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The toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease1,2. Accordingly, neurons invest considerable cellular resources in chaperones, protein degradation, autophagy, and mitophagy to maintain proteostasis and mitochondrial quality3,4. Complicating the challenges of neuroprotection, misfolded human disease proteins and mitochondria can move into neighboring cells via unclear mechanisms, which may promote pathology spread5,6. Here we document a previously unrecognized capacity of Caenorhabditis elegans adult neurons to extrude large (~4 μm) membrane-surrounded vesicles called “exophers” that can harbor protein aggregates and organelles. Inhibiting chaperone expression, autophagy, or the proteasome, as well as compromising mitochondrial quality, enhances exopher production. Proteotoxically-stressed neurons that extrude exophers subsequently function better than those that do not. The extruded exopher transits through a surrounding tissue where some contents appear degraded, but some non-degradable materials can be subsequently found in remote cells. Our observations suggest that exopher-genesis is a potential “garbage-removal” response to challenged proteostasis and organelle function. We propose that exophers are components of a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, which, when dysfunctional or diminished with age, might actively contribute to pathogenesis in human neurodegenerative disease and brain aging.
While studying age-associated dendritic restructuring in C. elegans neurons7, we noticed that fluorescent signals originating from neurons sometimes appeared situated outside of the cell in defined vesicle-like structures that we call exophers (Fig. 1a–c, Extended Data Fig. 1a–c, ,2g).2g). We first characterized exophers associated with the six gentle touch receptor neurons, for which cell bodies and dendrites are easily visualized. We found that exophers were comparable in size (average 3.8 μM) to neuronal somas (Extended Data Fig. 1d). The size of the vesicles, the morphological stages in their biogenesis (Fig. 1a–c), and the genetic requirements for their production (Extended Data Table 1a), distinguish them from much smaller exosomes (~30–100nm; Extended Data Table 2 compares exophers to characterized extracellular vesicles). Neuronal exophers do not appear to result from classical cell division: a) exophers did not stain with nuclear DNA indicator DAPI (Fig. 1b); b) cell division-inhibiting hydroxyurea8 did not change exopher levels (n > 30 per trial, three trials); and c) RNAi-mediated disruption of cell cycle genes did not change exopher detection (Extended Data Table 1b).
We found that exopher production is not restricted to a specific transgene reporter or line (examples in Fig. 1, Extended Data Fig. 1). Amphid neurons that are dye-filled due to openings to the outside environment9 (Extended Data Fig. 1e, f) can produce exophers, establishing that exophers can form under native/physiological cellular conditions. Exopher production differs dramatically among the six touch receptor neurons, with ALMR producing exophers most frequently (Fig. 1d). Multiple neuronal types can produce exophers, including dopaminergic PDE and CEP neurons (Extended Data Fig. 1g, h), FLP neurons (not shown), sensory ASER neurons (Extended Data Fig. 1i).
Time-lapse analyses (Supplemental Videos 1–2) revealed that exophers typically arise from the soma by asymmetrically amassing labeled protein to create a balloon-like extrusion via a pinching off event; the exopher compartment then moves outward from the neuronal cell body (extrusion ~15–100 minutes; Fig. 1a, Extended Data Fig. 1a). Plasma membrane reporter Pmec-4PH(plcDelta)::GFP (Extended Data Fig. 2a) and electron microscopy data (Extended Data Fig. 2) confirm that exophers are membrane-bound. Exophers can initially remain connected to the soma by a thin thread-like tube (Fig. 1c) that can allow transfer of tagged proteins and calcium into the attached exopher compartment (Extended Data Fig. 1a, ,3,3, Supplemental Video 2). Exophers ultimately disconnect from the originating neuronal soma (Extended Data Fig. 3).
Why might neurons produce exophers? Time-lapse studies indicated that aggregating mCherry often appeared preferentially concentrated into exophers, and we found that neurons expressing neurotoxic polyglutamine expansion protein huntingtinQ128CFP could also concentrate and extrude this aggregating protein in exophers (Fig. 2a, b). We therefore further queried the relationship of aggregating or toxic protein expression to exopher production. Strains expressing Q128 (toxic with high levels of apparent aggregation10,11) produced significantly more exophers compared to strains that did not express polyQ or expressed huntingtinQ19CFP (non-toxic and low aggregation) (Fig. 2c). Likewise, aggregating mCherry lines exhibited higher average exopher numbers over adult life as compared to lines expressing soluble GFP (example in Fig. 2d). High aggregate load in individual neurons was predictive of increased exopher production on the following day (Fig. 2e). Conversely, mCherry(RNAi) reduced exopher number by ~half in a line producing aggregating mCherry (Fig. 2f). Although our studies cannot determine the relative contribution of aggregate load from protein expression levels, they suggest that proteostatic challenges increase exopher production. Consistent with a potential role for exophers in elimination of potentially harmful neuronal contents, expression of amyloid-forming human Alzheimer’s disease fragment Aβ1–42 in ASER neurons increases exopher numbers (Fig. 2g). Our combined observations on exopher formation, contents, and frequency of detection suggest that exophers preferentially include aggregated, excess, or otherwise neurotoxic proteins for removal.
To address the hypothesis that aggregation-prone proteins might be selectively extruded in exophers, we constructed a line that expressed both an aggregation-prone mCherry (Is[Pmec4mCh1]) and a non-aggregating GFP (Is[pmec-4GFP]) and compared the red and green fluorescence distribution between exophers and somas (example in Fig. 2h, data in Fig. 2i). In 22/23 exophers, we found higher relative levels of mCherry in the exopher, and higher relative levels of GFP in the soma. That neurons appear to preferentially extrude aggregation-prone mCherry over soluble GFP suggests deleterious materials are identified and sorted for export during exopher-genesis.
To address the hypothesis that proteostatic challenges enhance the exopher production response, we manipulated the in vivo protein-folding milieu. We examined exopher production in an hsf-1(sy441) mutant deficient in core proteostasis transcription factor HSF-1 (and therefore chaperone expression) to record ~6-fold increase in exophers (Fig. 3a). We impaired autophagy by treating animals with a pharmacological inhibitor, Spautin-1 and by RNAi knockdown (lgg-1, atg-7, bec-1, lgg-1/2) in a strain expressing aggregation-prone mCherry and measured a significant increase in exopher incidence (Fig. 3b,c). Impairment of proteasome activity with inhibitor MG132 in strain Is[pmec-4mCh1] also increased exopher production (Fig. 3d). Given that inhibiting multiple facets of proteostasis increases exopher extrusion, we suggest that exophers may constitute a previously undescribed component of the proteostasis network, which may function as a backup or alternative response to rid cells of neurotoxic aggregates/proteins when proteostasis becomes overwhelmed by mounting intracellular proteotoxicity.
Exopher production occurs with a striking bimodal distribution over adult life: exophers are most commonly observed on adult days A2-A3, diminish in abundance A4-A8, and then reappear again later in life ~A10-A11 (Fig. 2d; similar young adult pattern with dye-filled amphid neurons, Extended Data Fig. 1f; and with a 1 day earlier onset in an hsf-1 mutant, Extended Data Fig. 1j). The distinctive temporal production profile suggests that conditions permissive for exopher production exist in young adulthood but can then be limited or remain below a threshold until late adulthood. The coincidence of the early peak with a transition in C. elegans young adult proteostasis management12–14 suggests the first wave of exopher-genesis may serve as a normal component of an orchestrated proteostasis reset in young adulthood that involves jettisoning neuronal garbage generated during development; the later adult increase in exopher production may be the consequence of age-associated decline in proteostatic robustness.
Rather than inducing neuronal death or dysfunction, exopher-genesis appears beneficial. First, in hundreds of longitudinal observations, we did not observe neuronal loss after exopher production: exophers are distinct from apoptotic bodies in their biogenesis (Fig. 1a, Extended Data Fig. 1a), and the soma of an exopher-producing neuron retains normal ultrastructural features (Extended Data Fig. 2e). Second, the relative functionality of proteotoxically-stressed neurons that have generated exophers is increased over neurons that did not extrude exophers. In blinded studies in a line expressing Q128CFP, which progressively impairs touch sensation10, we found that midlife touch sensitivity is greater when ALMR had definitely produced an exopher at A2, as compared to age-matched siblings in which ALMR had not produced an exopher (Fig. 3e). Third, we identified pod-1 and emb-8 as polarity genes required in adults for exopher-genesis (Fig. 3f), and found that adult RNAi knockdown impaired midlife touch sensitivity (Fig. 3g). Although we cannot rule out that pod-1 and emb-8 RNAi interventions might generally disrupt adult neuronal function, taken together our data are consistent with a model in which adult neurons that do not make exophers become functionally compromised compared to those neurons that extruded offending contents. Overall, despite a striking expulsion of cellular contents, adult neurons appear to be healthier after throwing out their trash.
Considering the large volume of exophers, we hypothesized they might include organelles. Indeed, both lysosomes (Extended Data Fig. 4) and mitochondria (Fig. 4a, b; Extended Data Fig. 5) can be extruded in exophers. Mitochondrially-localized GFP reporters revealed mitochondrial inclusion in budding and dissociated exophers, with punctate or filamentous morphology typical of adult mitochondrial networks (Fig. 4a, Extended Data Fig. 5a–c). To address whether impairing mitochondrial quality enhances the production of exophers, we genetically manipulated dct-1/BNIP3 (mitophagy), pink-1/PINK15 and pdr-1/Parkin16 (human Parkinson’s disease homologs implicated in mitochondrial maintenance), and ubl-517(mitochondrial unfolded protein response), (Fig. 4c, d). We conclude that multiple approaches toward genetic impairment of mitochondria can increase exopher-genesis.
To address the hypothesis that stressed or damaged mitochondria might be preferentially segregated to exophers, we utilized mitoROGFP, a mitochondrially localized reporter that changes its peak excitation wavelength from ~405 nm (oxidized) to 476 nm (reduced) according to the local oxidative environment18,19. We find a significant increase in the 405 nm (oxidized) / 476 nm (reduced) excitation ratio of mitochondria in exophers as compared to those in somas (Fig. 4e), roughly equivalent to the redox excitation ratio observed in C. elegans neurons subjected to H2O2-induced oxidative stress19. We confirmed higher oxidation scores using MitoTimer, an alternative reporter of mitochondrial matrix oxidation20 (Extended Data Fig. 5d). In addition, touch neurons of juglone-treated21 bzIs166[Pmec-4mCherry]; zhsEx17[Pmec-4mitoLS::ROGFP] animals had significantly higher numbers of mitochondria-including exophers than matched controls (Supplemental Data Fig. 5e). Although compromised mitochondrial health may impair neuronal proteostasis, thus increasing exopher production, our data establish that touch neurons can eject mitochondria via exophers, which raises the intriguing possibility that exopher-genesis may constitute a previously unappreciated removal-based mechanism of mitochondrial homeostasis.
What is the fate of the extruded exopher and its contents? With time, exopher fluorescence intensity diminishes or disappears (persistence times ~1–12 hours), possibly as exopher contents are degraded internally or digested by the neighboring hypodermis that fully surrounds the touch neuron and has degradative capabilities. Consequent to disruption of the C. elegans apoptotic engulfment genes ced-1 (homologue of mammalian CD91, LRP1 and MEGF10, and fly Draper), ced-6 (GULP) and ced-7 (ABC), ALMR neurons are associated with multiple exophers (Fig. 5a; Extended Data Fig. 6a). However, genetic manipulation of a parallel engulfment pathway comprising ced-2, ced-5, ced-10, ced-12, and psr-1, changed neither the frequency of exopher generation nor the detection of multiple exophers. Moreover, we did not detect the apoptotic “eat-me” signal phosphatidylserine (PS) on the exopher surface using a widely expressed PS-binding annexinV::GFP (0/43 exophers; Extended Data Fig. 6b). Our data suggest that hypodermal recognition/degradation of exophers and their contents occurs by mechanisms that are at least in part distinct from classical removal of apoptotic corpses, but involve the CED-1, CED-6, CED-7 proteins. Electron microscopy studies support that the hypodermis may mediate degradation of at least some of exopher contents (Extended Data Fig. 2d–f, h).
The lack of detectable PS “eat me” signal on exophers raised the question as to whether at least some exopher contents might be destined to elude hypodermal degradation. Indeed, fluorescent mCherry protein that was originally expressed specifically in touch neurons, or fluorescent DiI loaded into dye-filling neurons, appeared later in distant scavenger coelomocytes (Fig. 5b–d; Extended Data Fig. 6c). Blocking coelomocyte uptake capacity by cup-4 mutation22 caused fluorescent particles to accumulate outside neurons, possibly within the pseudocoelomic fluid (body cavity; Extended Data Fig. 6d, e). We conclude that some exopher contents transit the hypodermal tissue to be released into the pseudocoelom, from which materials can later be taken up by distant coelomocytes. Exophers can therefore mediate transfer of neuronal materials to remote cells.
Considerable excitement in the neurodegenerative disease field has been generated by the findings that mammalian neurons can extrude conformational disease proteins, including in Alzheimer’s, Parkinson’s and prion disease23. C. elegans exopher production constitutes a newly identified mechanism by which neurons can transfer cellular material (preferentially neurotoxic species) to other cells. Interestingly, in a C. elegans muscle model of prion toxicity, offending prion proteins were transferred among muscle cells and ultimately localized to coelomocytes24. We speculate that the basic mechanism we document here may correspond to a conserved pathway for the transfer of toxic contents out of multiple cell types. In this regard, it may be noteworthy that mammalian aggregated poly-Q expanded huntingtin can transfer between neurons via tunneling nanotubes25–27 that resemble thin connections between C. elegans somas and exophers, and that neuronal polyQ in Drosophila is transferred to glia via a process that requires the CED-1 homolog, DRAPER28.
Recent reports show mitochondria can transfer out of specific cells to contribute positive roles (mesenchymal stem cells via tunneling nanotubes29; astrocytes to neurons in a stroke model30), but our study underscores a generally underappreciated option for mitochondrial quality control: mitochondrial expulsion. The mito-expulsion we report in C. elegans touch neurons has a striking mammalian counterpart: mouse mitochondria originating in retinal ganglion cells can be extruded into neighboring astrocytes for degradation6 (with some intriguingly similar morphology to C. elegans exophers; see Fig. 1e of ref. 6). Although further study will be required to definitively establish the health status and fates of transferred mitochondria in the C. elegans model, it is tempting to speculate that transcellular degradation of mitochondria may be a more broadly utilized mechanism of mitochondrial quality control than currently appreciated, with associated potential importance in neuronal health.
Overall, although further experiments are needed to elucidate the detailed mechanisms at play, and validate the proposed functions of exophers in proteostasis and the removal of damaged organelles, we suggest that exopher production is a previously unrecognized mechanism for clearing out accumulating protein aggregates and dysfunctional organelles that threaten neuronal homeostasis (Extended Data Fig. 7). The analogous process in mammals could promote transfer of misfolded protein and/or dysfunctional mitochondria to neighboring cells, promoting human pathology in neurodegenerative disease if compromised. Mechanistic dissection of this novel facet of proteostasis and mitochondrial homeostasis should thus inform on fundamental mechanisms of neuronal maintenance and suggest novel targets for intervention in neurodegenerative disease.
C. elegans strains were cultured at 20 °C with standard methods31. Strains used were SK4005 zdIs5[Pmec-4GFP] (abbreviated in the text as Is[Pmec-4GFP]), ZB4065 bzIs166[Pmec-4mCherry1](abbreviated in the text as Is[Pmec-4mCh1]), ZB4066 bzIs167[Pmec-4mitogfp Pmec-4mCherry2] (abbreviated in the text as Is[Pmec-4mCh2]), ZB4067 bzIs167[Pmec-4mitogfp Pmec-4mCherry4]; igIs1[Pmec-7YFP Pmec-3htt57Q128::cfp lin-15+]10 (abbreviated in the text as Is[mCh2 ; Q128CFP]), sesIs2512[Pgcy-5GFP], sesIs25[Pflp-6 Aβ; Pgcy-5GFP]32, KWN176 rnyIs014[Pmec-4mCherry unc-119(+)], ZB4071 bzIs169[Pmec-18sid-1Psng-1YFP]; bzIs101[Pmec-4mCherry; Punc-119+], ZB4087 bzIs169[Pmec-18sid-1Psng-1YFP]; bzIs101[Pmec-4mCherry; Punc-119+]; hsf-1(sy441), BZ555 egIs1[Pdat-1GFP], ZB4070 bzIs168 [Pmec-7LMP-1::GFP], ZB4509 bzIs166[Pmec-4mCherry]; bzIs168[Pmec-7LMP-1::GFP], ZB4082 cup-4(ok837); bzIs166[pmec-4mCherry], ZB4083 smIS76 [Phsp-16ANV::GFP]33; bzIs166[Pmec-4mCherry], ZB4084 hsf-1(sy441); zdIs5[Pmec-4GFP], ZB4085 hsf-1(sy441); bzIs166 [Pmec-4mCherry], ZB4086 zdIs5[Pmec-4GFP]; bzIs166[Pmec-4mCherry], PTN73 pha-1(e2123); him-5(e1490); zhsEx17[Pmec-4mitoLS::ROGFP], RBW2834 rbw2834Si[Pmec-3::mitotimer::T54, CB-unc-119 + II ttTi5605] in unc-119 (ed3)20, QH3738 ced-1(e1735); zdIs5, QH3737ced-6(n1813); zdIs5, QH4623 ced-5(n1812); zdIs5, QH3768 ced-7(n2690); zdIs5, QH3130 ced-10(n3246); zdIs5, QH3533 psr-1(ok714); zdIs534, ZB4526 bzIs166[Pmec-4mCherry]; pdr-1(gk448), ZB4525 bzIs166[Pmec-4mCherry]; (pwIs979 [Pcup-4GFP::vps-29]cB-unc119), ZB4528 bzIs166[Pmec-4mCherry]; zhsEx17 [Pmec-4mitoLS::ROGFP], ZB4059 bzIs163 [Pmec-4::GCaMP3.0::SL2::mCherry], ZB4524 bzEx242 [Pmec-4::PH(plcDelta)::GFP]35, and wild type N2.
RNAi was administered through feeding animals with RNAi-expressing bacteria with standard methods36 with touch neurons RNAi-enhanced via SID-1 expression37. Exophers are readily visible at 400X total magnification, with high power dissecting microscopes. In general, exophers have the following features: a ~4 μm membrane-bound vesicle extruded from a neuron via a mechanism that temporarily includes a thin filamentous connection to the originating soma, but eventually breaks off. Contents of exophers can include neurotoxic proteins, mitochondria, and lysosomes; exophers are produced by native amphid neurons after dye-filling.
To synchronize animals, L4 stage hermaphrodites were selected and moved to test plates. The day after moving was considered adult day 1, and animals were scored on adult day 2 for the occurrence of exophers. For scoring of exophers, animals were immobilized by adding 100 μL of 10 mM tetramisole to the surface of the plate. Animals were measured on the plate with a Kramer dissecting scope with a 20x objective. The ALMR neuron was scored for the presence of an exopher, which was counted if greater than ¼ the size of the soma, as a threshold against inclusion of smaller species of extracellular vesicles. Exophers were also visible in live animals without anesthetic. RNAi experiments had a negative empty vector control. An mCherry knockdown was used to confirm RNAi had an effect in the neurons of interest. RNAi screens were performed with the strain bzIs169[Pmec-18sid-1 Psng-1YFP]; bzIs101[Pmec-4mCherry; unc-119+]. All genes were independently screened a minimum of three times.
For imaging, animals were mounted by placing them in a drop of cold, liquid 36% Pluronic F-127 with 1 mM tetramisole solution and pressed between two coverslips. The slides were brought to room temperature, solidifying the Pluronic F-127 gel and immobilizing the animals. Co-localization images were made using iVision software. Images were taken using a Zeiss Imager D1m upright compound microscope with a 40x dry objective. For confocal imaging, animals were immobilized by using 7.5% M9 agarose pads with 2.5 μl PolySciences 0.05 μm polystyrene microspheres. A Zeiss spinning disk confocal upright microscope with 100x oil immersion objective was used for select images to show additional details, including lysosomal imaging and connection imaging.
Adult day 2 PTN73 pha-1(e2123); him-5(e1490); zhsEx17[Pmec-4mitoLS::ROGFP] animals were mounted as above on a Leica SP5 II confocal microscope (Leica Microsystems, Exton, PA) with a 63x oil immersion lens. Samples were alternately excited with a 30% power 405 nm UV laser and a 30% power 476 nm visible laser with a sequential line scanning method. Emission was detected by HYD1 photon counting at 508–513 nm. Images were quantified using ImageJ. Images were thresholded to remove background. The 405 nm channel was divided by the 476 nm channel, and ROI measurement was used to quantify mean intensities.
MitoTimer encodes a dsRed derivative that fluoresces green when reduced (first synthesized), but irreversibly shifts tored fluorescence as it oxidizes 20. Adult day 2 rbw2834Si[Pmec-3::mitotimer::T54, CB-unc-119 + II ttTi5605] in unc-119 (ed3)20 animals were mounted as above on a Zeiss Imager D1m upright compound microscope with a 63x oil immersion lens. Samples were alternately measured under GFP and dsRed channels, keeping light intensity and exposure times constant between images. Images were quantified using ImageJ by selecting the ROI, subtracting the background, measuring red and green intensities, and calculating the red/green ratio.
Fluorescence quantification was performed in ImageJ by selecting the ROI, measuring the mean intensity, and subtracting background intensity.
Time-lapse imaging was performed with a 100x oil immersion objective with a motorized stage. 15 animals were mounted to a slide using a 7.5% M9 agarose pads with 2.5 μl PolySciences 0.05 μm polystyrene microspheres; coverslip was sealed with a 60:40 mix of Vaseline and paraffin wax. An iVision script was used to image selected locations every 8–15 minutes for 12 hours. Image analysis and video compilation were done manually.
Animals were washed off a plate into a 1.5 mL centrifuge tube with 1 mL M9 and 10 μL of 1 mM DiI. Animals were allowed to soak at room temperature for 3 hours. Animals were washed with M9 twice before mounting onto slides for imaging.
50 animals were synchronized at the L4 stage and 25 animals were measured on subsequent adult days, directly from the plate without anesthetics using a Kramer microscope. The animals were transferred to fresh plates every 2 days until adult day 8 to prevent crowding and starvation.
DAPI staining was performed after wash-harvesting with PBS and permeabilizing the membrane at −80 °C freezer for 10 minutes. After thawing, the supernatant was removed and animals were re-suspended in 1 mL cold methanol and incubated 5 minutes for fixation. Animals were washed with PBS twice and then stained in a 1 mL DAPI solution (200 ng/mL in PBS) for 30 minutes before mounting for microscopy.
Exopher and cell size was performed by measuring pixel length with Photoshop and comparing to a calibration scale for each objective used. Width was measured at the widest point.
MG132 (Sigma-Aldrich C2211) and Spautin-138 (Sigma-Aldrich SML0440) were dissolved in DMSO at 10 mM and 1 mM, respectively, and administered by placing 30 μL of the solution over the bacterial food lawn.
Juglone21 (Sigma-Aldrich 59990) was dissolved to a final concentration of 230 μM in a solution of 0.23% v/v ethanol in M9. Adult day 1 worms were transferred into either a 1 mL tube of the juglone solution or a 1 mL control tube of 0.23% v/v ethanol in M9 for 90 minutes. Animals were washed with M9 buffer, centrifuged, and recovered onto a microscope slide for imaging.
Hydroxyurea (Sigma-Aldrich H8627) was dissolved in distilled water to make a 1 M solution, of which 250 mL was added to a standard seeded NGM plate to reach a working concentration of 25 mM8. Plate was left at room temperature for 6 hours to allow for complete diffusion before transferring adult day 1 animals for measurement 24 hours later on adult day 2.
To assay for touch sensitivity, animals were stroked with a calibrated force probe on the anterior and posterior halves of the body. Reversal was an indication of a positive response. Animals responding to at least 3 out of 5 touches were considered sensitive. Animals responding to 2 or fewer touches were considered not sensitive.
Q128 aggregates can be visually distinguished in touch neuron somas with a 20x objective11,39. The aggregate exopher prediction experiment was done by separating day 1 adult animals into two populations, those that had one visible aggregate in the ALMR neuron and those that had two or more. The two populations were scored on the next day for exophers extruded from the ALMR neuron.
Young adults were screened by light microscopy to identify samples in which the ALM neurons had recently expelled an exopher. These animals were prepared for TEM analysis approximately 3 hours after initial selection by high pressure freezing and freeze substitution (HPF/FS) following a standard protocol for preservation of ultrastructure40. Briefly, after HPF, animals were exposed to 1% osmium tetroxide in acetone with 2% water added, kept at −90 °C for 5–6 days before slowly warming back to room temperature. Samples were rinsed in cold acetone and embedded in plastic resin before curing at high temperature for 1–2 days. Serial thin sections were collected on plastic-coated slot grids, post-stained with uranyl acetate, and examined with a Philips CM10 electron microscope. By looking in transverse sections for landmarks such as the 2nd bulb of the pharynx, it was possible to reach the vicinity of the ALM soma before collecting about 1,500 serial thin transverse sections. Having found the soma, one could then explore the region 30–50 μm posterior to the ALMR for evidence of the exopher.
Synchronized Is[Pmec-4mCh1] adult day 2 animals were immobilized on 7.5% M9 agarose pads with 2.5 μL PolySciences 0.05 μm polystyrene microspheres. Exopher centers were photo-bleached with 7 pulses of the MicroPoint pulsed nitrogen pumped dye laser (neutral density filter at position 9, Lumencor solid state light source) attached to a Zeiss Inverted Axio Observer microscope (100x 1.4 N.A. objective) on an anti-vibration table. 1 frame was recorded every 5 seconds using constant excitation intensity and exposure time with a Qimaging EXi Blue camera. Images were analyzed with ImageJ. Exopher fluorescence intensity was normalized to the intensity of the first data point in each series.
Adult day 4 bzIs163 [Pmec-4::GCaMP3.0::SL2::mCherry] worms expressing the genetically encoded calcium indicator GCaMP3.0 in the mechanosensory neurons were immobilized with 0.1% tetramisole on 3% agar pads. As described in Gabel et al.41, a Ti:Sapphire laser system was used to perform axotomy (10 KHz pulse rate, 15 nJ/pulse). Axons were cut 20 μm from the soma with five rapid exposures (0.25 seconds) to the laser beam, resulting in vaporization of the axon at the target point. Time-lapse images were taken 20 seconds before cutting and up to a minute following the cut, 1 frame/second. Two individuals with exophers connected to the soma and three individuals with exophers not connected to the soma were analyzed, with only the connected exophers showing any calcium response to axotomy.
Blinding was performed by lab members uninvolved in the relevant experiment. Strain and treatment information were recorded in a secret key and replaced with a symbol on the measurement plates. The data were unblinded following completion of the experiment. Animals were allocated to measurement plates randomly.
Sample size was established using G-power software to be able to detect moderate effects with 80% power at P = 0.05 after a replicate for routine measurements. For higher throughput, larger screens were designed to have an 80% power to meet the re-screening cutoff of P = 0.25. Data were considered normal by the Shapiro-Wilk normality test.
Because of variable RNAi outcomes in different trials, exopher numbers were always compared to the empty vector control for that particular experiment. Statistics were performed using a two tailed unpaired t-test between the trial means, considering neurons with an exopher as 1 and neurons without an exopher as 0. One-way ANOVA was performed with Dunnett’s test when multiple samples were compared to a single control, and with Tukey’s test when multiple samples were compared to each other. Details of statistics are described in figure legends.
The data that support the findings of this study are available from the corresponding author upon reasonable request.
|a. Exosome Biogenesis|
|Process Targeted||Gene Name||P-value|
|b. Cell Cycle-related|
|Gene Name||P-value||Gene Name||P-value|
|Diameter||30 nm–100 nm46||100 nm–1,000 nm46||500 nm–3,000 nm49||1,000 nm–7,800 nm|
|Timing of release||Tens of minutes47||Seconds47||40–200 minutes49||15–60 minutes|
|Mechanism of Release||Multi-vesicular bodies fuse to the cell membrane46||Outward budding and scission46||Expands at tip of retraction fibers49||Jettisoned from cell body|
|ESCRT Machinery involved||Yes47||ESCRT3; tsg-10147||Unknown||No|
|Attachment to releasing cell||No||No||Yes (via retraction fibers)49||Sometimes (via thin fiber)|
|Vesicular Contents||DNA, RNA, Proteins, Lipids46||DNA, RNA, Proteins, Lipids46||Cytosol, Proteins49||Mitochondria, Lysosomes, Protein Aggregates|
|Phosphatidylserine Distribution||Membrane outer leaflet46||Membrane outer leaflet48||Unknown||Not displayed on membrane outer leaflet|
We thank B. Grant for expert advice, C. Reina for time-lapse microscopy help; N. Kane and J. Kramer for confocal microscopy assistance; and H. Ushakov for construction of some genetic lines. We thank Geoff Perumal and Frank Macaluso for help with HPF fixations, and Chris Crocker for the cartoon in Extended Data Fig. 2b. Federico Sesti and Massimo Hilliard generously supplied C. elegans strains; Alex Mendenhall, Bryan Sands, Roger Brent constructed the MOSCI mitoTimer strain. Research was supported by the National Institutes of Health under award numbers 1R01NS086064 and 1R01AG046358. IM and RG were supported by the National Institute of General Medical Sciences under award number T32 GM008339. KN and DH were supported by NIH OD10943 (to DHH); Core EM facilities (Hall) NICHD P30 HD71593 for the RFK-IDDRC at Albert Einstein College of Medicine. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440) The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Author Contributions I.M., M.L.T., M.L.A., R.J.G. and G.H. conducted and designed experiments along with M.D. I.M. and M.D. wrote the manuscript with input from R.J.G., M.L.A. and M.L.T. C.V.G. and D.T. carried out calcium connection experiments. K.C.N. and D.H.H. carried out electron microscopy. J.A.P. and C.N. supplied the Q128 reagent and manuscript critiques.
Competing interests statement The authors declare that they have no competing financial interests.