that another JIP family member (JIP1) is important for regulating autophagosome retrograde transport in cultured vertebrate neurons (Fu et al., 2014). We now extend those findings and demonstrate an important role for unc-16/jip3 in regulating the retrograde trafficking of autophagosomes in vivo.
To examine how UNC-16-dependent retrograde transport contributes to the acidification and degradation of autophagosomes, we then tested if defects in unc-16 mutants altered the total number of autophagosomes in neurons. We observed that the total number of autophagosomes in neurons of unc-16/jip3 mutants is higher than seen in wild type animals (average from 4.0 in wild type neurons (n=65) to 6.4 in unc-16 mutant neurons (n=64)). Our findings indicate that the increased number of autophagosomes observed in the neurites of unc-16/jip3 mutants does not result from a simple redistribution of a fixed number of autophagosomes. Instead, retrograde transport, promoted by unc-16/jip3, appears to be necessary for the effective degradation of autophagosomes, and defects in transport in unc-16/jip3 mutants therefore result in an abnormal accumulation of autophagosomes. Our findings underscore the importance of regulated trafficking in the degradation of synaptic autophagosomes.
Mutant allele ola316 enhances the number of autophagosomes in the neurites of unc-16/jip3 mutants
Degradation of autophagosomes occurs upon fusion with lysosomes, an important and final step that is tightly regulated but poorly understood (Itakura et al., 2012; Nakamura and Yoshimori, 2017). To better understand the molecular machinery underlying coordinated trafficking and degradation of synaptic autophagosomes, we performed a visual forward genetic screen in the unc-16/jip3 mutant background. We reasoned that molecules important for regulated lysosomal fusion will result in an enhanced accumulation of autophagosomes, and screened for mutants with increased numbers of autophagosomes in the AIY synaptic regions. We uncovered a mutant allele, ola316, which displays a dramatic accumulation of autophagosomes in the neurites. While unc-16/jip3 mutants display an average of 2.6 autophagosomes abnormally accumulated in the synaptic regions (n=64 neurites), ola316;unc-16 double mutants enhance this phenotype and display an average of 9.4 autophagosomes in the synaptic regions (n=67 neurites) (Figures 4A-4D).
We then examined if the ola316 allele displayed a phenotype independent of the unc-16/jip3 mutant lesion. To achieve, this we outcrossed the unc-16/jip3 lesion and examined LGG-1 containing structures in the ola316 single mutants. We observed that, unlike the ola316;unc-16 double mutants, the ola316 single mutants did not display a significant accumulation of autophagosomes in the synaptic regions when compared with unc-16 or unc-16;ola316 double mutants (n= 64 neurites) (Figure 4D). However, we did observe an increased number of autophagosomes in the cell body of the ola316 single mutants, from an average of 3.9 autophagosomes in wild type (n=65) to 9.9 autophagosomes in ola316 mutants (n=64) (Figures 4E, 4G and 4H). We also observed an increase in the percentage of neurites with one or more autophagosomes in the synaptic regions, from 7% in wild type (n=68) to 58% in ola316 mutant neurites (n=78) (Figure S4A). The gene affected in the ola316 allele is important for most neurons in the nematode’s nervous system, as abnormal panneuronal accumulation of autophagosomes is observed for both the single mutants and the ola316;unc-16 double mutants (Figure S5). Together our data indicate that the ola316 allele affects a gene that is important for preventing the abnormal accumulation of autophagosomes both in the neuronal soma and, through cooperation with UNC-16/JIP3, in the neurites.
ola316 is a dominant allele of the autophagy gene atg-4.2
Genetic characterization of the ola316 allele revealed that the phenotype results from a single genetic dominant lesion (Figure 4E, green and red striped bar). To identify the causative lesion in ola316, we performed single-nucleotide polymorphism (SNP) mapping, whole genome sequencing, independent allele analyses and rescue experiments. We SNP-mapped ola316 to a 2.2 Megabase region between 10.1 and 12.3 Mb on chromosome IV. We then performed whole genome sequencing (Minevich et al., 2012; Sarin et al., 2008) and uncovered a missense lesion in exon 7 in the autophagy gene atg-4.2. Two independent alleles of atg-4.2 (gk430078 and gk628327), which result in early stop codons (Figure 4F), phenocopy the ola316 single mutant phenotype (Figure 4E (maroon and peach bars) and 4I). Moreover unc-16;atg-4.2(gk628327) double mutants also phenocopy the unc-16;ola316 double mutants isolated from the genetic screen (Figure 4D). Consistent with ola316 being an allele of atg-4.2, AIY-specific overexpression of the wildtype atg-4.2 cDNA in ola316 mutant animals rescues the ola316 mutant phenotype (Figures 4E (pink bar), 4H and 4J), while overexpression of mutant atg-4.2(ola316) cDNA in wild type animals is sufficient to induce the phenotype (Figures 4E (grey bars), 4K and 4L). Together our data indicate that ola316 is a dominant allele of the autophagy gene atg-4.2, and that ATG-4.2 acts cell autonomously in neurons to regulate the number of LGG-1 structures that otherwise accumulate in the cell body.
Atg4s are a conserved family of cysteine proteases that bind to and cleave Atg8/LC3/LGG-1 proteins to achieve two functions: 1) prime LGG-1 for conjugation onto the autophagosomal membrane; and 2) remove LGG-1 from the autophagosomal membrane (Kirisako et al., 2000; Maruyama and Noda, 2017). Conjugation of Atg8/LC3/LGG-1 to the membrane is necessary for autophagosome elongation and closure (Fujita et al., 2008; Kirisako et al., 1999; Manil-Segalen et al., 2014; Nakatogawa et al., 2007; Weidberg et al., 2010), and removal of Atg8/LC3/LGG-1 is thought to occur prior to degradation (Betin et al., 2013; Kimura et al., 2007; Yu et al., 2012). Therefore, the seemingly opposite roles for Atg4 protease activity on LGG-1 need to be coordinated at different stages of the autophagosome biogenesis and degradation process. How this duality of roles is achieved is not well understood.
In yeast one Atg4 gene performs both the priming and deconjugation events on Atg8/LGG-1 (Kirisako et al., 2000; Maruyama and Noda, 2017), while in higher metazoans multiple genes encode distinct Atg4 cysteine proteases, some with unknown function, specificity, or redundancy (Kauffman et al., 2018; Li et al., 2011; Marino et al., 2003; Wu et al., 2012; Zhang et al., 2016). Consistent with this, the two C. elegans atg-4 genes (atg-4.1 and atg-4.2) (Figure 5A) were shown to be largely redundant (Wu et al., 2012), with ATG-4.1 displaying enhanced proteolytic activity of a soluble pro-form of LGG-1/Atg8 as compared to ATG-4.2. The specific roles of ATG-4.2 in autophagy, if any, remain unknown.
ATG-4.2, but not ATG-4.1, is required to prevent autophagosome accumulation
The two C. elegans Atg4 homologs display 44% amino acid similarity, including conserved catalytic and regulatory sites (Wu et al., 2012). A previous study investigating the degradation of protein aggregates in embryos found that atg-4.1 is more efficient at the first LGG-1 cleavage event (priming), and more effective in removal of protein aggregates, than atg-4.2 (Wu et al., 2012). To examine if atg-4.1 also acts as atg-4.2 to prevent autophagosomal accumulation we examined two independent alleles of atg-4.1, including a confirmed null allele (bp501 (null) (Wu et al., 2012) and gk127286 (early stop codon)). Surprisingly we did not detect LGG-1 puncta accumulation in the examined atg-4.1 mutant animals (Figures 5D and 5F). Consistent with atg-4.1 having a distinct phenotype compared to atg-4.2, we observed that atg-4.1 did not enhance the unc-16 mutant phenotype (Figure 6H). Our findings indicate that atg-4.1 and atg-4.2 have distinct functions in vivo that result in different phenotypes regarding the accumulation of autophagosomes.
While we observe distinct phenotypes for atg-4.1 and atg-4.2 in the accumulation of autophagic structures, we also note that both atg-4.1 and atg-4.2 single mutant animals contain LGG-1 puncta in the neuron (Figures 5B-5D and 5F). This observation suggests that either protease is able to partially compensate for the other and perform the first priming cleavage necessary to conjugate LGG-1 onto the nascent autophagosome. Consistent with these two proteases performing partially redundant roles, we have previously shown that in AIY, where autophagy is also required for synapse development, the synaptic assembly phenotype is not seen for atg-4.1 or atg-4.2 single mutants, but is evident in the atg-4.1;atg-4.2 double mutants (Figure S6 and (Stavoe et al., 2016)). To further explore the partial redundancy of atg-4.1 and atg-4.2 in the accumulation of LGG-1 containing structures, we examined GFP::LGG-1 in the atg-4.1;atg-4.2 double mutants. We observed a reduction in the number of accumulated LGG-1 puncta for the atg-4.1;atg-4.2 double mutants as compared to either single mutant (Figures 5E and 5F). Our result indicates that atg-4.1 and atg-4.2 act in a partially redundant fashion, that atg-4.1 activity is necessary to achieve the autophagosome accumulation seen in atg-4.2 mutants, and that atg-4.2 activity is necessary in the atg-4.1 mutants to see normal levels of autophagosomes. Importantly, our findings indicate that atg-4.1 and atg-4.2 display distinct phenotypes in LGG-1 puncta accumulation, and that atg-4.2 is specifically necessary to prevent abnormal accumulation of autophagosomes in the neuronal soma.
ATG-4.2 promotes autophagosome maturation and removal from the cell body
Our observations that lesions in atg-4.2 result in an abnormal accumulation of autophagosomes in the neuronal cell body are consistent with a model where atg-4.2 acts late in the autophagy pathway to promote autophagosome maturation and degradation. Autophagosome maturation into an acidic autolysosome is mediated by fusion between autophagosomes and late endosomes or lysosomes in a process that requires the small GTPase Rab7 (Gutierrez et al., 2004; Hyttinen et al., 2013; Jager et al., 2004). We reasoned that if atg-4.2 mutants have a defect in lysosomal fusion, then loss of rab-7 function should phenocopy the atg-4.2 mutants. Since null lesions in rab-7 are lethal, we examined mutants for the rab-7 activator sand-1/Mon1 (Hegedus et al., 2016; Poteryaev et al., 2007), and the rab-7 effector, epg-5 (Wang et al., 2016). We observe that indeed epg-5 and sand-1 mutants display increased autophagosome accumulation in the cell body (averaging 13.6 autophagosomes in epg-5 mutants (n=42 neurons) and 12.7 autophagosomes in sand-1 mutants (n=69 neurons), compared to 12.1 autophagosomes in atg-4.2(ola316) mutants (n=78 neurons) and 3.6 autophagosomes in wild type animals (n=68 neurons); Figures 6A-6C, 6G). The observed phenotypes are specific to late blocks in autophagy, as mutants for genes involved in early events prior to autophagosome closure or trafficking, such as atg-9, atg-2, or unc-16 mutants, did not result in autophagosome accumulation in the cell body (Figure 6G). Moreover, sand-1;unc-16, like atg-4.2;unc-16, also dramatically enhanced the accumulation of autophagosomes in the synaptic regions (from an average of 20.2 autophagosomes in sand-1;unc-16 double mutants (n=46) compared to 8.1 autophagosomes in atg-4.2(ola316);unc-16 double mutants (n=58) and 4.7 autophagosomes in unc-16 single mutants (n=73); Figures 6D-6F and 6H). Importantly, early autophagy mutants like atg-2 (required for autophagosome closure) and atg-4.1 (required for LGG-1 priming) fail to enhance the neurite accumulation phenotype observed in unc-16 mutant animals (Figure 6H). Together our findings indicate that atg-4.2, like epg-5 and sand-1, acts in a late stage of autophagy prior to degradation.
We then tested the acidification state of the accumulated autophagosomes in atg-4.2 mutants by using the tandem LGG-1 marker. We observed that in atg-4.2 mutants, 83% of autophagosomes retained GFP signal (n=469 autophagosomes) compared to 48% in wild type neurons (n=266 autophagosomes), consistent with a defect in autophagosome maturation or acidification (Figure 6I-6O). Previous studies in yeast have observed Atg8/LGG-1 mislocalization onto the vacuole and other organelles in delipidation-defective Atg4 mutants (Nakatogawa et al., 2012; Yu et al., 2012). To test if the accumulated LGG-1 puncta in atg-4.2 mutants are acidification-defective lysosomes, we examined lysosomes in atg-4.2 mutants. We did not observe an accumulation of lysosomes in atg-4.2 mutants (Figure S4B), consistent with the accumulated structures representing immature autophagosomes. Together, our findings suggest that atg-4.2 mutants are defective in acidification, resulting in an accumulation of immature autophagosomes and a failure in degradation.
Our findings suggest that the ATG-4 proteases, while partially redundant, are also uniquely required for specific steps of autophagy, with loss of either gene resulting in distinct cell biological phenotypes of autophagy in neurons. Importantly our data uncover a novel role for ATG-4.2, and indicate that ATG-4.2 cooperates with retrograde transport mechanisms to promote autophagosome acidification and degradation in the cell soma.
DISCUSSION
Distinct steps of the autophagy pathway occur in different subcellular compartments. Previous studies demonstrated that autophagosome biogenesis in primary neurons occurs at the distal axon and follows a compartmentalized, ordered and spatially regulated process (Maday and Holzbaur, 2014). In vivo, autophagosome biogenesis occurs at presynaptic sites (Soukup et al., 2016; Stavoe et al., 2016) and the number of autophagosomes in the neurite correlates with the state of activity in the neuron (Figure S1 and (Shehata et al., 2012; Soukup et al., 2016; Wang et al., 2015). Autophagosomes then undergo retrograde trafficking prior to degradation (Maday et al., 2012), indicating a regional decoupling of biogenesis and degradation. In this study we extend these cell biological findings and demonstrate, using genetics, a requirement for distinct steps of autophagy in different subcellular compartments. We observe that defects in atg-2 and epg-6/WIP3/4, which are required for autophagosome closure, result in accumulated autophagosomes at the synapse, while defects in epg-5, required for autophagosome maturation, result in accumulated autophagosomes in the cell body. Our findings extend our understanding of the spatial organization of autophagy, and underscore the importance of coordination across cellular regions and in the context of the polarized structure of the neuron.
UNC-16/JIP3-dependent retrograde transport links autophagosome biogenesis at the synapse with autophagosome degradation in the cellular soma. In C. elegans, Drosophila and vertebrate neurons, axonal autophagosomes are transported towards the cell body via the dynein complex (Cheng et al., 2015; Ikenaka et al., 2013; Katsumata et al., 2010; Neisch et al., 2017). UNC-16/JIP3/Sunday Driver, a motor adaptor protein that interacts with dynein, plays important and conserved roles in the regulated localization and transport of late endosomes and lysosomes, as well as other organelles (Brown et al., 2009; Byrd et al., 2001; Drerup and Nechiporuk, 2013; Edwards et al., 2015; Edwards et al., 2013; Gowrishankar et al., 2017). Our findings extend the roles for UNC-16/JIP3 and now demonstrate that it is also required for autophagosome retrograde transport. In vertebrate neurons, autophagosome retrograde transport is promoted by JIP1, a motor adaptor protein that belongs to the same family as UNC-16/JIP3 (Fu and Holzbaur, 2014; Fu et al., 2014). While we did not detect a phenotype for autophagosome retrograde transport in C. elegans jip-1 mutants, we note that JIP1 and JIP3 share sequence and functional similarities, and are also known to cooperate in the transport of cargo (Hammond et al., 2008; Sun et al., 2017). Together, our findings ascribe an important and potentially conserved role for UNC-16/JIP3 in retrograde transport of autophagosomes in C. elegans neurons.
Impaired retrograde transport in unc-16/jip3 mutants results in accumulation of LGG-1 containing structures in the neurites. In vertebrate neurons, disruption of retrograde transport also causes accumulation of autophagosomes and lysosomes in swollen neuronal processes and synapses. This, in turn, results in autophagic stress and in neurodegeneration phenotypes similar to those seen in the neurons of Alzheimer’s disease patients (Ikenaka et al., 2013; Lee et al., 2011; Nixon et al., 2005; Takats et al., 2013). Specific disruption of JIP3 in vertebrate neurons has also been associated with an increase in soluble Aβ levels, plaque size, plaque abundance, and axonal dystrophy (Gowrishankar et al., 2017). Our findings in C. elegans neurons are consistent with these studies and underscore the importance of retrograde transport in linking the mechanisms of autophagosome biogenesis at the synapse with degradation in the cell body. Furthermore, our observations that combined defects in both retrograde transport and degradation machinery can enhance autophagosome accumulation in the neurites raises the question of whether the accumulated autophagosomes seen in progressive neurodegenerative diseases might result from an accruement of distinct dysfunctions in different stages of autophagy over time.
ATG-4.2 genetically cooperates with UNC-16/JIP3 in the clearance of autophagosomes from axons. Our forward genetic screens revealed that lesions in atg-4.2 enhance the autophagosome accumulation phenotype observed for unc-16/jip3 mutants (Figure 7B-7E). ATG-4.2 is one of two Atg4 cysteine proteases in C. elegans known to cleave LGG-1/Atg8 in autophagy (Wu et al., 2012). We find that the other Atg4 cysteine protease in the nematode, ATG-4.1, does not enhance the autophagosome accumulation phenotype observed for unc-16/jip3 mutants, suggesting a distinct role for ATG-4.2 in the clearance of autophagosomes from axons. Atg4 cysteine proteases are required during both early and late stages of autophagy to cleave LGG-1/Atg8, but to achieve two distinct functions: the first cleavage (termed priming) is required for the conjugation of LGG-1/Atg8 to the autophagosomal membrane to promote autophagosome elongation, while the second cleavage (termed delipidation) occurs prior to degradation, when LGG-1/Atg8 is removed from the autophagosome membrane (Kirisako et al., 2000; Maruyama and Noda, 2017). Our in vivo findings suggest that these distinct cleavage roles for Atg4 in LGG-1/Atg8 priming and delipidation might be regulated by distinct Atg4 proteases, and that the C. elegans ATG-4.2 has a specialized role in the delipidation of LGG-1/Atg8 from autophagosomal membranes. Consistent with this idea, a recent biochemical study examining mammalian Atg4 genes revealed that different Atg4s possess different activities for cleaving soluble or membrane-bound GABARAP/Atg8/LGG-1 (Kauffman et al., 2018). Moreover, biochemical characterizations of C. elegans atg-4.1 and atg-4.2 gene functions demonstrated that atg-4.2 mutants accumulate more lipidated LGG-1 compared to wild type or atg-4.1 mutant animals, consistent with distinct biochemical properties between ATG-4.1 and ATG-4.2, and with a preferential role for ATG-4.2 in delipidation (Wu et al., 2012). We now show that the different biochemical activities of ATG-4.1 and ATG-4.2 confer different requirements in vivo, resulting in distinct phenotypes regarding the accumulation of autophagosomes in neurons. The distinct ATG4 genes therefore differentially interact with unc-16/jip3 mutants in enhancing defects associated with clearance of autophagosomes from neurites.
ATG-4.2 is necessary for autophagosome maturation and degradation. In atg-4.2 mutants we observe the accumulation of non-acidic LGG-1 puncta in the cell bodies of neurons. These data further indicate a role for ATG-4.2 in the delipidation of LGG-1, and suggest that delipidation is necessary for the proper fusion of autophagosomes with lysosomes, and degradation. Our findings extend studies in yeast, where it has been hypothesized that delipidation of Atg8/LGG-1 from autophagosomes is necessary both for autophagosome biogenesis (Nair et al., 2012; Nakatogawa et al., 2012) and to promote fusion with lysosomes and degradation (Yu et al., 2012). Our findings further suggest that association of LGG-1 with the autophagosomal membrane, a process mediated by proteases ATG-4.1 and ATG-4.2, “bookends” the progression of autophagy. Our findings are consistent with a model (presented in Figure 7A) in which, upon biogenesis at the synapse, LGG-1 becomes conjugated to autophagosomes, a process that requires priming activity, likely performed by the priming protease ATG-4.1 to allow lipidation at the synapse. After transport to the cell body, ATG-4.2 then preferentially mediates removal of LGG-1 from the autophagosome membrane, a step that is necessary for maturation and degradation in the cell body. Therefore the specialized regulation of these proteases in distinct subcellular compartments contributes to the coordination of the autophagy pathway across the subcellular regions of the neuron.
EXPERIMENTAL PROCEDURES
Strains and genetics
All C. elegans strains were raised on NGM plates with OP50 Escherichia coli at 20°C, unless otherwise noted. We used N2 Bristol as the wild type reference strain. We obtained the following mutant strains through the Caenorhabditis Genetics Center (CGC): dnc-1(or404), unc-16(ju146), unc-16(n730), unc-16(e109), jip-1(gk466982), unc-116(e2310), unc-14(e57), atg-4.1(gk127286), atg-4.1(bp501), atg-4.2(gk430078), atg-4.2(gk628327), sand-1(or552), and unc-13(e450). We also obtained from the CGC, the balancer nT1[qIs51] isolated from VC3476 and used to assess escapers from olaIs35;atg-4.1;atg-4.2/nT1, which is sterile in its unbalanced state. We obtained mutants from Dr. Hong Zhang at the Institute of Biophysics, Chinese Academy of Sciences: atg-2(bp576) and epg-6(bp242). And we obtained from the Mitani laboratory at the Tokyo Women’s Medical University School of Medicine: epg-5(tm3425).
We also utilized atg-9(wy56), DCR4750 (olaIs35 [Pttx-3::egfp::lgg-1; Pttx-3::mCh]), and TV392 (wyIs45 [Pttx-3:gfp:rab-3]), as described in previous publications (Colon-Ramos et al., 2007; Stavoe et al., 2016).
Molecular biology and transgenic lines
The plasmids used in this study are derived from the pSM vector (Shen and Bargmann, 2003). We created transgenic strains using standard injection techniques. We used the following plasmids as co-injection markers: Punc-122::gfp (10-15ng/ul) and Punc-122::dsRed (30ng/ul), and generated these arrays: olaEx3986 [Pttx-3::mCh::rab-3 (30ng/ul)], olaEx3331 [Pttx-3::laat-1::gfp (1ng/uL), pttx-3::mCh (30ng/ul)], olaEx3013 [Pttx-3::mCh::egfp::lgg-1) (1ng/ul)], olaEx3015 [Pttx-3::mCh::egfp::lgg-1(G116A)) (1ng/ul)], olaEx3430 and olaEx3438 [pttx-3::atg-4.2 (30ng/ul)], olaEx3426 [Pttx-3::atg-4.2(G1117A) (30ng/ul)], olaEx3465 [Pmod-1::HisCl (30ng/ul)], olaEx3358 [Pttx-3::ebp-2::gfp (5ng/ul), pttx-3::mCh (30ng/ul)], olaEx1706 [Pttx-3::SP12::gfp (5ng/ul)], olaEx1700 [Pttx-3:aman-2:gfp (0.5 ng/ul)], olaEx1951 [Pttx-3::tom-20::gfp (30 ng/uL), pttx-3:mCh:rab-3 (30 ng/uL)], olaIs44 [Pttx-3::egfp::lgg-1 (15ng/ul)], olaIs58 [Paex-3::egfp::lgg-1 (15ng/ul)].
We also generated olaEx3293 [Pelt-7::gfp (10ng/ul)] to distinguish heterozygous cross offspring from self progeny for the dominant/recessive tests.
For the cDNA constructs generated for use in this study (ATG-4.2, LAAT-1 and EBP-2), we PCR amplified cDNA from a mixed stage population of C. elegans. A Q5 mutagenesis kit (NEB) was then used to introduce the DNA mutation G1117A (which corresponds to the allelic lesion in atg-4.2(ola316)) into the ATG-4.2 cDNA. The introduction of this lesion should result in expression of the mutant protein ATG-4.2(G373R). Detailed sub-cloning information is available upon request.
Fluorescence microscopy and confocal imaging
We used an UltraView VoX spinning disc confocal microscope with a 60x CFI Plan Apo VC, NA 1.4, oil objective on a NikonTi-E stand (PerkinElmer) with a Hammamatsu C9100-50 camera. We imaged the following fluorescently tagged fusion proteins, eGFP, GFP, RFP, and mCherry at 488 or 561 nm excitation wavelength. We anesthetized C. elegans at room temperature in 10mM levamisole (Sigma) or as indicated.
Images were obtained using Volocity software (Improvision by Perkin Elmer) and processed using Adobe Photoshop CS4 and (Fiji is Just) ImageJ (FIJI) software. Image processing included maximal projection, rotation, cropping, brightness/contrast, pseudo coloring, and making surface plots. Kymographs were made using the “reslice” function in FIJI. Between genotypes for compared groups the confocal laser and camera settings and the FIJI brightness/contrast settings were kept identical. All quantifications from confocal images were conducted on maximal projections of the raw data. All images are oriented anterior to the left and dorsal up.
SNP Mapping and Whole-Genome Sequencing
We obtained mutant allele atg-4.2(ola316) from a visual forward genetic enhancer screen in the unc-16(ju146);olaIs35 mutant background (integrated line olaIs35 contains Pttx-3::egfp::lgg-1; Pttx-3::mCh). Ethyl methanesulfonate (EMS) mutagenesis was performed and animals were screened for enhanced number of autophagosomes (visualized with GFP::LGG-1) in the AIY neurite. F2 progeny were viewed on a Leica DM 5000 B compound microscope with an HCX PL APO 63x/1.40-0.60 oil objective.
The novel lesion ola316 was out-crossed from the unc-16 lesion and found to have an independent dominant cell body accumulation of autophagosomes. We then used single-nucleotide polymorphism (SNP) mapping as described (Davis and Hammarlund, 2006; Davis et al., 2005) to map the ola316 lesion. Briefly, this involves crossing mutants to a divergent strain, Hawaiian CB4865, and detecting sites of recombination via comparison of dissimilar SNPs. To avoid selecting heterozygous recombinants of the dominant lesion, we mapped loss of the ola316 locus by selecting animals with the wild type phenotype. SNP mapping was then repeated for verification by selecting and confirming homozygous animals with the unc-16;ola316 enhanced phenotype.
We then performed whole-genome sequencing on both unc-16;ola316;olaIs35 and for comparison unc-16;olaIs35 animals at the Yale Center for Genome Analysis (YCGA), as previous (Sarin et al., 2008). We analyzed the results with www.usegalaxy.org, the “Cloudmap Unmapped Mutant workflow (w/ subtraction of other strains),” (Minevich et al., 2012) and verified lesions by Sanger sequencing.
Quantification of autophagosomes in AIY
Quantifications were performed taking into consideration best practices as suggested in (Landis et al., 2012), including randomization, blinding, and data handling procedures. Particulars for each assay as follows:
Tandem marker
Autophagosome maturation was assessed using extrachromosomal line olaEx3013 (Pttx-3::mCh::egfp::lgg-1).The tandem marker used in this study is based on similar probes used in mammalian neurons (Kimura et al., 2007) and in C. elegans tissues (Chang et al., 2017). Quantifications of puncta were performed on maximal projections of confocal micrographs and imaging settings were kept identical between compared groups. Puncta were scored as “Dual/GFP” (immature) if green puncta could be detected and as “mCh only” (mature) if the puncta displayed primarily red signal.
To ensure that the tandem labeled LGG-1 probe is lipidated onto membranes and to control for the possibility of marker aggregation, we also examined a mutant version of the probe olaEx3015 (Pttx-3::mCh::egfp::lgg-1(G116A)), which prevents LGG-1 from associating with the autophagosomal membrane (Mizushima et al., 2010; Zhang et al., 2015). As expected, we observed that in the LGG-1(G116A) mutant probe GFP was diffuse throughout the neuron (data not shown). We also observed mCherry puncta in the cell body (3.7 puncta on average, n=34 neurons), consistent with an accumulation of mCherry (which is stable at low pH (Shaner et al., 2004)), in a lysosomal compartment. Importantly our observations suggest that the tandem marker is lipidated at the G116 residue and is expected to associate with autophagosomes as described (Mizushima et al., 2010; Zhang et al., 2015).
Autophagosome dynamics
To assess autophagosome dynamics over time, we used the integrated line olaIs35 (Pttx-3::egfp::lgg-1; Pttx-3::mCh). From a dataset described previously (Stavoe et al., 2016), where confocal z-stacks were acquired once per minute for 30 minutes in wild type and autophagy mutant animals, we performed a new analysis, tracking individual autophagosomes over time and scoring the percentage of autophagosomes retained in the presynaptic region for the full 30 minutes. We also collected a new dataset, imaging at maximal speed for 5 minutes in wild type (n=71 neurons) and unc-16(ju146) mutant animals (n=73 neurons). These videos were scored for percentage of autophagosomes present in each of the three sub-neurite zones (Colon-Ramos et al., 2007), for anterograde and retrograde directionality of autophagosome trafficking in the neurite, and for percentage of autophagosomes leaving the cell body towards the neurite, and then returning to the cell body within the imaging window of 5 minutes.
Autophagosome accumulation in mutant neurites and cell bodies
To investigate the accumulation of autophagosomes in the AIY neuron, we tested mutant alleles in the olaIs35 background. For the temperature sensitive dnc-1(or404) allele (Koushika et al., 2004) and a wild type control, animals were held at either 20C (permissive) or 25C (restrictive) for 48 hours prior to examination. Other candidate transport mutants (unc-16, jip-1, unc-116, unc-14 and controls) were kept at 20C. Autophagosome accumulation in the neurite was then scored on a Leica DM 5000 B compound microscope. For each animal, the sum of autophagosomes in both AIY neurons was counted, then divided by two and reported as an average per neuron in Figure 3. Additional quantifications (presented in figures 4, 5, and 6) for the presence of autophagosomes in the cell bodies and neurites of mutant animals (unc-16, atg-4.2, atg-4.1, epg-5, sand-1 etc.), double mutant animals, and animals overexpressing ATG-4.2 arrays, were scored as follows: raw z-stacks were divided to separate individual AIY neurons, maximal projections were created, and images were randomized and scored blindly.
Activity-dependent autophagy
To assess synaptic autophagy, animals containing olaIs35 in a wild type, atg-9, atg-2, or unc-13 mutant background were grown at 20C and then shifted to 25C for variable lengths of time (as indicated) and assessed for number of autophagosomes in the AIY neurite on a Leica DM 5000 B compound microscope. For the HisCl experiments, the animals additionally contained the extrachromosomal array olaEx3465 (Pmod-1::HisCl) and were transferred to 10mM Histamine-containing or control NGM plates at 20C or 25C for 4 hours prior to assessment (Pokala et al., 2014). Animals were then mounted in either 5mM levamisole in M9 buffer (compared to 10mM used in all other experiments) or in M9 buffer containing 5mM levamisole and 10mM Histamine and scored blindly. Results are reported in Figure S1.
Other Quantifications in AIY
Lysosomes
To quantify lysosome number in AIY, we observed the extrachromosomal transgenic line olaEx3331 (Pttx-3::laat-1::gfp, pttx-3::mCh) in wild type or mutant backgrounds on a Leica DM 5000 B compound microscope. The number of GFP puncta in the two AIY neurons in the animal were counted by location, divided by two and then reported as an average per neuron.
Endoplasmic reticulum (ER), Golgi, and Mitochondria
For examination of ER, Golgi, and mitochondria the following extrachromosomal lines were used, respectively: olaEx1706 (Pttx-3::SP12::gfp), olaEx1700 (Pttx-3:aman-2:gfp), and olaEx1951 (Pttx-3::tom-20::gfp, pttx-3:mCh:rab-3) (Abe et al., 2000; Qi et al., 2012; Rolls et al., 2002) in wild type and unc-16(ju146) mutant backgrounds. Quantifications were performed on maximal projection confocal micrographs. Fluorescence intensity was measured by tracing the neurite and background in FIJI (Schindelin et al., 2012) and reported as an average of background-subtracted fluorescence intensity. The percent of the neurite occupied by mitochondria was calculated as a ratio of length of the neurite with background-subtracted fluorescence signal greater than zero divided by the total length of the neurite.
Presynaptic enrichment
To quantify presynaptic enrichment in AIY, we used the integrated transgenic line wyIs45, expressing pttx-3::gfp::rab-3 in wild type and mutant backgrounds, as described (Colon-Ramos et al., 2007; Stavoe et al., 2016). The AIY neurite is divided into three zones as follows, consistent with the C. elegans EM reconstruction (White et al., 1986): Zone 1 is proximal to the cell body and asynaptic; Zone 2 is defined morphologically as a ∼5-μm synapse-rich region at the dorsal turn of the neuron into the nerve ring; Zone 3 is the distal part of the neurite that traverses the nerve ring and contains punctate synaptic sites. We calculated the GFP::RAB-3 enrichment using maximal projection confocal micrographs and measured fluorescence intensity across presynaptic Zones 2 and 3 with the line scan function in FIJI (Schindelin et al., 2012). Fluorescence intensity was background subtracted. All settings, for the confocal microscope and camera, were kept identical between genotypes, and neurons were only quantified when all neurite fluorescence signal was within the measurable range. Synaptic enrichment was reported as signal in Zone 2 (the first 20% of the synaptic region) divided over Zones 2 and 3 (as described in (Stavoe and Colon-Ramos, 2012)) and reported as a percentage and shown in Figure S6.
Microtubule EBP-2 quantification
To examine microtubule polarity, we analyzed the direction of movement for EBP-2 comets (Baas and Lin, 2011; Maniar et al., 2011), which track with the growing ends of microtubules, along the AIY neurite using the extrachromosomal line olaEx3358 (Pttx-3::ebp-2::gfp, pttx-3::mCh). Confocal z-stacks were acquired at maximal speed for one minute and comets were assessed for movement towards the cell body (minus end out microtubule) or movement away from the cell body (plus end out microtubule) in wild type (n=18 animals) and unc-16 mutants (n=16 animals).
Statistical Analyses
Statistical analyses were conducted with PRISM software. For each case, the chosen statistical test is described in the figure legend. Briefly, for continuous data and for cases of counting structures, comparisons between two groups were determined by the Student’s t test, while groups of three or more were analyzed with a one-way ANOVA with post hoc analysis by Turkey’s multiple comparison test. Error bars were reported as standard errors of the mean (SEM). For categorical data, groups were compared with Fisher’s exact test, or for samples corresponding to large datasets, the chi-square test was used. Errors show a 95% confidence interval.
AUTHOR CONTRIBUTIONS
SEH and DACR designed the experiments; SEH performed the experiments and data analyses. SEH and DACR prepared the manuscript.
Author Information
The authors declare no competing financial interests. Correspondence and request for materials should be addressed to D.A.C-.R. (daniel.colon-ramos{at}yale.edu).
SUPPLEMENTAL FIGURE LEGENDS
Supplemental Movie S1. Time lapse video of GFP::LGG-1 in a wild type AIY neuron showing autophagosome biogenesis and retrograde transport from the synaptic region
Supplemental Movie S2. Time lapse video of GFP::LGG-1 in an AIY neuron in an atg-2(bp576) mutant animal, showing autophagosomes in the synaptic region
Supplemental Movie S3. Time lapse video of GFP::LGG-1 in a pair of AIY neurons in an epg-6(bp424) mutant animal, showing autophagosomes in the synaptic region
Supplemental Movie S4. Time lapse video of GFP::LGG-1 in a pair of AIY neurons in an epg-5(tm3425) mutant animal, showing autophagosome retrograde transport from the synaptic region
ACKNOWLEDGEMENTS
We thank members of the Colón-Ramos lab, the Thomas Melia lab (Yale University), Shawn Ferguson (Yale University) and Andrea Stavoe (University of Pennsylvania) for their thoughtful comments on the project. We thank undergraduate researchers Alec Rodriguez (Yale University) and Enrique Cruz-Reyes (Universidad de Puerto Rico, Cayey) and Sisi Yang (Colón-Ramos lab) for experimental assistance. We thank the Caenorhabditis Genetics Center (supported by the National Institutes of Health (NIH), the Office of Research Infrastructure Programs; P40 OD010440) for strains, the Mitani laboratory of the Tokyo Women’s Medical University School of Medicine for autophagy mutant strains and the Hong Zhang laboratory at the Institute of Biophysics, Chinese Academy of Sciences for autophagy mutant alleles. We thank Cori Bargmann for the HisCl construct (Pokala et al., 2014). We thank Z. Altun (www.wormatlas.org) for diagrams used in figures. We thank the Research Center for Minority Institutions program and the Instituto de Neurobiología de la Universidad de Puerto Rico for providing a meeting and brainstorming platform. This work was partially conducted at the Marine Biological Laboratories at Woods Hole under a Whitman research award to D.A.C.-R. Support for S.E.H. was provided by the Cellular and Molecular Biology Training grant T32-GM007223 from the NIH and the NSF Graduate Research Fellowship DGE-1122492. Research was supported by a Howard Hughes Medical Institute Scholar Award, by a National Science Foundation grant (NSF IOS 1353845) and the National Institutes of Health (R01NS076558).